Cost-Efficient Sequence-Based Nonextensible Oligonucleotide in Real-Time PCR and High-Throughput Sequencing.

Molecular detection of disease-associated mutations, especially those with low abundance, is essential for academic research and clinical diagnosis. Certain variant detection methods reach satisfactory sensitivity and specificity in detecting rare mutations based on the introduction of blocking oligos to prevent the amplification of wild-type or unwanted templates, thus selectively amplifying and enriching the mutations. These blocking oligos usually suppress PCR amplification through the 3' chemical modifications, with high price, slow synthesis, and reduced purity. Herein, we introduce chemistry-free designs to block enzymatic extension during PCR by the steric hindrance from the secondary structures attached to the 3' end of the oligos (nonextensible oligonucleotide, NEO). We demonstrated that NEO efficiently prohibited the extension of both Taq and high-fidelity DNA polymerases. By further applying NEO as blockers in blocker displacement amplification (BDA) qPCR, multiplex BDA (mBDA) NGS, and quantitative BDA (QBDA) NGS methods, we showed that NEO blockers had performance comparable with previously validated chemical modifications. Comparison experiments using QBDA with NEO blockers and droplet digital PCR (ddPCR) on clinical formalin-fixed paraffin-embedded (FFPE) samples exhibited 100% concordance. Lastly, the ability of NEO to adjust plex uniformity through changes of PCR amplification efficiency was demonstrated in an 80-plex NGS panel.

Global Burden of Respiratory Diseases Attributable to Ambient Particulate Matter Pollution: Findings From the Global Burden of Disease Study 2019.

Background: Exposure to ambient particulate matter pollution (APMP) is a global health issue that directly affects the human respiratory system. Thus, we estimated the spatiotemporal trends in the burden of APMP-related respiratory diseases from 1990 to 2019. Methods: Based on the Global Burden of Disease Study 2019, data on the burden of APMP-related respiratory diseases were analyzed by age, sex, cause, and location. Joinpoint regression analysis was used to analyze the temporal trends in the burden of different respiratory diseases over the 30 years. Results: Globally, in 2019, APMP contributed the most to chronic obstructive pulmonary disease (COPD), with 695.1 thousand deaths and 15.4 million disability-adjusted life years (DALYs); however, the corresponding age-standardized death and DALY rates declined from 1990 to 2019. Similarly, although age-standardized death and DALY rates since 1990 decreased by 24% and 40%, respectively, lower respiratory infections (LRIs) still had the second highest number of deaths and DALYs attributable to APMP. This was followed by tracheal, bronchus, and lung (TBL) cancer, which showed increased age-standardized death and DALY rates during the past 30 years and reached 3.78 deaths per 100,000 persons and 84.22 DALYs per 100,000 persons in 2019. Among children aged < 5 years, LRIs had a huge burden attributable to APMP, whereas for older people, COPD was the leading cause of death and DALYs attributable to APMP. The APMP-related burdens of LRIs and COPD were relatively higher among countries with low and low-middle socio-demographic index (SDI), while countries with high-middle SDI showed the highest burden of TBL cancer attributable to APMP. Conclusions: APMP contributed substantially to the global burden of respiratory diseases, posing a significant threat to human health. Effective actions aimed at air pollution can potentially avoid an increase in the PM2.5-associated disease burden, especially in highly polluted areas.

Mass spectrometric analysis of digesta does not improve the allergenicity assessment of GM crops.

An investigation of the potential allergenicity of newly expressed proteins in genetically modified (GM) crops comprises part of the assessment of GM crop safety. However, allergenicity is not completely predictable from a definitive assay result or set of protein characteristics, and scientific opinions regarding the data that should be used to assess allergenicity are continuously evolving. Early studies supported a correlation between the stability of a protein exposed to digestive enzymes such as pepsin and the protein's status as a potential allergen, but over time the conclusions of these earlier studies were not confirmed. Nonetheless, many regulatory authorities, including the European Food Safety Authority (EFSA), continue to require digestibility analyses as a component of GM crop risk assessments. Moreover, EFSA has recently investigated the use of mass spectrometry (MS), to make digestion assays more predictive of allergy risk, because it can detect and identify small undigested peptides. However, the utility of MS is questionable in this context, since known allergenic peptides are unlikely to exist in protein candidates intended for commercial development. These protein candidates are pre-screened by the same bioinformatics processes that are normally used to identify MS targets. Therefore, MS is not a standalone allergen identification method and also cannot be used to predict previously unknown allergenic epitopes. Thus, the suggested application of MS for analysis of digesta does not improve the poor predictive power of digestion assays in identifying allergenic risk.

