A sub-10-nm, folic acid-conjugated gold nanoparticle as self-therapeutic treatment of tubulointerstitial fibrosis.

Nanomedicines for treating chronic kidney disease (CKD) are on the horizon, yet their delivery to renal tubules where tubulointerstitial fibrosis occurs remains inefficient. We report a folic acid-conjugated gold nanoparticle that can transport into renal tubules and treat tubulointerstitial fibrosis in mice with unilateral ureteral obstruction. The 3-nm gold core allows for the dissection of bio-nano interactions in the fibrotic kidney, ensures the overall nanoparticle (~7 nm) to be small enough for glomerular filtration, and naturally inhibits the p38α mitogen-activated protein kinase in the absence of chemical or biological drugs. The folic acids support binding to selected tubule cells with overexpression of folate receptors and promote retention in the fibrotic kidney. Upon intravenous injection, this nanoparticle can selectively accumulate in the fibrotic kidney over the nonfibrotic contralateral kidney at ~3.6% of the injected dose. Delivery to the fibrotic kidney depends on nanoparticle size and disease stage. Notably, a single injection of this self-therapeutic nanoparticle reduces tissue degeneration, inhibits genes related to the extracellular matrix, and treats fibrosis more effectively than standard Captopril therapy. Our data underscore the importance of constructing CKD nanomedicines based on renal pathophysiology.

Eosinophils regulate intra-adipose axonal plasticity.

Sympathetic innervation regulates energy balance, and the nerve density in the adipose tissues changes under various metabolic states, resulting in altered neuronal control and conferring resilience to metabolic challenges. However, the impact of the immune milieu on neuronal innervation is not known. Here, we examined the regulatory role on nerve plasticity by eosinophils and found they increased cell abundance in response to cold and produced nerve growth factor (NGF) in the white adipose tissues (WAT). Deletion of Ngf from eosinophils or depletion of eosinophils impairs cold-induced axonal outgrowth and beiging process. The spatial proximity between sympathetic nerves, IL-33-expressing stromal cells, and eosinophils was visualized in both human and mouse adipose tissues. At the cellular level, the sympathetic adrenergic signal induced calcium flux in the stromal cells and subsequent release of IL-33, which drove the up-regulation of IL-5 from group 2 innate lymphoid cells (ILC2s), leading to eosinophil accretion. We propose a feed-forward loop between sympathetic activity and type 2 immunity that coordinately enhances sympathetic innervation and promotes energy expenditure.

Using Portuguese BRCA pathogenic variation as a model to study the impact of human admixture on human health.

BACKGROUND: Admixture occurs between different ethnic human populations. The global colonization in recent centuries by Europeans led to the most significant admixture in human history. While admixture may enhance genetic diversity for better fitness, it may also impact on human health by transmitting genetic variants for disease susceptibility in the admixture population. The admixture by Portuguese global exploration initiated in the 15th century has reached over 20 million of Portuguese-heritage population worldwide. It provides a valuable model to study the impact of admixture on human health. BRCA1 and BRCA2 (BRCA) are two of the important tumor suppressor genes. The pathogenic variation (PV) in BRCA is well determined to cause high risk of hereditary breast and ovarian cancer. Tracing the distribution of Portuguese BRCA PV in Portuguese-heritage population will help to understand the impact of admixture on cancer susceptibility in modern humans. In this study, we analyzed the distribution of the Portuguese-originated BRCA variation in Brazilian population, which has high degree Portuguese-heritage. METHODS: By comprehensive data mining, standardization and annotation, we generated a Portuguese-derived BRCA variation dataset and a Brazilian-derived BRCA variation dataset. We compared the two BRCA variation datasets to identify the BRCA variants shared between the two populations. RESULTS: The Portuguese-derived BRCA variation dataset consists of 220 BRCA variants including 78 PVs from 11,482 Portuguese cancer patients, 93 (42.2%) in BRCA1 and 127 (57.7%) in BRCA2. Of the 556 Portuguese BRCA PV carriers carrying the 78 PVs, 331 (59.5%) carried the three Portuguese-BRCA founder PVs of BRCA1 c.2037delinsCC, BRCA1 c.3331_3334del and BRCA2 c.156_157insAlu. The Brazilian-derived BRCA variation dataset consists of 255 BRCA PVs from 7,711 cancer patients, 136 (53.3%) in BRCA1 and 119 (46.6%) in BRCA2. We developed an open database named dbBRCA-Portuguese ( https://genemutation.fhs.um.edu.mo/dbbrca-portuguese/ ) and an open database named dbBRCA-Brazilian ( https://genemutation.fhs.um.edu.mo/dbbrca-brazilian ) to host the BRCA variation data from Portuguese and Brazilian populations. We compared the BRCA PV datasets between Portuguese and Brazilian populations, and identified 29 Portuguese-specific BRCA PVs shared between Portuguese and Brazilian populations, 14 in BRCA1 including the Portuguese founder BRCA1 c.3331_3334del and BRCA1 c.2037delinsCC, and 15 in BRCA2 including the Portuguese founder BRCA2 c.156_157insAlu. Searching the 78 Portuguese BRCA PVs in over 5,000 ancient human genomes identified evolution origin for only 8 PVs in Europeans dated between 37,470 and 3,818 years before present, confirming the Portuguese-specificity of Portuguese BRCA PVs; comparing the 78 Portuguese BRCA PVs Portuguese, 255 Brazilian BRCA PVs, and 134 African BRCA PVs showed little overlapping, ruling out the possibility that the BRCA PVs shared between Portuguese and Brazilian may also be contributed by African. CONCLUSION: Our study provides evidence that the admixture in recent human history contributed to cancer susceptibility in modern humans.

