Evidence-based Care Bundles for Preventing Surgical Site Infections in Spinal Instrumentation Surgery.

STUDY DESIGN: A retrospective study, using prospectively collected data. OBJECTIVE: The aim of this study was to evaluate the impact of evidence-based care bundles for preventing surgical site infections (SSIs) in spinal instrumentation surgery. SUMMARY OF BACKGROUND DATA: About half of all SSIs are preventable via evidence-based methods. For successful SSI prevention, the bacterial load must be minimized, and methicillin-resistant Staphylococcus aureus (MRSA) protection must be maximized. However, it is difficult to cover all of these requirements by single preventative method. METHODS: We screened consecutive patients scheduled for spinal instrumentation surgeries at a single tertiary referral hospital for high surgical, SSI, and MRSA colonization risks. Evidence-based care bundles were implemented for high-risk patients and included 1) additional vancomycin prophylaxis, 2) diluted povidone-iodine irrigation, and 3) nasal and body decontamination. Patient demographics, comorbidities, operative features, and SSIs reported to the Japanese Nosocomial Infections Surveillance system were prospectively obtained in the same method by the same assessor and were used for the analyses. The results were compared before and after the application of the bundle. RESULTS: There were 1042 spinal instrumentation surgeries (741 before and 301 after care bundles) performed from November 2010 to December 2015. Of 301 surgeries, 57 cases (18.9%) received care bundles. There were no significant differences in patient backgrounds before and after the intervention. The SSI rate decreased significantly from 3.8% to 0.7% (P < 0.01) after the intervention, with an overall 82% relative risk reduction. A significant protective effect was observed in the multivariate analysis (adjusted odds ratio 0.18, 95% confidence interval: 0.04-0.77, P = 0.02). There were no MRSA-related SSIs among those that received care bundles, even though MRSA was the predominant pathogen in the study population. CONCLUSION: Evidence-based care bundles, applied in selected high-risk spinal instrumentation cases, minimized bacterial load, maximized MRSA protection, and significantly reduced SSI rates without topical vancomycin powder. LEVEL OF EVIDENCE: 4.

Biosynthesis-inspired deracemizative production of d-luciferin by combining luciferase and thioesterase.

Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.

Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3.

Reporter assays that use luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of luciferase protein in the cells, which results in an increase of sensitivity of the luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST (t1/2=9.8 to 2.8h), the half-life of C9-fused ELuc was significantly shorter (t1/2=1.0h) than that of PEST-fused ELuc when measurements were conducted at 37°C. In addition, firefly luciferase (luc2) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence.

ABCG2 dysfunction causes hyperuricemia due to both renal urate underexcretion and renal urate overload.

Gout is a common disease which results from hyperuricemia. We have reported that the dysfunction of urate exporter ABCG2 is the major cause of renal overload (ROL) hyperuricemia, but its involvement in renal underexcretion (RUE) hyperuricemia, the most prevalent subtype, is not clearly explained so far. In this study, the association analysis with 644 hyperuricemia patients and 1,623 controls in male Japanese revealed that ABCG2 dysfunction significantly increased the risk of RUE hyperuricemia as well as overall and ROL hyperuricemia, according to the severity of impairment. ABCG2 dysfunction caused renal urate underexcretion and induced hyperuricemia even if the renal urate overload was not remarkable. These results show that ABCG2 plays physiologically important roles in both renal and extra-renal urate excretion mechanisms. Our findings indicate the importance of ABCG2 as a promising therapeutic and screening target of hyperuricemia and gout.

Common dysfunctional variants in ABCG2 are a major cause of early-onset gout.

Gout is a common disease which mostly occurs after middle age, but more people nowadays develop it before the age of thirty. We investigated whether common dysfunction of ABCG2, a high-capacity urate transporter which regulates serum uric acid levels, causes early-onset gout. 705 Japanese male gout cases with onset age data and 1,887 male controls were genotyped, and the ABCG2 functions which are estimated by its genotype combination were determined. The onset age was 6.5 years earlier with severe ABCG2 dysfunction than with normal ABCG2 function (P = 6.14 × 10(-3)). Patients with mild to severe ABCG2 dysfunction accounted for 88.2% of early-onset cases (twenties or younger). Severe ABCG2 dysfunction particularly increased the risk of early-onset gout (odds ratio 22.2, P = 4.66 × 10(-6)). Our finding that common dysfunction of ABCG2 is a major cause of early-onset gout will serve to improve earlier prevention and therapy for high-risk individuals.

In vivo imaging demonstrates ATP release from murine keratinocytes and its involvement in cutaneous inflammation after tape stripping.

