m6A-induced TRIB3 regulates Hippo pathway through interacting with LATS1 to promote the progression of lung adenocarcinoma.

Recent studies have indicated that dysregulation of the Hippo/Yes-associated protein (YAP) axis is associated with tumor progression and therapy resistance in various cancer types, including lung adenocarcinoma (LUAD). Understanding the regulation of Hippo signaling in LUAD is of great significance. Elevated levels of TRIB3, a pseudo kinase, have been observed in certain lung malignancies and are associated with an unfavorable prognosis. Our research aims to investigate whether increased TRIB3 levels enhance the malignant characteristics of LUAD cells and tumor progression through its interaction with the Hippo signaling pathway. In this study, we reported a positive correlation between elevated expression of TRIB3 and LUAD progression. Additionally, TRIB3 has the ability to enhance TEAD luciferase function and suppress Hippo pathway activity. Moreover, TRIB3 increases total YAP protein levels and promotes YAP nuclear localization. Mechanistic experiments revealed that TRIB3 directly interacts with large tumor suppressor kinase 1 (LATS1), thereby suppressing Hippo signaling. Moreover, the decrease in METTL3-mediated N6-methyladenosine modification of TRIB3 results in a substantial elevation of its expression levels in LUAD cells. Collectively, our research unveils a novel discovery that TRIB3 enhances the growth and invasion of LUAD cells by interacting with LATS1 and inhibiting the Hippo signaling pathway. TRIB3 may serve as a potential biomarker for an unfavorable prognosis and a target for novel treatments in YAP-driven lung cancer.

Activation of CHPF by transcription factor NFIC promotes NLRP3 activation during the progression of colorectal cancer.

Given the role of chondroitin polymerizing factor (CHPF) in several cancers, we investigated its role in the progression of colorectal cancer (CRC) and its association with NLRP3 inflammasome activation. High expression of CHPF in CRC predicted poor patient prognosis. Using colony formation, EdU staining, wound healing, Transwell invasion, and flow cytometry assays, we revealed that the downregulation of CHPF inhibited the malignant behavior of CRC cells. CHPF promoted NLRP3 inflammasome activation by inducing the MAPK signaling pathway, as evidenced by enhanced expression of Phos-ERK1/2, Phos-MEK1, Phos-MEK2, and NLRP3. Additionally, nuclear factor 1 C-type (NFIC) was revealed as a potential upstream transcription factor of CHPF in the modulation of CRC, and the anti-tumor effects elicited through its knockdown were compromised by CHPF in vitro and in vivo. In summary, we demonstrated that NFIC promoted NLRP3 activation to support CRC development via the CHPF-mediated MAPK signaling.

Aging under endocrine hormone regulation.

Aging is a biological process in which the environment interacts with the body to cause a progressive decline in effective physiological function. Aging in the human body can lead to a dysfunction of the vital organ systems, resulting in the onset of age-related diseases, such as neurodegenerative and cardiovascular diseases, which can seriously affect an individual's quality of life. The endocrine system acts on specific targets through hormones and related major functional factors in its pathways, which play biological roles in coordinating cellular interactions, metabolism, growth, and aging. Aging is the result of a combination of many pathological, physiological, and psychological processes, among which the endocrine system can achieve a bidirectional effect on the aging process by regulating the hormone levels in the body. In this paper, we explored the mechanisms of growth hormone, thyroid hormone, and estrogen in the aging process to provide a reference for the exploration of endocrine mechanisms related to aging.

A Single-Domain Antibody-Based Anti-PSMA Recombinant Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Therapy.

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

Preclinical evaluation of a novel anti-mesothelin immunotoxin based on a single domain antibody as the targeting ligand.

