Development of simultaneous quantitation method for 20 free advanced glycation end products using UPLC-MS/MS and clinical application in kidney injury.

Advanced glycation end products (AGEs), derived from the non-enzymatic glycation reaction, are defined as glycotoxins in various diseases including aging, diabetes and kidney injury. Exploring AGEs as potential biomarkers for these diseases holds paramount significance. Nevertheless, the high chemical structural similarity and great heterogeneity among AGEs present a formidable challenge when it comes to the comprehensive, simultaneous, and accurate detection of multiple AGEs in biological samples. In this study, an UPLC/MS/MS method for simultaneous quantification of 20 free AGEs in human serum was firstly established and applied to quantification of clinical samples from individuals with kidney injury. Simple sample preparation method through protein precipitation without derivatization was used. Method performances including imprecision, accuracy, sensitivity, linearity, and carryover were systematically validated. Intra- and inter- imprecision of 20 free AGEs were 1.93-5.94 % and 2.30-8.55 %, respectively. The method accuracy was confirmed with good recoveries ranging from 96.40 % to 103.25 %. The LOD and LOQ were 0.1-3.13 ng/mL and 0.5-6.25 ng/mL, respectively. Additionally, the 20 free AGEs displayed excellent linearity (R2 >0.9974) across a wide linear range (1.56-400 ng/mL). Finally, through simultaneous quantitation of 20 Free AGEs in 100 participants including kidney injury patient and healthy controls, we identified six free AGEs, including N6-carboxyethyl-L-arginine (CEA), N6-carboxymethyl-L-lysine (CML), methylglyoxal-derived hydroimidazolones (MG-H), N6-formyl-lysine, N6-carboxymethyl-L-arginine (CMA), and glyoxal-derived hydroimidazolone (G-H), could well distinguish kidney injury patients and healthy individuals. Among them, the levels of four free AGEs including CML, CEA, MG-H, and G-H strongly correlate with traditionally clinical markers of kidney disease. The high area under the curve (AUC) values (AUC=0.965) in receiver operating characteristic (ROC) curve indicated that these four free AGEs can be served as combined diagnostic biomarkers for the diagnosis of kidney disease.

Tanshinone IIA modulates cancer cell morphology and movement via Rho GTPases-mediated actin cytoskeleton remodeling.

Actin filaments form unique structures with robust actin bundles and cytoskeletal networks affixed to the extracellular matrix and interact with neighboring cells, which are crucial structures for cancer cells to acquire a motile phenotype. This study aims to investigate a novel antitumor mechanism by which Tanshinone IIA (Tan IIA) modulates the morphology and migration of liver cancer cells via actin cytoskeleton regulation. 97H and Huh7 exhibited numerous tentacle-like protrusions that interacted with neighboring cells. Following treatment with Tan IIA, 97H and Huh7 showed a complete absence of cytoplasmic protrusion and adherens junctions, thereby effectively impeding their migration capability. The fluorescence staining of F-actin and microtubules indicated that these tentacle-like protrusions and cell-cell networks were actin-based structures that led to morphological changes after Tan IIA treatment by retracting and reorganizing beneath the membrane. Tan IIA can reverse the actin depolymerization and cell morphology alterations induced by latrunculin A. Tan IIA down-regulated actin and Rho GTPases expression significantly, as opposed to inducing Rho signaling activation. Preventing the activity of proteasomes and lysosomes had no discernible impact on the modifications in cellular structure and protein expression induced by Tan IIA. However, as demonstrated by the puromycin labeling technique, the newly synthesized proteins were significantly inhibited by Tan IIA. In conclusion, Tan IIA can induce dramatic actin cytoskeleton remodeling by inhibiting the protein synthesis of actin and Rho GTPases, resulting in the suppression of tumor growth and migration. Targeting the actin cytoskeleton of Tan IIA is a promising strategy for HCC treatment.

Understanding the Effects of News-Finds-Me Perception on Health Knowledge and Information Seeking During Public Health Crises.

