QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

R-2HG Exhibits Anti-tumor Activity by Targeting FTO/m6A/MYC/CEBPA Signaling.

R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling.

Histone H3 trimethylation at lysine 36 guides m6A RNA modification co-transcriptionally.

DNA and histone modifications have notable effects on gene expression1. Being the most prevalent internal modification in mRNA, the N6-methyladenosine (m6A) mRNA modification is as an important post-transcriptional mechanism of gene regulation2-4 and has crucial roles in various normal and pathological processes5-12. However, it is unclear how m6A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. We show that m6A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m6A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m6A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation.

A clearer vision: unveiling the importance of cycloplegic refraction and the pseudomyopia prevalence in Chinese preschoolers.

BACKGROUND: This study aimed to investigate the difference between cycloplegic and noncycloplegic refraction and evaluate the pseudomyopia prevalence in Chinese preschool children during the outbreak of COVID-19. METHODS: A cross-sectional study was conducted in the Tongzhou District of Beijing, China. Refractive error was measured under both noncycloplegic and cycloplegic conditions with autorefraction. The difference between noncycloplegic and cycloplegic spherical equivalent refraction (SER) and pseudomyopia prevalence were analyzed. Pseudomyopia was defined as SER ≤-0.50D in precycloplegic assessments and >-0.50D in post-cycloplegic assessments. RESULTS: Out of the 1487 participants who were enrolled in the study, 1471 individuals (98.92%) between the ages of 3-6 years completed all required procedures. A statistically significant difference in refraction was observed between noncycloplegic and cycloplegic measurements, the median of difference in spherical equivalent refraction (SER) of 0.88D (dioptre)(0.50,1.38). There was a high intraclass correlation (ICC) between these two methods for cylinders (ICC = 0.864; 95% CI, 0.850-0.877). The median DSE for myopia, emmetropia and hyperopia were 0.25D (0.00, 0.38),0.25D (0.06, 0.50) and 1.00D (0.62, 1.38), an hypermetropes showed considerably greater differences than myopes and emmetropes (Kruskal-Wallis test, H = 231.023, P = 0.000). Additionally, girls displayed a greater DSE than boys. Furthermore, when comparing against-the-rule (ATR) and oblique astigmatism, it was found that with-the-rule (WTR) astigmatism had the largest DSE. The study found varying prevalence rates of myopia, emmetropia, and hyperopia with and without cycloplegia, which were 1.90% vs. 10.06%, 11.49% vs. 50.31%, and 86.61% vs. 39.63%, respectively. Additionally, the overall prevalence of pseudomyopia was determined to be 8.29%. Participants with pseudomyopia had a significantly higher mean difference in SER (DSE) compared to non-pseudomyopic participants. CONCLUSIONS: Cycloplegic refraction is more sensitive than a noncycloplegic one for measuring refractive error in preschool children. Pseudomyopia is prevalent in preschool children during the COVID-19 outbreak period. Our study indicates the possibility that cycloplegic refraction should be performed in preschool children routinely.

Assessment of Key Factors Impacting Variability in AAV Vector Genome Titration by Digital PCR.

Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze-thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.

De novo disruptive heterozygous MMP21 variants are potential predisposing genetic risk factors in Chinese Han heterotaxy children.

BACKGROUND: Heterotaxy syndrome (HTX) is caused by aberrant left-right patterning early in embryonic development, which results in abnormal positioning and morphology of the thoracic and abdominal organs. Currently, genetic testing discerns the underlying genetic cause in less than 20% of sporadic HTX cases, indicating that genetic pathogenesis remains poorly understood. In this study, we aim to garner a deeper understanding of the genetic factors of this disease by documenting the effect of different matrix metalloproteinase 21 (MMP21) variants on disease occurrence and pathogenesis. METHODS: Eighty-one HTX patients with complex congenital heart defects and 89 healthy children were enrolled, and we investigated the pathogenetic variants related to patients with HTX by exome sequencing. Zebrafish splice-blocking Morpholino oligo-mediated transient suppression assays were performed to confirm the potential pathogenicity of missense variants found in these patients with HTX. RESULTS: Three MMP21 heterozygous non-synonymous variants (c.731G > A (p.G244E), c.829C > T (p.L277F), and c.1459A > G (p.K487E)) were identified in three unrelated Chinese Han patients with HTX and complex congenital heart defects. Sanger sequencing confirmed that all variants were de novo. Cell transfection assay showed that none of the variants affect mRNA and protein expression levels of MMP21. Knockdown expression of mmp21 by splice-blocking Morpholino oligo in zebrafish embryos revealed a heart looping disorder, and mutant human MMP21 mRNA (c.731G > A, c.1459A > G, heterozygous mRNA (wild-type&c.731G > A), as well as heterozygous mRNA (wild-type& c.1459A > G) could not effectively rescue the heart looping defects. A patient with the MMP21 p.G244E variant was identified with other potential HTX-causing missense mutations, whereas the patient with the MMP21 p.K487E variant had no genetic mutations in other causative genes related to HTX. CONCLUSION: Our study highlights the role of the disruptive heterozygous MMP21 variant (p.K487E) in the etiology of HTX with complex cardiac malformations and expands the current mutation spectrum of MMP21 in HTX.