Pancreatic cancer, as one of the most aggressive and lethal malignancies in the world, is lack of effective treatment. Constructing immunotoxin molecules to target the mesothelin (MSLN) receptor is a potential therapeutic strategy for pancreatic cancer and other related malignant tumors, with some molecules being tested in clinical trials. However, currently, there are still some limitations in its applications, such as the difficulty of the preparation of drug molecules, the limited effectiveness of drugs, and the inadequacy of drug safety and immunogenicity. In this study, we constructed a novel type of anti-MSLN immunotoxin, A1-PE24X7, in which a single domain antibody (sdAb) molecule was used as the target ligand and an improved PE24X7 toxin with reduced off-target toxicity and immunogenicity was used as the effector. Unlike conventional immunotoxins, the designed A1-PE24X7 could be easily expressed in the E. coli system in the form of a soluble protein with a good yield (15--20 mg/L), avoiding the complex process of denaturation and refolding of inclusion bodies, and it can be conveniently stored in PBS solution for more than 7 days at 4 °C, showing high storage stability. Cell-based experiments showed that A1-PE24X7 entered MSLN-expressing tumor cells in a receptor-mediated manner and killed these cells with an EC50 in the low nanomolar range (0.13 nM against NCI-N87 cells and 0.79 nM against AsPC-1 cells) and it showed ideal selectivity for the MSLN receptor (>100 nM against receptor negative PC3 cells). In animal-based experiments, A1-PE24X7 had tumor enrichment ability in relation to MSLN-positive tumors and showed strong tumor killing and inhibition in mouse models of pancreatic cancer and gastric cancer. Five injections of 3.0 mg/kg A1-PE24X7 significantly reduced the tumor volume of gastric NCI-N87 cancer and also significantly inhibited the growth of pancreatic AsPC-1 cancer. In addition, the maximum tolerable dosage (MSD) of A1-PE24X7 to mice was higher than 15 mg/kg, showing that A1-PE24X7 has a relatively broad therapeutic window. These preclinical results indicate that this strategy has good potential for application to the treatment of pancreatic cancer and other tumors with high MSLN expression.

Genome-wide association analysis reveals regulation of at-risk loci by DNA methylation in prostate cancer.

Epigenetic changes are potentially important for the ontogeny and progression of tumors but are not usually studied because of the complexity of analyzing transcript regulation resulting from epigenetic alterations. Prostate cancer (PCa) is characterized by variable clinical manifestations and frequently unpredictable outcomes. We performed an expression quantitative trait loci (eQTL) analysis to identify the genomic regions that regulate gene expression in PCa and identified a relationship between DNA methylation and clinical information. Using multi-level information published in The Cancer Genome Atlas, we performed eQTL-based analyses on DNA methylation and gene expression. To better interpret these data, we correlated loci and clinical indexes to identify the important loci for both PCa development and progression. Our data demonstrated that although only a small proportion of genes are regulated via DNA methylation in PCa, these genes are enriched in important cancer-related groups. In addition, single nucleotide polymorphism analysis identified the locations of CpG sites and genes within at-risk loci, including the 19q13.2-q13.43 and 16q22.2-q23.1 loci. Further, an epigenetic association study of clinical indexes detected risk loci and pyrosequencing for site validation. Although DNA methylation-regulated genes across PCa samples are a small proportion, the associated genes play important roles in PCa carcinogenesis.

PEG Linker Improves Antitumor Efficacy and Safety of Affibody-Based Drug Conjugates.

Antibody drug conjugates (ADCs) have become an important modality of clinical cancer treatment. However, traditional ADCs have some limitations, such as reduced permeability in solid tumors due to the high molecular weight of monoclonal antibodies, difficulty in preparation and heterogeneity of products due to the high drug/antibody ratio (4-8 small molecules per antibody). Miniaturized ADCs may be a potential solution, although their short circulation half-life may lead to new problems. In this study, we propose a novel design strategy for miniaturized ADCs in which drug molecules and small ligand proteins are site-specifically coupled via a bifunctional poly(ethylene glycol) (PEG) chain. The results showed that the inserted PEG chains significantly prolonged the circulation half-life but also obviously reduced the cytotoxicity of the conjugates. Compared with the conjugate ZHER2-SMCC-MMAE (HM), which has no PEG insertion, ZHER2-PEG4K-MMAE (HP4KM) and ZHER2-PEG10K-MMAE (HP10KM) with 4 or 10 kDa PEG insertions have 2.5- and 11.2-fold half-life extensions and 4.5- and 22-fold in vitro cytotoxicity reductions, respectively. The combined effect leads to HP10KM having the most ideal tumor therapeutic ability at the same dosages in the animal model, and its off-target toxicity was also reduced by more than 4 times compared with that of HM. These results may indicate that prolonging the half-life is very helpful in improving the therapeutic capacity of miniaturized ADCs. In the future, the design of better strategies that can prolong half-life without affecting cytotoxicity may be useful for further improving the therapeutic potential of these molecules.

