The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli.

Small proteins (<50 amino acids) are emerging as ubiquitous and important regulators in organisms ranging from bacteria to humans, where they commonly bind to and regulate larger proteins during stress responses. However, fundamental aspects of small proteins, such as their molecular mechanism of action, downregulation after they are no longer needed, and their evolutionary provenance, are poorly understood. Here, we show that the MntS small protein involved in manganese (Mn) homeostasis binds and inhibits the MntP Mn transporter. Mn is crucial for bacterial survival in stressful environments but is toxic in excess. Thus, Mn transport is tightly controlled at multiple levels to maintain optimal Mn levels. The small protein MntS adds a new level of regulation for Mn transporters, beyond the known transcriptional and post-transcriptional control. We also found that MntS binds to itself in the presence of Mn, providing a possible mechanism of downregulating MntS activity to terminate its inhibition of MntP Mn export. MntS is homologous to the signal peptide of SitA, the periplasmic metal-binding subunit of a Mn importer. Remarkably, the homologous signal peptide regions can substitute for MntS, demonstrating a functional relationship between MntS and these signal peptides. Conserved gene neighborhoods support that MntS evolved from the signal peptide of an ancestral SitA protein, acquiring a life of its own with a distinct function in Mn homeostasis.

Downscaling platelet cryopreservation: Are platelets frozen in tubes comparable to standard cryopreserved platelets?

BACKGROUND: Platelet cryopreservation extends the shelf-life to at least 2 years. However, platelets are altered during the freeze/thaw process. Downscaling platelet cryopreservation by freezing in tubes would enable rapid screening of novel strategies to improve the quality of cryopreserved platelets (CPPs). The aim of this study was to characterize the effect of freezing conditions on the in vitro phenotype and function of platelets frozen in a low volume compared to standard CPPs. METHODS: Platelets were prepared for cryopreservation using 5%-6% DMSO and processed using standard protocols or aliquoted into 2 mL tubes. Platelets were hyperconcentrated to 25 mL (standard CPPs) or 200 µL (tubes) before freezing at -80°C (n = 8). Six insulators/controlled rate freezing containers were used to vary the freezing rate of platelets in tubes. Platelets were thawed, resuspended in plasma, and then assessed by flow cytometry and thromboelastography. RESULTS: The use of different insulators for tubes changed the freezing rate of platelets compared to platelets frozen using the standard protocol (p < .001). However, this had no impact on the recovery of the platelets (p = .87) or the proportion of platelets expressing GPIbα (p = .46) or GPVI (p = .07), which remained similar between groups. A lower proportion of platelets frozen in tubes externalized phosphatidylserine compared to standard CPPs (p < .001). The clot-forming ability (thromboelastography) of platelets was similar between groups (p > .05). CONCLUSION: Freezing platelets in tubes modified the freezing rate and altered some platelet characteristics. However, the functional characteristics remained comparable, demonstrating the feasibility of downscaling platelet cryopreservation for high-throughput exploratory investigations.

Regulatory RNAs in bacteria.

Bacteria possess numerous and diverse means of gene regulation using RNA molecules, including mRNA leaders that affect expression in cis, small RNAs that bind to proteins or base pair with target RNAs, and CRISPR RNAs that inhibit the uptake of foreign DNA. Although examples of RNA regulators have been known for decades in bacteria, we are only now coming to a full appreciation of their importance and prevalence. Here, we review the known mechanisms and roles of regulatory RNAs, highlight emerging themes, and discuss remaining questions.

The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element.

The highly structured, cis-encoded RNA elements known as riboswitches modify gene expression upon binding a wide range of molecules. The yybP-ykoY motif was one of the most broadly distributed and numerous bacterial riboswitches for which the cognate ligand was unknown. Using a combination of in vivo reporter and in vitro expression assays, equilibrium dialysis, and northern analysis, we show that the yybP-ykoY motif responds directly to manganese ions in both Escherichia coli and Bacillus subtilis. The identification of the yybP-ykoY motif as a manganese ion sensor suggests that the genes that are preceded by this motif and encode a diverse set of poorly characterized membrane proteins have roles in metal homeostasis.