MicroRNA-31-5p Exacerbates Lipopolysaccharide-Induced Acute Lung Injury via Inactivating Cab39/AMPKα Pathway.

Acute lung injury (ALI) and the subsequent acute respiratory distress syndrome remain devastating diseases with high mortality rates and poor prognoses among patients in intensive care units. The present study is aimed at investigating the role and underlying mechanisms of microRNA-31-5p (miR-31-5p) on lipopolysaccharide- (LPS-) induced ALI. Mice were pretreated with miR-31-5p agomir, antagomir, and their negative controls at indicated doses for 3 consecutive days, and then they received a single intratracheal injection of LPS (5 mg/kg) for 12 h to induce ALI. MH-S murine alveolar macrophage cell lines were cultured to further verify the role of miR-31-5p in vitro. For AMP-activated protein kinase α (AMPKα) and calcium-binding protein 39 (Cab39) inhibition, compound C or lentiviral vectors were used in vivo and in vitro. We observed an upregulation of miR-31-5p in lung tissue upon LPS injection. miR-31-5p antagomir alleviated, while miR-31-5p agomir exacerbated LPS-induced inflammation, oxidative damage, and pulmonary dysfunction in vivo and in vitro. Mechanistically, miR-31-5p antagomir activated AMPKα to exert the protective effects that were abrogated by AMPKα inhibition. Further studies revealed that Cab39 was required for AMPKα activation and pulmonary protection by miR-31-5p antagomir. We provide the evidence that endogenous miR-31-5p is a key pathogenic factor for inflammation and oxidative damage during LPS-induced ALI, which is related to Cab39-dependent inhibition of AMPKα.

Template-based peptide modeling for celiac risk assessment of newly expressed proteins in GM crops.

Newly expressed proteins in genetically modified (GM) crops are subject to celiac disease risk assessment according to EFSA guidelines. Amino acid identity matches between short peptides (9aa) and known celiac restricted epitopes are required to be further evaluated through peptide modeling; however, validated methods and criteria are not yet available. In this investigation, several structures of HLA-DQ2.5/peptide/TCR (T-cell receptor) complexes were analyzed and two template-based peptide molding software packages were evaluated using various peptides including ones not associated with celiac disease. Structural characterization indicates that residues at P(position)1, P2, P5, P8, and P9 in the 9aa restricted epitopes also contribute to the binding of celiac peptides to the HLA-DQ2.5 antigen in addition to the presence of the motif Q/EX1PX2 starting at P4 or P6. The recognition of the HLA-DQ2.5/peptide complex by TCR is through specific interactions between the residues in the restricted epitopes and some loop structures in the TCR. The template-based software package GalaxyPepDock seems to be suitable for the application of peptide modeling when an estimated accuracy value of >0.95 combined with >160 interaction similarity score are used as a threshold for biologically meaningful in silico binding. Nevertheless, caution should be exercised when applying peptide modeling to celiac disease risk assessment until methods are rigorously validated and further evaluated to demonstrate its value in the risk assessment of newly expressed proteins in GM crops.

[Cost-Effectiveness Analysis of Different Chemotherapy Regimens in the Treatment of Patients with Multiple Myeloma].

OBJECTIVE: To compare the pharmaco-economic effect of 3 chemotherapeutic regimens in the treatment of patients with multiple myeloma(MM). METHODS: One hundred and thirty-eight newly diagnosed cases of MM in our hospital were analyzed retrospectively, and then MM patients were divided into group A, B and C group according to therapeutic regimen. Group A was treated with VCD therapeutic regimen (bortezomib + cyclophosphamide + dexamethasone, 63 cases), The patients in group B was treated with BiCTD therapeutic regimen (clarithromycin+cyclophosphamide+thalidomide+dexamethasone, 44 cases), The patients in group C was treated with CTD therapeutic regimen (cyclophosphamide+ thalidomide+dexamethasone, 33 cases). The clinical efficacy, adverse reaction, cost-effectiveness were observed and analysed after 4 courses of treatment among 3 groups. RESULTS: The overall response rates of group A, B and C were 96.83%, 81.82% and 64.52% with statistical significant difference (P<0.01). The high efficiency response rates of 3 groups were 82.5%, 59.09%, 32.26% with very significant statistical difference (P<0.01). The infection rate of group A was statistically and significantly higher than other 2 groups (P=0.048), and the constipation rate in group A was statistically and significantly higer than that in group B and C (P<0.05). The cost-effectiveness ratios of 3 groups were 69567.44, 20765.12 and 21475.48, respectively. The incremental cost-effectiveness ratio of group A and B were 183933.21 and 22259.09, as compared with group C. The result was in accordance with sensitivity test. CONCLUSION: Clinicial efficacy of group A is the best,but group B has advantages on cost-effectiveness ratio as compared with other groups, otherwise, group B has low incidence of adverse reaction. In the view of safety, therapeutic efficacy and pharmacoeconomics for treatment of patients with MM, the BiCTD regimen has been confirmed to be superior to the other 2 groups.

