Search

Here, the bacterial diversity in chronic suppurative otitis media (CSOM) and middle-ear cholesteatoma (MEC) was analysed. Nine and twenty-nine patients with CSOM and MEC, respectively, were recruited. Middle-ear lesion tissue was collected intraoperatively after opening the tympanic sinus and mastoid cavity under general anaesthesia and sterile conditions. The full-length 16S rRNA genome sequenced using third-generation sequencing (TGS) was then used to profile the bacterial community of each patient. Principal coordinate analysis showed that PC1 and PC2 could explain more than 50% of the between-group differences. Similarity analysis using the binary Jaccard distance matrix indicated that between-group differences were greater than within-group differences (P Staphylococcus aureus was the most common strain in both groups. At the species level, the abundance of Anaerococcus_octavius was significantly different between both groups (P Alphaproteobacteria and Bacillus were abundant in the CSOM group, respectively. Peptoniphilus_grossensis and Peptostreptococcaceae_bacterium_oral_taxon_929 were abundant at the species level in the MEC group (P P

In this study, the bacterial diversity of acquired middle ear cholesteatoma (MEC) was evaluated to reveal its pathogenesis and provides a guide for the use of antibiotics. Twenty-nine cases of acquired MEC and eight cases of healthy middle ears undergoing cochlear implantation (CI) were evaluated. Full-length 16S rRNA gene sequencing was performed to profile the bacterial communities in lesions and healthy tissues of the middle ear. ACE (P = 0.043) and Chao1 (P = 0.039) indices showed significant differences in alpha diversity (P < 0.05). Analysis of PERMANOVA/Anosim using the Bray–Curtis distance matrix results suggested that the between-group differences were greater than the within-group differences (R = 0.238, P < 0.05, R2 = 0.066, P < 0.05). Bacterial community analysis revealed that Alphaproteobacteria at the class level and Caulobacterales and Sphingomonadales at the order level were significantly different (P < 0.05). In the LefSe (Linear discriminant analysis effect size) analysis, Porphyromonas bennonis was elevated, and Bryum argenteum and unclassified Cyanobacteriales were reduced at the species level in MEC (P < 0.05). Fifteen metabolic pathways were found to be significantly different between the two groups by analysing the abundance of metabolic pathways in level 2 of the Kyoto Encyclopaedia of Genes and Genomes (KEGG). Seven and eight metabolic pathways were significantly elevated in the MEC and control groups, respectively (P < 0.05). The role of bacteria in the pathogenesis of acquired MEC was further refined through analysis of metabolic pathways. These findings indicate that the acquired MEC and healthy middle ear contain more diverse microbial communities than previously thought.

Gout nodules (tophi) are formed by a chronic inflammatory reaction in tissues resulting in deposition of urate crystals. They are commonly seen in joints and surrounding tissues, subcutaneous tissues, as well as the pinna and kidney, and are characteristic manifestations of gout. Vocal cord tophi are rarely reported in the literature, and patients often present with hoarseness, progressive dysphagia, and other symptoms. We report a case of a vocal cord mass found by gastroscopy in a patient with a history of gout for more than 20 years. Postoperative pathological findings were vocal cord tophi. Tophi can have serious consequences and should be included in the differential diagnosis of laryngeal masses in patients with a history of gout. Therapy should involve a combination of systemic uric acid-lowering treatment and surgery to improve symptoms and reduce recurrence.

To better understand how amino acid sequence encodes protein structure, we engineered mutational pathways that connect three common folds (3α, β−grasp, and α/β−plait). The structures of proteins at high sequence-identity intersections in the pathways (nodes) were determined using NMR spectroscopy and analyzed for stability and function. To generate nodes, the amino acid sequence encoding a smaller fold is embedded in the structure of an ~50% larger fold and a new sequence compatible with two sets of native interactions is designed. This generates protein pairs with a 3α or β−grasp fold in the smaller form but an α/β−plait fold in the larger form. Further, embedding smaller antagonistic folds creates critical states in the larger folds such that single amino acid substitutions can switch both their fold and function. The results help explain the underlying ambiguity in the protein folding code and show that new protein structures can evolve via abrupt fold switching.

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins., Chen et al. describe a rational design of subtilisin mutants that degrade active RAS by cleaving a conserved sequence in switch 2. They further modified the active site to be dependent on a cofactor to generate high target specificity. Proteases engineered to cleave this region degraded RAS in vitro and in cells with a promise of adaptability for other target proteins too.

Aims: To determine the clinical characteristics and genetic cause of Waardenburg syndrome type 1 (WS1) in a Chinese family. Materials and Methods: Evaluations, including history, clinical features, and audiological tests, were performed on the proband and her parents. Genetic analyses were performed targeting 144 known deafness genes using a next-generation sequencing panel. Bioinformatic analyses were used to analyze the candidate mutation. Results: The proband and her parents suffered from congenital bilateral profound hearing loss. Her mother exhibited bilateral blue irides. WS1 was diagnosed in the proband and her mother according to the Waardenburg syndrome consortium criteria: the calculated W index of the proband was 2.39 and that of her mother was 2.31. A novel mutation c.1076_1077del (p.Thr359fs) in exon 7 of the PAX3 gene (paired box 3) was identified in the proband and her mother that was absent in the father and controls. Conclusion: Mutations in exon 7 of the PAX3 gene are rare. We identified a novel frameshift mutation in exon 7 of the PAX3 gene that we determined was responsible for WS1 in this family.

Spontaneous cerebrospinal fluid (CSF) leak is a condition that commonly presents with unilateral watery drainage from the nose or ear, tinnitus, and stuffy ears or hearing loss. Spontaneous CSF rhinorrhea and otorrhea together are rare. A 64-year-old woman presented at our department with complaints of clear watery rhinorrhea and hearing loss on the right side persisting for 10 months. Imaging and surgery were used to diagnose the condition. Through surgical treatment, she was eventually cured. Review of the literature has shown that patients with both nasal and aural CSF leaks are rare. When a patient presents with both unilateral watery drainage from both the nose and ear, a diagnosis of CSF rhinorrhea and otorrhea should be considered. This case report will benefit clinicians by providing more information to assist with diagnosing the disease.

