Methanol-Dependent Carbon Fixation for Irreversible Synthesis of d-Allulose from d-Xylose by Engineered Escherichia coli.

d-Allulose is a rare hexose with great application potential, owing to its moderate sweetness, low energy, and unique physiological functions. The current strategies for d-allulose production, whether industrialized or under development, utilize six-carbon sugars such as d-glucose or d-fructose as a substrate and are usually based on the principle of reversible Izumoring epimerization. In this work, we designed a novel route that coupled the pathways of methanol reduction, pentose phosphate (PP), ribulose monophosphate (RuMP), and allulose monophosphate (AuMP) for Escherichia coli to irreversibly synthesize d-allulose from d-xylose and methanol. After improving the expression of AlsE by SUMO fusion and regulating the carbon fluxes by knockout of FrmRAB, RpiA, PfkA, and PfkB, the titer of d-allulose in fed-batch fermentation reached ≈70.7 mM, with a yield of ≈0.471 mM/mM on d-xylose or ≈0.512 mM/mM on methanol.

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation.

D-Allulose is an ultra-low calorie sweetener with broad market prospects. As an alternative to Izumoring, phosphorylation-dephosphorylation is a promising method for D-allulose synthesis due to its high conversion of substrate, which has been preliminarily attempted in enzymatic systems. However, in vitro phosphorylation-dephosphorylation requires polyphosphate as a phosphate donor and cannot completely deplete the substrate, which may limit its application in industry. Here, we designed and constructed a metabolic pathway in Escherichia coli for producing D-allulose from D-fructose via in vivo phosphorylation-dephosphorylation. PtsG-F and Mak were used to replace the fructose phosphotransferase systems (PTS) for uptake and phosphorylation of D-fructose to fructose-6-phosphate, which was then converted to D-allulose by AlsE and A6PP. The D-allulose titer reached 0.35 g/L and the yield was 0.16 g/g. Further block of the carbon flux into the Embden-Meyerhof-Parnas (EMP) pathway and introduction of an ATP regeneration system obviously improved fermentation performance, increasing the titer and yield of D-allulose to 1.23 g/L and 0.68 g/g, respectively. The E. coli cell factory cultured in M9 medium with glycerol as a carbon source achieved a D-allulose titer of ≈1.59 g/L and a yield of ≈0.72 g/g on D-fructose.

Methemoglobin-Based Biological Dose Assessment for Human Blood.

Methemoglobin is an oxidative form of hemoglobin in erythrocytes. The authors' aim was to develop a new biological dosimeter based on a methemoglobin assay. Methemoglobin in peripheral blood (of females or males) that was exposed to a Co source (0.20 Gy min) was quantified using an enzyme-linked immunosorbent assay. The dose range was 0.5-8.0 Gy. In a time-course experiment, the time points 0, 0.02, 1, 2, 3, 7, 15, 21, and 30 d after 4-Gy irradiation of heparinized peripheral blood were used. Methemoglobin levels in a lysed erythrocyte pellet from the irradiated blood of females and males increased with the increasing dose. Methemoglobin levels in female blood irradiated with γ-doses more than 4 Gy were significantly higher than those in male samples at the same doses. Two dose-response relations were fitted to the straight line: one is with the correlation coefficient of 0.98 for females, and the other is with the correlation coefficient of 0.99 for males. The lower limit of dose assessment based on methemoglobin is about 1 Gy. Methemoglobin levels in blood as a result of auto-oxidation increase after 7-d storage at -20 °C. The upregulation of methemoglobin induced by γ-radiation persists for ∼3 d. The absorbed doses that were estimated using the two dose-response relations were close to the actual doses. The results suggest that methemoglobin can be used as a rapid and accurate biological dosimeter for early assessment of absorbed γ-dose in human blood.

An investigation of drug-resistant Acinetobacter baumannii infections in a comprehensive hospital of East China.