News-finds-me (NFM) perception is a belief that, in the era of social media, individuals can remain adequately well-informed about current events even if they do not actively seek news. While it has been examined in the context of general and political news, NFM perception has not been explored in the context of other genres of news. Through an online survey involving 1,001 Singaporeans, with the Planned Risk Information Seeking Model, this study examines how NFM perception is related to information seeking and COVID-19 knowledge. An issue-specific NFM perception was also proposed and tested in order to determine whether NFM perception and its associated effects differ when operationalized as general news exposure or issue-specific news relating to COVID-19. The negative relationship between general NFM perception and knowledge and the mediating role of information seeking on social media in this relationship are detected. It is also found that when the NFM perception is issue-specific (i.e. COVID-NFM perception), information insufficiency and intentions of information seeking on social media fully mediated the relationship between NFM perception and knowledge. Theoretical and practical implications are discussed.

A novel homozygous CYP17A1 mutation causes partial 17 α-hydroxylase/17,20-lyase deficiency in 46,XX: a case report and literature review.

Aim: 17 α-hydroxylase/17,20-lyase deficiency (17-OHD) is an extremely rare autosomal recessive disorder that typically causes hypertension, hypokalaemia, primary amenorrhoea, and the absence of secondary sex characteristics in 46,XX individuals. Partial 17-OHD is even rarer than complete 17-OHD and is prone to missed diagnosis due to its subtler symptoms. The aim of this study was to help early detection and diagnosis of partial 17-OHD.Methods: We present a case of a 41-year-old female (46,XX) patient with partial 17-OHD caused by a novel missense CYP17A1 mutation, c.391 A > C (p.T131P). This patient experienced hypertension, hypokalaemia and adrenal hyperplasia, but did not present with primary amenorrhoea or absence of secondary sex characteristics. Initially, she was misdiagnosed and underwent right and left adrenalectomy, but the procedures were ineffective. Afterward, she received a one-month treatment of 0.5 mg dexamethasone, which greatly relieved her symptoms. Additionally, we reviewed reports of thirteen other patients with partial 17-OHD in 46,XX individuals from the literature, totalling fourteen probands.Results: We found that primary amenorrhoea, hypertension, hypokalaemia, and ovarian cysts accounted for 15.4%, 42.9%, 38.5%, and 72.7% of these patients, respectively. In contrast, elevated serum progesterone was present in all patients.Conclusion: Based on our literature review, the absence of primary amenorrhoea, hypertension or hypokalaemia cannot rule out suspicion for 17-OHD in 46,XX individuals. However, an elevation in serum progesterone levels is a highly sensitive indicator for diagnosing 17-OHD.

What is the context?17-OHD is a rare cause of secondary hypertension, often with hypokalaemia, primary amenorrhoea and absence of secondary sex characteristics.Partial 17-OHD is an even rarer subtype of 17-OHD, with subtler symptoms.There are few reports concerning partial 17-OHD, especially in 46,XX patients.What is new?We reported a case of a 46,XX patient with partial 17-OHD caused by a novel missense CYP17A1 mutation, c.391 A > C (p.T131P).We also conducted a literature review to summarise the clinical, hormonal and genetic characteristics of fourteen 46,XX probands with partial 17-OHD.From the literature review, we found that:Most 46,XX patients with partial 17-OHD presented with partial pubic hair, breast development, oligomenorrhea or secondary amenorrhoea, normotension, and/or normokalemia.All 46,XX patients with partial 17-OHD presented with elevated serum progesterone.However, the relationship between in vitro enzyme activities of the 17-hydroxylase and/or17,20-lyase and clinical severity is still unclear.What is the impact?The current study can help early detection and diagnosis of partial 17-OHD.

A Functional Polymorphism Downstream of Vitamin A Regulator Gene CYP26B1 Is Associated with Hand Osteoarthritis.