OBJECTIVE: To evaluate the preliminary value of the cross-sectional area and morphological changes of the external ear canal opening after the two-flap auriculoplasty through the I shaped posterior incision. METHODS: One hundred and thirty-seven patients(a total of 155 ears) who received open radical mastoidectomy in the department of otolaryngology in the First Affiliated Hospital of Kunming Medical University were treated with I shaped incision and two-flap auriculoplasty. Vertical diameter(D1) and horizontal diameter(D2) of the external ear canal were measured at the completion of surgery, 1 month and 6 months post-operation, respectively. The cross-sectional area(S=1/4πD1×D2) of the external ear canal was calculated according to the two diameters. The dry ear time and intraoperative lumen epithelialization time were observed after operation. At 6 months after operation, the morphology of the external ear canal opening was analyzed. RESULTS: The postoperative dry ear duration was 18-61 days(27.32±7.52) days. The time to complete epithelialization of the operative cavity was 24-70 days(32.18±10.36) days. Six months after the operation, the morphological classification of 155 outer ear meatal openings was as follows: 117 ears(75.48%) were round(the difference between vertical diameter and horizontal diameter was within 2 mm); Oval(oval appearance, difference between vertical diameter and horizontal diameter greater than 2 mm) 35 ears(22.58%), triangle 3 ears(1.94%); Irregular ear canal orifice was not observed in all cases. During the operation, and at 1 month and 6 months after the operation, the cross-sectional area of the external ear canal was(2.51±0.48) cm(2), (2.45±0.35) cm(2), (2.41±0.43) cm(2), respectively. And no significant differences were observed. (P>0.05). CONCLUSION: The I shaped posterior auricular incision and two-flap auricular lumenoplasty is not compex and easy to perform. The morphology of the external ear opening is regular after the operation, which can effectively match the ventilation of the operative cavity.

Waardenburg syndrome (WS) is a congenital hereditary disease, attributed to the most common symptoms of sensorineural deafness and iris hypopigmentation. It is also known as the hearing-pigmentation deficient syndrome. Mutations on SOXl0 gene often lead to congenital deafness and has been shown to play an important role in the pathogenesis of WS. We investigated one family of five members, with four patients exhibiting the classic form of WS2, whose DNA samples were analyzed by the technique of Whole-exome sequencing (WES). From analysis of WES data, we found that both the mother and all three children in the family have a heterozygous mutation on the Sex Determining Region Y - Box 10 (SOX10) gene. The mutation was c.298_300delinsGG in exon 2 of SOX10 (NM_006941), which leads to a frameshift of nine nucleotides, hence the amino acids (p. S100Rfs*9) are altered and the protein translation may be terminated prematurely. Further flow cytometry confirmed significant down-regulation of SOX10 protein, which indicated the SOX10 gene mutation was responsible for the pathogenesis of WS2 patients. In addition, we speculated that some other mutated genes might be related to disease phenotype in this family, which might also participate in promoting the progression of WS2.

We have engineered switches between the three most common small folds, 3α, 4β+α, and α/β−plait, referred to here as A, B, and S, respectively. Mutations were introduced into the natural S protein until sequences were created that have a stable S-fold in their longer (∼90 amino acid) form and have an alternative fold (either A or B) in their shorter (56 amino acid) form. Five sequence pairs were designed and key structures were determined using NMR spectroscopy. Each protein pair is 100% identical in the 56 amino acid region of overlap. Several rules for engineering switches emerged. First, designing one sequence with good native state interactions in two folds requires care but is feasible. Once this condition is met, fold populations are determined by the stability of the embedded A- or B-fold relative to the S-fold and the conformational propensities of the ends that are generated in the switch to the embedded fold. If the stabilities of the embedded fold and the longer fold are similar, conformation is highly sensitive to mutation so that even a single amino acid substitution can radically shift the population to the alternative fold. The results provide insight into why dimorphic sequences can be engineered and sometimes exist in nature, while most natural protein sequences populate single folds. Proteins may evolve toward unique folds because dimorphic sequences generate interactions that destabilize and can produce aberrant functions. Thus, two-state behavior may result from nature’s negative design rather than being an inherent property of the folding code.Significance StatementWe establish general rules for designing protein fold switches by engineering dimorphic sequences that link the three most common small folds. The fact that switches can be engineered in arbitrary and common protein folds, sheds light on several important questions: 1) What is the generality of fold switching? 2) What types of folds are amenable to switching? 3) What properties are shared by sequences that can fold into two completely different structures? This work has implications for understanding how amino acid sequence encodes structure, how proteins evolve, how mutation is related to disease, and how function is annotated to sequences of unknown structure.ClassificationBiological Sciences: Biochemistry; Physical Sciences: Biophysics and Computational Biology