Adenosine 5'-triphosphate (ATP) release from keratinocytes has been observed in various stress models in vitro, but studies demonstrating epidermal ATP release in vivo are limited. To visualize extracellular ATP (eATP) in vivo, we developed enhanced green-emitting luciferase immobilized on agarose beads (Eluc-agarose). Subcutaneous injection of Eluc-agarose together with ATP into the dorsal skin of BALB/c mice following intraperitoneal luciferin injection produced detectable and measurable bioluminescence using an in vivo imaging system. Using Eluc-agarose, we demonstrated in vivo that bright bioluminescence was observed from 1 to 20 minutes after repeated tape stripping of murine skin. This bioluminescence was suppressed by the local administration of apyrase. Eluc-agarose bioluminescence was observed only in tape-stripped skin with transepidermal water loss (TEWL) between 100 and 140 g m(2) h(-1), indicating a loss of bioluminescence with excessive tape stripping (TEWL>140 g m(-2) h(-1)). Histologically, tape-stripped skin with detectable eATP had a viable epidermis and a subepidermal neutrophil infiltrate, and administration of apyrase reduced the inflammatory infiltrate. Neither a viable epidermis nor an upper dermal neutrophil infiltrate was observed after excessive tape stripping. These results suggest that tape stripping prompts ATP release from viable keratinocytes, which facilitates inflammatory cell migration. Eluc-agarose may be useful in the in vivo detection of eATP in murine models of skin diseases.

The origin of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase): luciferin stereoselectivity as a switch for the oxygenase activity.

Beetle luciferases evolved from AMP/CoA-ligases. However, it is unclear how the new luciferase activity evolved. In order to clarify this question, we compared the luminescence and catalytic properties of a recently cloned luciferase-like enzyme from Zophobas mealworm, an AMP/CoA-ligase displaying weak luminescence activity, with those of cloned luciferases from the three main families of luminescent beetles: Phrixthrix hirtus railroad worm; Pyrearinus termitilluminans click beetle and Photinus pyralis firefly. The catalytic constant of the mealworm enzyme was 2-4 orders of magnitude lower than that of beetle luciferases, but 3 orders of magnitude above the non-catalyzed chemiluminescence of luciferyl-adenylate in buffer. Studies with D- and L-luciferin and their adenylates show that the luminescence reaction of the luciferase-like enzyme and beetle luciferases are stereoselective for D-luciferin and its adenylate, and that the selectivity is determined mainly at the adenylation step. Modelling studies showed that the luciferin binding site cavity of this enzyme is smaller and more hydrophobic than that of beetle luciferases. Therefore Zophobas mealworm enzyme displays true luciferase activity, keeping the attributes of an ancient protoluciferase. These results suggest that stereoselectivity for D-luciferin may have been a key event for the origin of oxygenase/luciferase activity in AMP/CoA-ligases, and that efficient luciferase activity may have further evolved mainly by increasing the catalytic constant of the oxidative reaction and the quantum yield of bioluminescence.

Applications of luciferin biosynthesis: Bioluminescence assays for l-cysteine and luciferase.

Based on the biosynthetic pathway of firefly bioluminescence substrate D-luciferin, the concentration of L-cysteine can be quantified using a simple protocol and a conventional luminescence detector. The lower limit of quantification (signal/noise ratio [S/N]=10) was 0.26 microM. Using our method, the total amount of free/reduced and disulfide/oxidized L-cysteine could be measured successfully in human serum. In addition, biosynthetic precursors such as 2-cyano-6-hydroxybenzothiazole and L-luciferin could replace d-luciferin in the cell-based luciferase assay. Our results suggest that the bioluminescence reaction associated with the biosynthesis of bioluminescence substrates can provide a fast and cost-effective assay method.

Common defects of ABCG2, a high-capacity urate exporter, cause gout: a function-based genetic analysis in a Japanese population.

Gout based on hyperuricemia is a common disease with a genetic predisposition, which causes acute arthritis. The ABCG2/BCRP gene, located in a gout-susceptibility locus on chromosome 4q, has been identified by recent genome-wide association studies of serum uric acid concentrations and gout. Urate transport assays demonstrated that ABCG2 is a high-capacity urate secretion transporter. Sequencing of the ABCG2 gene in 90 hyperuricemia patients revealed several nonfunctional ABCG2 mutations, including Q126X. Quantitative trait locus analysis of 739 individuals showed that a common dysfunctional variant of ABCG2, Q141K, increases serum uric acid. Q126X is assigned to the different disease haplotype from Q141K and increases gout risk, conferring an odds ratio of 5.97. Furthermore, 10% of gout patients (16 out of 159 cases) had genotype combinations resulting in more than 75% reduction of ABCG2 function (odds ratio, 25.8). Our findings indicate that nonfunctional variants of ABCG2 essentially block gut and renal urate excretion and cause gout.

Stereoisomeric bio-inversion key to biosynthesis of firefly D-luciferin.

The chirality of the luciferin substrate is critical to the luciferin-luciferase reaction producing bioluminescence. In firefly, the biosynthetic pathway of D-luciferin is still unclear, although it can be synthesized in vitro from D-cysteine. Here, we show that the firefly produces both D- and L-luciferin, and that the amount of active D-luciferin increases gradually with maturation stage. Studies of firefly body extracts indicate the possible conversion of L-cysteine via L-luciferin into D-luciferin, suggesting that the biosynthesis is enzymatically regulated by stereoisomeric bio-inversion of L-luciferin. We conclude that the selection of chirality in living organisms is not as rigid as previously thought.