Phenylalanine ammonia lyase (PAL) can catalyze L-phenylalanine to produce trans-cinnamic acid, which is widely used in the fields of pharmacy, food and agriculture. In particular, phenylalanine ammonia lyase from Anabaena variabilis (AvPAL) is the only protein drug for the treatment of phenylketonuria. However, the poor activity and low stability limit the application in industry of AvPAL. In this study, the key amino acids of substrate-binding cavity in AvPAL were identified by screening the single site saturation mutagenesis library. Subsequently, the impact of replacing M222 with the additional 19 amino acids on activity was also evaluated by site-directed mutagenesis. It was found that the k_cat values of mutants M222L and M222V were 90% and 60% higher than that of AvPAL, and the k_cat/K_m was 1.4 and 1.5 times as that of AvPAL. Molecular docking results revealed that the higher activity of M222L and M222V may be due to the increase of hydrophobicity favorable for the substrate-binding cavity. This study is important for elucidating the structure-function relationship of AvPAL.

Objective: To summarize and investigate the characteristics, prognosis and treatments of chronic lymphocytic leukemia (CLL) patients with trisomy 12 by using FISH (CEP12). Methods: Clinical data of 330 CLL patients were analyzed retrospectively by using FISH (CEP12) to detect trisomy 12 from May 2003 to April 2015. The clinical data and laboratory characteristics of CEP12 positive patients (70 cases) were compared with those CEP12 negative patients (260 cases). Results: Compared with CEP12 negative CLL patients, the proportion of hepatomegaly (13.6% vs 4.0%, P=0.011) and LDH>247 U/L (43.3% vs 18.5%, χ(2)=15.892, P<0.001) in CEP12 positive CLL patients were much higher, respectively. There were no significant differences between age, sex, clinical stage, β(2)-microglobulin level, IGHV mutation ratio and splenomegaly/lymphadenopathy in these two subgroups. However, compared with CEP12 negative patients, CEP12 positive patients had higher ratio of FMC7 (23.8% vs 12.7%, χ(2)=4.730, P=0.030), and lower ratio of CD23 (95.2% vs 99.6%, P=0.033). The overall response rates (ORR) in Fludarabine (without Rituximab), Rituximab (with or without Fludarabine) and the traditional chemotherapy group (chlorambucil, CHOP or CHOP-like) were 77.5% (31/40), 84.8% (56/66) and 45.4% (50/110), respectively. The ORR of the traditional chemotherapy group was lower than that of the Fludarabine group and Rituximab group. For CEP12 positive patients, the ORR was inferior to CEP12 negative patients when only using Fludarabine (P<0.05). However, when using Rituximab, the difference could be eliminated, and the ORR was even a little higher in CEP12 negative patients (91.7% vs 81.0%, P=0.306). Compared with CEP12 negative patients, there were no significant differences in progression-free survival (PFS) (χ(2)=0.410, P=0.478) and overall survival (OS) (χ(2)=0.052, P=0.180) for CEP12 positive patients whom the median time from diagnosis to start treatment and OS time was 22.6 (95%CI 15.4-31.7) and 118.5 (95%CI 74.5-162.4) month while the 5-year PFS and OS were (52.9±7.6)% and (74.8±6.6)%. Conclusions: CEP12 positive CLL patients are more common in hepatomegaly and higher level of LDH. The traditional chemotherapy treatment had the lowest efficacy, and the curative effect of single use of fludarabine is not as good as that of CEP12 negative patients, however, when using Ritaximab, the efficacy could be comparable.
Antibodies, Monoclonal, Murine-Derived ; Antineoplastic Combined Chemotherapy Protocols ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; Retrospective Studies ; Rituximab ; Trisomy ; Vidarabine

OBJECTIVETo investigate the effect of premature rupture of the membrane (PROM) on neonatal complications in premature infants.

METHODSThe registration information of 7684 preterm infants with gestational age <37 weeks were collected from the cooperative units in the task group between January 1, 2014 to December 31, 2014. Specially trained personnel from each cooperative units filled in the unified form in a standardized format to record the gender, gestational age, birth weight, PROM, placental abruption, antenatal corticosteroid, Apgar score, amniotic fluid pollution, and complications of the infants. The data were analyzed comparatively between the cases with PROM and those without (control).

RESULTSThe preterm mortality rate was significantly lower but the incidences of ICH, NEC, ROP and BPD were significantly higher in PROM group than in the control group (P<0.05). The 95% confidence interval of the OR value was <1 for mortality, and was >1 for ICH, NEC, ROP and BPD. After adjustment for gestational age, birth weight, gender, mode of delivery, placental abruption, placenta previa, prenatal hormones, gestational diabetes mellitus (GDM), gestational period hypertension and 5-min Apgar score <7, the incidences of NEC, ROP and BPD were significantly different between the two groups (P<0.05) with 95% confidence interval of OR value >1, but the mortality rate and incidence of ICH were not significantly different between the two groups (P>0.05).