Matrix metalloproteinase (MMP)-9 is a key enzyme responsible for extracellular matrix degradation and contributes to the progressive histological changes observed in lower respiratory tract infections. Integrin β1 and α-tubulin are potential MMP-9-interacting proteins, and microRNA (miR)-29b-3p can regulate MMP-9 expression. MMP-9 is highly expressed in chronic rhinosinusitis with nasal polyps (CRSwNPs), regardless of its effects on miR-29b-3p, integrin β1 and α-tubulin expression. In the present study, samples from 100 patients with CRSwNPs were examined via reverse transcription-quantitative PCR to assess the mRNA expression of miR-29b-3p, and western blotting was performed to assess the protein expression of MMP-2, MMP-9, acetyl-α-tubulin, integrin β1 and tissue inhibitor of metalloproteinase 1 (TIMP-1). A dual-luciferase reporter assay was used to verify the direct binding of miR-29b-3p and MMP-2/MMP-9. Co-immunoprecipitation (Co-IP) and GST pull-down assays showed that integrin β1 and α-tubulin were MMP-9-interacting proteins. Cell viability, apoptosis and inflammatory cytokine levels were determined via a Cell Counting Kit-8 assay, flow cytometry and ELISA, respectively. miR-29b-3p expression was found to be positively correlated with MMP-2 and MMP-9 expression. Whereas, TIMP-1 expression was negatively correlated with MMP-2 and MMP-9 expression. The dual-luciferase assay revealed that miR-29b-3p targeted the 3′ untranslated region of MMP-2/MMP-9. The Co-IP and GST pull-down assays showed that MMP-9 could directly bind to integrin β1 and indirectly bind to α-tubulin. Finally, the overexpression of miR-29b-3p decreased the expression of MMP-9 and increased the levels of acetyl-α-tubulin. By contrast, the knockdown of miR-29b-3p increased the expression of MMP-9 and decreased the levels of acetyl-α-tubulin. Additionally, MMP-9 expression was found to be negatively correlated with acetyl-α-tubulin expression. Of note, the expression of integrin β1 did not change following the overexpression and knockdown of MMP-9. Finally, the overexpression of miR-29b-3p not only decreased MMP-9 expression, but also alleviated lipopolysaccharide-induced inflammation in NP69 cells. The results showed that the downregulation of miR-29b-3p promoted α-tubulin deacetylation by increasing the number of MMP-9-integrin β1 complexes in CRSwNPs, thus targeting miR-29b-3p/MMP-9 may be a potential novel strategy for the clinical treatment of CRSwNPs.

Acute otitis media (AOM) is a common infectious disease in children that is accompanied by signs and symptoms of middle ear inflammation and infection. Previous studies have shown that the long non-coding (lnc)RNA nuclear-enriched abundant transcript 1(NEAT1) participates in various inflammatory conditions and plays an important regulatory role. The focus of the present study was the biological function of NEAT1 and underlying molecular mechanism in lipopolysaccharide (LPS)-induced human middle ear epithelial cells (HMEECs). The expression of NEAT1, miR-301b-3p and toll-like receptor 4 (TLR4) protein were determined by reverse transcription-quantitative PCR and western blot assays, respectively. Dual-luciferase reporter assay was performed to investigate the combination of miR-301b-3p and NEAT1 or TLR4. In addition, cell viability, apoptosis and the levels of pro-inflammatory factors (IL-1β, TNF-α and IL-6) were measured by Cell Counting Kit-8 assay, flow cytometry and ELISA, respectively. Cell viability was significantly decreased, whereas apoptosis and inflammation were increased in LPS-stimulated HMEECs. Functional analyses demonstrated that NEAT1 was upregulated following LPS treatment, whereas knockdown of NEAT1 significantly increased cell viability and alleviated apoptosis and inflammation. Mechanistically, NEAT1 directly bound to and negatively regulated miR-301b-3p expression, whereas miR-301b-3p inhibitors abolished the inhibitory effect of NEAT1 knockdown on cell apoptosis and inflammation. As a target of miR-301b-3p, TLR4 was regulated by NEAT1 and miR-301b-3p. TLR4 overexpression alleviated NEAT1 silencing-induced inflammatory suppression. Rescue experiments demonstrated that NEAT1 promoted TLR4 expression by inhibiting miR-301b-3p. Collectively, the results of the present study suggested that NEAT1 may attenuate LPS-induced inflammation and apoptosis in HMEECs by modulating the miR-301b-3p/TLR4 axis, and may provide a new therapeutic target for the clinical treatment of AOM.

Gasification is a thermochemical process which can be used as a low-emission and highly efficient method to produce syngas and chemicals such as biomethanol and dimethyl ether (DME). In this paper,...

Objectives To determine the clinical characteristics and genetic causes of Waardenburg syndrome type 1 (WS1) present in a Chinese Han family. Methods Evaluations, including the familial history, clinical features and audiological tests, were performed on the proband and her parents. Genetic analyses were conducted using targeted next-generation sequencing of 144 known deafness genes, and confirmed by Sanger sequencing. Bioinformatics analyses of the candidate variant were performed. Results The proband suffered from moderate hearing loss of the right ear, and her mother suffered from profound congenital bilateral hearing loss. The proband exhibited a left blue iris. The calculated W index of the proband was 2.61, while her mother's W index was 2.12. The proband and her mother were diagnosed with WS1 according to the Waardenburg Syndrome Consortium criteria. A novel missense variant NM_181457.3: c.127G > T; p.(Gly43Cys) in exon 2 in Paired Box 3 (PAX3) was identified in the proband and her mother, but this variant was not detected in the father and the controls. This variant was not reported in the HGMD, ClinVar, 1000G and ESP6500 databases. Conclusion We identified a novel missense variant in exon 2 of PAX3 as the genetic cause of WS1 in this two-generation family, which broadened the genetic spectrum of WS1.

Biomass is a renewable source of energy, which refers to plant-based organic materials such as agricultural residue, woody biomass, and algal biomass, which can be directly used as a feedstock for energy systems gas engines, and gas turbines or as a fuel for the production of other light fuels such as hydrogen gas. In this paper, an equilibrium model of gasification process was developed to study the economic properties of biomass gasification such as hydrogen production cost (HPC) and fixed capital investment (FCI). When the plant size increased from 1500 to 2500 (dry metric tonne per day), HPC decreased from 3.2 to 2.4 €/kg for agricultural residue, from 3.45 to 2.5 €/kg for green algae, and from 4.4 to 3.85 €/kg for RDF. Simulated results also showed as the stoichiometric ratio increases from 0.23 to 0.45, concentrations of H2, CO, and CH4 decrease, while CO2 increases because of the thermal cracking of heavier hydrocarbons and char combustion at higher stoichiometric ratios.