ZnGeSe2 monolayer: water-splitting photocatalyst with ultrahigh solar conversion efficiency.

Hydrogen production through solar water-splitting offers a clean and renewable solution to tackle the ongoing issues of energy scarcity and environmental pollution. Here, the solar water-splitting performance of the ZnGeSe2 monolayer was explored via first-principles calculations. Our calculated results reveal that the ZnGeSe2 monolayer embodies stable configurations and semiconducting properties with direct bandgaps ranging from 1.23 to 1.60 eV under the biaxial strain from -1% to +2%. The generated holes and electrons of the ZnGeSe2 monolayer are separately distributed because of the intrinsic dipole. The calculated band edges of the ZnGeSe2 monolayer are demonstrated to be favorable for solar water-splitting. Additionally, the ZnGeSe2 monolayer exhibits strong optical absorption in the whole visible region. The hydrogen and oxygen evolution reactions can be accomplished without cocatalysts. Of particular significance, the solar to hydrogen (STH) efficiency of the ZnGeSe2 monolayer reaches up to 32%, far exceeding the economic value (10%). In light of these hallmarks, the ZnGeSe2 monolayer is demonstrated as an excellent water-splitting photocatalyst.

Refraction and ocular biometric parameters of preschool children in the Beijing whole childhood eye study: the first-year report.

BACKGROUND: Prevention of myopia should begin before school age. However, few population-based cohort studies have investigated refractive status in preschool children with cycloplegia. This study aimed to investigate the post-COVID-19 refraction and ocular biometric parameters of preschool children in Beijing Tongzhou District. METHODS: A population-based cohort study of kindergarten children in Tongzhou District, Beijing, commenced in November 2021. The present study reports data from the first year of the aforementioned population-based study. We selected children aged 3-6 years from nine kindergartens. Biometric parameters, including axial length (AL), anterior chamber depth (ACD), and corneal radius of curvature (CR), were collected before cycloplegia. Cycloplegic refraction was also measured. The spherical equivalent (SE), lens power (LP), and AL-to-CR ratio were calculated. Multiple linear regression analysis was used to analyse the correlation between refraction and ocular biometric parameters. RESULTS: A total of 1,505 children completed the examination, and a mean SE of 1.24 ± 0.91 D was found. The overall prevalence of myopia was 1.93%. The mean AL, ACD, CR, LP, and AL-to-CR ratio were 22.24 ± 0.70 mm, 3.28 ± 0.26 mm, 7.77 ± 0.26 mm, 26.01 ± 1.56 D, and 2.86 ± 0.07, respectively. Longer AL, deeper ACD, larger AL-to-CR ratio, and lower LP were associated with older age; the CR was not significantly different among different ages. In the multiple linear regression analysis, after adjusting for sex and age, the model that included AL, CR, and LP explained 87% of the SE variation. No differences were observed in the prevalence of myopia or the SE in this particular age range. CONCLUSION: The findings of this study suggest that a large proportion of preschool children in Beijing are mildly hyperopic, with a considerably low prevalence of myopia. In preschool children, refractive development was found to present mild hyperopia rather than emmetropia or myopia, a phenomenon that is characteristic of this age range.

Observation of Spin-Tensor Induced Topological Phase Transitions of Triply Degenerate Points with a Trapped Ion.

