The Rhodanese PspE Converts Thiosulfate to Cellular Sulfane Sulfur in Escherichia coli.

Hydrogen sulfide (H2S) and its oxidation product zero-valent sulfur (S0) play important roles in animals, plants, and bacteria. Inside cells, S0 exists in various forms, including polysulfide and persulfide, which are collectively referred to as sulfane sulfur. Due to the known health benefits, the donors of H2S and sulfane sulfur have been developed and tested. Among them, thiosulfate is a known H2S and sulfane sulfur donor. We have previously reported that thiosulfate is an effective sulfane sulfur donor in Escherichia coli; however, it is unclear how it converts thiosulfate to cellular sulfane sulfur. In this study, we showed that one of the various rhodaneses, PspE, in E. coli was responsible for the conversion. After the thiosulfate addition, the ΔpspE mutant did not increase cellular sulfane sulfur, but the wild type and the complemented strain ΔpspE::pspE increased cellular sulfane sulfur from about 92 µM to 220 µM and 355 µM, respectively. LC-MS analysis revealed a significant increase in glutathione persulfide (GSSH) in the wild type and the ΔpspE::pspE strain. The kinetic analysis supported that PspE was the most effective rhodanese in E. coli in converting thiosulfate to glutathione persulfide. The increased cellular sulfane sulfur alleviated the toxicity of hydrogen peroxide during E. coli growth. Although cellular thiols might reduce the increased cellular sulfane sulfur to H2S, increased H2S was not detected in the wild type. The finding that rhodanese is required to convert thiosulfate to cellular sulfane sulfur in E. coli may guide the use of thiosulfate as the donor of H2S and sulfane sulfur in human and animal tests.

Optimization of a Method for Detecting Intracellular Sulfane Sulfur Levels and Evaluation of Reagents That Affect the Levels in Escherichia coli.

Sulfane sulfur is a class of compounds containing zero-valent sulfur. Most sulfane sulfur compounds are reactive and play important signaling roles. Key enzymes involved in the production and metabolism of sulfane sulfur have been characterized; however, little is known about how to change intracellular sulfane sulfur (iSS) levels. To accurately measure iSS, we optimized a previously reported method, in which reactive iSS reacts with sulfite to produce thiosulfate, a stable sulfane sulfur compound, before detection. With the improved method, several factors were tested to influence iSS in Escherichia coli. Temperature, pH, and osmotic pressure showed little effect. At commonly used concentrations, most tested oxidants, including hydrogen peroxide, tert-butyl hydroperoxide, hypochlorous acid, and diamide, did not affect iSS, but carbonyl cyanide m-chlorophenyl hydrazone increased iSS. For reductants, 10 mM dithiothreitol significantly decreased iSS, but tris(2-carboxyethyl)phosphine did not. Among different sulfur-bearing compounds, NaHS, cysteine, S2O32- and diallyl disulfide increased iSS, of which only S2O32- did not inhibit E. coli growth at 10 mM or less. Thus, with the improved method, we have identified reagents that may be used to change iSS in E. coli and other organisms, providing tools to further study the physiological functions of iSS.

Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning Efficiencies.

Escherichia coli recA - strains are usually used for cloning to prevent insert instability via RecA-dependent recombination. Here, we report that E. coli BW25113 (recA +) competent cells prepared by using a previously reported transformation and storage solution (TSS) had 100-fold or higher transformation efficiency than the commonly used E. coli cloning strains, including XL1-Blue MRF'. The cloning success rates with E. coli BW25113 were 440 to 1,267-fold higher than those with E. coli XL1-Blue MRF' when several inserts were assembled into four vectors by using a simple DNA assembly method. The difference was in part due to RecA, as the recA deletion in E. coli BW25113 reduced the transformation efficiency by 16 folds and cloning success rate by about 10 folds. However, the transformation efficiency and the cloning success rate of the recA deletion mutant of E. coli BW25113 are still 12- and >48-fold higher than those of E. coli XL1-Blue MRF', which is a commonly used cloning strain. The cloned inserts with different lengths of homologous sequences were assembled into four vectors and transformed into E. coli BW25113, and they were stably maintained in BW25113. Thus, we recommend using E. coli BW25113 for efficient cloning and DNA assembly.

The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study.

The recombination of short homologous ends in Escherichia coli has been known for 30 years, and it is often used for both site-directed mutagenesis and in vivo cloning. For cloning, a plasmid and target DNA fragments were converted into linear DNA fragments with short homologous ends, which are joined via recombination inside E. coli after transformation. Here this mechanism of joining homologous ends in E. coli was determined by a linearized plasmid with short homologous ends. Two 3'-5' exonucleases ExoIII and ExoX with nonprocessive activity digested linear dsDNA to generate 5' single-strand overhangs, which annealed with each other. The polymerase activity of DNA polymerase I (Pol I) was exclusively employed to fill in the gaps. The strand displacement activity and the 5'-3' exonuclease activity of Pol I were also required, likely to generate 5' phosphate termini for subsequent ligation. Ligase A (LigA) joined the nicks to finish the process. The model involving 5' single-stranded overhangs is different from established recombination pathways that all generate 3' single-stranded overhangs. This recombination is likely common in bacteria since the involved enzymes are ubiquitous.