TP53 R249S mutation in hepatic organoids captures the predisposing cancer risk.

BACKGROUND AND AIMS: Major genomic drivers of hepatocellular carcinoma (HCC) are nowadays well recognized, although models to establish their roles in human HCC initiation remain scarce. Here, we used human liver organoids in experimental systems to mimic the early stages of human liver carcinogenesis from the genetic lesions of TP53 loss and L3 loop R249S mutation. In addition, chromatin immunoprecipitation sequencing (ChIP-seq) of HCC cell lines shed important functional insights into the initiation of HCC consequential to the loss of tumor-suppressive function from TP53 deficiency and gain-of-function activities from mutant p53. APPROACH AND RESULTS: Human liver organoids were generated from surgical nontumor liver tissues. CRISPR knockout of TP53 in liver organoids consistently demonstrated tumor-like morphological changes, increased in stemness and unrestricted in vitro propagation. To recapitulate TP53 status in human HCC, we overexpressed mutant R249S in TP53 knockout organoids. A spontaneous increase in tumorigenic potentials and bona fide HCC histology in xenotransplantations were observed. ChIP-seq analysis of HCC cell lines underscored gain-of-function properties from L3 loop p53 mutants in chromatin remodeling and overcoming extrinsic stress. More importantly, direct transcriptional activation of PSMF1 by mutant R249S could increase organoid resistance to endoplasmic reticulum stress, which was readily abrogated by PSMF1 knockdown in rescue experiments. In a patient cohort of primary HCC tumors and genome-edited liver organoids, quantitative polymerase chain reaction corroborated ChIP-seq findings and verified preferential genes modulated by L3 mutants, especially those enriched by R249S. CONCLUSIONS: We showed differential tumorigenic effects from TP53 loss and L3 mutations, which together confer normal hepatocytes with early clonal advantages and prosurvival functions.

Pathogenic variants in human DNA damage repair genes mostly arose in recent human history.

BACKGROUND: Genome stability is maintained by the DNA damage repair (DDR) system composed of multiple DNA repair pathways of hundreds of genes. Germline pathogenic variation (PV) in DDR genes damages function of the affected DDR genes, leading to genome instability and high risk of diseases, in particular, cancer. Knowing evolutionary origin of the PVs in human DDR genes is essential to understand the etiology of human diseases. However, answer to the issue remains largely elusive. In this study, we analyzed evolutionary origin for the PVs in human DDR genes. METHODS: We identified 169 DDR genes by referring to various databases and identified PVs in the DDR genes of modern humans from ClinVar database. We performed a phylogenetic analysis to analyze the conservation of human DDR PVs in 100 vertebrates through cross-species genomic data comparison using the phyloFit program of the PHAST package and visualized the results using the GraphPad Prism software and the ggplot module. We identified DDR PVs from over 5000 ancient humans developed a database to host the DDR PVs ( https://genemutation.fhs.um.edu.mo/dbDDR-AncientHumans ). Using the PV data, we performed a molecular archeological analysis to compare the DDR PVs between modern humans and ancient humans. We analyzed evolution selection of DDR genes across 20 vertebrates using the CodeML in PAML for phylogenetic analysis. RESULTS: Our phylogenic analysis ruled out cross-species conservation as the origin of human DDR PVs. Our archeological approach identified rich DDR PVs shared between modern and ancient humans, which were mostly dated within the last 5000 years. We also observed similar pattern of quantitative PV distribution between modern and ancient humans. We further detected a set of ATM, BRCA2 and CHEK2 PVs shared between human and Neanderthals. CONCLUSIONS: Our study reveals that human DDR PVs mostly arose in recent human history. We propose that human high cancer risk caused by DDR PVs can be a by-product of human evolution.