While genetic analyses have revealed ~100 risk loci associated with osteoarthritis (OA), only eight have been linked to hand OA. Besides, these studies were performed in predominantly European and Caucasian ancestries. Here, we conducted a genome-wide association study in the Han Chinese population to identify genetic variations associated with the disease. We recruited a total of 1136 individuals (n = 420 hand OA-affected; n = 716 unaffected control subjects) of Han Chinese ancestry. We carried out genotyping using Axiom Asia Precisi on Medicine Research Array, and we employed the RegulomeDB database and RoadMap DNase I Hypersensitivity Sites annotations to further narrow down our potential candidate variants. Genetic variants identified were tested in the Geisinger's hand OA cohort selected from the Geisinger MyCode community health initiative (MyCode®). We also performed a luciferase reporter assay to confirm the potential impact of top candidate single-nucleotide polymorphisms (SNPs) on hand OA. We identified six associated SNPs (p-value = 6.76 × 10-7-7.31 × 10-6) clustered at 2p13.2 downstream of the CYP26B1 gene. The strongest association signal identified was rs883313 (p-value = 6.76 × 10-7, odds ratio (OR) = 1.76), followed by rs12713768 (p-value = 1.36 × 10-6, OR = 1.74), near or within the enhancer region closest to the CYP26B1 gene. Our findings showed that the major risk-conferring CC haplotype of SNPs rs12713768 and rs10208040 [strong linkage disequilibrium (LD); D' = 1, r2 = 0.651] drives 18.9% of enhancer expression activity. Our findings highlight that the SNP rs12713768 is associated with susceptibility to and severity of hand OA in the Han Chinese population and that the suggested retinoic acid signaling pathway may play an important role in its pathogenesis.

Locating the Mandibular Lingula Using Cone-Beam Computed Tomography: A Literature Review.

This study aimed to review the literature on adult mandibular lingula (ML) locations and related distances determined using cone-beam computed tomography (CBCT). A search was conducted for studies on CBCT using the following databases: PubMed, Web of Science, and Embase. The search results were limited to studies published between 1970 and 2021. The inclusion criteria were the investigation of ML location, CBCT, and participants aged ≥18 years. Eligible studies were examined for the distances from the lingual tip to the anterior ramus border, posterior ramus border, sigmoid notch, inferior ramus border, and occlusal plane. Eight studies on CBCT qualified for inclusion in the study. The mean distances from the ML to the anterior ramus border were 15.57 to 20 mm. In most of these, the ML was located above the occlusal plane. No significant differences were observed in the location and related distances for the ML among patients of different sexes, ethnicities, or skeletal patterns.

Gold Nanorods Inhibit Tumor Metastasis by Regulating MMP-9 Activity: Implications for Radiotherapy.

Dysregulation of matrix metalloproteinase (MMP) is strongly implicated in tumor invasion and metastasis. Nanomaterials can interact with proteins and have impacts on protein activity, which provides a potential strategy for inhibiting tumor invasion and metastasis. However, the regulation of MMP activity by nanomaterials has not been fully determined. Herein, we have found that gold nanorods (Au NRs) are able to induce the change of the secondary structure of MMP-9 and thereby inhibit their activity. Interestingly, the inhibition of MMP-9 activity is highly dependent on the aspect ratio of Au NRs, and an aspect ratio of 3.3 shows the maximum inhibition efficiency. Molecular dynamics simulations combined with mathematical statistics algorithm reveal the binding behaviors and interaction modes of MMP-9 with Au NRs in atomic details and disclose the mechanism of aspect ratio-dependent inhibition effect of Au NRs on MMP-9 activity. Au NRs with an aspect ratio of 3.3 successfully suppress the X-ray-activated invasion and metastasis of tumor by inhibiting MMP-9 activity. Our findings provide important guidance for the modulation of MMP-9 activity by tuning key parameters of nanomaterials and demonstrate that gold nanorods could be developed as potential MMP inhibitors.

Enrichment of Prevotella intermedia in human colorectal cancer and its additive effects with Fusobacterium nucleatum on the malignant transformation of colorectal adenomas.