P22 virus-like particles as an effective antigen delivery nanoplatform for cancer immunotherapy.

As a new strategy for cancer immunotherapy, therapeutic cancer vaccines have been greatly improved in recent years. However, addressing the needs to quickly and efficiently elicit a high-intensity immune response against neoantigen peptides, especially to induce an effective cytotoxic lymphocyte (CTL) reaction, remain challenges in this field. In this study, virus-like particles (VLPs) derived from the phage P22 were adopted to load peptide antigens on the surface, to test whether VLP technology can be used as a platform for efficient peptide antigen delivery by therapeutic cancer vaccines. The B and T epitopes (OVAB peptide and OVAT peptide) of ovalbumin (OVA) were used here as model antigens and fused individually at the C terminus of the coat protein (CP), which allowed display on the surface of P22 particles to form two types of vaccine particles (VLP-OVAB and VLP-OVAT). Subsequent experiments showed that VLP-OVAB induced an antibody titer against the peptide antigen as high as 5.0 × 105 and that VLP-OVAT induced highly effective cross-presentation and then strongly activated a T epitope-specific CTL response. Mouse tumor model experiments showed that VLP-OVAT could significantly inhibit tumor growth by increasing the proportions of CD4+ T cells, CD8+ T cells and effector memory T cells (TEM cells) and lowering the proportion of myeloid-derived suppressor cells (MDSCs) among tumor-infiltrating lymphocytes and splenocytes. Compared with other chemically synthesized nanomaterials, VLPs have obvious advantages as vaccine carriers due to their clear chemical composition, fixed spatial structure, excellent biocompatibility, and relatively high potential for clinical translation. Therefore, this platform may lay a solid foundation for the design and preparation of personalized therapeutic vaccines based on neoantigen peptides.

TRIB3 Promotes Lung Adenocarcinoma Progression via an Enhanced Warburg Effect.

BACKGROUND: The pseudokinase Tribbles 3 (TRIB3) is involved in many cellular processes and various cancers. In recent years, the importance of metabolic transformation in the maintenance of malignant tumors has become increasingly prominent. Abnormal metabolism of cancer cells is considered a hallmark of cancer. However, the exact role and molecular mechanism of TRIB3 in lung adenocarcinoma (LUAD) cell reprogramming is largely unknown. METHODS: The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells were examined with a Seahorse XF Extracellular Flux Analyzer. In vitro and in vivo RT-qPCR, Western blotting, and functional assays were performed to explore the functional roles of TRIB3 in LUAD. RESULTS: In the present study, we demonstrated that TRIB3 is remarkably upregulated in LUAD cell lines as well as tissues. TRIB3 knockdown significantly inhibited LUAD cell growth and suppressed LUAD cell invasion, while TRIB3 overexpression conferred the opposite effects. Moreover, silencing TRIB3 suppressed the tumorigenesis and metastatic ability of LUAD cells. Mechanistically, we demonstrated that silencing TRIB3 significantly impaired aerobic glycolysis ability in LUAD cells. Furthermore, our data indicated that TRIB3 knockdown decreased hypoxia-inducible factor (HIF)1α levels and targeted the glycolytic genes regulated by HIF1α. CONCLUSION: Together, our findings revealed a previously unappreciated function of TRIB3 in cancer cell metabolism and tumor progression, illustrating that TRIB3 could be considered a valuable therapeutic target for LUAD patients.

KRAS G12C mutations in Asia: a landscape analysis of 11,951 Chinese tumor samples.