BACKGROUND: To investigate the drug resistant gene profiles and molecular typing of Acinetobacter baumannii isolates collected from clinical specimens in a comprehensive hospital, Jiangsu province. METHODS: This study included 120 patients in a comprehensive hospital with drug-resistant A. baumannii infections on clinical specimens from October 2011 to December 2013. Antibiotic susceptibility test was determined by Vitek 2 Compact system. OXA-51, OXA-23, OXA-24, OXA-58, VIM, IMP, SHV, GES, TEM, AmpC, qacEΔ1-sul1, intI l, CarO, aac(6')-Ib, and aac(6')-II were analyzed by PCR. The analysis of molecular typing for 50 multidrug resistant A. baumannii isolates was performed by PFGE. RESULTS: A total of 64(53%) isolates were multidrug-resistant A.baumannii. The antibiotic susceptibility tests showed that the resistant rates to common antibiotics of mutidrug-resistant A. baumannii were extremely high, most of which over 60%. One hundred and ten isolates harbored OXA-51 (91.7%), 100 for OXA-23(83.3%), 103 for VIM-1(85.8%), 90 for AmpC(75.00%), 50 for aac(6')-Ib(41.7%), 77 for the loss of CarO (64.2%), 85 for intl1(70.8%), and 64 for qacEΔ1-sul1(53.33%), while OXA-24 was undetected. Fifty multidrug-resistant A. baumannii isolates belong to 14 clones according to the PFGE DNA patterns. Main clone A includes 24 isolates, while clone B and clone C includes 6 and 9 isolates, respectively and others with no common source identified. CONCLUSION: There is high morbidity of A. baumannii infections in the hospital, especially in ICU and sputum is the most common sample type.The mainly drug-resistant genes of A. baumannii are OXA-51, OXA-23, and VIM-1 in the hospital. Clonal dissemination provides evidence for the prevalence of multidrug-resistant A. baumannii among clinical isolates. It is suggested that there is an urgent need for effective control and prevention measures.

[Effects of chlorides on Cd transformation in a simulated grate incinerator during sludge incineration process ].

The effects of organic chloride-PVC and inorganic chloride-NaCl on Cd partitioning during sludge incineration with adding Cd(CH3COO)2 . 2H2O to the real sludge were investigated using a simulated tubular incineration furnace. And transformation and distribution of Cd were studied in different sludge incineration operation conditions. The results indicated that the partitioning of Cd tended to be enhanced in the fly ash and fule gas as the chloride content increasing. The migration and transformation of Cd-added sludge affected by different chloride were not obvious with the increasing of chloride content. With increasing temperature, organic chloride (PVC) and inorganic chloride (NaC1) can reduce the Cd distribution in the bottom ash. However, the effect of chlorides, the initial concentration and incineration time on Cd emissions had no significant differences. Using SEM-EDS and XRD technique, different Cd compounds including CdCl2, Na2CdCl4, K2CdCl6, K2CdSiO4 and NaCdO2 were formed in the bottom ash and fly ash after adding NaCl to the sludge. In contrast, after adding PVC to the sludge, the Na2CdCl4 and CdCl2 were the main forms of Cd compounds, at the same time, K4CdCI6 and K6CdO4 were also formed. The two different mechanisms of chlorides effects on Cd partitioning were affected by the products of Cd compound types and forms.

[Effects of males' age on sperm apoptosis and DNA integrity].

OBJECTIVE: To explore the correlation of males'age with sperm apoptosis, sperm DNA integrity and other seminal parameters. METHODS: We collected 104 semen samples and divided them into three groups according to the males' age: <35 yr (n = 43), 35 -39 yr (n = 31), and > or = 40 yr (n = 30). Based on the WHO Laboratory Manual (4th ed), we detected the seminal parameters, calculated the percentage of apoptotic sperm by flow cytometry (FCM), determined sperm DNA integrity by Acridine orange staining, and compared the results among the three groups. RESULTS: There were no statistically significant differences among the < 35 yr, 35 -39 yr and > or = 40 yr groups in semen volume ([2.87 +/- 0.89] ml vs [2.98 +/- 1.09] ml vs [2.65 +/- 0.95] ml), sperm concentration ([60.40 +/- 25.43] x 10(6)/ml vs [69.74 +/- 28.33] x 10(6)/ml vs [55.97 +/- 27.22] x 10(6)/ml) (P>0.05). The percentage of progressively motile sperm was significantly lower in the > or = 40 yr ([39.00 +/- 8.35 %) than in the <35 and 35 -39 yr groups ([48.73 +/- 9.89]% and [45.65 +/- 10.55]%) (P<.0.1), and so was the percentage of morphologically normal sperm in the > or = 40 yr than in the < 35 yr group ([11.11 +/- 8.26]% vs [16.43 +/- 8.75 ]%, P<0.01). The percentage of apoptotic sperm was markedly higher in the > or = 40 yr than in the <35 yr group ([11.82 +/- 5.77]% vs [7.04 +/- 3.50]%, P<0.01), while the sperm DNA integrity significantly reduced in the > or = 40 yr group ([75.52 +/- 10.60]%) as compared with the <35 yr ([86.55 +/- 5.60])% and 35 -39 yr group ( [81.39 +/- 8.94]%) (P<0.01). The males' age was correlated positively with the rate of sperm apoptosis (P<0.01), and negatively with sperm DNA integrity and the percentage of progressively motile sperm (P<0.01). CONCLUSION: The advance in males' age increases sperm apoptosis and reduces sperm progressive motility, normal morphology and DNA integrity.