Triply degenerate points (TDPs), which correspond to new types of topological semimetals, can support novel quasiparticles possessing effective integer spins while preserving Fermi statistics. Here by mapping the momentum space to the parameter space of a three-level system in a trapped ion, we experimentally explore the transitions between different types of TDPs driven by spin-tensor-momentum couplings. We observe the phase transitions between TDPs with different topological charges by measuring the Berry flux on a loop surrounding the gap-closing lines, and the jump of the Berry flux gives the jump of the topological charge (up to a 2π factor) across the transitions. For the Berry flux measurement, we employ a new method by examining the geometric rotations of both spin vectors and tensors, which lead to a generalized solid angle equal to the Berry flux. The controllability of a multilevel ion offers a versatile platform to study high-spin physics, and our Letter paves the way to explore novel topological phenomena therein.

Cell division cycle associated 8: A novel diagnostic and prognostic biomarker for hepatocellular carcinoma.

The cell division cycle associated 8 (CDCA8) is a crucial component of the chromosome passenger complex (CPC). It has been implicated in the regulation of cell dynamic localization during mitosis. However, its role in hepatocellular carcinoma (HCC) is not clearly known. In this study, data of 374 patients with HCC were retrieved from the Cancer Genome Atlas (TCGA) database. Pan analysis of Gene Expression Profiling Interactive Analysis (GEPIA) database was performed to profile the mRNA expression of CDCA8 in HCC. Then, the Kaplan-Meier plotter database was analysed to determine the prognostic value of CDCA8 in HCC. In addition, samples of tumour and adjacent normal tissues were collected from 88 HCC patients to perform immunohistochemistry (IHC), reverse transcription-quantitative polymerase chain reaction (qRT-PCR) and Western blotting. The results obtained from bioinformatic analyses were validated through CCK-8 assay, EdU assay, colony formation assay, cell cycle assays and Western blotting experiments. Analysis of the Kaplan-Meier plotter database showed that high expression of CDCA8 may lead to poor overall survival (OS, p = 4.06e-05) in patients with HCC. For the 88 patients with HCC, we found that stages and grades appeared to be strongly linked with CDCA8 expression. Furthermore, the high expression of CDCA8 was found to be correlated with poor OS (p = 0.0054) and progression-free survival (PFS, p = 0.0009). In vitro experiments revealed that inhibition of CDCA8 slowed cell proliferation and blocked the cell cycle at the G0/G1 phase. In vivo experiments demonstrated that inhibition of CDCA8 inhibited tumour growth. Finally, blockade of CDCA8 reduced the expression levels of cyclin A2, cyclin D1, CDK4, CDK6, Ki67 and PCNA. And, there is an interaction between CDCA8 and E2F1. In conclusion, this research demonstrates that CDCA8 may serve as a biomarker for early diagnosis and prognosis prediction of HCC patients. In addition, CDCA8 could be an effective therapeutic target in HCC.

A Comparison of Subconjunctival Wound Healing between Different Methods of Dissecting Subconjunctival Tissues.

OBJECTIVE: To compare different methods for dissecting subconjunctival tissues by developing subconjunctival wound healing models. METHODS: New Zealand white rabbits were separated into 3 groups based on the method by which the rabbit subconjunctival wound healing model was generated: subconjunctival tissues were dissected episclerally (EPI) or subepithelially (SUB), with a corresponding blank control (CON). All the cases in the experimental groups were surgically prepared with conjunctival flaps, and they were sacrificed on the third postoperative day. At the surgical sites, the protein levels of hypoxia-inducible factor-1 (HIF-1)-α, vascular endothelial growth factor (VEGF)-A, and matrix metalloproteinase (MMP)-2 were detected by Western blot, morphological vascularity was measured by Adobe Photoshop, and subconjunctival fibrosis was assessed by histology. RESULTS: Compared with the CON group, both the EPI and SUB groups showed significantly upregulated protein levels of HIF-1α, VEGF-A, and MMP-2. In addition, the protein levels of HIF-1α, VEGF-A, and MMP-2 were higher in the EPI group than in the SUB group. Morphological vascularity was significantly elevated in the EPI group compared with the SUB and CON groups. Collagen content was markedly increased in the EPI group compared with the SUB and CON groups. CONCLUSIONS: Dissecting subconjunctival tissues subepithelially inhibits subconjunctival fibrosis, which may be instructive in tenonectomy in filtration surgery.

Homoharringtonine exhibits potent anti-tumor effect and modulates DNA epigenome in acute myeloid leukemia by targeting SP1/TET1/5hmC.