BACKGROUND: Kirsten rat sarcoma vial oncogene (KRAS) is one of the most prevalent oncogenes in multiple cancer types, but the incidence is different between the Asian and non-Asian populations. The recent development of KRAS G12C targeting drug has shown great promise. It is thus important to understand the genomic landscape of KRAS G12C in a specific population. METHODS: Sequencing data of 11,951 tumor samples collected from 11/2016 to 7/2019 from multiple centres in China were analyzed for KRAS mutation status. Concomitant genomic aberrations were further analyzed in tumors with KRAS G12C mutations, which were sequenced with comprehensive cancer panel including over 450 cancer-related genes. Smoking status and its correlation with KRAS were analyzed in 2,235 lung cancer cases within this cohort. RESULTS: KRAS mutations were identified in 1978 (16.6%) patient samples. Specifically, KRAS G12C accounted for 14.5% (n=286) of all KRAS mutations. G12C was most commonly seen in lung cancer (4.3%), followed by colorectal cancer (2.5%) and biliary cancer (2.3%). Almost all patients (99.6%) with G12C mutations had concomitant genomic aberrations. These were most commonly associated with the RAS/RTK pathway including BRAF and PI3KCA mutations. Moreover, KRAS mutation was positively correlated with smoking status in lung adenocarcinomas. CONCLUSIONS: The overall incidence of KRAS G12C mutations remains low in the Chinese population. The most common tumor types harboring KRAS G12C mutations are in patients suffering from lung, colorectal and biliary cancers.

Gut transcriptomic changes during hibernation in the greater horseshoe bat (Rhinolophus ferrumequinum).

BACKGROUND: The gut is the major organ for nutrient absorption and immune response in the body of animals. Although effects of fasting on the gut functions have been extensively studied in model animals (e.g. mice), little is known about the response of the gut to fasting in a natural condition (e.g. hibernation). During hibernation, animals endure the long term of fasting and hypothermia. RESULTS: Here we generated the first gut transcriptome in a wild hibernating bat (Rhinolophus ferrumequinum). We identified 1614 differentially expressed genes (DEGs) during four physiological states (Torpor, Arousal, Winter Active and Summer Active). Gene co-expression network analysis assigns 926 DEGs into six modules associated with Torpor and Arousal. Our results reveal that in response to the stress of luminal nutrient deficiency during hibernation, the gut helps to reduce food intake by overexpressing genes (e.g. CCK and GPR17) that regulate the sensitivity to insulin and leptin. At the same time, the gut contributes energy supply by overexpressing genes that increase capacity for ketogenesis (HMGCS2) and selective autophagy (TEX264). Furthermore, we identified separate sets of multiple DEGs upregulated in Torpor and Arousal whose functions are involved in innate immunity. CONCLUSION: This is the first gut transcriptome of a hibernating mammal. Our study identified candidate genes associated with regulation of food intake and enhance of innate immunity in the gut during hibernation. By comparing with previous studies, we found that two DEGs (CPE and HSPA8) were also significantly elevated during torpor in liver and brain of R. ferrumequinum and several DEGs (e.g. TXNIP and PDK1/4) were commonly upregulated during torpor in multiple tissues of different mammals. Our results support that shared expression changes may underlie the hibernation phenotype by most mammals.

Association between microRNA 25 expression in serum and lung cancer: A protocol for systematic review and meta-analysis.

BACKGROUND: This study aims to identify the association between microRNA 25 (mRNA 25) expression in serum and lung cancer (LC). METHODS: This planned study will cover all eligible case-controlled studies that report association between mRNA 25 expression in serum and LC. It will include published studies from inception to the present in Cochrane Library, PUBMED, EMBASE, Web of Science, Allied and Complementary Medicine Database, VIP database, and China National Knowledge Infrastructure regardless language and geographical location. We will also search other sources, such as conference abstracts and reference lists of related known studies and experts in the domain consulted to avoid missing potential studies. Two contributors will independently examine and select studies, collect all necessary data, and judge study quality for all included studies. We will perform statistical analysis using RevMan V.5.3 software and Stata V.12.0 software. RESULTS: This study will summarize current evidence to present first systematic review of research on the association between mRNA 25 expression in serum and LC. CONCLUSION: This study will present comprehensive evidence to determine whether mRNA 25 expression in serum is associated with LC, and will provide helpful evidence for the future studies. SYSTEMATIC REVIEW REGISTRATION: INPLASY202040056.