[Influence of male age on the outcome of conventional IVF-ET].

OBJECTIVE: To study the influence of male age on the outcome of conventional IVF-ET. METHODS: Based on male age, 170 couples undergoing conventional IVF-ET were divided into three groups: <35 yr (n = 60), 35 -39 yr (n = 77) and > or = 40 yr (n = 33). We observed the rates of fertilization, cleavage, good quality embryo, implantation, clinical pregnancy and abortion in different groups. RESULTS: There were no significant differences among the three groups in semen volume ([3.10 +/- 1.22] ml vs [2.84 +/- 1.05] ml vs [2.80 +/- 0.79] ml), sperm concentration ([54.23 +/- 26.07] x 10(6)/ml vs [60.27 +/- 24.80] x 10(6)/ml vs [60.21 +/- 27.42] x 10(6)/ml) and sperm viability ([53.93 +/- 13.25]% vs [56.10 +/- 16.58]% vs [51.82 +/- 15.45]%) (P>0.05). The men of the > or = 40 yr group showed a significantly lower percentage of grade a + b sperm ([40.97 +/- 11.91]%) than those of the <35 and 35 - 39 yr groups ([48.47 +/- 11.78]% and [46.84 +/- 13.51]%) (P<0.05), and morphologically normal sperm ([11.76 +/- 5.97]%) than those of the <35 yr group ([15.25 +/- 6.94]% (P<0.05). The rates of fertilization, cleavage, good quality embryo, implantation, clinical pregnancy were 81.52%, 82.61%, 52.33%, 18.06% and 33.33% in the > or = 40 yr group, with no significant differences from those of the <35 yr group (83.18%, 82.68%, 56.99%, 22.40% and 40.00%) and the 35 - 39 yr group (78.78%, 80.66%, 55.01%, 21.74% and 38.96%) (P>0.05), while the abortion rate was markedly increased in the > or = 40 yr group as compared with the <35 yr group (36.36% vs 8.33%, P>0.05). CONCLUSION: Increasing male age is related with decreasing percentages of progressively motile sperm and morphologically normal sperm, but not obviously with the rates of fertilization, good quality embryo, implantation, pregnancy and abortion.

[Expression of SKP2 protein in lung carcinoma and its implication for prognosis].

OBJECTIVE: To investigate the characteristics of SKP2 protein expression in lung carcinoma tissues and its implication for prognosis. METHODS: The expression of SKP2 protein was detected in 89 NSCLC, 13 SCLC, 5 benign lung neoplasms, 5 normal bronchus and lung tissues by tissue chip and immunohistochemical techniques. RESULTS: The positive rate of SKP2 staining was (23.52 +/-13.57)% in NSCLC tissues and (53.85 +/- 12.26)% in SCLC tissues, significantly higher than (2.91 +/- 1.27)% in benign lung neoplasms and normal bronchus and lung tissues. Its expression was highest in SCLC tissues and lowest in benign lung tissues, with a significant difference between them (P <0.01). The expressive level of SKP2 protein in lung carcinoma tissues was closely related to cell differentiation and lymph node metastasis, but not to age, sex, smoking history, tumor site and size, and TNM staging, etc. The survival analysis revealed that the 5-year survival rate of lung carcinoma patients was much lower in SKP2 protein positive expression group than that in negative expression group (P < 0.01). CONCLUSION: The positive expression of SKP2 protein is higher in lung carcinoma than in benign or normal lung tissues, in particular, much higher in SCLC tissue. Moreover, it may be an independent factor to exert negative influence on prognosis of patients with lung carcinoma.

[Nuclear gene involves in phenotype of non-syndromic deafness associated with mitochondrial 12S rRNA mutation].