The in vitro quality of X-irradiated platelet components in PAS-E is equivalent to gamma-irradiated components.

BACKGROUND: Blood components are irradiated to inactivate lymphocytes in an effort to prevent transfusion-associated graft versus host disease. Although gamma irradiators are commonly used, they are subjected to rigorous health, safety, and compliance regulations, compared with X-irradiators which have the advantage of only emitting radiation while the machine is switched on. While the effects of gamma irradiation on platelet components are well known, there is little or no data comparing the effects of X- and gamma-irradiation on the quality of these components. Therefore, this study examined the in vitro quality of platelet components (pooled and apheresis) following X- or gamma-irradiation. STUDY DESIGN AND METHODS: Whole-blood-derived (pooled) and apheresis platelet components in platelet additive solution (n = 20 pairs for each type) were irradiated (X vs. gamma). In vitro platelet quality was tested prior to irradiation (day 1) and subsequently on days 2, 5, and 7. Non-irradiated components were tested on day 5 in parallel as reference controls. Metabolic parameters, surface expression of glycoproteins and activation markers (CD62P and annexin-V binding), and agonist-induced aggregation were measured. RESULTS: All components met Council of Europe specifications. There were no statistical differences in any in vitro quality measurements between X- and gamma-irradiated pooled or apheresis platelet components. CONCLUSION: X- and gamma-irradiation have similar effects on the in vitro quality of stored blood components, indicating that either technology represents a suitable option for irradiation of platelet components.

Calcium chelation: a novel approach to reduce cryopreservation-induced damage to frozen platelets.

BACKGROUND: Cryopreserved platelets are phenotypically and functionally different to conventionally stored platelets. Calcium may be released from internal stores during the freeze-thaw process, initiating signaling events which lead to these alterations. It was hypothesized that the addition of a calcium chelator prior to cryopreservation may mitigate some of these changes. METHODS: Buffy coat-derived platelets that had been pooled and split were tested fresh and following cryopreservation (n = 8 per group). Platelets were cryopreserved using 5%-6% dimethylsulfoxide (DMSO) or were supplemented with increasing concentrations of the internal calcium chelator, BAPTA-AM (100 µM, 200 µM, or 400 µM), prior to storage at -80°C. RESULTS: Supplementation of platelets with BAPTA-AM prior to freezing improved platelet recovery in a dose response manner (400 µM: 84 ± 2%) compared to standard DMSO cryopreserved platelets (70 ± 4%). There was a loss of GPIbα, GPVI, and GPIIb/IIIa receptors on platelets following cryopreservation, which was rescued when platelets were supplemented with BAPTA-AM (400 µM: p < 0.0001 for all). Platelet activation markers, such as phosphatidylserine and P-selectin, were externalized on platelets following cryopreservation. However, the addition of BAPTA-AM significantly reduced the increase of these activation markers on cryopreserved platelets (400 µM: p < 0.0001 for both). Both cryopreserved platelet groups exhibited similar functionality as assessed by thromboelastography, forming clots at a faster rate than fresh platelets. CONCLUSIONS: This study demonstrates that calcium plays a crucial role in mediating cryopreservation-induced damage to frozen platelets. The addition of the calcium chelator, BAPTA-AM, prior to cryopreservation reduces this damage.

Freezing expired platelets does not compromise in vitro quality: An opportunity to maximize inventory potential.