Background To analyze the clinical phenotypes and genetic variants of a Chinese family with Waardenburg syndrome (WS) and to explore the possible molecular pathogenesis of WS. Methods The clinical data from a patient and his family were collected. The genomic DNA of the patient and his family was purified from their peripheral blood. All exons and flanking sequences of the MITF, PAX3, SOX10, SNAI2, END3, and EDNRB genes were investigated through high‐throughput sequencing. Based on the results of high‐throughput sequencing, genetic variants in the patient and his family were verified and analyzed by Sanger sequencing. Results The patient was diagnosed with typical WS1 that manifested in hearing impairment, inner canthus ectopia and heterochromic iris. Sanger sequencing revealed the pathogenic heterozygous c.420‐424de1CGCGGinsTTAC mutation in the PAX3 gene in the proband, which is a frameshift mutation that changed the amino acid sequence of the PAX3 protein from AVCDRNTVPSV to YSVIETPCRQ* (* refers to a stop codon) from amino acids 141–151. The stop codon induced by this mutation resulted in the truncation of the PAX3 protein. The same mutation sites were also found in the mother and younger sister of the proband. No previous report of this mutation was found in the Human Gene Mutation Database. Conclusion The novel heterozygous c.420‐424de1CGCGGinsTTAC mutation is the molecular pathological cause for WS1 in our patient. The clinical and genetic characterization of this family with WS1 elucidated the genetic heterogeneity of PAX3 in WS1. Moreover, the mutation detected in this case has expanded the database of PAX3 mutations.

Waardenburg syndrome type 2 (WS2) is a rare genetic disorder, characterized by bright blue eyes, moderate to profound hearing loss and pigmental abnormalities of the hair and skin. Between 10 and 20 mutations in the SRY‑box 10 (SOX10) gene were previously identified to be associated with WS2. The present study aimed to identify the genetic causes of WS2 in a Chinese family. Clinical and molecular analyses were performed to genetically characterize a Chinese family with two cases of WS2. The clinical data of the proband were collected using a questionnaire. The genomic DNA was extracted from peripheral blood samples of each individual in the family, and 168 candidate genes associated with hearing loss were sequenced using the Illumina HiSeq 2000 and confirmed by Sanger sequencing. A heterozygous nonsense mutation [substitution; position 127; cytosine to thymine (c.127C>T)] was identified in exon 2 of SOX10 (transcript ID: NM_006941.3) in the proband and the mother; however, not in other family members or healthy controls. The novel nonsense heterozygous mutation may cause the replacement of codon 43 [arginine (Arg)] with a stop codon (Arg43stop), leading to premature termination of protein translation. The novel nonsense heterozygous mutation c.127C>T in the SOX10 gene was considered to be the cause of WS2 in the family. This mutation has not been identified in any databases, to the best of the authors' knowledge, including The Single Nucleotide Polymorphism Database, The Human Gene Mutation Database, 1000 Genomes Project and ClinVar and Exome Sequencing Project v. 6500.

The cover image based on Original Article A novel mutation of the PAX3 gene in a Chinese family with Waardenburg syndrome type I by Jing Ma et al., DOI: 10.1002/mgg3.798. [Image: see text]

Objective Waardenburg syndrome is a congenital genetic disorder. It is the most common type of syndromic hearing impairment with highly genetic heterogeneity and proved to be related by 6 genes as follows: PAX3, MITF, SNAI2, EDN3, EDNRB and SOX10. This article aims to identify the genetic causes of a Chinese WS child patient. Methods A Chinese WS child was collected for clinical data collection by questionnaire survey. DNA samples of proband and his parents were extracted from peripheral blood samples. Six candidate genes were sequenced by the Trusight One sequencing panel on the illumina NextSeq 500 platform. Results A novel nonsense heterozygous mutation was found in the coding region of exon 2 in the SOX10 gene of proband. The novel nonsense heterozygous mutation could cause the replacement of the 55th lysine codon by stop codon (484T > C, C142R) and further more possibly cause terminating the protein translation in advance. However, both proband's parents had no mutation of genes above mentioned. Conclusion The gene mutation of SOX10 [NM_006941.3 c.163A > T] is a novel nonsense mutation. No record of this mutation has been found in dbSNP, HGMD, 1000 Genomes Project, ClinVar and ESP6500 databases. It meets the condition of PS2 of strong evidence in 2015 ACMG Standards and Guidelines.

Plasmodium subtilisin 2 (Sub2) is a multidomain protein that plays an important role in malaria infection. Here, we describe the solution NMR structure of a conserved region of the inhibitory prodomain of Sub2 from Plasmodium falciparum, termed prosub2. Despite the absence of any detectable sequence homology, the protozoan prosub2 has structural similarity to bacterial and mammalian subtilisin-like prodomains. Comparison with the three-dimensional structures of these other prodomains suggests a likely binding interface with the catalytic domain of Sub2 and provides insights into the locations of primary and secondary processing sites in Plasmodium prodomains.

Moxifloxacin was generally well tolerated and highly effective in the treatment of acute bacterial rhinosinusitis (ABS). The incidence of adverse events (AEs) and adverse drug reactions (ADRs) was low. The effectiveness, safety, and tolerability information collected in this study confirm the clinical safety profile of moxifloxacin and its benefit as a treatment option for ABS.To assess the effectiveness, safety, and tolerability of moxifloxacin under daily life treatment conditions in patients with ABS.The study was carried out in China between September 2005 and May 2007. Patients with ABS were treated with moxifloxacin tablets 400 mg once daily for a duration that was left to the physician's discretion. Data were collected on demography, diagnosis of infection, pretreatment, concomitant diseases and medications, moxifloxacin therapy, course of symptoms during investigations, and final assessment of therapy.In all, 578 patients with ABS treated with moxifloxacin were valid for effectiveness analysis. An improvement was observed in 98.8% (n = 571/578) of the patients. Cure was documented in 89.4% (n = 517/578) of the patients. The physicians' overall tolerability rating was 'very good' or 'good' in 92.9% (n = 537/578) of patients. The incidence rates of AEs and ADRs were 1.5% (n = 10/681) and 0.6% (n = 4/681), respectively. No serious AE was reported.