The human mitochondrial 12S rRNA gene mutation at position 1555 associated with non-syndromic deafness and aminoglycoside-induced deafness. Family of Huaiyin in Jiangsu is one of the biggest non-syndromic deafness family in the world. In this family, deafness is maternally inherited. After establishing immortal lymphoblastoid cell lines of the family by EB virus, we analysed 17 lymphoblastoid cell lines derived, respectively, from symptomatic, asymptomatic and controll members of the family. Compared with control members, symptomatic and asymptomatic members both exhibited significant decreases in the rate of growth as well as in the rates of mitochondrial protein synthesis. But the extent of decreases is different and the severity of mitochondrial defect is related with its clinical phenotype. These results supported that the nuclear factor involves in the phenotypic manifestation of the non-syndromic deafness associated with the A1555G mutation.

[Sequence analysis of the connexin 26 genes from a deafness family with A1555G mutation in Huaiyin].

OBJECTIVE: To ascertain whether connexin 26 (Cx26) gene was a nuclear modifier gene in an extensive family with matrilineal nonsyndromic deafness associated with A1555G mutation in Huaiyin, China. METHODS: Following PCR-restriction fragment length polymorphism (PCR-RFLP) with ApaI restriction enzyme, Cx26 genes from 26 cases, with A1555G mitochondrial mutations in this family, and 62 controls (including 2 patrilineal relatives, 10 spouse controls and 50 unrelated controls), were sequenced. RESULTS: Compared with the reference sequence of Cx26 gene, totally four kinds of nucleotide changes,79G -->A, 109G-->A, 341G-->A and 235delC, were detected in a heterozygous form. However, the former three were previously reported polymorphisms, and only the 235delC was a previously described recessive mutation associated with most autosomal nonsyndromic sensorineural hearing loss in Japan and China. Further study showed that the heterozygous 235delC mutation existed in both one individual with mild hearing loss and two individuals with normal hearing. Clinical characterization showed that 235delC mutation did not seem to modify the deafness phenotype due to the A1555G mutation. Moreover, this 235delC mutation was deduced to derive from a married-in control. Finally, there were no co-segregation between the phenotypes of hearing loss and the genotypes for Cx26 genes based on the four kinds of nucleotide changes. CONCLUSIONS: The heterozygous 235delC mutation of the Cx26 gene may not modulate the severity of hearing loss associated with A1555G mutation and Cx26 gene is unlikely to be a modifier gene for hearing loss due to A1555G mitochondrial mutation in this Chinese family.

Sequence analysis of the mitochondrial genome from a large family with maternally inherited nonsyndromic deafness.

OBJECTIVE: To ascertain whether other variations coexist with 1555(A--> G) mutation in the mitochondrial DNA and may aggravate the severity of hearing loss or increase the penetrance of 1555(A--> G) mutation in a large family with maternally inherited nonsyndromic deafness in Huaiyin, Jiangsu province. METHODS: PCR-restriction fragment length polymorphism (PCR-RFLP) was used to screen both the nt1555 and the nt7445 of the mitochondrial DNA from 27 maternal members in the core family; and then the mitochondrial genomes from two maternal members, and the 12S rRNA genes MTRNR1 and tRNA-Ser(UCN) gene MTTS1 from the others, were amplified by PCR-RFLP and were sequenced. RESULTS: 1555(A--> G) mutation in the mitochondrial DNA was reverified to be one of the major factors which cause maternally inherited nonsyndromic deafness and the cosegregation of 955-960(insC) and 1555(A--> G) was present in this family. Moreover, 7449 (insG), a novel homoplasmic mutation in the tRNA-Ser(UCN) gene, was found to co-exist with 1555(A--> G) mutation in two maternal members. CONCLUSION: The cosegregation of 955-960(insC) and 1555(A--> G) implies that 955-960(insC) may synergistically cause hearing loss in the presence of an 1555(A--> G) mutation, serving as an aggravating factor to enhance the sensitivity to aminoglycosides, and may sometimes increase the penetrance of 1555(A--> G) mutation.

[Establishment of Immortal Lymphoblastoid Cell Lines of Non-syndromic Deafness Family and Several Methods fro Transforming Human B Lyphocyte by EB virus.].

Reservation of rare family materials is the base for us to do further research. Family of Jiang-Su Huai-Yin is one of the biggest non-syndromic deafness families in the world. In this family,deafness is maternally inherited and all the sufferers have the mitochondrial DNA 12s RNA A1555G mutation. Four methods are used in the experiments for establishing immortal lymphoblastoid cell lines of the family with non-syndromic deafness. Results were as follows: 1 cell line was from small amout of whole blood method, 1 cell line from frozen whole blood method, 14 cell lines from frozen leukocyte method, and 36 cell lines from cyclosporin A method. In this paper, we will discuss these four methods through our experiments of establishing cell lines.