Anti-HER2 Affibody-Conjugated Photosensitizer for Tumor Targeting Photodynamic Therapy.

Antibody-coupled photosensitive molecules can achieve an ideal tumor-specific photodynamic therapy (PDT) and show strong clinical application potential. However, some inherent disadvantages, such as long circulation half-life, poor permeation into solid tumors, and difficulty in obtaining uniform coupling products, present potential problems to clinical applications. In this study, we propose a novel design of targeting photosensitizers, based on a very small targeting protein (an affibody molecule) coupled with photosensitive compounds, to address these problems. In the synthesis, photosensitive pyropheophorbide-a (Pyro) is modified with a PEG linker (molecular weight of 727 Da) and then site specifically coupled to the anti-HER2 ZHER2:2891 affibody protein to provide a homogeneous protein-coupled photosensitizer via a convenient process. In vitro and in vivo experiments show that this molecule has an ideal selectivity for binding and photocytotoxicity against HER2-positive cells (more than 50-fold selectivity between HER2-high expression and HER2-low expression cells) and highly specific tumor accumulation; at a relatively low dose, it effectively eliminated HER2-high expression NCI-N87 tumors in a mouse model. It is worth noting that Pyro only has a moderate photodynamic activity; however, the affibody-coupled Pyro molecule (Pyro-Linker-ZHER2) still shows excellent tumor therapeutic function. The more ideal tumor permeability of small ligands may be helpful to enhance the drug concentration in the tumor site and the ability to penetrate deeply inside the tumor. Coupling photosensitive compounds with affibody proteins may provide a new way for targeting PDT of tumors.

HER2-specific immunotoxins constructed based on single-domain antibodies and the improved toxin PE24X7.

Human epidermal growth factor receptor 2 (HER2) is an attractive target for cancer therapy, although a large fraction of tumors that express HER2 may still resist first-line therapies. Immunotoxins with antibodies that are armed with extremely potent cytotoxic toxin molecules may provide an important solution to this problem. In this work, we constructed three new anti-HER2 immunotoxins by using single-domain antibody (sdAb) molecules as the targeting moiety and the improved toxin PE24X7 as the effector with the aim of simplifying the preparation and reducing the off-target toxicity of the immunotoxins. Due to the beneficial outcomes of sdAb molecules, the synthesized immunotoxins were efficiently expressed in soluble form, avoiding the refolding process required by the common immunotoxin design and having high solubility and stability. Cell toxicity experiments showed that they have high cytotoxicity against various HER2-positive tumor cells and good selectivity (more than 1000-fold) towards HER2-positive rather than HER2-negative cells. Importantly, in vivo treatment experiments showed that one of the new immunotoxins could efficiently halt tumor growth at doses lower than 0.75 mg/kg, and it had a maximum tolerated dose (MTD) higher than 8.0 mg/kg, showing a substantially improved MTD and a broadened therapeutic window than the previously reported anti-HER2 immunotoxins. Given that PE toxin-based immunotoxins have been approved for clinical cancer therapy, the unique characteristics of the immunotoxins presented here make them promising for use in the development of anti-HER2 cancer therapeutics.

Somatic genetic aberrations in gallbladder cancer: comparison between Chinese and US patients.

