Advancing Desulfurization in the Model Biocatalyst Rhodococcus qingshengii IGTS8 via an In Locus Combinatorial Approach.

Biodesulfurization poses as an ideal replacement to the high cost hydrodesulfurization of the recalcitrant heterocyclic sulfur compounds, such as dibenzothiophene (DBT) and its derivatives. The increasingly stringent limits on fuel sulfur content intensify the need for improved desulfurization biocatalysts, without sacrificing the calorific value of the fuel. Selective sulfur removal in a wide range of biodesulfurization strains, as well as in the model biocatalyst Rhodococcus qingshengii IGTS8, occurs via the 4S metabolic pathway that involves the dszABC operon, which encodes enzymes that catalyze the generation of 2-hydroxybiphenyl and sulfite from DBT. Here, using a homologous recombination process, we generate two recombinant IGTS8 biocatalysts, harboring native or rearranged, nonrepressible desulfurization operons, within the native dsz locus. The alleviation of sulfate-, methionine-, and cysteine-mediated dsz repression is achieved through the exchange of the native promoter Pdsz, with the nonrepressible Pkap1 promoter. The Dsz-mediated desulfurization from DBT was monitored at three growth phases, through HPLC analysis of end product levels. Notably, an 86-fold enhancement of desulfurization activity was documented in the presence of selected repressive sulfur sources for the recombinant biocatalyst harboring a combination of three targeted genetic modifications, namely, a dsz operon rearrangement, a native promoter exchange, and a dszA-dszB overlap removal. In addition, transcript level comparison highlighted the diverse effects of our genetic engineering approaches on dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. IMPORTANCE Rhodococcus is perhaps the most promising biodesulfurization genus and is able to withstand the harsh process conditions of a biphasic biodesulfurization process. In the present work, we constructed an advanced biocatalyst harboring a combination of three genetic modifications, namely, an operon rearrangement, a promoter exchange, and a gene overlap removal. Our homologous recombination approach generated stable biocatalysts that do not require antibiotic addition, while harboring nonrepressible desulfurization operons that present very high biodesulfurization activities and are produced in simple and low-cost media. In addition, transcript level quantification validated the effects of our genetic engineering approaches on recombinant strains' dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. Based on these findings, the present work can pave the way for further strain and process optimization studies that could eventually lead to an economically viable biodesulfurization process.

A systematic re-examination of processing of MHCI-bound antigenic peptide precursors by endoplasmic reticulum aminopeptidase 1.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mature antigenic peptides for presentation by major histocompatibility complex class I (MHCI) molecules and regulates adaptive immune responses. ERAP1 has been proposed to trim peptide precursors both in solution and in preformed MHCI-peptide complexes, but which mode is more relevant to its biological function remains controversial. Here, we compared ERAP1-mediated trimming of antigenic peptide precursors in solution or when bound to three MHCI alleles, HLA-B*58, HLA-B*08, and HLA-A*02. For all MHCI-peptide combinations, peptide binding onto MHCI protected against ERAP1-mediated trimming. In only a single MHCI-peptide combination, trimming of an HLA-B*08-bound 12-mer progressed at a considerable rate, albeit still slower than in solution. Results from thermodynamic, kinetic, and computational analyses suggested that this 12-mer is highly labile and that apparent on-MHC trimming rates are always slower than that of MHCI-peptide dissociation. Both ERAP2 and leucine aminopeptidase, an enzyme unrelated to antigen processing, could trim this labile peptide from preformed MHCI complexes as efficiently as ERAP1. A pseudopeptide analogue with high affinity for both HLA-B*08 and the ERAP1 active site could not promote the formation of a ternary ERAP1/MHCI/peptide complex. Similarly, no interactions between ERAP1 and purified peptide-loading complex were detected in the absence or presence of a pseudopeptide trap. We conclude that MHCI binding protects peptides from ERAP1 degradation and that trimming in solution along with the dynamic nature of peptide binding to MHCI are sufficient to explain ERAP1 processing of antigenic peptide precursors.

Deciphering the biodesulfurization potential of two novel Rhodococcus isolates from a unique Greek environment.

Sustainable biodesulfurization (BDS) processes require the use of microbial biocatalysts that display high activity against the recalcitrant heterocyclic sulfur compounds and can simultaneously withstand the harsh conditions of contact with petroleum products, inherent to any industrial biphasic BDS system. In this framework, the functional microbial BDS-related diversity in a naturally oil-exposed ecosystem, was examined through a 4,6-dimethyl-dibenzothiophene based enrichment process. Two new Rhodococcus sp. strains were isolated, which during a medium optimization process revealed a significantly enhanced BDS activity profile when compared to the model strain R. qingshengii IGTS8. In biocatalyst stability studies conducted in biphasic mode using partially hydrodesulfurized diesel under various process conditions, the new strains also presented an enhanced stability phenotype. In these studies, it was also demonstrated for all strains, that the BDS activity losses were decoupled from the overall cells' viability, in addition to the fact that the use of whole-broth biocatalyst positively affected BDS performance.