Evaluation of global sequence comparison and one-to-one FASTA local alignment in regulatory allergenicity assessment of transgenic proteins in food crops.

To address the high false positive rate using >35% identity over 80 amino acids in the regulatory assessment of transgenic proteins for potential allergenicity and the change of E-value with database size, the Needleman-Wunsch global sequence alignment and a one-to-one (1:1) local FASTA search (one protein in the target database at a time) using FASTA were evaluated by comparing proteins randomly selected from Arabidopsis, rice, corn, and soybean with known allergens in a peer-reviewed allergen database (http://www.allergenonline.org/). Compared with the approach of searching >35%/80aa+, the false positive rate measured by specificity rate for identification of true allergens was reduced by a 1:1 global sequence alignment with a cut-off threshold of ≧30% identity and a 1:1 FASTA local alignment with a cut-off E-value of ≦1.0E-09 while maintaining the same sensitivity. Hence, a 1:1 sequence comparison, especially using the FASTA local alignment tool with a biological relevant E-value of 1.0E-09 as a threshold, is recommended for the regulatory assessment of sequence identities between transgenic proteins in food crops and known allergens.

An outpatient antibacterial stewardship intervention during the journey to JCI accreditation.

BACKGROUND: Antibacterial overuse, misuse and resistance have become a major global threat. The Joint Commission International (JCI) accreditation standards include quality improvement and patient safety, which is exemplified by antimicrobial stewardship. There are currently few reports on interventions to improve the quality of outpatient antibacterial prescribing. METHODS: A before-after intervention study, aiming at antibacterial use in outpatients, was performed in a university-affiliated hospital with 2.8 million outpatient visits annually during the journey to JCI accreditation (March of 2012 - March of 2013). Comprehensive intervention measures included formulary adjustment, classification management, motivational, information technological, educational and organizational measures. A defined daily dose (DDD) methodology was applied. Pharmacoeconomic data and drug-related problems (DRPs) were statistically compared between the two phases. RESULTS: The variety of antibacterials available in outpatient pharmacy decreased from 38 to 16. The proportion of antibacterial prescriptions significantly decreased (12.7% versus 9.9%, P < 0.01). The proportion of prescriptions containing the restricted antibacterials was 30.4% in the second phase, significantly lower than the value of 44.7% in the first phase (P < 0.01). The overall proportion of oral versus all antibacterial prescriptions increased (94.0% to 100%, P < 0.01) when measured as defined daily doses. Statistically significant increases in relative percentage of DDDs of oral antibacterials (i.e., DDDs of individual oral antibacterial divided by the sum of DDDs of all antibacterials) were observed with moxifloxacin, levofloxacin, cefuroxime axetil, ornidazole, clindamycin palmitate, cefaclor, amoxicillin and clarithromycin. Occurrence rate of DRPs decreased from 13.6% to 4.0% (P < 0.01), with a larger decrease seen in surgical clinics (surgical: 19.5% versus 5.6%; internal medicine: 8.4% versus 2.8%, P < 0.01). The total expenditure on antibacterials for outpatients decreased by 34.7% and the intervention program saved about 6 million Chinese Yuan Renminbi (CNY) annually. CONCLUSION: The one-year intervention program on outpatient antibacterial use during the journey to JCI accreditation reduced the expenditure on antibacterials, improved the appropriateness of antibacterial prescriptions. Quality improvements need integrated multifaceted intervention measures and long-term adherence to the antibiotic stewardship. Approach of i.v. to oral antibacterial switch, classification management, and motivational measures may play the most efficient role in changing antibacterial prescription practices.

[Significance of low molecular weight urinary protein for assessment of early renal damage in patients with multiple myeloma].