Bacillus subtilisin has been a popular model protein for engineering altered substrate specificity. Although some studies have succeeded in increasing the specificity of subtilisin, they also demonstrate that high specificity is difficult to achieve solely by engineering selective substrate binding. In this paper, we analyze the structure and transient state kinetic behavior of Sbt160, a subtilisin engineered to strongly prefer substrates with phenylalanine or tyrosine at the P4 position. As in previous studies, we measure improvements in substrate affinity and overall specificity. Structural analysis of an inactive version of Sbt160 in complex with its cognate substrate reveals improved interactions at the S4 subsite with a P4 tyrosine. Comparison of transient state kinetic behavior against an optimal sequence (DFKAM) and a similar, but suboptimal, sequence (DVRAF) reveals the kinetic and thermodynamic basis for increased specificity, as well as the limitations of this approach. While highly selective substrate binding is achieved in Sbt160, several factors cause sequence specificity to fall short of that observed with natural processing subtilisins. First, for substrate sequences which are nearly optimal, the acylation reaction becomes faster than substrate dissociation. As a result, the level of discrimination among these substrates diminishes due to the coupling between substrate binding and the first chemical step (acylation). Second, although Sbt160 has 24-fold higher substrate affinity for the optimal substrate DFKAM than for DVRAF, the increased substrate binding energy is not translated into improved transition state stabilization of the acylation reaction. Finally, as interactions at subsites become stronger, the rate-determining step in peptide hydrolysis changes from acylation to product release. Thus, the release of the product becomes sluggish and leads to a low k(cat) for the reaction. This also leads to strong product inhibition of substrate turnover as the reaction progresses. The structural and kinetic analysis reveals that differences in the binding modes at subsites for substrates, transition states, and products are subtle and difficult to manipulate via straightforward protein engineering. These findings suggest several new strategies for engineering highly sequence selective enzymes.

To evaluate the species, quantity and diffusion of allergenic pollens as well as the relationship among allergic rhinitis (AR), pollen species and meteorological conditions in Kunming area.The exposure film was used to investigate the species of airborne pollen, the total number of these pollens during December 2009 to November 2010 by means of gravitational sedimentation, at the same time, AR patients were registered. The species of allergic pollen in Kunming area during the same period, and the statistic analysis was made with the meteorological elements, using the correlation analysis methods of Excel for data analysis.From December 2009 to November 2010, a total of 60 exposure films of pollen species was found, including 26 147 pollens. The annual airborne pollen showed two scattering peak, respectively: February to April and September to October; the number of advantage pollen was 17 species. The number of year-round drifting pollen was 13 species while 2 species showed two peaks on spring and autumn. There was another 26 new species added to the results of 2009-2010, compared with 1987-1989, while the original 12 species were not seen. The drifting peak was the same. The number of allergen intradermal tests (AIT) positive AR patients was positively correlated with 16 pollen species, respectively, oleaceae, alnus nepalensis, walnut, palm, pig weed, fennel, Amaranthaceae, artemisia, rosa, Albizia julibrissin Durazz, gramineae, castor, Fraxinus L, populus, Morus alba L, hemp humulus, (r value were 0.689 7, 0.550 1, 0.742 1, 0.755 2, 0.771 2, 0.663 3, 0.556 1, 0.568 3, 0.659 8, 0.607 4, 0.834 7, 0.664 3, 0.745 9, 0.634 7, 0.718 3, 0.777 2, all P0.05). The population of AIT positive AR patients was positively correlated with average temperature, average wind speed and other meteorological factors (r values were 0.552 0, 0.883 5, both P0.05), and was negatively correlated with average relative humidity (r = -0.714 7, P0.05).Kunming had a large number of airborne pollen species which showed two peaks annually, respectively, spring and autumn. The relationship among the number of AIT positive AR patients, the meteorological factors and the number of airborne pollen were close.

To research the correlation between development of sphenoid sinus and age in normal children and adolescents by 3-D CT images.Review of CT images of the skulls of 120 children and adolescents aged 0~20 years old. They were divided into five groups. Then reconstructed 3-D shape of the sphenoid sinus, and measureed the maximal anteroposterior diameter, longitudinal diameter, and transverse diameter of the bilateral sphenoid sinus by the 2-D and 3-D approach respectively, then process statistical analysis.Pearson analyses revealed a positive correlation between various diameters of sphenoid sinus measured by 2-D and 3-D approach and age (P0. 05).The development of sphenoid sinus has a linear correlation with age, and increases with age and growth in Yunnan region. It helps to know the three-dimensional CT images of the sphenoid sinus in children and adolescents by Simplant software, thereby allow the clinicians to understand the current situation of the development of sphenoid sinus. Meanwhile, it contributes to preoperative evaluation of sinus problems.

To investigate the quantification and significance of Msx2, topoII-α; HPV16 and VEGF in sinonasal inverted papilloma(SNIP), to study the correlation among the four factors,and to discover the relationship between Msx2 and topoII-α in the process of SNIP malignant transfomation.Real-time quantitative Polymerase Chain Reaction (RT-qPCR) was used to detect the expression of Msx2, topoII-α, HPV16 and VEGF in 13 cases of sinonasal inverted papilloma (SNIP), 10 cases of sinonasal squamous cell carcinoma(NSCC) and 10 cases of inflammatory nasal polyp paraffin (INP)tissues. According to the pathology results SNIP were divided into mild dysplasia, moderate dysplasia and severe dysplasia. All the data were analysised by SPSS17. 0, P0. 05 was refered to statistically significant difference.The mRNA level of Msx2, topoII-α, VEGF and HPV16 in SNIP, NSCC tissues were significantly higher than in the INP tissues (P0. 05). The expression differences of Msx2, topoII-α, HPV16 and VEGF mRNA level in SNIP tissues which were divided into three groups according to their pathological results,were all statistically significantly different between any two of the three groups (P0. 05). Using Pearson correlation coefficient analysis,we found positive correlation between any two of the mRNA level of Msx2, topoII-α, VEGF and HPV16 (P0. 05).Msx2 and topoII-α may play an important role in the process of SNIP Malignant transformation,which may be new targets for gene therapy of SNIP and NSCC.