Distinct severity of phenotype in Hajdu-Cheney syndrome: a case report and literature review.

BACKGROUND: Hajdu-Cheney syndrome (HCS) is a rare inherited skeletal disorder caused by pathogenic mutations in exon 34 of NOTCH2. Its highly variable phenotypes make early diagnosis challenging. In this paper, we report a case of early-onset HCS with severe phenotypic manifestations but delayed diagnosis. CASE PRESENTATION: The patient was born to non-consanguineous, healthy parents of Chinese origin. She presented facial anomalies, micrognathia and skull malformations at birth, and was found hearing impairment, congenital heart disease and developmental delay during her first year of life. Her first visit to our center was at 1 year of age due to cardiovascular repair surgery for patent ductus arteriosus (PDA) and ventricular septal defect (VSD). Skull X-ray showed wormian bones. She returned at 7 years old after she developed progressive skeletal anomalies with fractures. She presented with multiple wormian bones, acro-osteolysis, severe osteoporosis, bowed fibulae and a renal cyst. Positive genetic test of a de novo heterozygous frameshift mutation in exon 34 of NOTCH2 (c.6426dupT) supported the clinical diagnosis of HCS. CONCLUSION: This is the second reported HCS case caused by the mutation c.6426dupT in NOTCH2, but presenting much earlier and severer clinical expression. Physicians should be aware of variable phenotypes so that early diagnosis and management may be achieved.

Two novel mutations in the ALPL gene of unrelated Chinese children with Hypophosphatasia: case reports and literature review.

OBJECTIVE: Hypophosphatasia (HPP) is an inherited disorder of defective skeletal mineralization caused by mutations in the ALPL gene that encodes the Tissue Non-specific Alkaline Phosphatase (TNSALP). It is subdivided into six forms depending on the age of onset: perinatal lethal, prenatal benign, infantile, childhood, adult, and odonto HPP. Among these, infantile HPP is characterized by early onset and high frequency of lethal outcome. Few studies have reported the phenotype and genetic characteristics of HPP in Chinese children. CASE PRESENTATION: Three forms of HPP were identified in four unrelated patients from four different Chinese families, including one lethal infantile (patient 1), two childhood (patient 2 and 3) and one odonto HPP (patient 4). Six variants in the ALPL gene were identified, including five missense mutations and one frameshift mutation. Of which, none were reported previously in the Chinese population, and two were novel (c.359G > C: p.G120A and c.1017dupG: p.H340AfsX3). Patient 1 carrying a novel homozygous (c.359G > C) mutation showed respiratory distress and pneumonia at first day of his life. He presented nearly negligible level of serum ALP activity, overall skeletal hypominaralization and died at 3 months old. Patient 2, 3 and 4 were compound heterozygotes with decreased serum ALP activity. Patient 2 and 3 presented premature loss of deciduous teeth, muscle weakness and bone pain, whereas patient 4 had early loss of deciduous teeth only. All four pedigrees exhibited autosomal recessive pattern of inheritance. CONCLUSIONS: In this study, six mutations in the ALPL gene were found in four Chinese HPP patients, two of which were novel: c.359G > C in exon 5 and c.1017dupG in exon 10. Our results strongly indicated that the novel mutation c.359G > C might be disease-causing and associated with severe infantile form of HPP.

BAPTA-AM Nanoparticle for the Curing of Acute Kidney Injury Induced by Ischemia/Reperfusion.

Ischemia-reperfusion (I/R) is a major cause of acute kidney injury (AKI), which is associated with unacceptably high mortality rates in ICU. This research was designed to explore the therapeutic effect of BAPTA-AM (1,2-Bis(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid tetrakis(acetoxymethyl ester)) nanoparticle (BA-N) on AKI. BA-N was developed by liposome strategy and characterized by standard methods. The rat model was selected and the rats were randomly allocated into four groups: (1) Normal group; (2) Sham-operated group; (3) Model group (I/R + NS); (4) BA-N treatment group (I/R + BA-N). AKI model was established via clipping the bilateral renal artery with a microvascular clamp for 45 min. After reperfusion, serum cystatin C (Cys C), creatinine (Cr), blood urea nitrogen (BUN), lactate dehydrogenase (LDH) and caspase 3 levels were determined for the assessment of renal function. Kidney samples were then collected for the measurement of renal malondialdehyde (MDA) level and superoxide dismutase (SOD) activity. The assays of histological examination, ELISA, immunohistochemistry, western blot, TUNEL and RT-PCR were utilized for the detection of apoptosis. The results demonstrated that AKI model caused a significant decreasing in SOD activity, accompanied by a remarkable increase in Cys C, Cr, BUN, LDH, MDA, caspase 3 and cytochrome c (Cyt C) level, compared to the control group. BA-N (100 µg/kg i.v.) significantly improved renal function and histopathological appearance, restored MDA level and SOD activity, decreased Bax/Bcl-2 ratio, caspase 3 activity, Cyt C release and TUNEL positive apoptotic cells. Our studies indicated that BA-N plays a renal-protective role, probably through antiapoptotic and antioxidant mechanisms. BA-N may regulate mitochondria pathway via decreasing Bax/Bcl-2 ratio, inhibiting caspase 3 expression and Cyt C release. Overall, BA-N may have potentials as an anti-AKI drug.