First Insight into Fetal Exposure to Legacy and Emerging Plasticizers Revealed by Infant Hair and Meconium: Occurrence, Biotransformation, and Accumulation.

Epidemiological studies have demonstrated the embryonic and developmental toxicity of plasticizers. Thus, understanding the in utero biotransformation and accumulation of plasticizers is essential to assessing their fate and potential toxicity in early life. In the present study, 311 infant hair samples and 271 paired meconium samples were collected at birth in Guangzhou, China, to characterize fetal exposure to legacy and emerging plasticizers and their metabolites. Results showed that most of the target plasticizers were detected in infant hair, with medians of 9.30, 27.6, and 0.145 ng/g for phthalate esters (PAEs), organic phosphate ester (OPEs), and alternative plasticizers (APs), and 1.44, 0.313, and 0.066 ng/g for the metabolites of PAEs, OPEs, and APs, respectively. Positive correlations between plasticizers and their corresponding primary metabolites, as well as correlations among the oxidative metabolites of bis(2-ethylhexyl) phthalate (DEHP) and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH), were observed, indicating that infant hair retained the major phase-I metabolism of the target plasticizers. While no positive correlations were found in parent compounds or their primary metabolites between paired infant hair and meconium, significant positive correlations were observed among secondary oxidative metabolites of DEHP and DINCH in hair and meconium, suggesting that the primary metabolites in meconium come from hydrolysis of plasticizers in the fetus but most of the oxidative metabolites come from maternal-fetal transmission. The parent compound/metabolite ratios in infant hair showed a decreasing trend across pregnancy, suggesting in utero accumulation and deposition of plasticizers. To the best of our knowledge, this study is the first to report in utero exposure to both parent compounds and metabolites of plasticizers by using paired infant hair and meconium as noninvasive biomonitoring matrices and provides novel insights into the fetal biotransformation and accumulation of plasticizers across pregnancy.

First Report of Meloidogyne enterolobii Infecting Yellow pitaya (Selenicereus megalanthus) in China.

Yellow pitaya, Selenicereus megalanthus, is a night-blooming, climbing cacti of tropical origin, which has received increasing attention for its potential as a new exotic fruit crop (Lichtenzveig et al. 2000). The crop is grown extensively in Hainan Province, China (3000 ha). In October 2021, a survey was conducted on a farm located in Changjiang (19°21'4″N, 108°47'2″S), Hainan Province, China. Some yellow pitaya plants were found that were stunted and chlorotic, with abnormally thin stems (Fig. 1B), and no symptoms on healthy plants (Fig. 1A). Dead plants were also observed. Many galls and females with egg masses were observed on roots (Figs. 1C & 1D). This is typical of root-knot nematode (RKN) infections, and the incidence of infection was 36.7%. Meloidogyne sp. females and egg masses were dissected from roots of the infected plants. The perineal pattern of females (n= 5) was round to oval-shaped with a high dorsal arch (Figs. 1I & 1J). Second-stage juveniles (J2s) had truncated lips (Figs. 1E & 1F) and long-conical tails with bluntly rounded tips (Figs. 1G & 1H). The J2s body length (n= 24) averaged 416.79 µm (349.21 to 472.76 µm) with a mean width of 15.36 µm (12.47 to 17.52 µm); mean stylet length was 11.16 µm (10.10 to 13.23 µm); tail length averaged 53.73 µm (43.46 to 65.90 µm). The morphological characteristics matched the original description of M. enterolobii (Yang and Eisenback 1983). Males were not found. Genomic DNA was extracted from eight single J2s, and the mitochondrial (mtDNA) region between COII and 16S rRNA gene was amplified with primers C2F3/1108 (Powers and Harris 1993). A 652-bp DNA fragment was obtained, for which the sequence (GenBank accession no. OP122499) was 100% identical to the sequences of M. enterolobii isolates from China（MN269947）and the USA (MN809527). Furthermore, species identification was also confirmed using M. enterolobii specific primers Me-F/Me-R. An amplicon size of ∼230 bp was obtained, which is consistent with those previously reported for M. enterolobii (Fig. 2) (Long et al. 2006). Therefore, this population was identified as M. enterolobii based on morphological and molecular characteristics. Pathogenicity tests were performed in the greenhouse at 26℃ and 80% relative humidity with a 14-h/10-h light/dark photoperiod. Ten RKN-free S. megalanthus seedlings were transplanted into pots containing sterilized soil. After 3 weeks, the roots of 5 plants were inoculated with 3,000 eggs and J2s of M. enterolobii per plant. Five uninoculated plants were used as control plants. After 2 months, no galling or symptoms were observed on the control plants. All inoculated plants had galled roots similar to those observed in the field. Females and egg masses were obtained by dissecting galls. The nematode reproduction factor (RF= final population/initial population) was 1.9. Adult females (n= 5) dissected from inoculated plants were identified as M. enterolobii with sequence-specific primers Me-F/Me-R, thus confirming pathogenicity. The pathogenicity test was carried out twice with similar results. M. enterolobii is one of the most damaging species of RKN, due to its wide host range, high level of pathogenicity, and ability to develop and reproduce on several crops with resistance genes to other RKN (Castagnone-Sereno 2012). To our knowledge, this is the first report of S. megalanthus (yellow pitaya) as a host of M. enterolobii in China. Further studies are needed to develop and evaluate integrated management strategies.