To evaluate the efficacy of standardized medication for patients with chronic rhinosinusitis.According to the diagnosis and treatment guidelines on chronic rhinosinusitis formulated in 2008, by means of prospective study, we studied 54 patients suffering from chronic rhinosinusitis treated with standardized medication including, a combination of local intranasaI corticosteroids, macrolides, mucus discharging agent and nasal irrigation treatment and followed up for 3 months. Visual analogue scale (VAS), sino nasal outcome test-20 Chinese version scales (SNOT-20 CV), Lund-Mackay CT and Lund-Kennedy endoscopy methods were employed to conduct the subjective and objective assessment and comprehensively evaluate the clinical efficacy before and after treatment.(1) After three months of standardized medication, the patients' total scores of VAS, SNOT-20 CV, CT and endoscopy were improved significantly compared with those before-treatment (P0.01 for all these scoring systems). (2) There was statistically significant difference between the clinical efficacies of chronic rhinosinusitis patients with and without nasal polyps groups (P0.01). After 3 months of standardized medication, the effective rates of the CRSwNP group evaluated by subjective assessment and CT evaluation were 66.7% and 94.4% respectively, while those of the CRSsNP groups were 91.7% and 97.2% respectively. (3) Betwecn CRSwNP and CRSsNP groups, there was no significant difference in the improvement rate or inefficiency rate in subjective assessment except for the cure rate, while there were significant differences in both cure rate and improvement rate in CT evaluation. (4) The CRS patients' self-testing-based questionnaires results showed positive correlation with objective assessments.The standardized medication with combination of intranasal local glucocorticoid, macrolides (14-membered ring), the mucus discharging agent and nasal irrigation on CRS was effective.

Stability is a property of subtilisin which has proven particularly amenable to enhancement via random mutagenesis and screening, yet the effects of most stabilizing mutations are not understood in structural and energetic detail. This paper seeks to explain the longstanding observation that stabilizing mutations are usually calcium-dependent in their stabilizing effect, irrespective of their proximity to the calcium binding sites. Stabilizing mutations in subtilisin fall into one of three classes. The largest class of mutations stabilize only in the presence of excess calcium. A smaller number of mutations stabilize independently of [calcium], and a few mutations stabilize only in the presence of chelating agents, such as EDTA. This study compares the effects of mutations from each class when introduced into subtilisin BPN' and two calcium-free versions of subtilisin. The calcium-dependent effects of mutations can be explained by considering subtilisin to be in conformational equilibrium between two structurally similar but energetically distinct states: N and N*. The equilibrium from the N* to the N state can be altered either by calcium binding to site A or by mutation. Mutations which stabilize only in the presence of calcium stabilize the N state relative to N*. Mutations which stabilize only in the presence of chelants stabilize the N* state relative to N. As a byproduct of this analysis, we have developed a hyperstable variant of subtilisin whose inactivation at high temperature in the presence of EDTA is 10(5) times slower than wild-type subtilisin.

In vitro folding of mature subtilisin is extremely slow. The isolated pro-domain greatly accelerates in vitro folding of subtilisin in a bimolecular reaction whose product is a tight complex between folded subtilisin and folded pro-domain. In our studies of subtilisin, we are trying to answer two basic questions: why does subtilisin fold slowly without the pro-domain and what does the pro-domain do to accelerate the folding rate? To address these general questions, we are trying to characterize all the rate constants governing individual steps in the bimolecular folding reaction of pro-domain with subtilisin. Here, we report the results of a series of in vitro folding experiments using an engineered pro-domain mutant which is independently stable (proR9) and two calcium-free subtilisin mutants. The bimolecular folding reaction of subtilisin and proR9 occurs in two steps: an initial binding of proR9 to unfolded subtilisin, followed by isomerization of the initial complex into the native complex. The central findings are as follows. First, the independently stable proR9 folds subtilisin much faster than the predominantly unfolded wild-type pro-domain. Second, at micromolar concentrations of proR9, the subtilisin folding reaction becomes limited by the rate at which prolines in the unfolded state can isomerize to their native conformation. The simpliest mechanism which closely describes the data includes two denatured forms of subtilisin, which form the initial complex with proR9 at the same rate but which isomerize to the fully folded complex at much different rates. In this model, 77% of the subtilisin isomerizes to the native form slowly and the remaining 23% isomerizes more rapidly (1.5 s-1). The slow-folding population may be unfolded subtilisin with the trans form of proline 168, which must isomerize to the cis form during refolding. Third, in the absence of proline isomerization, the rate of subtilisin folding is rapid and at [proR9]/= 20 microM is limited by the rate at which the proR9 forms a collision complex with unfolded subtilisin. Without proline isomerization, the rate of the isomerization of the initial collision complex to the folded complex is3 s-1. The implications of these results concerning why subtilisin folds slowly without the pro-domain are discussed.

We have devised a procedure using monovalent phage display to select for stable mutants in the pro-domain of the serine protease, subtilisin BPN'. In complex with subtilisin, the pro-domain assumes a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated, however, the pro-domain is 97% unfolded. These experiments use combinatorial mutagenesis to select for stabilizing amino acid combinations at a particular structural locus and determine how many combinations are close to the maximum protein stability. The selection for stability is based on the fact that the independent stability of the pro-domain is very low and that binding to subtilisin is thermodynamically linked to folding. Two libraries of mutant pro-domains were constructed and analyzed to determine how many combinations of amino acids at a particular structural locus result in the maximum stability. A library comprises all combinations of four amino acids at a structural locus. Previous studies using combinatorial genetics have shown that many different combinations of amino acids can be accommodated in a selected locus without destroying function. The present results indicate that the number of sequence combinations at a structural locus, which are close to the maximum stability, is small. The most striking example is a selection at an interior locus of the pro-domain. After two rounds of phagemid selection, one amino acid combination is found in 40% of sequenced mutants. The most frequently selected mutant has a deltaG(unfolding) = 4 kcal/mol at 25 degrees C, an increase of 6 kcal/mol relative to the naturally occurring sequence. Some implications of these results on the amount of sequence information needed to specify a unique tertiary fold are discussed. Apart from possible implications on the folding code, the phage display selection described here should be useful in optimizing the stability of other proteins, which can be displayed on the phage surface.