BACKGROUND: Gallbladder cancer (GBC) is often diagnosed at an advanced stage with limited therapeutic options and poor prognosis. The five-year survival rate of this cancer when diagnosed at an advanced stage is below 5%, and the median survival time is less than a year with standard gemcitabine-based chemotherapy. Survival benefit with second-line treatment is unknown. Thus, there is an urgent need for novel treatment strategies and targeted therapy based on next generation sequencing (NGS) may be of value. METHODS: Comprehensive genomic profiling (CGP) was performed with NGS panel on paraffin-embedded tumors from a cohort of 108 Chinese and 107 US GBC patients. Clinical data were collected using an IRB approved protocol from a single-center in US and from China. RESULTS: In Chinese and US GBC cohorts, an average of 6.4 vs. 3.8 genomic alterations (GAs) were identified per patient. The most frequent alterations were TP53 (69.4%), CDKN2A/B (26%), ERBB2 (18.5%), PIK3CA (17%) and CCNE1 (13%) in Chinese cohort, TP53 (57.9%), CDKN2A/B (25%), SMAD4 (17%), ARID1A (14%), PIK3CA (14%) and ERBB2 (13.1%) in US patients. NFE2L2 mutations were present in 6.5% of Chinese patients and not observed in the US cohort. Interestingly, ERBB2 genetic aberrations were significantly associated with better pathological tumor differentiation and tended to co-occurrence with CDKN2A/B mutations in both the Chinese and US GBC cases. Out of the top 9 dysregulated genetic pathways in cancer, Chinese patients harbored more frequent mutations in ERBB genes (30.6% vs. 19.0%, P=0.04). High frequency of PI3K/mTOR pathway variations was observed in both Chinese (37%) and US cohort (33%) (P=0.5). Additionally, both Chinese and US GBC patients exhibited a relatively high tumor mutational burden (TMB) (17.6% and 17.0%, respectively). In the Chinese cohort, a significant association was seen between direct repair gene alterations and TMB ≥10 muts/Mb (P=0.004). CONCLUSIONS: In our study, over 83% Chinese and 68% US GBC patients had actionable alterations that could potentially guide and influence personalized treatment options. The identification of high TMB, ERBB2, CDKN2A/B, PI3K/mTOR pathway and DNA repair mutations indicated that both Chinese and US GBC patients may benefit from targeted or immune checkpoint inhibitors.

Gut microbial diversity in two insectivorous bats: Insights into the effect of different sampling sources.

The gut microbiota is now known as a key factor in mammalian physiology and health. Our understanding of the gut microbial communities and their effects on ecology and evolution of their hosts is extremely limited in bats which represent the second largest mammalian order. In the current study, gut microbiota of three sampling sources (small intestine, large intestine, and feces) were characterized in two sympatric and insectivorous bats (Rhinolophus sinicus and Myotis altarium) by high-throughput sequencing of the V3-V4 region of the 16S rRNA gene. Combining with published studies, this work reveals that Gammaproteobacteria may be a dominant class in the whole Chiroptera and Fusobacteria is less observed in bats although it has been proven to be dominant in other mammals. Our results reveal that the sampling source influences alpha diversity of the microbial community in both studied species although no significant variations of beta diversity were observed, which support that fecal samples cannot be used as a proxy of the microbiota in other gut regions in wild animals.

cRGD Peptide-Conjugated Pyropheophorbide-a Photosensitizers for Tumor Targeting in Photodynamic Therapy.

Pyropheophorbide-a (Pyro) is a highly promising photosensitizer for tumor photodynamic therapy (PDT), although its very limited tumor-accumulation ability seriously restricts its clinical applications. A higher accumulation of photosensitizers is very important for the treatment of deeply seated and larger tumors. The conjugation of Pyro with tumor-homing peptide ligands could be a very useful strategy to optimize the physical properties of Pyro. Herein, we reported our studies on the conjugation of Pyro with a cyclic cRGDfK (cRGD) peptide, an integrin binding sequence, to develop highly tumor-specific photosensitizers for PDT application. To further reduce the nonspecific uptake and, thus, reduce the background distribution of the conjugates in normal tissues, we opted to add a highly hydrophilic polyethylene glycol (PEG) chain and an extra strongly hydrophilic carboxylic acid group as the linker to avoid the direct connection of the strongly hydrophobic Pyro macrocycle and cRGD ligand. We reported here the synthesis and characterization of these conjugates, and the influence of the hydrophilic modification on the biological function of the conjugates was carefully studied. The tumor-accumulation ability and photodynamic-induced cell-killing ability of these conjugates were evaluated through both in vitro cell-based experiment and in vivo distribution and tumor therapy experiments with tumor-bearing mice. Thus, the synthesized conjugate significantly improved the tumor enrichment and tumor selectivity of Pyro, as well as abolished the xenograft tumors in the murine model through a one-time PDT treatment.

The little brown bat nuclear genome contains an entire mitochondrial genome: Real or artifact?

Nuclear mitochondrial DNA sequences (NUMTs) have been documented in almost all eukaryotic genomes studied. Recently, with the number of sequenced genomes increasing, extremely large NUMTs, even a nearly entire mitochondrial genome, have been reported in some plants and animals. However, few such studies provided strong experimental evidences for these important discoveries. In this study using a computer-based search method an entire mitochondrial genome (NUMT-1) was found in the nuclear genome of a bat species (Myotis lucifugus). This super-large NUMT shared a same scaffold with a 754bp nuclear genomic sequence and a second NUMT (NUMT-2, 3292bp). If NUMT-1 was real, it will be the largest NUMT found in animals and this finding will provide valuable insights into the mode of generation of NUMTs in the nuclear genome. Unfortunately, although the initial sequencing technology of the published M. lucifugus genome makes the possibility of artifact less likely, our results from both the PCR amplification followed by Sanger sequencing and mapping method based on the whole-genome resequencing datasets suggested that the scaffold containing the entire mitochondrial genome was artifact possibly due to a misassembly of mitochondrial and the nuclear DNA sequences. Our current study highlights the necessity to validate the authenticity of extremely large NUMTs identified in previous searches on whole-genome sequence assemblies.

The complete mitochondrial genome of the Thomas's horseshoe bat (Rhinolophus thomasi) using next-generation sequencing and Sanger sequencing.

The Thomas's horseshoe bat (Rhinolophus thomasi) is restricted to southeast Asia and few studies have been conducted on this species. Here we characterized the complete mitochondrial genome of R. thomasi using next-generation sequencing and Sanger sequencing. The whole mitogenome is 16,899 bp in length and contains 22 tRNA, 2 rRNA, 13 protein-coding genes and a non-coding control region. The tissue used in this study was taken from a sample collected 13 years ago and the genomic DNA was seriously degraded. Our study adds to a growing number of studies indicating that next-generation sequencing is powerful in generating genetic/genomic resources from museum samples.

The implications and mechanisms of the extra-nuclear nucleolin in the esophageal squamous cell carcinomas.

In recent decades, the multi-functional protein nucleolin (NCL) has been reported to express outside the nucleus of many cancer cells. However, the expression and role of the extra-nuclear NCL in esophageal squamous cell carcinoma (ESCC) were not well characterized. Here, NCL was detected by immunohistochemistry and Western blotting in 60 ESCC tissues. Further, the associations of NCL, EGFR, CXCR4 and Ki67 were analyzed by in vitro assays. Our results showed that NCL expression in all 40 cases of ESCC tissues with metastasis was extensively located in the nucleus, cytoplasm and cell membrane (extra-nucleus), while NCL expression in all 20 cases of ESCC without metastasis was merely limited into the nucleus (intra-nucleus).The extra-nuclear NCL expression was positively correlated with the expression of EGFR, CXCR4 and Ki67 and serves as an independent prognostic factor for ESCC patients. In vitro, NCL siRNA (si-NCL) efficaciously affected the expression of EGF or SDF-1-induced p-AKT, p-ERK and Ki67. Also, NCL siRNA inhibited the capacity of migration and invasion of ECA109 cells. In conclusions, our study suggests that NCL is implicated in the initiation and transduction of EGFR and CXCR4 signaling and further up-regulates Ki67 expression to modulate the biological behaviors of ESCC. Clinically, the extra-nuclear NCL expression can be used as an important indicator to determine metastasis and predict the prognosis, which help develop new therapeutic strategies against ESCC.