Unique molecular characteristics of NAFLD-associated liver cancer accentuate ß-catenin/TNFRSF19-mediated immune evasion.

BACKGROUND & AIMS: The hepatic manifestation of the metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), can lead to the development of hepatocellular carcinoma (HCC). Despite a strong causative link, NAFLD-HCC is often underrepresented in systematic genome explorations. METHODS: Herein, tumor-normal pairs from 100 patients diagnosed with NAFLD-HCC were subject to next-generation sequencing. Bioinformatic analyses were performed to identify key genomic, epigenomic and transcriptomic events associated with the pathogenesis of NAFLD-HCC. Establishment of primary patient-derived NAFLD-HCC culture was used as a representative human model for downstream in vitro investigations of the underlying CTNNB1 S45P driver mutation. A syngeneic immunocompetent mouse model was used to further test the involvement of CTNNB1mutand TNFRSF19 in reshaping the tumor microenvironment. RESULTS: Mutational processes operative in the livers of patients with NAFLD inferred susceptibility to tumor formation through defective DNA repair pathways. Dense promoter mutations and dysregulated transcription factors accentuated activated transcriptional regulation in NAFLD-HCC, in particular the enrichment of MAZ-MYC activities. Somatic events common in HCCs arising from NAFLD and viral hepatitis B infection underscore similar driver pathways, although an incidence shift highlights CTNNB1mut dominance in NAFLD-HCC (33%). Immune exclusion correlated evidently with CTNNB1mut. Chromatin immunoprecipitation-sequencing integrated with transcriptome and immune profiling revealed a unique transcriptional axis, wherein CTNNB1mut leads to an upregulation of TNFRSF19 which subsequently represses senescence-associated secretory phenotype-like cytokines (including IL6 and CXCL8). This phenomenon could be reverted by the Wnt-modulator ICG001. CONCLUSIONS: The unique mutational processes in the livers of patients with NAFLD and NAFLD-HCC allude to a "field effect" involving a gain-of-function role of CTNNB1 mutations in immune exclusion. LAY SUMMARY: The increasing prevalence of metabolic syndrome in adult populations means that NAFLD is poised to be the major cause of liver cancer in the 21st century. We showed a strong "field effect" in the livers of patients with NAFLD, wherein activated ß-catenin was involved in reshaping the tumor-immune microenvironment.

The Association between Spermidine/Spermine N1-Acetyltransferase (SSAT) and Human Malignancies.

Spermidine/spermine N1-acetyltransferase (SSAT) functions as a critical enzyme in maintaining the homeostasis of polyamines, including spermine, spermidine, and putrescine, in mammalian cells. SSAT is a catalytic enzyme that indirectly regulates cellular physiologies and pathways through interaction with endogenous and exogenous polyamines. Normally, SSAT exhibits only at a low cellular level, but upon tumorigenesis, the expression, protein level, and activities of SSAT are altered. The alterations induce cellular damages, including oxidative stress, cell cycle arrest, DNA dynamics, and proliferation by influencing cellular mechanisms and signaling pathways. The expression of SSAT has been reported in various studies to be altered in different cancers, and it has been correlated with tumor development and progression. Tumor grades and stages are associated with the expression levels of SSAT. SSAT can be utilized as a target for substrate binding, and excreted metabolites may be used as a novel cancer biomarker. There is also potential for SSAT to be developed as a therapeutic target. Polyamine analogs could increase SSAT expression and increase the cytotoxicity of chemotherapy to tumor cells. Drugs targeting polyamines and SSAT expression have the potential to be developed into new cancer treatments in the future.

Genomic and Epigenomic Features of Primary and Recurrent Hepatocellular Carcinomas.