CONCLUSIONPROM is a risk factor for NEC, ROP and BPD in preterm infants, and adequate intervention of PROM can reduce the incidences of such complications as NEC, ROP and BPD in the infants.

Apgar Score ; Birth Weight ; Female ; Fetal Membranes, Premature Rupture ; pathology ; Gestational Age ; Humans ; Incidence ; Infant, Newborn ; Infant, Newborn, Diseases ; etiology ; Infant, Premature ; Pregnancy ; Risk Factors

OBJECTIVETo analyze the whole microbial structure in a case of rampant caries to provide evidence for its prevention and treatment.

METHODSClinical samples including blood, supragingival plaque, plaque in the caries cavity, saliva, and mucosal swabs were collected with the patient's consent. The blood sample was sent for routine immune test, and the others samples were stained using Gram method and cultured for identifying colonies and 16S rRNA sequencing. DNA was extracted from the samples and tested for the main cariogenic bacterium (Streptococcus mutans) with qPCR, and the whole microbial structure was analyzed using DGGE.

RESULTSThe patient had a high levels of IgE and segmented neutrophils in his blood. Streptococci with extremely long chains were found in the saliva samples under microscope. Culture of the samples revealed the highest bacterial concentration in the saliva. The relative content of hemolytic bacterium was detected in the samples, the highest in the caries cavity; C. albicans was the highest in the dental plaque. In addition, 33 bacterial colonies were identified by VITEK system and 16S rDNA sequence phylogenetic analysis, and among them streptococci and Leptotrichia wade were enriched in the dental plaque sample, Streptococcus mutans, Fusobacterium nucleatum, and Streptococcus tigurinus in the caries cavity, and Lactobacillus in the saliva. S. mutans was significantly abundant in the mucosal swabs, saliva and plaque samples of the caries cavity as shown by qPCR. Compared to samples collected from a healthy individual and another two patients with rampant caries, the samples from this case showed a decreased bacterial diversity and increased bacterial abundance shown by PCR-DGGE profiling, and multiple Leptotrichia sp. were detected by gel sequencing.

CONCLUSIONThe outgrowth of such pathogenic microorganisms as S. mutans and Leptotrichia sp., and dysbiosis of oral microbial community might contribute to the pathogenesis of rampant caries in this case.

Abnormalities, Multiple ; Dental Caries ; microbiology ; Dental Plaque ; microbiology ; Fusobacterium ; isolation & purification ; Humans ; Immunoglobulin E ; blood ; Lactobacillus ; isolation & purification ; Leptotrichia ; isolation & purification ; Limb Deformities, Congenital ; Microbiota ; Mouth Mucosa ; microbiology ; Neutrophils ; cytology ; Phylogeny ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; genetics ; Saliva ; microbiology ; Streptococcus ; isolation & purification ; Tooth Abnormalities

OBJECTIVE: To assess the lesion conspicuity and image quality in CT evaluation of small (< or = 3 cm) hepatocellular carcinomas (HCCs) using automatic tube voltage selection (ATVS) and automatic tube current modulation (ATCM) with or without iterative reconstruction. MATERIALS AND METHODS: One hundred and five patients with 123 HCC lesions were included. Fifty-seven patients were scanned using both ATVS and ATCM and images were reconstructed using either filtered back-projection (FBP) (group A1) or sinogram-affirmed iterative reconstruction (SAFIRE) (group A2). Forty-eight patients were imaged using only ATCM, with a fixed tube potential of 120 kVp and FBP reconstruction (group B). Quantitative parameters (image noise in Hounsfield unit and contrast-to-noise ratio of the aorta, the liver, and the hepatic tumors) and qualitative visual parameters (image noise, overall image quality, and lesion conspicuity as graded on a 5-point scale) were compared among the groups. RESULTS: Group A2 scanned with the automatically chosen 80 kVp and 100 kVp tube voltages ranked the best in lesion conspicuity and subjective and objective image quality (p values ranging from < 0.001 to 0.004) among the three groups, except for overall image quality between group A2 and group B (p = 0.022). Group A1 showed higher image noise (p = 0.005) but similar lesion conspicuity and overall image quality as compared with group B. The radiation dose in group A was 19% lower than that in group B (p = 0.022). CONCLUSION: CT scanning with combined use of ATVS and ATCM and image reconstruction with SAFIRE algorithm provides higher lesion conspicuity and better image quality for evaluating small hepatic HCCs with radiation dose reduction.
Adult ; Aged ; Aged, 80 and over ; Algorithms ; Carcinoma, Hepatocellular/*radiography ; Contrast Media ; Female ; Fluoroscopy ; Humans ; Image Enhancement/*methods ; Liver Neoplasms/*radiography ; Male ; Middle Aged ; Prospective Studies ; Radiation Dosage ; Radiographic Image Interpretation, Computer-Assisted/*methods ; Tomography, X-Ray Computed/*methods ; Young Adult