We have expressed the RecD subunit of the RecBCD enzyme from Escherichia coli as a fusion protein with a 31-amino acid NH2-terminal extension including 6 consecutive histidine residues (HisRecD). The overexpressed fusion protein can be purified in urea-denatured form by metal chelate affinity chromatography. The mixture of renatured HisRecD protein and the RecB and RecC proteins has a high level of ATP-dependent nuclease activity with either single- or double-stranded DNA, enhanced DNA unwinding activity, enhanced ATP hydrolysis activity in the presence of a small DNA oligomer cosubstrate, and chi-cutting activity. These are all characteristics of the RecBCD holoenzyme. The HisRecD protein by itself hydrolyzes ATP in the presence of high concentrations of single-stranded DNA (polydeoxythymidine). The activity is unstable at 37 degrees C, but is measurable at room temperature (about 23 degrees C). The HisRecD has very little ATPase activity in the presence of a much shorter single-stranded DNA (oligodeoxy(thymidine)12). HisRecD hydrolyzes ATP more efficiently than GTP and UTP, and has very little activity with CTP. We also purified a fusion protein containing a Lys to Gln mutation in the putative ATP-binding site of RecD. This mutant protein has no ATPase activity, indicating that the observed ATP hydrolysis activity is intrinsic to the RecD protein itself.

To investigate which aeroallergens were prevalent in patients with allergic rhinitis in Yunnan.Retrospective analysis of the medical records of intradermal test performed in 1893 AR patients. The samples were divided into four age groups: 4-17-year-olds,17-35-year-olds,35-50-year-olds and50-70-year-olds. According to gender,the samples were decided into two groups: the males and the females. The positive rate of aeroallergens were compared among the groups.(1)The total positive rate of intradermal test was 70.1%. The top ten allergens were as follow: dermatophagoides pteronyssinus, dermatophagoides farina, house dust, pollen of the summer and autumn, alnus nepalensis, chenopodiaceae, mugwort, poaceae, brassica and cockroach. (2) In all groups,patients with triple positive allergens were the most common,followed by dual positive allergens and single positive allergen. (3) Among the top ten allergens, the top three positive ones in all groups were dermatophagoides pteronyssinus, dermatophagoides farina and house dust. And there were significant differences between 4-17-year-olds group and the other age groups (P0.01). (4) There was no significant difference between male and female patients in distribution of aeroallergens except House dust and mite.The most common allergen in patients with allergic rhinitis in Yunnan is mite,the follow are pollen of the summer and autumn, alnus nepalensis, chenopodiaceae, mugwort, poaceae, and brassica. The distribution of aeroallergens are different among the age groups, but has no evident correlation with gender.

To investigate the characteristics and significant of mutations of GJB2 gene, SLC26A4 gene and mitochondrial 12S rRNA in deaf children who received cochlear implantation (CI) in Yunnan and to provide the data for diagnoses and research of recovery in C1 children.Genomic DNA was extracted from the peripheral blood samples collected from 46 children and their parents (110 cases). All the children received the CI. Their parents had normal auditory phenotype. PCR was performed and the products were sequenced by automated DNA sequencer to detect the hot spots of mutations.The detection rates of GJB2 235delC (13.0%) and 109GA (24.0%) mutations were significantly higher than other mutations. SLC26A was the secondary major mutation (13.0%). We found out that no patient carried the mitochondrial 12S rRNA mutations. Leukoencephalopathy, hyperbilirubinemia and hypoxic-ischemic injure were disclosed in 7 patients (15.2%). The rate of mutations in parents was 36.0% (23/64). There had no difference between Han and other racial minorities (P0.05).The CI recipients in Yunnan with a high frequency of 235 delC and 109 GA mutation, IVS7-2AG (6.5%) is also a common mutation related hearing loss; aminoglycoside antibiotics may not be the main reason which induced congenital deaf in CI children; environment facts was suggested to contribute another important cause. The hot-spots gene screening for the C1 children could offer an accurate genetic counseling for early diagnosis and treatment, it also provide evidences for the clinical analysis between mutations and curative effect.

A three-dimensional morphology of the maxillary sinus was reconstructed. We studied the relationship between sinusitis and three-dimensional morphology, volume as well as gasification coefficients.One hundred and fifty adult subjects were selected in this study, and divided into three groups: normal bilateral maxillary sinus, unilateral maxillary sinusitis and bilateral maxillary sinusitis, with fifty cases in each group. Use Siemens helix CT for sequential scanning of the nasal-sinus. After scanning, the DICOM data was recorded in DVD-R and transferred into another computer for reconstruction and measurement.The volume of the normal maxillary sinus group was (15 018.64 +/- 473.36) mm3. The volume of the maxillary sinusitis group was (14 971.86 +/- 360.93)mm3. There was no significant difference between the values of volumes for the normal maxillary sinus group and maxillary sinusitis group. The gasification coefficient was 0.345 +/- 0.071 for the normal maxillary sinus group, 0.252 +/- 0.057 for the maxillary sinusitis group. There was significant difference between the two groups.Maxillary sinus reconstruction permits more vivid visualization of the three-dimensional structure and three-dimensional shape of the maxillary sinus. The occurrence of the maxillary sinusitis is closely related to the three-dimensional shape of the maxillary sinus and the gasification coefficient measured. The more regular the three-dimensional shape of the maxillary sinus, with the gasification coefficient0.300, the lower the probability of the maxillary sinusitis. Otherwise, the probability increases. Adult inflammation of maxillary sinus may be originated from a relatively larger volume of maxillary sinus in childhood and adolescence.