Homoharringtonine, a plant alkaloid, has been reported to suppress protein synthesis and has been approved by the US Food and Drug Administration for the treatment of chronic myeloid leukemia. Here we show that in acute myeloid leukemia (AML), homoharringtonine potently inhibits cell growth/viability and induces cell cycle arrest and apoptosis, significantly inhibits disease progression in vivo, and substantially prolongs survival of mice bearing murine or human AML. Strikingly, homoharringtonine treatment dramatically decreases global DNA 5-hydroxymethylcytosine abundance through targeting the SP1/TET1 axis, and TET1 depletion mimics homoharringtonine's therapeutic effects in AML. Our further 5hmC-seq and RNA-seq analyses, followed by a series of validation and functional studies, suggest that FLT3 is a critical down-stream target of homoharringtonine/SP1/TET1/5hmC signaling, and suppression of FLT3 and its downstream targets (e.g. MYC) contributes to the high sensitivity of FLT3-mutated AML cells to homoharringtonine. Collectively, our studies uncover a previously unappreciated DNA epigenome-related mechanism underlying the potent antileukemic effect of homoharringtonine, which involves suppression of the SP1/TET1/5hmC/FLT3/MYC signaling pathways in AML. Our work also highlights the particular promise of clinical application of homoharringtonine to treat human AML with FLT3 mutations, which accounts for more than 30% of total cases of AML.

Expression and prognostic analyses of the insulin-like growth factor 2 mRNA binding protein family in human pancreatic cancer.

BACKGROUND: Despite advances in early diagnosis and treatment, cancer remains the leading cause of mortality worldwide. The insulin-like growth factor 2 mRNA binding protein (IGF2BP) family has been reported to be involved in a variety of human malignant tumours. However, little is known about their expression and prognostic value in human pancreatic cancer. Therefore, we performed a detailed cancer versus normal differential analysis. METHODS: The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) databases were used to analyse the mRNA expression levels of the IGF2BP family in various cancers, including pancreatic cancer. Then, the LinkedOmics and GEPIA databases were used to assess the relation between the expression levels of IGF2BPs and overall survival (OS). Then, univariate and multivariate Cox regression analyses were performed, and subgroups based on grade and stage were analysed. The signalling pathways associated with IGF2BP2 and IGF2BP3 were then investigated via gene set enrichment analysis (GSEA). RESULTS: IGF2BP2 and IGF2BP3 were associated with each subset of OS based on grade and stage. Further clinical correlation analysis of IGF2BP2 and IGF2BP3 confirmed that IGF2BP2 and IGF2BP3 are fundamental factors in promoting pancreatic cancer progression. CONCLUSION: IGF2BP2 and IGF2BP3 are key factors in promoting the progression of pancreatic cancer and are closely related to overall survival.

Theoretical Characterization of Catalytically Active Species in Reductive Hydroxymethylation of Styrene with CO2 over a Bisphosphine-Ligated Copper Complex.

In this work, a density functional theory (DFT) study was performed to identify the catalytically active species in the copper-catalyzed three-component reductive hydroxymethylation of styrene with CO2 and hydrosilane. The calculations reveal that the dimeric copper(I) hydride species, formed in a mixture of the bisphosphine ligand, Cu(OAc)2, and hydrosilane, probably acts as the catalyst precursor. In the beginning, this species is catalytically competent to trigger the hydrocupration of styrene, along with the formation of the dimeric copper(I) alkyl intermediate. Subsequently, CO2 insertion into the dimeric copper(I) alkyl intermediate occurs, which is accompanied by the cleavage of the Cu-Cu bond and the generation of the monomeric copper(I) carboxylate intermediate. In the end, the sequential reduction of the monomeric copper(I) carboxylate intermediate with the hydrosilane produces the monomeric copper(I) hydride species as the actual catalyst and turns on the catalytic cycle. On the other hand, the monomeric copper(II) hydride species, yielded as the kinetic product in the initial reaction of the bisphosphine ligand, Cu(OAc)2, and hydrosilane, is also reactive for the hydrocupration of styrene. However, the resulting monomeric copper(II) alkyl intermediate is found to be the catalyst resting state, because of the much higher energy barrier demanded for the subsequent nucleophilic attack toward CO2. On the basis of the results of an activation-strain model (ASM) analysis and charge decomposition analysis (CDA), the low activity of the monomeric copper(II) alkyl intermediate can be ascribed to the more crowded environment around the central copper(II) ion and the weaker nucleophilicity of the alkyl moiety. Furthermore, all of the possible CuH species generated in the system are competent to promote the two-component hydrosilylation of CO2 with hydrosilane, which is an inevitable side reaction along with the reductive hydroxymethylation of styrene with CO2 and hydrosilane.