This study was purposed to evaluate the clinical significance of low molecular weight urinary proteins for diagnosis of early renal damage in patients with multiple myeloma (MM). Medical records of 278 patients with MM in Nanjing School of Clinical Medicine from January 2004 to May 2012 were analyzed retrospectively. These patients were divided into 3 groups: glomerular damage group (n = 143), tubular damage group (n = 114) and normal group (n = 21). The clinical and laboratorial data were compared among them. The correlations of urinary retinol-binding protein (RBP) or urinary N-acetyl-ß-D-amino-glucosaminidase (NAG) with blood urea nitrogen (BUN), Scr, blood cystatin-C (Cys-C), clearance of creatinine (Ccr), 24 h protein uria and 24 h urine light chains were further analyzed, and the correlation of renal tubulointerstitial lesion scores with low molecular weight urinary proteins in 61 patients were also analyzed. The area under curve (ROC curve) was used to evaluate and compare the discrimination of urinary RBP and urinary NAG. The results showed that glomerular damage group had higher urinary RBP than tubular damage group. However, glomerular damage group had lower urinary NAG than tubular damage group. The two groups had higher urinary RBP and urinary NAG than that in normal group. Urinary RBP related positively to the level of Scr, BUN, Cys-C, 24 h proteinurias and related negatively to the level of Ccr. Urinary NAG related positively to the level of 24 h proteinurias, Ccr and related negatively to the level of Cys-C. Renal tubulointerstitial lesions were significantly correlated with urinary RBP, but weakly correlated with urinary NAG. It is concluded that urinary RBP significantly correlates with renal tubular damage. Compared with urinary NAG, urinary RBP can better assess the extent of renal damage, and has higher specificity.

Neuroendoscopic fenestration of the septum pellucidum for monoventricular hydrocephalus.

OBJECTIVES: Monoventricular hydrocephalus (MH) is a rare condition in which the site of obstruction is located around one of the interventricular foramen. In this paper, the authors offer their experiences in the neuroendoscopic management of this uncommon type of hydrocephalus. PATIENTS AND METHODS: The authors retrospectively reviewed 12 neuroendoscopic procedures performed between July 2003 and June 2011 with MH. Clinical and radiological charts were reviewed and analysed. RESULTS: The operative course is a simple and successful procedure, and the postoperative complaints are mild. The postoperative radiological findings showed maintenance of ventricular enlargement in four cases and a significant decrease in enlargement in eight patients. However, upon shifting of the septum pellucidum, the periventricular transudation disappeared in all patients. CONCLUSIONS: Fenestration of the septum pellucidum by neuroendoscope is the best treatment for patients with monoventricular hydrocephalus. The approach for fenestration of the septum pellucidum is based on the experience of the neurosurgeon and the preoperative planning.

[Multi-state model in the evaluation of outcome on mild cognitive impairment to Alzheimer's disease].

OBJECTIVE: The aim of this study was to introduce the multi-state Markov model for the prediction of mild cognitive impairment (MCI) to Alzheimer's disease (AD) and to find out the related factors for AD prevention and early intervention among the elderly. METHODS: MCI, moderate to severe cognitive impairment, and AD were defined as state 1, 2 and 3, respectively. A three-state homogeneous model with discrete states and discrete times from data on six follow-up visits was constructed to explore factors for various progressive stages from MCI to AD. Transition probability and survival curve were made after the model fit assessment. RESULTS: At the level of 0.05, data from the multivariate analysis showed that gender (HR=1.23, 95%CI: 1.12-1.38), age (HR=1.37, 95%CI: 1.07-1.72), hypertension (HR=1.54, 95%CI: 1.31-2.19) were statistically significant for the transition from state 1 to state 2, while age (HR=0.78, 95%CI: 0.69-0.98), education level (HR=1.35, 95%CI: 1.09-1.86) and reading (HR=1.20, 95%CI: 1.01-1.41) were statistically significant for transition from state 2 to state 1, and gender (HR=1.59, 95%CI: 1.33-1.89), age (HR=1.33, 95%CI: 1.02-1.64), hypertension (HR=1.22, 95%CI: 1.11-1.43), diabetes (HR=1.52, 95%CI: 1.12-2.00), ApoEe4 (HR=1.44, 95%CI: 1.09-1.68) were statistically significant for transition from state 2 to state 3. Based on the fitted model, the three-year transition probabilities during each state at average covariate level were estimated. CONCLUSION: To delay the disease progression of MCI, phase by phase prevention measures could be adopted based on the main factors of each stage. Multi-state Markov model could imitate the natural history of disease and showed great advantage in dynamically evaluating the development of chronic diseases with multi-states and multi-factors.