BACKGROUND: Owing to the heterogeneity of microbiota among individuals and populations, only Fusobacterium nucleatum and Bacteroides fragilis have been reported to be enriched in colorectal cancer (CRC) in multiple studies. Thus, the discovery of additional bacteria contributing to CRC development in various populations can be expected. We aimed to identify bacteria associated with the progression of colorectal adenoma to carcinoma and determine the contribution of these bacteria to malignant transformation in patients of Han Chinese origin. METHODS: Microbiota composition was determined through 16S rRNA V3-V4 amplicon sequencing of autologous adenocarcinomas, adenomatous polyps, and non-neoplastic colon tissue samples (referred to as "tri-part samples") in patients with CRC. Enriched taxa in adenocarcinoma tissues were identified through pairwise comparison. The abundance of candidate bacteria was quantified through genomic quantitative polymerase chain reaction (qPCR) in tissue samples from 116 patients. Associations of candidate bacteria with clinicopathological features and genomic and genetic alterations were evaluated through odds ratio tests. Additionally, the effects of candidate bacteria on CRC cell proliferation, migration, and invasion were evaluated through the co-culture of CRC cells with bacterial cells or with conditioned media from bacteria. RESULTS: Prevotella intermedia was overrepresented in adenocarcinomas compared with paired adenomatous polyps. Furthermore, co-abundance of P. intermedia and F. nucleatum was observed in tumor tissues. More notably, the coexistence of these two bacteria in adenocarcinomas was associated with lymph node involvement and distant metastasis. These two bacteria also exerted additive effects on the enhancement of the migration and invasion abilities of CRC cells. Finally, conditioned media from P. intermedia promoted the migration and invasion of CRC cells. CONCLUSION: This report is the first to demonstrate that P. intermedia is enriched in colorectal adenocarcinoma tissues and enhances the migration and invasion abilities of CRC cells. Moreover, P. intermedia and F. nucleatum exert additive effects on the malignant transformation of colorectal adenomas into carcinomas. These findings can be used to identify patients at a high risk of malignant transformation of colorectal adenomas or metastasis of CRC, and they can accordingly be provided optimal clinical management.

Coordination of leaf functional traits under climatic warming in an arid ecosystem.

BACKGROUND: Climatic warming is increasing regionally and globally, and results concerning warming and its consequent drought impacts have been reported extensively. However, due to a lack of quantitative analysis of warming severities, it is still unclear how warming and warming-induced drought influence leaf functional traits, particularly how the traits coordinate with each other to cope with climatic change. To address these uncertainties, we performed a field experiment with ambient, moderate and severe warming regimes in an arid ecosystem over 4 years. RESULTS: Severe warming significantly reduced the specific leaf area and net photosynthetic rate with a relatively stable change and even enhancement under moderate warming, especially showing species-specific performance. The current results largely indicate that a coordinated trade-off can exist between plant functional traits in plant communities in a dryland ecosystem under ambient temperature conditions, which is strongly amplified by moderate warming but diminished or even eliminated by severe warming. Based on the present findings and recent results in the relevant literature, we advance the ecological conceptual models (e.g., LES and CSR) in the response to climatic warming in arid grassland communities, where the few key species play a crucial role by balancing their functional performances to cope with environmental change. CONCLUSION: Our results highlight the importance of coordination and/or trade-off between leaf functional traits for understanding patterns of climatic change-induced vegetation degradation and suggest that the plant community composition in these drylands could be shifted under future climate change.

Delayed inhibition of ERK and p38 attenuates neuropathic pain without affecting motor function recovery after peripheral nerve injury.