A novel putative betapartitivirus isolated from the plant-pathogenic fungus Rhizoctonia solani.

In this study, we describe the genome sequence of a novel double-stranded RNA (dsRNA) mycovirus, designated as "Rhizoctonia solani partitivirus 15" (RsPV15), from the phytopathogenic fungus Rhizoctonia solani. RsPV15 consists of two genomic double-stranded RNA segments, dsRNA-1 and dsRNA-2, which are 2433 bp and 2350 bp long, respectively. Each of the dsRNA segments contains a single open reading frame, encoding the putative RNA-dependent RNA polymerase and coat protein, respectively. Homology searches and phylogenetic analysis suggested that RsPV15 is a new member of the genus Betapartitivirus within the family Partitiviridae.

Development and Validation of a Reporter-Cell-Line-Based Bioassay for Therapeutic Soluble gp130-Fc.

Soluble glycoprotein 130 kDa (sgp130)-Fc fusion protein, an innovative therapeutic bio-macromolecular drug specifically targeting IL-6 trans-signaling, proved to have good potential for application in the treatment of chronic inflammatory diseases. A simple and quick bioassay for sgp130-Fc was developed in this study. First, a stable reporter cell line was obtained by transfecting CHO-K1 cells with a sis-inducible element (SIE)-driving luciferase reporter gene (CHO/SIE-Luc). Sgp130-Fc could inhibit the expression of luciferase induced by IL-6/sIL-6Rα complex, and the dose-response curve fitted the four-parameter logistic model, with 50% inhibitive concentration (IC50) being about 500 ng/mL and detection range between 40 and 5000 ng/mL. Both the intra-assay and inter-assay coefficient of variation (CV) were below 10.0%, and the accuracy estimates ranged from 94.1% to 106.2%. The assay indicated a good linearity (R² = 0.99) in the range of 50% to 150% of optimized initial concentration. No significant difference was found between the test results of new assay and BAF3/gp130 proliferation assay (unpaired t test, p = 0.4960, n = 6). The dose-response effect and copy number of the luciferase gene was basically unchanged after long-term culture (up to passage 60), demonstrating the stability of CHO/SIE-Luc cells. These results suggested that the new reporter assay was suited to routine potency determination of therapeutic sgp130-Fc.

A Cell-Based Strategy for Bioactivity Determination of Long-Acting Fc-Fusion Recombinant Human Growth Hormone.

The long-acting growth hormone (LAGH) is a promising alternative biopharmaceutical to treat growth hormone (GH) deficiency in children, and it was developed using a variety of technologies by several pharmaceutical companies. Most LAGH preparations, such as Fc fusion protein, are currently undergoing preclinical study and clinical trials. Accurate determination of bioactivity is critical for the efficacy of quality control systems of LAGH. The current in vivo rat weight gain assays used to determine the bioactivity of recombinant human GH (rhGH) in pharmacopoeias are time-consuming, expensive, and imprecise, and there are no recommended bioassays for LAGH bioactivity in pharmacopoeias. Therefore, we developed a cell-based bioassay for bioactivity determination of therapeutic long-acting Fc-fusion recombinant human growth hormone (rhGH-Fc) based on the luciferase reporter gene system, which is involved in the full-length human GH receptor (hGHR) and the SG (SIE and GAS) response element. The established bioassay was comprehensively validated according to the International Council for Harmonization (ICH) Q2 (R1) guidelines and the Chinese Pharmacopoeia, and is highly precise, time-saving, simple, and robust. The validated bioassay could be qualified for bioactivity determination during the research, development, and manufacture of rhGH-Fc, and other LAGH formulations.

Bioactivity Determination of a Therapeutic Recombinant Human Keratinocyte Growth Factor by a Validated Cell-based Bioassay.