BACKGROUND & AIMS: Intratumor heterogeneity and divergent clonal lineages within and among primary and recurrent hepatocellular carcinomas (HCCs) produce challenges to patient management. We investigated genetic and epigenetic variations within liver tumors, among hepatic lesions, and between primary and relapsing tumors. METHODS: Tumor and matched nontumor liver specimens were collected from 113 patients who underwent partial hepatectomy for primary or recurrent HCC at 2 hospitals in Hong Kong. We performed whole-genome, whole-exome, or targeted capture sequencing analyses of 356 HCC specimens collected from multiple tumor regions and matched initial and recurrent tumors. We performed parallel DNA methylation profiling analyses of 95 specimens. Genomes and epigenomes of nontumor tissues that contained areas of cirrhosis or fibrosis were analyzed. We developed liver cancer cell lines that endogenously expressed a mutant form of TP53 (R249S) or overexpressed mutant forms of STAT3 (D170Y, K348E, and Y640F) or JAK1 (S703I and L910P) and tested the abilities of pharmacologic agents to reduce activity. Cells were analyzed by immunoblotting and chromatin immunoprecipitation with quantitative polymerase chain reaction. RESULTS: We determined the monoclonal origins of individual tumors using a single-sample collection approach that captured more than 90% of mutations that are detected in all regions of tumors. Phylogenetic and phyloepigenetic analyses showed interactions and codependence between the genomic and epigenomic features of HCCs. Methylation analysis showed a field effect in cirrhotic liver tissues that predisposes them to tumor development. Comparisons of genetic features showed that 52% of recurrent HCCs derive from the clonal lineage of the initial tumor. The clonal origin of recurrent HCCs allowed construction of a temporal map of genetic alterations that were associated with tumor recurrence. Activation of JAK signaling to STAT was a characteristic of HCC progression via mutations that are associated with response to drug sensitivity. The combination of a mutation that increases the function of TP53 and the 17p chromosome deletion might provide liver cancer cells with a replicative advantage. Chromatin immunoprecipitation analysis of TP53 with the R249S substitution showed its interaction with genes that encode chromatin regulators (MLL1 and MLL2). We validated MLL1 and MLL2 as direct targets of TP53R249S and affirmed their association in the cancer genome atlas data set. The MLL-complex antagonists MI-2-2 (inhibitor of protein interaction) and OICR-9492 (inhibitor of activity) specifically inhibited proliferation of HCC cells that express TP53R249S at nanomolar concentrations. CONCLUSIONS: We performed a systematic evaluation of intra- and intertumor genetic heterogeneity in HCC samples and identified genetic and epigenetic changes that are associated with tumor progression and recurrence. We identified chromatin regulators that are up-regulated by mutant TP53 in HCC cells and inhibitors that reduce proliferation of these cells. DNA methylation patterns in cirrhotic or fibrotic liver tissues might be used to identify those at risk of HCC development.

Genomic and Epigenomic Features of Primary and Recurrent Hepatocellular Carcinomas.

BACKGROUND & AIMS: Intratumor heterogeneity and divergent clonal lineages within and among primary and recurrent hepatocellular carcinomas (HCCs) produce challenges to patient management. We investigated genetic and epigenetic variations within liver tumors, among hepatic lesions, and between primary and relapsing tumors. METHODS: Tumor and matched nontumor liver specimens were collected from 113 patients who underwent partial hepatectomy for primary or recurrent HCC at 2 hospitals in Hong Kong. We performed whole-genome, whole-exome, or targeted capture sequencing analyses of 356 HCC specimens collected from multiple tumor regions and matched initial and recurrent tumors. We performed parallel DNA methylation profiling analyses of 95 specimens. Genomes and epigenomes of nontumor tissues that contained areas of cirrhosis or fibrosis were analyzed. We developed liver cancer cell lines that endogenously expressed a mutant form of TP53 (R249S) or overexpressed mutant forms of STAT3 (D170Y, K348E, and Y640F) or JAK1 (S703I and L910P) and tested the abilities of pharmacologic agents to reduce activity. Cells were analyzed by immunoblotting and chromatin immunoprecipitation with quantitative polymerase chain reaction. RESULTS: We determined the monoclonal origins of individual tumors using a single sample collection approach that captured more than 90% of mutations that are detected in all regions of tumors. Phylogenetic and phylo-epigenetic analyses revealed interactions and codependence between the genomic and epigenomic features of HCCs. Methylation analysis revealed a field effect in cirrhotic liver tissues that predisposes them to tumor development. Comparisons of genetic features revealed that 52% of recurrent HCCs derive from the clonal lineage of the initial tumor. The clonal origin if recurrent HCCs allowed construction of a temporal map of genetic alterations that associated with tumor recurrence. Activation of JAK signaling to STAT was a characteristic of HCC progression via mutations that associate with response to drug sensitivity. The combination of a mutation that increases the function of TP53 and the 17p chromosome deletion might provide liver cancer cells with a replicative advantage. Chromatin immunoprecipitation analysis of TP53 with the R249S substitution revealed its interaction with genes that encode chromatin regulators (MLL1 and MLL2). We validated MLL1 and MLL2 as direct targets of TP53R249S and affirmed their association in the Cancer Genome Atlas dataset. The MLL-complex antagonists MI-2-2 (inhibitor of protein interaction) and OICR-9492 (inhibitor of activity) specifically inhibited proliferation of HCC cells that express TP53R249S at nanomolar concentrations. CONCLUSIONS: We performed a systematic evaluation of intra- and intertumor genetic heterogeneity in HCC samples and identified genetic and epigenetic changes that associate with tumor progression and recurrence. We identified chromatin regulators that are upregulated by mutant TP53 in HCC cells and inhibitors that reduce proliferation of these cells. DNA methylation patterns in cirrhotic or fibrotic liver tissues might be used to identify those at risk of HCC development.