Peripheral nerve injuries (PNIs) often result in persistent neuropathic pain, seriously affecting quality of life. Existing therapeutic interventions for PNI-induced neuropathic pain are far from satisfactory. Extracellular signal-regulated kinases (ERKs) and p38 have been found to participate in triggering and maintaining PNI-induced neuropathic pain. However, ERK and p38 also contribute to axonal regeneration and motor function recovery after PNI, making it difficult to inhibit ERK and p38 for therapeutic purposes. In this study, we simultaneously characterized neuropathic pain and motor function recovery in a mouse sciatic nerve crush injury model to identify the time window for therapeutic interventions. We further demonstrated that delayed delivery of a combination of ERK and p38 inhibitors at three weeks after PNI could significantly alleviate PNI-induced neuropathic pain without affecting motor function recovery. Additionally, the combined use of these two inhibitors could suppress pain markedly better than either inhibitor alone, possibly reducing the required dose of each inhibitor and alleviating the side effects and risks of the inhibitors when used individually.

Postoperative Changes in Tongue Area and Pharyngeal Airway Space following Mandibular Setback Surgery through Intraoral Vertical Ramus Osteotomy.

PURPOSE: The aim of this study was to determine changes in the tongue area and pharyngeal airway space (PAS) after intraoral vertical ramus osteotomy (IVRO). MATERIALS AND METHODS: Serial lateral cephalograms of 40 patients with mandibular prognathism who underwent IVRO were evaluated before (T1), immediately after (T2), and more than 1 year after (T3) surgery. Paired t-tests and Pearson's correlation analysis were used to evaluate the postoperative changes in the mandible, nasopharyngeal airway (NOP), retropalatal pharyngeal airway (RPP), retroglossal pharyngeal airway (RGP), hypopharyngeal airway (HOP), PAS, and tongue area (TA). The null hypothesis states that there are no significant correlations among the extent of mandibular setback and the changes in the TA and PAS after IVRO. RESULTS: Immediately after the operation (T12), the mandible was set back by 12.6 mm. The NOP, HOP, and PAS were significantly reduced by 35.7 mm2, 116 mm2, and 185 mm2, respectively. The TA was increased by 69.6 mm2. The changes in PAS and TA revealed no significant difference between female and male patients at T12, T23, and T13. Moreover, no significant correlations were found among the extent of mandibular setback, TA changes, and PAS changes after IVRO. Thus, the null hypothesis was accepted. CONCLUSIONS: At the final follow-up (T13), no significant change was found in the PAS (including NOP, RPP, RGP, and HOP) and TA. The changes in PAS and TA revealed no significant difference between female and male patients at T12, T23, and T13.

Using multi-disciplinary teams to treat obese patients helps improve clinical efficacy: the general practitioner's perspective.

OBJECTIVE: This paper aims to explore the influences of multi-disciplinary teams (MDT) from the general practitioner's (GP's) perspective on the clinical efficacy of treating obese patients. METHODS: Admitted to our hospital from January 2018 to October 2019, 127 obese patients were divided into two groups based on the different models of diagnosis and treatment each underwent. The routine diagnostic and treatment model was administered to the patients in the control group (60 cases), and the MDT model was administered to the patients in the research group (67 cases). The weight loss success rates in both groups were observed. Before and after the treatment, the blood glucose, blood lipid, tumor necrosis factor-α (TNF-α), adiponectin (APN), leptin (LP), and recombinant human fibroblast growth factor 21 (FGF-21) levels were measured. The SAS and SDS scores were evaluated. RESULTS: After the treatment, the weight loss success rate in the research group was significantly higher than it was in the control group, and the FPG and the 2hPBG levels were significantly lower in the research group. Compared with the control group, the TC, TG, and LDL-C levels were remarkably lower in the study group, and the HDL-C levels were remarkably higher in the research group. The TNF-α, LP, and FGF-21 levels were significantly lower in the research group, and the APN levels were significantly higher. The research group had significantly lower SAS and SDS scores and higher GSES scores. CONCLUSION: MDTs from the GP's perspective are conducive to increasing the weight loss success rate and improving the blood glucose, blood lipid and adipokine levels in obese patients.

Fiber optic nanogold-linked immunosorbent assay for rapid detection of procalcitonin at femtomolar concentration level.