Plasmodium subtilisin 2 (Sub2) is a multidomain protein that plays an important role in malaria infection. Here, we describe the solution NMR structure of a conserved region of the inhibitory prodomain of Sub2 from Plasmodium falciparum, termed prosub2. Despite the absence of any detectable sequence homology, the protozoan prosub2 has structural similarity to bacterial and mammalian subtilisin-like prodomains. Comparison with the three-dimensional structures of these other prodomains suggests a likely binding interface with the catalytic domain of Sub2 and provides insights into the locations of primary and secondary processing sites in Plasmodium prodomains.

To investigate the expression and significance of muscle segment homeobox2 (Msx2) and topo II-alpha in sinonasal inverted papilloma (SNIP), and the relationship in the process of malignant transformation of SNIP.Immunohistochemical method was used to detect the expression of Msx2 and topo II-alpha in 32 cases of SNIP, 30 cases of inflammatory nasal polyp (INP) and 30 cases of SNIP with carcinoma. According to the pathology results, SNIP were divided into mild atypical hyperplasia, moderate atypical hyperplasia and severe atypical hyperplasia.The mean optical density of Msx2 in SNIP and SNIP with carcinoma tissues were 0.2183 +/- 0.0598 and 0.2521 +/- 0.0761,which were significantly higher than 0.1878 +/- 0. 0372 in the INP tissue (P0.05 or 0.01). The mean optical density of topo II-alpha in SNIP and SNIP with carcinoma tissues were 0.2303 +/- 0.0397 and 0.2666 +/- 0.0483, which were significantly higher than 0.1978 +/- 0.0388 in the NIP tissue (P0.01). There were significant difference of Msx2 and topo II-alpha in SNIP between any two of the three groups divided according to pathological morphology (P0.01 or 0.05). The expression of Msx2 and topo II-alpha in SNIP were positively correlated (P0.05).Msx2 and topo II-alpha may play an important role in the occurrence and development of SNIP. So it can be used as new therapeutic targets.

To investigate the maxillary sinus development and provide data for sinus surgery on children.Two thousand two hundred and thirty-eight children were randomly selected among those who underwent skull and nasal sinus CT scanning because of certain symptoms and signs. Patients were divided into eighteen age cohorts based on their age at the time of the scan. Anterior-posterior, transverse diameters and vertical height of the maxillary sinus were measured and statistically analyzed.The maxillary sinus volume was enlarging even in adult, hut the growth of maxillary sinus was relatively stable in adolescent. The difference of transverse and anterior-posterior diameters of maxillary sinus had no statistical significance between female and male (P0.05), while there was statistically significant difference in the vertical height of maxillary sinus (P0.05).The results will aid the physicians when correlating the clinical and radiographic findings of pediatric patients who are being evaluated for sinus disease and potential surgical intervention.

Residue-specific exchange rates of 223 amide protons in free and prodomain-complexed subtilisin were determined in order to understand how the prodomain binding affects the energetics of subtilisin folding. In free subtilisin, amide protons can be categorized according to exchange rate: 74 fast exchangers (ratesor = 1 h(-1)); 52 medium exchangers (rates between 1 h(-1) and 1 day(-1)); 31 slow exchangers (rates between 1 day(-1) and 0.001 day(-1)). The remaining 66 amide proteins did not exchange detectibly over 9 months (k(obs)year(-1)) and were denoted as core protons. Core residues occur throughout the main structural elements of subtilisin. Prodomain binding results in high protection factors (100-1000) in the central beta-sheet, particularly in the vicinity of beta-strands S5, S6, and S7 and the connecting loops between them. These connecting loops provide the ligands to the cation at metal site B. Overall, prodomain binding seems to facilitate the organization of the entire central beta-sheet and alpha-helix C in the left-handed crossover connection between beta-strands two and three. It also appears to facilitate the isomerization of multiple prolines late in folding, allowing the formation of metal site B. The gain of stability region around site B comes at the cost of stability in regions more distal to prodomain binding: the C-terminal alpha-helix H and the N-terminal alpha-helices A and B. The acceleration of exchange in these regions by prodomain binding reveals an antagonism between the folding intermediate and the full native structure. This antagonism helps to explain why the prodomain is needed to stabilize the folding intermediate as well as why the unfolding of free subtilisin seldom occurs via this intermediate.

Like many secreted proteases, subtilisin is kinetically stable in the mature form but unable to fold without assistance from its prodomain. The existence of high kinetic barriers to folding challenges many widely accepted ideas, namely, the thermodynamic determination of native structure and the sufficiency of thermodynamic stability to determine a pathway. The purpose of this article is to elucidate the physical nature of the kinetic barriers to subtilisin folding and to show how the prodomain overcomes these barriers. To address these questions, we have studied the bimolecular folding reaction of the subtilisin prodomain and a series of subtilisin mutants, which were designed to explore the steps in the folding reaction. Our analysis shows that inordinately slow folding of the mature form of subtilisin results from the accrued effects of two slow and sequential processes: (1) the formation of an unstable and topologically challenged intermediate and (2) the proline-limited isomerization of the intermediate to the native state. The low stability of nascent folding intermediates results in part from subtilisin's high dependence on metal binding for stability. Native subtilisin is thermodynamically unstable in the absence of bound metals. Because the two metal binding sites are formed late in folding, however, they contribute little to the stability of folding intermediates. The formation of productive folding intermediates is further hindered by the topological challenge of forming a left-handed crossover connection between beta-strands S2 and S3. This connection is critical to propagate the folding reaction. In the presence of the prodomain, folding proceeds through one major intermediate, which is stabilized by prodomain binding, independent of metal concentration and proline isomerization state. The prodomain also catalyzes the late proline isomerizations needed to form metal site B. Rate-limiting proline isomerization is common in protein folding, but its effect in slowing subtilisin folding is amplified because of the instability of the intermediate and an apparent need for simultaneous isomerization of multiple prolines in order to create metal site B. Thus, the kinetically controlled folding reaction of subtilisin, although unusual, is explained by the accrued effects of events found in other proteins.