Speeding up in Vitro Discovery of Structure-Switching Aptamers via Magnetic Cross-Linking Precipitation.

We report here a modified aptamer selection method, magnetic cross-linking precipitation (MCP)-SELEX, for highly efficient library enrichment and aptamer isolation. MCP-SELEX isolates bound aptamers via highly efficient chemical cross-linking between amino groups of target proteins and activated carboxylic acid groups on magnetic beads (>90% coupling efficiency). Importantly, MCP-SELEX avoids surface interferences in conventional target-fixed methods and substantially minimizes nonspecific binding. The enrichment efficiencies of MCP-SELEX for various proteins (PD-L1, ubiquitin, thrombin, and HSA) were all greatly higher than those of the conventional target-bound magnetic bead based-SELEX (MB-SELEX). Antithrombin aptamer with KD of 33 nM was successfully isolated by four rounds of MCP-SELEX. MCP-SELEX also enabled the efficient aptamer isolation by coupling with MB-SELEX or falling-off-SELEX. We identified structure-switching aptamers (SSAs) that specifically bind to HSA with low nanomolar dissociation constant via three rounds of MCP-SELEX and 1 round of falling-off-SELEX. Our HSA SSAs also have ∼3-fold higher specificity against streptavidin relative to thrombin SSAs discovered through falling-off-SELEX only. The enriched library has ∼78-fold higher signal-to-noise ratio (the number of DNAs eluted by 50 nM HSA divided by the number of DNAs self-dissociated in blank buffer) than that obtained by 4 rounds of direct falling-off-SELEX. We finally demonstrated the application of the selected SSA in fluorescent detection of HSA in urine with diagnostic required sensitivity and dynamic range. We expect that MCP-SELEX may be coupled with other selection methods to substantially accelerate aptamer discovery.

Forward and reverse mutations in stages of cancer development.

BACKGROUND: Massive occurrences of interstitial loss of heterozygosity (LOH) likely resulting from gene conversions were found by us in different cancers as a type of single-nucleotide variations (SNVs), comparable in abundance to the commonly investigated gain of heterozygosity (GOH) type of SNVs, raising the question of the relationships between these two opposing types of cancer mutations. METHODS: In the present study, SNVs in 12 tetra sample and 17 trio sample sets from four cancer types along with copy number variations (CNVs) were analyzed by AluScan sequencing, comparing tumor with white blood cells as well as tissues vicinal to the tumor. Four published "nontumor"-tumor metastasis trios and 246 pan-cancer pairs analyzed by whole-genome sequencing (WGS) and 67 trios by whole-exome sequencing (WES) were also examined. RESULTS: Widespread GOHs enriched with CG-to-TG changes and associated with nearby CNVs and LOHs enriched with TG-to-CG changes were observed. Occurrences of GOH were 1.9-fold higher than LOH in "nontumor" tissues more than 2 cm away from the tumors, and a majority of these GOHs and LOHs were reversed in "paratumor" tissues within 2 cm of the tumors, forming forward-reverse mutation cycles where the revertant LOHs displayed strong lineage effects that pointed to a sequential instead of parallel development from "nontumor" to "paratumor" and onto tumor cells, which was also supported by the relative frequencies of 26 distinct classes of CNVs between these three types of cell populations. CONCLUSIONS: These findings suggest that developing cancer cells undergo sequential changes that enable the "nontumor" cells to acquire a wide range of forward mutations including ones that are essential for oncogenicity, followed by revertant mutations in the "paratumor" cells to avoid growth retardation by excessive mutation load. Such utilization of forward-reverse mutation cycles as an adaptive mechanism was also observed in cultured HeLa cells upon successive replatings. An understanding of forward-reverse mutation cycles in cancer development could provide a genomic basis for improved early diagnosis, staging, and treatment of cancers.

Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs.