Preliminary safety assessment of a membrane-bound delta 9 desaturase candidate protein for transgenic oilseed crops.

A gene encoding delta 9 desaturase (D9DS), an integral membrane protein, is being considered for incorporation into oilseed crops to reduce saturated fatty acids and thus improve human nutritional value. Typically, a safety assessment for transgenic crops involves purifying heterologously produced transgenic proteins in an active form for use in safety studies. Membrane-bound proteins have been very difficult to isolate in an active form due to their inherent physicochemical properties. Described here are methods used to derive enriched preparations of the active D9DS protein for use in early stage safety studies. Results of these studies, in combination with bioinformatic results and knowledge of the mode of action of the protein, along with a history of safe consumption of related proteins, provides a weight of evidence supporting the safety of the D9DS protein in food and feed.

Bioinformatics and the allergy assessment of agricultural biotechnology products: industry practices and recommendations.

Bioinformatic tools are being increasingly utilized to evaluate the degree of similarity between a novel protein and known allergens within the context of a larger allergy safety assessment process. Importantly, bioinformatics is not a predictive analysis that can determine if a novel protein will ''become" an allergen, but rather a tool to assess whether the protein is a known allergen or is potentially cross-reactive with an existing allergen. Bioinformatic tools are key components of the 2009 CodexAlimentarius Commission's weight-of-evidence approach, which encompasses a variety of experimental approaches for an overall assessment of the allergenic potential of a novel protein. Bioinformatic search comparisons between novel protein sequences, as well as potential novel fusion sequences derived from the genome and transgene, and known allergens are required by all regulatory agencies that assess the safety of genetically modified (GM) products. The objective of this paper is to identify opportunities for consensus in the methods of applying bioinformatics and to outline differences that impact a consistent and reliable allergy safety assessment. The bioinformatic comparison process has some critical features, which are outlined in this paper. One of them is a curated, publicly available and well-managed database with known allergenic sequences. In this paper, the best practices, scientific value, and food safety implications of bioinformatic analyses, as they are applied to GM food crops are discussed. Recommendations for conducting bioinformatic analysis on novel food proteins for potential cross-reactivity to known allergens are also put forth.

[Dynamic changes of Ruoergai Plateau wetland ecosystem service value].

Based on the satellite remote sensing data acquired in 1975 and 2006, and by using the assessment method of ecosystem service value, the dynamic changes of physical production value, gas regulation value, and water storage value of Ruoergai Plateau wetland ecosystem in 1975-2006 were studied. During study period, the total value of the three services decreased from 19.59 billion RMB Yuan to 12.38 billion Yuan RMB, among which, physical production value increased by 0.302 billion RMB Yuan, while the gas regulation and water storage value decreased by 7.507 billion RMB Yuan. The benefit from the increase of physical production was much less than the loss of ecosystem degradation. Overgrazing induced the biomass reduction and soil deterioration, resulting in the decline of Ruoergai Plateau wetland ecosystem service value and service capacity.

Molecular cloning and sequence analysis of full-length growth hormone cDNAs from six important economic fishes.

In this study,the full-length cDNAs of GH (Growth Hormone) gene was isolated from six important economic fishes, Siniperca kneri, Epinephelus coioides, Monopterus albus, Silurus asotus, Misgurnus anguillicaudatus and Carassius auratus gibelio Bloch. It is the first time to clone these GH sequences except E. coioides GH. The lengths of the above cDNAs are as follows: 953 bp, 1 023 bp, 825 bp, 1 082 bp, 1 154 bp and 1 180 bp. Each sequence includes an ORF of about 600 bp which encodes a protein of about 200 amino acid: S. kneri, E. coioides and M. albus GHs of 204 amino acid, S. asotus GH of 200 amino acid, M. anguillicaudatus and C. auratus gibelio GHs of 210 amino acid. Then detailed sequence analysis of the six GHs with many other fish sequences was performed. The six sequences all showed high homology to other sequences, especially to sequences within the same order, and many conserved residues were identified, most localized in five domains. The phylogenetic trees (MP and NJ) of many fish GH ORF sequences (including the new six) with Amia calva as outgroup were generally resolved and largely congruent with the morphology-based tree though some incongruities were observed, suggesting GH ORF should be paid more attention to in teleostean phylogeny.