The ERAP1 active site cannot productively access the N-terminus of antigenic peptide precursors stably bound onto MHC class I.

Processing of N-terminally elongated antigenic peptide precursors by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) is a key step in antigen presentation and the adaptive immune response. Although ERAP1 can efficiently process long peptides in solution, it has been proposed that it can also process peptides bound onto Major Histocompatibility Complex I molecules (MHCI). In a previous study, we suggested that the occasionally observed "ontο MHCI" trimming by ERAP1 is likely due to fast peptide dissociation followed by solution trimming, rather than direct action of ERAP1 onto the MHCI complex. However, other groups have proposed that ERAP1 can trim peptides covalently bound onto MHCI, which would preclude peptide dissociation. To explore this interaction, we constructed disulfide-linked MHCI-peptide complexes using HLA-B*08 and a 12mer kinetically labile peptide, or a 16mer carrying a phosphinic transition-state analogue N-terminus with high-affinity for ERAP1. Kinetic and biochemical analyses suggested that while both peptides could access the ERAP1 active site when free in solution, they were unable to do so when tethered in the MHCI binding groove. Our results suggest that MHCI binding protects, rather than promotes, antigenic peptide precursor trimming by ERAP1 and thus solution trimming is the more likely model of antigenic peptide processing.

Muscular strength and jumping performance after handball training versus physical education program for pre-adolescent children.

The purpose was to compare a 6-mo. specific handball training program and a typical physical education program on various strength and jumping skills. The participants (M age= 13.7 yr., SD= 1.5) were divided into the Handball Group (n=51) and the Physical Education Group (n=70). The latter performed 3 sessions/ week (60 min.) including ball-handling drills, horizontal and vertical jump shots, fast break, and several defensive skills. The former performed the program provided by the Ministry of Education including track and field and other team sport drills. Analyses of covariance showed that the handball group displayed greater improvement in explosive strength of upper limbs, jumping performance, maximum isometric force of right grip, and 10-m running velocity. Handball training can significantly improve pre-adolescent performance with upper and lower limbs. Inclusion of specific handball drills in the physical education program is recommended.

Primary omental torsion in children: ten-year experience.

Primary omental torsion (POT), is a rare cause of acute abdomen commonly affecting obese male adults, whereas it is extremely rare in children. In this retrospective study, we present our experience regarding the management of five children with POT and discuss the diagnostic and therapeutic implications of this entity. We retrospectively reviewed the medical records of children diagnosed for POT, from January 1996 to July 2006 at our department. Among 2,734 children operated for acute appendicitis, five patients were diagnosed with POT (ratio 1:587 or 0.18%). Clinical presentation, laboratory findings, diagnostic imaging results as well as surgical and histological findings were reviewed. There were four boys and one girl, M/F ratio 4:1, with a mean age of 9.5 years (range 7.2-10.3). All subjects were obese and their weight percentages were over 85% for their age group. On admission the clinical symptoms and laboratory findings were similar to those of acute appendicitis. They were submitted to laparotomy and the surgical findings were; free serosanguineous fluid in the peritoneal cavity, normal appendix and an ischemic twisted mass of the omentum at the right side of the abdomen. The mass and the appendix were excised and the postoperative course was uneventful. The histological examination of the specimens revealed hemorrhagic ischemic necrosis of the omentum and normal appendix. POT is very rare in children. In the pediatric age group the clinical presentation and the laboratory findings are similar to those of acute appendicitis and it is extremely difficult to be diagnosed preoperatively. Obesity seems to be an important predisposing high-risk factor. Excision of the twisted omentum is the treatment of choice.

Multiple failed urethroplasties: definitive repair with the Duckett island-flap technique.

BACKGROUND/PURPOSE: The correction of postoperative complications after hypospadias repair presents frequently serious problems, and the results are not always satisfactory. The aim of this study is to present our experience with the tubularized island flap technique (Duckett procedure) in patients who had been submitted to repeated unsuccessful attempts for the repair of hypospadias and its complications. METHODS: During an 8-year period (1994 through 2001), 21 patients, aged 4 to 18 years, presented with severe recurrent hypospadias (3 to 13 times). In all patients, the urethral orifice was quite proximal because of disruption of the neourethra and was associated with severe penile ventriflexion. In addition, there were diverticula with hair ingrowth in 5 boys and lack of the prepuce in 4. All patients underwent the Duckett island-flap technique. The inner layer of the prepuce was used in 17. The dorsal penile skin was used for the island flap formation in the remaining 4 patients in whom the prepuce had been resected during the previous operations. RESULTS: After repair, the meatus was located at the top of the glans in all patients. There were 5 complications (24%): distal fistula near the glans (n = 1), meatal stenosis (n = 1), diverticula at the proximal anastomosis (n = 2), and anastomotic stenosis (n = 1). All of them were treated successfully by meatotomy, fistula closure, diverticula tapering, and dilatations, respectively. CONCLUSIONS: The island-flap technique gives satisfactory results in patients with multiple failed urethroplasties; it is applicable even in boys with resected prepuce and has an acceptable complication rate.