Exosomes are increasingly recognized as important mediators of cell-cell communication in cancer progression through the horizontal transfer of RNAs and proteins to neighboring or distant cells. Hepatocellular carcinoma (HCC) is a highly malignant cancer, whose metastasis is largely influenced by the tumor microenvironment. The possible role of exosomes in the interactions between HCC tumor cell and its surrounding hepatic milieu are however largely unknown. In this study, we comprehensively characterized the exosomal RNA and proteome contents derived from three HCC cell lines (HKCI-C3, HKCI-8 and MHCC97L) and an immortalized hepatocyte line (MIHA) using Ion Torrent sequencing and mass spectrometry, respectively. RNA deep sequencing and proteomic analysis revealed exosomes derived from metastatic HCC cell lines carried a large number of protumorigenic RNAs and proteins, such as MET protooncogene, S100 family members and the caveolins. Of interest, we found that exosomes from motile HCC cell lines could significantly enhance the migratory and invasive abilities of non-motile MIHA cell. We further demonstrated that uptake of these shuttled molecules could trigger PI3K/AKT and MAPK signaling pathways in MIHA with increased secretion of active MMP-2 and MMP-9. Our study showed for the first time that HCC-derived exosomes could mobilize normal hepatocyte, which may have implication in facilitating the protrusive activity of HCC cells through liver parenchyma during the process of metastasis.

Nanoprobe-Enhanced, Split Aptamer-Based Electrochemical Sandwich Assay for Ultrasensitive Detection of Small Molecules.

It is quite challenging to improve the binding affinity of antismall molecule aptamers. We report that the binding affinity of anticocaine split aptamer pairs improved by up to 66-fold by gold nanoparticles (AuNP)-attached aptamers due to the substantially increased local concentration of aptamers and multiple and simultaneous ligand interactions. The significantly improved binding affinity enables the detection of small molecule targets with unprecedented sensitivity, as demonstrated in nanoprobe-enhanced split aptamer-based electrochemical sandwich assays (NE-SAESA). NE-SAESA replaces the traditional molecular reporter probe with AuNPs conjugated to multiple reporter probes. The increased binding affinity allowed us to use 1,000-fold lower reporter probe concentrations relative to those employed in SAESA. We show that the near-elimination of background in NE-SAESA effectively improves assay sensitivity by ∼1,000-100,000-fold for ATP and cocaine detection, relative to equivalent SAESA. With the ongoing development of new strategies for the selection of aptamers, we anticipate that our sensor platform should offer a generalizable approach for the high-sensitivity detection of diverse targets. More importantly, we believe that NE-SAESA represents a novel strategy to improve the binding affinity between a small molecule and its aptamer and potentially can be extended to other detection platforms.

Kinetic adsorption profile and conformation evolution at the DNA-gold nanoparticle interface probed by dynamic light scattering.

The kinetic adsorption profile at the DNA-gold nanoparticle (AuNP) interface is probed by following the binding and organization of thiolated linear DNA and aptamers of varying chain lengths (15, 30, 44, and 51 mer) to the surface of AuNPs (13.0 ± 1.0 nm diameter). A systematic investigation utilizing dynamic light scattering has been performed to directly measure the changes in particle size during the course of a typical aging-salting thiolated DNA/AuNP preparation procedure. We discuss the effect of DNA chain length, composition, salt concentration, and secondary structure on the kinetics and conformation at the DNA-AuNP interface. The adsorption kinetics are chain-length dependent, composition independent, and not diffusion rate limited for the conditions we report here. The kinetic data support a mechanism of stepwise adsorption of thiols to the surface of AuNPs and reorganization of the thiols at the interface. Very interestingly, the kinetic increases of the particle sizes are modeled accurately by the pseudo-second-order rate model, suggesting that DNA could possess the statistically well-defined conformational evolution. Together with other experimental evidence, we propose a dynamic inner-layer and outer-tail (DILOT) model to describe the evolution of the DNA conformation after the initial adsorption of a single oligonucleotide layer. According to this model, the length of the tails that extend from the surface of AuNPs, capable for hybridization or molecular recognition, can be conveniently calculated. Considering the wide applications of DNA/AuNPs, the results should have important implications in sensing and DNA-directed nanoparticle assembly.

KfoE encodes a fructosyltransferase involved in capsular polysaccharide biosynthesis in Escherichia coli K4.

Escherichia coli K4 synthesizes a capsular polysaccharide (CPS) consisting of a fructose-branched chondroitin (GalNAc-GlcA(fructose)n), which is a biosynthetic precursor of chondroitin sulfate. Here, the role of kfoE in the modification of the chondroitin backbone was investigated using knock-out and recombinant complementation experiments. kfoE disruption and complementation had no significant effect on cell growth. CPS production was increased by 15 % in the knock-out strain, and decreased by 21 % in the knock-out strain complemented with recombinant kfoE. CPS extracted from the knock-out strain was chondroitin, whereas CPS extracted from the complemented strain was a fructose-branched chondroitin. The results demonstrated that the kfoE gene product altered the fructose group at the C3 position of the GlcA residue during production of K4CPS.