A rapid and ultrasensitive biosensing method based on fiber optic nanogold-linked immunosorbent assay is reported. The method employs an immobilized capture probe on the fiber core surface of an optical fiber and a detection probe conjugated to gold nanoparticles (AuNPs) in a solution. Introduction of a sample containing an analyte and the detection probe into a biosensor chip leads to the formation of a sandwich-like complex of capture probe-analyte-detection probe on the fiber core surface, through which nanoplasmonic absorption of the fiber optic evanescent wave occurs. The performance of this method has been evaluated by its application to the detection of procalcitonin (PCT), an important biomarker for sepsis. In this study, anti-PCT capture antibody is functionalized on an unclad segment of an optical fiber to yield a fiber sensor and anti-PCT detection antibody is conjugated to AuNPs to afford nanoplasmonic probes. The method provides a wide linear response range from 1 pg/mL to 100 ng/mL (5 orders) and an extremely low limit of detection of 95 fg/mL (7.3 fM) for PCT. In addition, the method shows a good correlation in determining PCT in blood plasma with the clinically validated electrochemiluninescent immunoassay. Furthermore, the method is quick (analysis time ≤15 min), requires low-cost instrumentation and sensor chips, and is also potentially applicable to the detection of many other biomarkers.

Overexpression of P2X4 receptor in Schwann cells promotes motor and sensory functional recovery and remyelination via BDNF secretion after nerve injury.

Of the seven P2X receptor subtypes, P2X4 receptor (P2X4R) is widely distributed in the central nervous system, including in neurons, astrocytes, and microglia. Accumulating evidence supports roles for P2X4R in the central nervous system, including regulating cell excitability, synaptic transmission, and neuropathic pain. However, little information is available about the distribution and function of P2X4R in the peripheral nervous system. In this study, we find that P2X4R is mainly localized in the lysosomes of Schwann cells in the peripheral nervous system. In cultured Schwann cells, TNF-a not only enhances the synthesis of P2X4R protein but also promotes P2X4R trafficking to the surface of Schwann cells. TNF-a-induced BDNF secretion in Schwann cells is P2X4R dependent. in vivo experiments reveal that expression of P2X4R in Schwann cells of injured nerves is strikingly upregulated following nerve crush injury. Moreover, overexpression of P2X4R in Schwann cells by genetic manipulation promotes motor and sensory functional recovery and accelerates nerve remyelination via BDNF release following nerve injury. Our results suggest that enhancement of P2X4R expression in Schwann cells after nerve injury may be an effective approach to facilitate the regrowth and remyelination of injured nerves.

Analysis of virulence diversity of 73 Helicobacter pylori strains isolated in Guizhou province, China.

The present study aimed to investigate the virulence diversity of Helicobacter pylori (H. pylori) in major ethnic groups residing in Guizhou province, China, and its association with clinical outcomes. Gastric mucosal biopsies were collected from the pylorus of patients with gastrointestinal disorders. H. pylori was identified by colonial morphology, Gram staining, a urease test and H. pylori­specific 16S rRNA gene fragment PCR amplification. DNA was extracted from pure culture and used for virulence gene analysis. The cytotoxin associated gene A (cagA), vacuolating cytotoxin A (vacA) and induced by contact with epithelium gene A (iceA) genes were analyzed by polymerase chain reaction analysis. The cagA gene was further analyzed through sequencing of the C­terminal region containing EPIYA motifs, and phylogenetic analysis of the cagA C­terminal variable region was performed using MEGA 6.0 software. In the present study, 73 H. pylori strains were isolated from clinical samples. cagA genotypes were detected in all strains, namely cagA­AB, ­ABC, ­ABD and ­BD genotypes were found in five (6.85%), three (4.11%), 63 (86.30%) and two (2.74%) isolates, respectively. Phylogenetic analysis showed that there was a clustering association between the cagA­AB and cagA­ABC genotypes, and between the cagA­ABD and cagA­BD genotypes. In terms of the frequency of the four EPIYA or EPIYA­like motifs, the most predominant was EPIYA (92.92%), followed by EPIYT (3.77%), ESIYA (2.83%) and ESIYT (0.47%). The predominant vacA genotype was s1c/m2 (65.75%), and the predominant iceA genotype was iceA1 (79.45%). There were no associations between the H. pylori cagA, vacA or iceA genotypes and clinical outcomes. No significant difference was found in the distribution of these genotypes according to the age, ethnicity or location of residence of patients. In conclusion, H. pylori isolated from patients in Guizhou region, China, showed a unique genotype, which was mainly East Asia­type cagA (ABD), vacA s1c/m2 genotype or iceA1­postiive. These results provide important information on the distribution of H. pylori virulence genotypes in Guizhou province, China.

