An Escherichia coli strain that cannot scavenge hydrogen peroxide has been used to identify the cell processes that are most sensitive to this oxidant. Low micromolar concentrations of H 2 O 2 completely blocked the biosynthesis of leucine. The defect was tracked to the inactivation of isopropylmalate isomerase. This enzyme belongs to a family of [4Fe-4S] dehydratases that are notoriously sensitive to univalent oxidation, and experiments confirmed that other members were also inactivated. In vitro and in vivo analyses showed that H 2 O 2 directly oxidized their solvent-exposed clusters in a Fenton-like reaction. The oxidized cluster then degraded to a catalytically inactive [3Fe-4S] form. Experiments indicated that H 2 O 2 accepted two consecutive electrons during the oxidation event. As a consequence, hydroxyl radicals were not released; the polypeptide was undamaged; and the enzyme was competent for reactivation by repair processes. Strikingly, in scavenger-deficient mutants, the H 2 O 2 that was generated as an adventitious by-product of metabolism (<1 M) was sufficient to damage these [4Fe-4S] enzymes. This result demonstrates that aerobic organisms must synthesize H 2 O 2 scavengers to avoid poisoning their own pathways. The extreme vulnerability of these enzymes may explain why many organisms, including mammals, deploy H 2 O 2 to suppress microbial growth.

Highlights d Cities possess a consistent ''core'' set of non-human microbes d Urban microbiomes echo important features of cities and city-life d Antimicrobial resistance genes are widespread in cities d Cities contain many novel bacterial and viral species

SummaryEnvironmental H2O2 creates several injuries in Escherichia coli, including the oxidative conversion of dehydratase [4Fe-4S] clusters to an inactive [3Fe-4S] form. To protect itself, H2O2-stressed E. coli activates the OxyR regulon. This regulon includes the suf operon, which encodes an alternative to the housekeeping Isc iron-sulphur cluster assembly system. Previously studied [3Fe-4S] clusters are repaired by an Isc/Suf-independent pathway, so the rationale for Suf induction was not obvious. Using strains that cannot scavenge H2O2, we imposed chronic low-grade stress and found that suf mutants could not maintain the activity of isopropylmalate isomerase, a key iron-sulphur dehydratase. Experiments showed that its damaged cluster was degraded in vivo beyond the [3Fe-4S] state, presumably to an apoprotein form, and thus required a de novo assembly system for reactivation. Surprisingly, submicromolar H2O2 poisoned the Isc machinery, thereby creating a requirement for Suf both to repair the isomerase and to activate nascent Fe-S enzymes in general. The IscS and IscA components of the Isc system are H2O2-resistant, suggesting that oxidants disrupt Isc by oxidizing clusters as they are assembled on or transferred from the IscU scaffold. Consistent with these results, organisms that are routinely exposed to oxidants rely upon Suf rather than Isc for cluster assembly.

SummaryThe small RNA RyhB has recently been shown to negatively regulate a number of mRNAs encoding dispensable iron-using proteins in Escherichia coli. The resulting decrease in the synthesis of iron-using proteins is thought to spare iron in order to ensure its availability for iron-requiring proteins that are indispensable. Indeed, the expression of RyhB from a heterologous promoter activates the iron-sensing repressor Fur, which suggests an increase in the pool of free intracellular iron (iron-sparing). In accordance with these observations, we report here that RyhB expression increases the concentration of free intracellular iron, as shown by direct measurements of the metal in whole cells by electron paramagnetic resonance spectroscopy. Our data also suggest that ironsparing originates from rapid uptake of extracellular iron and not from already internalized metal. Furthermore, RyhB is shown to be essential for normal bacterial growth and survival during iron starvation, which is consistent with previous data describing the function of the small RNA. Overall, our data demonstrate that, by regulating synthesis of nonessential iron-using proteins, the small RNA RyhB ensures that the iron is directed towards the iron-requiring enzymes that are indispensable.

The aims of this study were to analyze the range of cartilage damage and patellofemoral joint (PFJ) degeneration in degenerative osteoarthritis (OA) and determine the duration for the positive effects of platelet-rich plasma (PRP) injection. This study included 65 patients suffering from OA that were treated with intra-articular PRP injection. The patients were prospectively evaluated at 1, 3, 6, 9, and 12 months after the procedure using a visual analogue scale (VAS) score and an International Knee Documentation Committee (IKDC) score. Clinical improvement in the average VAS score from 7.4 before the procedure to 4.2 at 6 months post-procedure had been reported, but the symptoms tended to deteriorate to 5.0 1 year after injection. The IKDC score also showed statistical significance (P < 0.05). Patients reported relapsed pain 8.8 months after the procedure. Developing degeneration according to the Kellgren-Lawrence grade reduced the clinical effects of PRP (P < 0.05) and also accelerated the time for feeling relapsed pain (P < 0.05). There was a statistically significant negative correlation between patient age and the PRP potential in the VAS score (slope = 0.1667) and IKDC score (slope = 1.3333). The presence of PFJ degeneration is expected to produce a worse outcome (P < 0.05). While intra-articular PRP injection can be used for the treatment of early OA, increasing age, and developing degeneration result in a decreased potential for PRP injection therapy.