Neuroimmune regulation in the pancreas.

The pancreas exerts endocrine and exocrine functions in energy balance. The neural innervation and immune milieu are both crucial in supporting pancreatic homeostasis. The neuronal network connects the pancreas with the central nervous system (CNS) and the enteric nervous system (ENS) and sustains metabolic activities. The nerves in the pancreas are categorized as spinal sensory afferent fibers, vagal sensory afferent nerves, autonomic fibers of both sympathetic and parasympathetic divisions, and fibers from the ENS and intrapancreatic ganglia. They innervate different regions and various cell types, which collectively determine physiological functions. Studies have established that the diverse pathological conditions, including pancreatitis, diabetes, and pancreatic tumor, are attributed to aberrant immune reactions; however, it is largely not clear how the neuronal network may influence the disease conditions. Enlightened by the recent advances illuminating the organ-wide neuronal architecture and the dysfunctions in pancreatic disorders, this review will highlight emerging opportunities to explore the cellular interrelationship, particularly the neuroimmune components in pancreatic health and diseases.

Pathogenic variants in human DNA damage repair genes mostly arose after the latest human out-of-Africa migration.

Introduction: The DNA damage repair (DDR) system in human genome is pivotal in maintaining genomic integrity. Pathogenic variation (PV) in DDR genes impairs their function, leading to genome instability and increased susceptibility to diseases, especially cancer. Understanding the evolution origin and arising time of DDR PV is crucial for comprehending disease susceptibility in modern humans. Methods: We used big data approach to identify the PVs in DDR genes in modern humans. We mined multiple genomic databases derived from 251,214 modern humans of African and non-Africans. We compared the DDR PVs between African and non-African. We also mined the DDR PVs in the genomic data derived from 5,031 ancient humans. We used the DDR PVs from ancient humans as the intermediate to further the DDR PVs between African and non-African. Results and discussion: We identified 1,060 single-base DDR PVs across 77 DDR genes in modern humans of African and non-African. Direct comparison of the DDR PVs between African and non-African showed that 82.1% of the non-African PVs were not present in African. We further identified 397 single-base DDR PVs in 56 DDR genes in the 5,031 ancient humans dated between 45,045 and 100 years before present (BP) lived in Eurasian continent therefore the descendants of the latest out-of-Africa human migrants occurred 50,000-60,000 years ago. By referring to the ancient DDR PVs, we observed that 276 of the 397 (70.3%) ancient DDR PVs were exclusive in non-African, 106 (26.7%) were shared between non-African and African, and only 15 (3.8%) were exclusive in African. We further validated the distribution pattern by testing the PVs in BRCA and TP53, two of the important genes in genome stability maintenance, in African, non-African, and Ancient humans. Our study revealed that DDR PVs in modern humans mostly emerged after the latest out-of-Africa migration. The data provides a foundation to understand the evolutionary basis of disease susceptibility, in particular cancer, in modern humans.

CHHM: a Manually Curated Catalogue of Human Histone Modifications Revealing Hotspot Regions and Unique Distribution Patterns.

Histone modification is one of the key elements in epigenetic control and plays important roles in regulation of biological processes and disease development. Currently, records of human histone modifications with various levels of confidence in evidence are scattered in various knowledgebases and databases. In the present study, a curated catalogue of human histone modifications, CHHM, was obtained by manual retrieval, evidence assessment, and integration of modification records from 10 knowledgebases/databases and 3 complementary articles. CHHM contains 6612 nonredundant modification entries covering 31 types of modifications (including 9 types of emerging modifications) and 2 types of histone-DNA crosslinks, that were identified in 11 H1 variants, 21 H2A variants, 21 H2B variants, 9 H3 variants, and 2 H4 variants. For ease of visualization and accessibility, modification entries are presented with aligned protein sequences in an Excel file. Confidence level in evidence is provided for each entry. Acylation modifications contribute to the highest number of modification entries in CHHM. This supports that cellular metabolic status plays a very important role in epigenetic control. CHHM reveals modification hotspot regions and uneven distribution of the modification entries across the histone families. Such uneven distribution may suggest that a particular histone family is more susceptible to certain types of modifications. CHHM not only serves as an important and user-friendly resource for biomedical and clinical researches involving histone modifications and transcriptional regulation, but also provides new insights for basic researches in the mechanism of human histone modifications and epigenetic control.