A genome-wide association study links small-vessel ischemic stroke to autophagy.

Genome-wide association studies (GWAS) can serve as strong evidence in correlating biological pathways with human diseases. Although ischemic stroke has been found to be associated with many biological pathways, the genetic mechanism of ischemic stroke is still unclear. Here, we performed GWAS for a major subtype of stroke-small-vessel occlusion (SVO)-to identify potential genetic factors contributing to ischemic stroke. GWAS were conducted on 342 individuals with SVO stroke and 1,731 controls from a Han Chinese population residing in Taiwan. The study was replicated in an independent Han Chinese population comprising an additional 188 SVO stroke cases and 1,265 controls. Three SNPs (rs2594966, rs2594973, rs4684776) clustered at 3p25.3 in ATG7 (encoding Autophagy Related 7), with P values between 2.52 × 10-6 and 3.59 × 10-6, were identified. Imputation analysis also supported the association between ATG7 and SVO stroke. To our knowledge, this is the first GWAS to link stroke and autophagy. ATG7, which has been implicated in autophagy, could provide novel insights into the genetic basis of ischemic stroke.

A genome-wide association study identifies a novel susceptibility locus for the immunogenicity of polyethylene glycol.

Conjugation of polyethylene glycol (PEG) to therapeutic molecules can improve bioavailability and therapeutic efficacy. However, some healthy individuals have pre-existing anti-PEG antibodies and certain patients develop anti-PEG antibody during treatment with PEGylated medicines, suggesting that genetics might play a role in PEG immunogenicity. Here we perform genome-wide association studies for anti-PEG IgM and IgG responses in Han Chinese with 177 and 140 individuals, defined as positive for anti-PEG IgM and IgG responses, respectively, and with 492 subjects without either anti-PEG IgM or IgG as controls. We validate the association results in the replication cohort, consisting of 211 and 192 subjects with anti-PEG IgM and anti-PEG IgG, respectively, and 596 controls. We identify the immunoglobulin heavy chain (IGH) locus to be associated with anti-PEG IgM response at genome-wide significance (P = 2.23 × 10-22). Our findings may provide novel genetic markers for predicting the immunogenicity of PEG and efficacy of PEGylated therapeutics.Some individuals develop antibodies against the polyethylene glycol that is commonly used in therapeutic preparations. Here the authors conduct a GWAS in Han Chinese and find the IGH locus is associated with anti-PEG IgM.

Autophagy mediates cell cycle response by regulating nucleocytoplasmic transport of PAX6 in limbal stem cells under ultraviolet-A stress.