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
Blastocyst ; CRISPR-Cas Systems ; Hemoglobins, Abnormal ; genetics ; metabolism ; Humans ; Zygote

Objective To investigate risk factors of the prognosis of patients with severe fever with thrombocytopenia syndrome (SFTS).Methods From May 2012 to July 2014,17 cases of severe fever with thrombocytopenia syndrome in Renmin Hospital of Wuhan University were treated.Clinical data including history of epidemiology,clinical manifestations,complications,physical examination and laboratory test results on admission and the third day after admission were retrospectively analyzed and compared with the death group and recovery group by application of Spearman correlation analysis.Results Elderly male patients with neuropsychiatric symptoms,or abnormal liver function,or abnormal blood clotting function had higher risk of the poor prognosis.In SFTS patients,AST,ALT was significantly increased,AST 539 U/L (229.73,545.4) U/L (r =0.597,P =0.015) was a risk factor affecting prognosis.Elevated blood ammonia indicated serious liver dysfunction and neurological dysfunction which were manifested as irritability,delirium,and trembling limbs.In SFTS patients,platelets were significantly decreased accompanied with mouth ulcers / bleeding gums,gastrointestinal bleeding.PLT 24.88 × 10 9/L-1 (12.75,35.00) ×10 9/L-1 (r=0.557,P=0.005) or APTT 86.06 s (66.88,114.18) (r=0.798,P=0.001) or D-dimmer 9.79 mg / L (4.09,16.51) mg/L (r =0.597,P =0.015) are risk factors affecting poor prognosis.Conclusions On the third days after admission,AST,WBC,PLT,APTT,Ddimmer are risk factors for prognosis of patients with severe fever with thrombocytopenia syndrome infected by a novel bunyavirus.

Objective To construct the head nurses performance evaluation system in order to measure their performance fairly and equitably and to enhance their responsibility,crisis and sense of competition.Methods Many methods were used such as the balanced scorecard four elements were used to construct the basic framework of the performance evaluation system and Delphi method was used to determine the various index,analytic hierarchy process was used to identify the index of value at all levels.Results Two wheels consultation were used and the enthusiasm of experts was high with the coefficient was 88％ and authoritative coefficient was 0.76.Four primary indexes of performance evaluation system were consisted,such as finance,client,interior process,learn and study. It included sixteen secondary indexes,such as nursing service projects,nursing equipments,patients and families,nurses,doctors,and so on.Conclusions Performance evaluation system construction of head nurses based on the Delphi method can measure their performance fairly and equitably and it is worth to applyingin nursing.

OBJECTIVEIt is clear that PAI-1 is a very important factor inhibiting extracellular matrix (ECM) degradation in the pathology process of IgA nephropathy (IgAN), so we design to investigate the relationship between PAI-1 promoter 4G/5G polymorphism and IgAN pathogenesis and progression.

METHODSClinical baseline data such as blood pressure, urinary protein excretion, serum profile, and extent of renal tissue damage at the time of renal biopsy were collected. The genotypes of PAI-1 were profiled by PCR-RFLP.

RESULTS(1) The distributions of genotype 4G 4G, 4G5G, 5G5G in PAI-1 gene promoter showed no significant difference between the IgAN group (0.33, 0.19, 0.48) and control group (0.3, 0.23, 0.47; chi-square =1.63, P>0.05); (2) There is an increasing frequency of 4G4G homozygote in the IgAN group who had severe pathology change proved by biopsy (0.39 vs 0.28; chi-square =7.86, P<0.05); in the patients group,the ones who carried 4G4G genotype got lower Ccr than 4G5G genotype cases did (P<0.05).

CONCLUSIONThe data here suggest that the 4G/5G polymorphism of PAI-1 is not a risk factor in IgAN etiology, but may facilitate the process of IgAN to end stage renal disease.

Adolescent ; Adult ; Child ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; genetics ; Genotype ; Glomerulonephritis, IGA ; genetics ; pathology ; Homozygote ; Humans ; Male ; Middle Aged ; Plasminogen Activator Inhibitor 1 ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Young Adult