The therapeutic recombinant human keratinocyte growth factor 1 (rhKGF-1) was approved by the FDA for oral mucositis resulting from hematopoietic stem cell transplantation for hematological malignancies in 2004. However, no recommended bioassay for rhKGF-1 bioactivity has been recorded in the U.S. Pharmacopoeia. In this study, we developed an rhKGF-1-dependent bioassay for determining rhKGF-1 bioactivity based on HEK293 and HaCat cell lines that stably expressed the luciferase reporter driven by the serum response element (SRE) and human fibroblast growth factor receptor (FGFR2) IIIb. A good responsiveness to rhKGF-1 and rhKGF-2 shared by target HEK293/HaCat cell lines was demonstrated. Our stringent validation was completely focused on specificity, linearity, accuracy, precision, and robustness according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, AAPS/FDA Bioanalytical Workshop and the Chinese Pharmacopoeia. We confirmed the reliability of the method in determining rhKGF bioactivity. The validated method is highly timesaving, sensitive, and simple, and is especially valuable for providing information for quality control during the manufacture, research, and development of therapeutic rhKGF.

Effect of Huaier granule on recurrence after curative resection of HCC: a multicentre, randomised clinical trial.

OBJECTIVE: There is little evidence that adjuvant therapy after radical surgical resection of hepatocellular carcinoma (HCC) improves recurrence-free survival (RFS) or overall survival (OS). We conducted a multicentre, randomised, controlled, phase IV trial evaluating the benefit of an aqueous extract of Trametes robinophila Murr (Huaier granule) to address this unmet need. DESIGN AND RESULTS: A total of 1044 patients were randomised in 2:1 ratio to receive either Huaier or no further treatment (controls) for a maximum of 96 weeks. The primary endpoint was RFS. Secondary endpoints included OS and tumour extrahepatic recurrence rate (ERR). The Huaier (n=686) and control groups (n=316) had a mean RFS of 75.5 weeks and 68.5 weeks, respectively (HR 0.67; 95% CI 0.55 to 0.81). The difference in the RFS rate between Huaier and control groups was 62.39% and 49.05% (95% CI 6.74 to 19.94; p=0.0001); this led to an OS rate in the Huaier and control groups of 95.19% and 91.46%, respectively (95% CI 0.26 to 7.21; p=0.0207). The tumour ERR between Huaier and control groups was 8.60% and 13.61% (95% CI -12.59 to -2.50; p=0.0018), respectively. CONCLUSIONS: This is the first nationwide multicentre study, involving 39 centres and 1044 patients, to prove the effectiveness of Huaier granule as adjuvant therapy for HCC after curative liver resection. It demonstrated a significant prolongation of RFS and reduced extrahepatic recurrence in Huaier group. TRIAL REGISTRATION: NCT01770431; Post-results.

iTRAQ-based quantitative analysis of age-specific variations in salivary proteome of caries-susceptible individuals.

BACKGROUND: Human saliva is a protein-rich, easily accessible source of potential biomarkers for the diagnosis of oral and systemic diseases. However, little is known about the changes in salivary proteome associated with aging of patients with dental caries. Here, we applied isobaric tags for relative and absolute quantitation (iTRAQ) in combination with multiple reaction monitoring mass spectrometry (MRM-MS) to characterize the salivary proteome profiles of subjects of different ages, presenting with and without caries, with the aim of identifying age-related biomarkers for dental caries. METHODS: Unstimulated whole saliva samples were collected from 40 caries-free and caries-susceptible young adults and elderly individuals. Salivary proteins were extracted, reduced, alkylated, digested with trypsin and then analyzed using iTRAQ-coupled LC-MS/MS, followed by GO annotation, biological pathway analysis, hierarchical clustering analysis, and protein-protein interaction analysis. Candidate verification was then conducted using MRM-MS. RESULTS: Among 658 salivary proteins identified using tandem mass spectrometry, 435 proteins exhibited altered expression patterns in different age groups with and without caries. Of these proteins, 96 displayed age-specific changes among caries-susceptible adults and elderly individuals, and were mainly associated with salivary secretion pathway, while 110 age-specific proteins were identified among healthy individuals. It was found that the age factor caused significant variations and played an important role in both healthy and cariogenic salivary proteomes. Subsequently, a total of 136 target proteins with complex protein-protein interactions, including 14 age-specific proteins associated with caries, were further successfully validated using MRM analysis. Moreover, non-age-specific proteins (histatin-1 and BPI fold-containing family B member 1) were verified to be important candidate biomarkers for common dental caries. CONCLUSIONS: Our proteomic analysis performed using the discovery-through-verification pipeline revealed distinct variations caused by age factor in both healthy and cariogenic salivary proteomes, highlighting the significance of age in the great potential of saliva for caries diagnosis and biomarker discovery.