Limbal stem cells (LSC) account for homeostasis and regeneration of corneal epithelium. Solar ultraviolet A (UVA) is the major source causing oxidative damage in the ocular surface. Autophagy, a lysosomal degradation mechanism, is essential for physiologic function and stress defense of stem cells. PAX6, a master transcription factor governing corneal homeostasis by regulating cell cycle and cell fate of LSC, responds to oxidative stress by nucleocytoplasmic shuttling. Impaired autophagy and deregulated PAX6 have been reported in oxidative stress-related ocular surface disorders. We hypothesize a functional role for autophagy and PAX6 in LSC's stress response to UVA. Therefore, human LSC colonies were irradiated with a sub-lethal dose of UVA and autophagic activity and intracellular reactive oxygen species (ROS) were measured by CYTO-ID assay and CM-H2DCFDA live staining, respectively. Following UVA irradiation, the percentage of autophagic cells significantly increased in LSC colonies while intracellular ROS levels remained unaffected. siRNA-mediated knockdown (KD) of ATG7 abolished UVA-induced autophagy and led to an excessive accumulation of ROS. Upon UVA exposure, LSCs displayed nuclear-to-cytoplasmic translocation of PAX6, while ATG7KD or antioxidant pretreatment largely attenuated the intracellular trafficking event. Immunofluorescence showing downregulation of proliferative marker PCNA and induction of cell cycle regulator p21 indicates cell cycle arrest in UVA-irradiated LSC. Abolishing autophagy, adenoviral-assisted restoration of nuclear PAX6 or antioxidant pretreatment abrogated the UVA-induced cell cycle arrest. Adenoviral expression of an ectopic PAX gene, PAX7, did not affect UVA cell cycle response. Furthermore, knocking down PAX6 attenuated the cell cycle progression of irradiated ATG7KD LSC by de-repressing p21 expression. Collectively, our data suggest a crosstalk between autophagy and PAX6 in regulating cell cycle response of ocular progenitors under UVA stress. Autophagy deficiency leads to impaired intracellular trafficking of PAX6, perturbed redox balance and uncurbed cell cycle progression in UVA-stressed LSCs. The coupling of autophagic machinery and PAX6 in cell cycle regulation represents an attractive therapeutic target for hyperproliferative ocular surface disorders associated with solar radiation.

Pharmacogenetic dosing of warfarin in the Han-Chinese population: a randomized trial.

AIM: This study aimed to determine clinical utility of genotype-guided dosing for warfarin in Han-Chinese. METHODS: A total of 320 patients were randomly assigned International Warfarin Pharmacogenetic Consortium algorithm, Taiwan algorithm and optimal clinical care arms. The primary outcome of the study was the percentage of time in the therapeutic range during the first 90 days of treatment. RESULTS: The percentage of time in the therapeutic range of the clinical care group in the first 2 weeks was significantly higher than the algorithm groups. This difference was no longer observed after 4 weeks. No difference in excessive anticoagulation (international normalized ratio ≥4.0) and adverse events was observed. CONCLUSION: Genotype-guided dosing did not provide significant benefit. Loading dose with frequent international normalized ratio monitoring could provide sufficient control of anticoagulation.

Tyrosinase-Cre-Mediated Deletion of the Autophagy Gene Atg7 Leads to Accumulation of the RPE65 Variant M450 in the Retinal Pigment Epithelium of C57BL/6 Mice.

Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of Atg7f/f Tyr-Cre mice (on a C57BL/6 background), in which Cre recombinase is expressed under the control of the tyrosinase promoter to delete the autophagy gene Atg7. In line with pigment cell-directed blockade of autophagy, the RPE and the melanocytes of the choroid showed strong accumulation of the autophagy adaptor and substrate, sequestosome 1 (Sqstm1)/p62, relative to the levels in control mice. Immunofluorescence and Western blot analysis demonstrated that the RPE, but not the choroid melanocytes, of Atg7f/f Tyr-Cre mice also had strongly increased levels of retinoid isomerohydrolase RPE65, a pivotal enzyme for the maintenance of visual perception. In contrast to Sqstm1, genes involved in retinal regeneration, i.e. Lrat, Rdh5, Rgr, and Rpe65, were expressed at higher mRNA levels. Sequencing of the Rpe65 gene showed that Atg7f/f and Atg7f/f Tyr-Cre mice carry a point mutation (L450M) that is characteristic for the C57BL/6 mouse strain and reportedly causes enhanced degradation of the RPE65 protein by an as-yet unknown mechanism. These results suggest that the increased abundance of RPE65 M450 in the RPE of Atg7f/f Tyr-Cre mice is, at least partly, mediated by upregulation of Rpe65 transcription; however, our data are also compatible with the hypothesis that the RPE65 M450 protein is degraded by Atg7-dependent autophagy in Atg7f/f mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms.