Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (FcgammaRIII) on effector cells to an Fv specific for mouse or human CD32B (FcgammaRIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitt's lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity.

Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, protease-rich environment of the lysosome. Potential artifacts include degradation or release of the tag and acid quenching of fluorescence. Tagging can also affect protein folding, glycosylation and/or trafficking. To specifically investigate the use of fluorescent tags to reveal lysosomal localization, we tested mCherry derivatives as C-terminal tags for Niemann-Pick disease type C protein 2 (NPC2), a luminal lysosomal protein. Full-length mCherry was released from the NPC2 chimera while deletion of the 11 N-terminal residues of mCherry generated a cleavage-resistant (cr) fluorescent variant. Insertion of proline linkers between NPC2 and crmCherry had little effect while Gly-Ser linkers promoted cleavage. The NPC2-crmCherry fusion was targeted to the lysosome and restored function in NPC2-deficient cells. Fusion of crmCherry to known and candidate lysosomal proteins revealed that the linkers had different effects on lysosomal localization. Direct fusion of crmCherry impaired mannose 6-phosphorylation and lysosomal targeting of the lysosomal protease tripeptidyl peptidase I (TPP1), while insertion of linkers corrected the defects. Molecular modeling suggested structural bases for the effects of different linkers on NPC2 and TPP1 fusion proteins. While mCherry fusion proteins can be useful tools for studying the lysosome and related organelles, our findings underscore the potential artifacts associated with such applications.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.

Protégé was a phase 3, randomized, double-blind, parallel, placebo-controlled 2-year study of three intravenous teplizumab dosing regimens, administered daily for 14 days at baseline and again after 26 weeks, in new-onset type 1 diabetes. We sought to determine efficacy and safety of teplizumab immunotherapy at 2 years and to identify characteristics associated with therapeutic response. Of 516 randomized patients, 513 were treated, and 462 completed 2 years of follow-up. Teplizumab (14-day full-dose) reduced the loss of C-peptide mean area under the curve (AUC), a prespecified secondary end point, at 2 years versus placebo. In analyses of prespecified and post hoc subsets at entry, U.S. residents, patients with C-peptide mean AUC >0.2 nmol/L, those randomized ≤6 weeks after diagnosis, HbA1c <7.5% (58 mmol/mol), insulin use <0.4 units/kg/day, and 8-17 years of age each had greater teplizumab-associated C-peptide preservation than their counterparts. Exogenous insulin needs tended to be reduced versus placebo. Antidrug antibodies developed in some patients, without apparent change in drug efficacy. No new safety or tolerability issues were observed during year 2. In summary, anti-CD3 therapy reduced C-peptide loss 2 years after diagnosis using a tolerable dose.

There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded in vitro. Primary xenografts and metastatic lesions derived from these cells in NSG mice fully recapitulate both the adenocarcinoma and squamous features of the patient tumor. Interestingly, while the CSLC all co-expressed cytokeratins 5 and 7, most xenograft cells expressed either one, or neither, with <10% remaining double positive. We also demonstrated the potential of the CSLC to differentiate to multi-lineage structures with branching lung morphology expressing bronchial, alveolar and neuroendocrine markers in vitro. Taken together the properties of these ASC-derived CSLC suggests that ASC may arise from a primitive lung stem cell distinct from the bronchial-alveolar or basal stem cells.

Many phylogenetic questions in the Ciconiiformes remain unresolved and complete mitogenome data are urgently needed for further molecular investigation. In this work, we determined the complete mitogenome sequence of the little egret (Egretta garzetta). The genome was 17,361 bp in length and the gene organization was typical of other avian mtDNA. In protein-coding genes (PCGs), a C insertion was found in ND3, and COIII and ND4 terminated with incomplete stop codons (T). tRNA-Val and tRNA-Ser (AGY) were unable to fold into canonical cloverleaf secondary structures because they had lost the DHU arms. Long repetitive sequences consisting of five types of tandem repeats were found at the 3' end of Domain III in the control region. A phylogenetic analysis of 11 species of Ciconiiformes was done using complete mitogenome data and 12 PCGs. The tree topologies obtained with these two strategies were identical, which strongly confirmed the monophyly of Ardeidae, Threskiorothidae and Ciconiidae. The phylogenetic analysis also revealed that Egretta was more closely related to Ardea than to Nycticorax in the Ardeidae, and Platalea was more closely related to Threskiornis than to Nipponia in the Threskiornithidae. These findings contribute to our understanding of the phylogenetic relationships of Ciconiiformes based on complete mitogenome data.

This article was aimed to study the FOXF2 effects on cervical cancer. Tumor tissues and adjacent tissues of 41 cervical cancer patients were collected. Human endometrial epithelial cells (hEEC) and Hela cells were cultured. FOXF2 expression vector and its empty vector were transfected into Hela cells, and named as pcDNA 3.1-FOXF2 group and Vector group, respectively. Hela cells without any treatment were set as Blank group. qRT-PCR was used to detect mRNA expression. Nude mouse xenograft assay was performed to test Hela cells proliferation ability in vivo FOXF2 and β-catenin positive cell numbers were detected by immunohistochemistry. Protein expression was analyzed by Western blot. Cells migration and invasion were conducted by Transwell. Tumor tissues and Hela cells FOXF2 expression were lower than that in adjacent tissues and hEEC (P<0.01). Low FOXF2 expression predicted poor outcomes of cervical cancer patients. Compared with Blank group and Vector group, Hela cells of pcDNA 3.1-FOXF2 group were with higher FOXF2 expression, lower OD495 value, migrated and invaded cells, higher E-cadherin expression, lower Vimentin and Snail expression, smaller tumor volume in nude mice, lower c-Myc, CyclinDl, MMP9, Lgr5, and nuclear β-catenin expression (all P<0.01). FOXF2 inhibits Hela cells proliferation, migration, and invasion through regulating Wnt signaling pathway.

Mammals cannot see light over 700 nm in wavelength. This limitation is due to the physical thermodynamic properties of the photon-detecting opsins. However, the detection of naturally invisible near-infrared (NIR) light is a desirable ability. To break this limitation, we developed ocular injectable photoreceptor-binding upconversion nanoparticles (pbUCNPs). These nanoparticles anchored on retinal photoreceptors as miniature NIR light transducers to create NIR light image vision with negligible side effects. Based on single-photoreceptor recordings, electroretinograms, cortical recordings, and visual behavioral tests, we demonstrated that mice with these nanoantennae could not only perceive NIR light, but also see NIR light patterns. Excitingly, the injected mice were also able to differentiate sophisticated NIR shape patterns. Moreover, the NIR light pattern vision was ambient-daylight compatible and existed in parallel with native daylight vision. This new method will provide unmatched opportunities for a wide variety of emerging bio-integrated nanodevice designs and applications. VIDEO ABSTRACT.

Patients with end-stage renal disease (ESRD) have an impaired immune response with a prematurely aged T-cell system. Mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK) and p38, regulate diverse cellular programs by transferring extracellular signals into an intracellular response. T cell receptor (TCR)-induced phosphorylation of ERK (pERK) may show an age-associated decline, which can be reversed by inhibiting dual specific phosphatase (DUSP) 6, a cytoplasmic phosphatase with substrate specificity to dephosphorylate pERK. The aim of this study was to assess whether ESRD affects TCR-mediated signaling and explore possibilities for intervening in ESRD-associated defective T-cell mediated immunity.

Objective: Obesity/metabolic syndrome and hyperuricemia are clinically associated; however, the association of obesity/metabolic syndrome-related genetic variants with hyperuricemia is not clear. Therefore, we assessed this association in Chinese men diagnosed with hyperuricemia in comparison to a non-hyperuricemia group. Methods: We genotyped 47 single nucleotide polymorphisms (SNPs) previously identified to be associated with obesity or metabolic syndrome in 474 adult males (aged ≥ 18 years) using multiplex polymerase chain reaction. Multivariate logistic regression was used to investigate the association between the genetic variations and hyperuricemia. Stratified analyses were applied to further assess the associations. Results: The obesity-related SNP in MSRA rs545854 significantly affected serum uric acid levels. In addition, the G-allele of rs545854 was positively associated with the risk of hyperuricemia [odds ratio (OR) = 2.80, 95% confidence interval (CI) = 1.19-6.64, P = 0.0188]. After adjusting the model for body mass index and central obesity, rs545854 was shown to be an independent factor increasing the risk of hyperuricemia (OR = 2.81, 95%CI = 1.18-6.70, P = 0.0196). Stratified analyses also showed a significant association between rs545854 and hyperuricemia among meat eaters (OR = 2.62, 95%CI = 1.09-6.26, P = 0.0308). Conclusion: The obesity-related SNP rs545854 was correlated with the serum uric acid level and risk of hyperuricemia in a male Chinese population. Therefore, men carrying this SNP could benefit from limiting their meat consumption to prevent hyperuricemia. These findings suggest an underlying genetic link between obesity and hyperuricemia worthy of further exploration.

The bromodomain and extraterminal (BET) family of proteins comprises four members-BRD2, BRD3, BRD4 and the testis-specific isoform BRDT-that largely function as transcriptional coactivators and play critical roles in various cellular processes, including the cell cycle, apoptosis, migration and invasion. BET proteins enhance the oncogenic functions of major cancer drivers by elevating the expression of these drivers, such as c-Myc in leukemia, or by promoting the transcriptional activities of oncogenic factors, such as AR and ERG in prostate cancer. Pathologically, BET proteins are frequently overexpressed and are clinically linked to various types of human cancer; they are therefore being pursued as attractive therapeutic targets for selective inhibition in patients with cancer. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed and have shown promising outcomes in early clinical trials. Although resistance to BET inhibitors has been documented in preclinical models, the molecular mechanisms underlying acquired resistance are largely unknown. Here we report that cullin-3SPOP earmarks BET proteins, including BRD2, BRD3 and BRD4, for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the degradation of BET proteins, leading to their elevated abundance in SPOP-mutant prostate cancer. As a result, prostate cancer cell lines and organoids derived from individuals harboring SPOP mutations are more resistant to BET-inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor-suppressor role of SPOP in prostate cancer in which it acts as a negative regulator of BET protein stability and also provide a molecular mechanism for resistance to BET inhibitors in individuals with prostate cancer bearing SPOP mutations.

Cytokinin fulfills its diverse roles in planta through a series of transcriptional responses. We identify the in vivo DNA binding site profiles for three genetically redundant type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs): ARR1, ARR10, and ARR12. The expression and genome-wide DNA binding locations of the three B-ARRs extensively overlap. Constructing a primary cytokinin response transcriptional network reveals a recurring theme of widespread cross-regulation between the components of the cytokinin pathway and other plant hormone pathways. The B-ARRs are found to have similar DNA binding motifs, though sequences flanking the core motif were degenerate. Cytokinin treatments amalgamate the three different B-ARRs motifs to identical DNA binding signatures (AGATHY, H(a/t/c), Y(t/c)) which suggests cytokinin may regulate binding activity of B-ARR family members. Furthermore, we find that WUSCHEL, a key gene required for apical meristem maintenance, is a cytokinin-dependent B-ARR target gene, demonstrating the importance of the cytokinin transcription factor network in shoot development.

Isothiocyanates are a group of compounds that exist in the majority of cruciferous plants. A number of isothiocyanates have been demonstrated to exhibit anticancer effects; however, antitumor properties of propyl isothiocyanate (PITC) have not been evaluated previously. In this study, the possible effects of PITC on gastric cancer (GC) cells were investigated, and the potential underlying mechanisms were explored. The results demonstrated that PITC inhibited cell viability of two GC cell lines and induced cell cycle arrest and apoptosis. Treatment with PITC promoted total glutathione depletion in GC cell lines, leading to reactive oxygen species accumulation and DNA damage, which activated the mitochondria-dependent and p53 signaling pathways to trigger apoptosis in GC cells. The effects of PITC were reversed by N-Acetyl-L-cysteine. The results of the present study revealed the potential mechanisms of PITC on apoptosis induction in GC cells, which may be mediated by mitochondria-dependent apoptosis and DNA damage.

Dalbergia benthami Prain (D.benthami) is an important legume species of the Dalbergia family, due to the use of its trunk and root heart in traditional Chinese medicine (TCM). In the present study, we reported the isolation, characterization and pharmacological activities of robustic acid (RA) from the ethyl acetate extract of D. benthami Prain. The SwissADME prediction showed that the RA satisfied the Lipinski's rule of five (molecule weight (MW): 380.39 g/mol, lipid-water partition coefficient (log P): 3.72, hydrogen bond donors (Hdon): 1, hydrogen bond acceptors (Hacc): 6, rotatable bonds (Rbon): 3. Other chemical and pharmacological properties of this RA were also evaluated, including topological polar surface area (TPSA) = 78.13 Å and solubility (Log S) = -4.8. The probability values of the antineoplastic, anti-free radical activities and topoisomerase I (TopoⅠ) inhibitory activity were found to be 0.784, 0.644 and 0.379, respectively. The molecular docking experiment using the Surflex-Dock showed that the Total Score and C Score of RNA binding with the human DNA-Topo I complex were 7.80 and 4. The MTS assay experiment showed that the inhibitory rates of RA on HL-60, MT4, Hela, HepG2, SK-OV-3 and MCF-7 cells were 37.37%, 97.41%, 81.22%, 34.4%, 32.68% and 51.4%, respectively. In addition, RA exhibited an inhibitory effect on the angiogenesis of zebrafish embryo, a good TopoⅠ inhibitory activity at a 10 mM concentration and in a dose-dependent manner, excellent radical scavenging in the DPPH and ABTS assays, and the free radical scavenging rate was close to the positive control (BHT) at different concentrations (0.5-2.0 mg/mL). Furthermore, 18 potential targets were found for this RA by PharmMapper, including ANXA3, SRC, FGFR2, GSK3B, CSNK2B, YARS, LCK, EPHA2, MAPK14, RORA, CRABP2, PPP1CC, METAP2, MME, TTR, MET and KDR. The GO and KEGG pathway analysis revealed that the "protein tyrosine kinase activity", "rap1 signaling pathway" and "PI3K-Akt signaling pathway" were significantly enriched by the RA target genes. Our results will provide new insights into the pharmaceutical use of this species. More importantly, our data will expand our understanding of the molecular mechanisms of RA functions.

Low-density lipoprotein receptor-related protein 10 (LRP10) is associated with a series of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease which share genetic risk factors and pathophysiological processes with amyotrophic lateral sclerosis (ALS). To investigate whether LRP10 variants could cause a predisposition to ALS, we screened rare, pathogenic LRP10 variants among a cohort of 584 patients with ALS from mainland China and performed burden analysis using data from a large external database. A total of 7 rare, pathogenic variants in LRP10, of which one (c.1182A>T, p.R394S) was novel, were identified in 11 unrelated patients. Burden analysis revealed significant associations between ALS and LRP10 at both the gene and single-variant levels (c.1721G>A, p.R574Q; c.1182A>T, p.R394S; and c.1681C>T, p.R561C). Interestingly, patients with sporadic ALS carrying variant c.1721G>A tended to have a bulbar onset, increased phenotype severity, and a worse prognosis. Our findings first provide independent evidence that rare, pathogenic LRP10 variants may be risk factors for ALS and delineate a special phenotype in patients with sporadic ALS carrying variant c.1721G>A.

Vascular dysfunctions are a common feature of multiple age-related diseases. However, modeling healthy and pathological aging of the human vasculature represents an unresolved experimental challenge. Here, we generated induced vascular endothelial cells (iVECs) and smooth muscle cells (iSMCs) by direct reprogramming of healthy human fibroblasts from donors of different ages and Hutchinson-Gilford Progeria Syndrome (HGPS) patients. iVECs induced from old donors revealed upregulation of GSTM1 and PALD1, genes linked to oxidative stress, inflammation and endothelial junction stability, as vascular aging markers. A functional assay performed on PALD1 KD VECs demonstrated a recovery in vascular permeability. We found that iSMCs from HGPS donors overexpressed bone morphogenetic protein (BMP)-4, which plays a key role in both vascular calcification and endothelial barrier damage observed in HGPS. Strikingly, BMP4 concentrations are higher in serum from HGPS vs. age-matched mice. Furthermore, targeting BMP4 with blocking antibody recovered the functionality of the vascular barrier in vitro, hence representing a potential future therapeutic strategy to limit cardiovascular dysfunction in HGPS. These results show that iVECs and iSMCs retain disease-related signatures, allowing modeling of vascular aging and HGPS in vitro.

This study aimed to compare clinical features and health-related quality of life (HRQoL) in the Chinese chronic kidney disease (CKD) 3 population and determined the necessity of the subdivision of CKD3 in Chinese patients with CKD.

Breast cancer (BRCA) ranks among the top most common female malignancies and was regarded as incurable when combined with bone and distant metastasis. Alternative splicing events (ASEs) together with splicing factors (SFs) were considered responsible for the development and progression of tumors.

Colitis-associated colorectal cancer (CAC) develops from active colonic inflammation, which is characterized by the production of proinflammatory cytokines that can induce mutations. IL-6 is produced by multiple cell types located within the tumor microenvironment including tumor-infiltrating immune cells, stromal cells, and the tumor cells themselves. The aim of our study was to explore the mechanism of Feng-Liao-Chang-Wei-Kang (FLCWK) and 5-fluorouracil (5-FU) in treating CAC.

Because old age is the greatest risk factor for dementia, a successful therapy will require an understanding of the physiological changes that occur in the brain with aging. Here, two structurally distinct Alzheimer's disease (AD) drug candidates, CMS121 and J147, were used to identify a unique molecular pathway that is shared between the aging brain and AD. CMS121 and J147 reduced cognitive decline as well as metabolic and transcriptional markers of aging in the brain when administered to rapidly aging SAMP8 mice. Both compounds preserved mitochondrial homeostasis by regulating acetyl-coenzyme A (acetyl-CoA) metabolism. CMS121 and J147 increased the levels of acetyl-CoA in cell culture and mice via the inhibition of acetyl-CoA carboxylase 1 (ACC1), resulting in neuroprotection and increased acetylation of histone H3K9 in SAMP8 mice, a site linked to memory enhancement. These data show that targeting specific metabolic aspects of the aging brain could result in treatments for dementia.