BACKGROUND AND OBJECTIVES: Cryopreservation provides an option for long-term storage of platelet concentrates. While platelets are usually frozen as soon as practical after collection (within 2 days), the ability to freeze units at a later stage of the shelf life may improve inventory management. As such, the aim of this study was to determine the impact of freezing platelets approaching expiry (Day 5/6). MATERIALS AND METHODS: Two ABO-matched buffy coat-derived platelets (30% plasma/70% platelet additive solution) were pooled and split to produce matched pairs (n = 8 pairs). Platelets were frozen on Day 1 after collection (cryopreserved platelets [CPPs]) or Day 5 or 6 (expired-CPPs) at -80°C with 5% to 6% dimethyl sulfoxide. In vitro platelet quality was tested before freezing and after thawing and reconstitution in plasma. RESULTS: The majority of prefreeze parameters were equivalent for all platelet units (Day 1 vs. Day 5 or 6). Expired-CPPs had a higher mean postthaw platelet recovery (82 ± 4%) compared to CPPs (75 ± 4%; p = 0.0021). Cryopreservation resulted in a loss of surface glycoproteins (glycoprotein (GP) Ibα, GPIIb, GPVI), an increase in activation markers (phosphatidylserine and P-selectin) and microparticle release, compared to unfrozen platelets. However, the cryopreservation-induced changes were equivalent in CPPs and expired-CPPs. Functionality was measured by thromboelastography and was similar between expired-CPPs (R-time: 5.3 ± 0.3) and CPPs (R-time: 5.4 ± 0.5; p = 0.7094). CONCLUSION: The phenotype and functional profile of platelets frozen at expiry were similar to platelets frozen 1 day following collection. These data suggest that expired platelets may represent a suitable starting material for cryopreservation.

Characterizing the ability of an ice recrystallization inhibitor to improve platelet cryopreservation.

Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5-6% dimethyl sulfoxide (Me2SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at -80 °C. Alternatively, platelets were frozen with 5-6% Me2SO at -30 °C or with 3% Me2SO at -80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me2SO (p = 0.0461) but not at -30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me2SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at -30 °C or with 3% Me2SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at -30 °C or with 3% Me2SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage.

Structure-function analysis of manganese exporter proteins across bacteria.

Manganese (Mn) is an essential trace nutrient for organisms because of its role in cofactoring enzymes and providing protection against reactive oxygen species (ROS). Many bacteria require manganese to form pathogenic or symbiotic interactions with eukaryotic host cells. However, excess manganese is toxic, requiring cells to have manganese export mechanisms. Bacteria are currently known to possess two widely distributed classes of manganese export proteins, MntP and MntE, but other types of transporters likely exist. Moreover, the structure and function of MntP is not well understood. Here, we characterized the role of three structurally related proteins known or predicted to be involved in manganese transport in bacteria from the MntP, UPF0016, and TerC families. These studies used computational analysis to analyze phylogeny and structure, physiological assays to test sensitivity to high levels of manganese and ROS, and inductively coupled plasma-mass spectrometry (ICP-MS) to measure metal levels. We found that MntP alters cellular resistance to ROS. Moreover, we used extensive computational analyses and phenotypic assays to identify amino acids required for MntP activity. These negatively charged residues likely serve to directly bind manganese and transport it from the cytoplasm through the membrane. We further characterized two other potential manganese transporters associated with a Mn-sensing riboswitch and found that the UPF0016 family of proteins has manganese export activity. We provide here the first phenotypic and biochemical evidence for the role of Alx, a member of the TerC family, in manganese homeostasis. It does not appear to export manganese, but rather it intriguingly facilitates an increase in intracellular manganese concentration. These findings expand the available knowledge about the identity and mechanisms of manganese homeostasis proteins across bacteria and show that proximity to a Mn-responsive riboswitch can be used to identify new components of the manganese homeostasis machinery.

Cryopreservation of UVC pathogen-inactivated platelets.

BACKGROUND: Extending the platelet (PLT) shelf life and enhancing product safety may be achieved by combining cryopreservation and pathogen inactivation (PI). Although studied individually, limited investigations into combining these treatments has been performed. The aim of this study was to investigate the effect of PI treating PLTs before cryopreservation on in vitro PLT quality and function. STUDY DESIGN AND METHODS: ABO-matched buffy coat-derived PLTs in PLT additive solution (SSP+; Macopharma) were pooled and split to form matched pairs (n = 8). One unit remained untreated and the other was treated with the THERAFLEX UV-Platelets System (UVC; Macopharma). For cryopreservation, 5% to 6% dimethyl sulfoxide was added to the PLTs, and they were frozen at -80°C. After being thawed, untreated cryopreserved PLTs (CPPs) and UVC-treated CPPs (UVC-CPPs) were resuspended in plasma. In vitro quality was assessed immediately after thawing and after 24 hours of room temperature storage. RESULTS: UVC-CPPs had lower in vitro recovery compared to CPPs. By flow cytometry, PLTs demonstrated a similar abundance of GPIX (CD42a), GPIIb (CD41a), and GPIbα (CD42b-HIP1), while the activation of GPIIb/IIIa (PAC-1) was increased in UVC-CPPs compared to CPPs. UVC-CPPs demonstrated greater phosphatidylserine exposure (annexin V) and microparticle shedding but similar P-selectin (CD62P) abundance compared to CPPs. UVC-CPPs displayed similar functionality to CPPs when assessed using aggregometry, thromboelastography, and thrombin generation. CONCLUSIONS: This study demonstrates the feasibility of cryopreserving UVC-PI-treated PLT products. UVC-PI treatment may increase the susceptibility of PLTs to damage caused during cryopreservation, but this is more pronounced during postthaw storage at room temperature.

The impact of refrigerated storage of UVC pathogen inactivated platelet concentrates on in vitro platelet quality parameters.

BACKGROUND AND OBJECTIVES: Refrigeration (cold-storage) of pathogen inactivated (PI) platelet components may increase the shelf-life and safety profile of platelet components, compared to conventional room-temperature (RT) storage. Whilst there is substantial knowledge regarding the impact of these individual treatments on platelets, the combined effect has not been assessed. MATERIALS AND METHODS: Using a pool-and-split study design, paired buffy-coat derived platelets in 70% platelet additive solution (SSP+; MacoPharma) were left untreated or PI-treated using the THERAFLEX UV-Platelets System (UVC; MacoPharma). Units from each pair were split and stored at room temperature (20-24°C) or cold-stored (2-6°C) to yield RT, cold, RT-UVC and cold-UVC study groups (n = 8 in each group). In vitro quality and function was tested over 9 days. RESULTS: Cold-storage of UVC-treated platelets reduced glycolytic metabolism (glucose consumption and lactate production) compared to RT-UVC units. Cold-UVC platelets demonstrated complete abrogation of HSR by day 5, increased externalisation of phosphatidylserine (annexin-V binding) and activation of the GPIIb/IIIa receptor (PAC-1 binding) above the levels observed with the individual treatments. Aggregation responses (ADP and collagen) were enhanced in the cold-UVC platelets compared to both RT groups, but this was primarily mediated by cold-storage. Haemostatic function, as measured using TEG, was similar between the groups. CONCLUSION: Cold-storage of UVC-treated platelets reduced PI-induced acceleration of glycolytic metabolism. However, combining cold-storage and UVC-treatment resulted in additional phenotypic changes compared to each treatment individually. Further work is required to understand the impact of these changes in clinical efficacy.

The Escherichia coli small protein MntS and exporter MntP optimize the intracellular concentration of manganese.

Escherichia coli does not routinely import manganese, but it will do so when iron is unavailable, so that manganese can substitute for iron as an enzyme cofactor. When intracellular manganese levels are low, the cell induces the MntH manganese importer plus MntS, a small protein of unknown function; when manganese levels are high, the cell induces the MntP manganese exporter and reduces expression of MntH and MntS. The role of MntS has not been clear. Previous work showed that forced MntS synthesis under manganese-rich conditions caused bacteriostasis. Here we find that when manganese is scarce, MntS helps manganese to activate a variety of enzymes. Its overproduction under manganese-rich conditions caused manganese to accumulate to very high levels inside the cell; simultaneously, iron levels dropped precipitously, apparently because manganese-bound Fur blocked the production of iron importers. Under these conditions, heme synthesis stopped, ultimately depleting cytochrome oxidase activity and causing the failure of aerobic metabolism. Protoporphyrin IX accumulated, indicating that the combination of excess manganese and iron deficiency had stalled ferrochelatase. The same chain of events occurred when mutants lacking MntP, the manganese exporter, were exposed to manganese. Genetic analysis suggested the possibility that MntS exerts this effect by inhibiting MntP. We discuss a model wherein during transitions between low- and high-manganese environments E. coli uses MntP to compensate for MntH overactivity, and MntS to compensate for MntP overactivity.

Cryopreserved platelets demonstrate reduced activation responses and impaired signaling after agonist stimulation.

BACKGROUND: Room temperature-stored (20-24°C) platelets (PLTs) have a shelf life of 5 days, making it logistically challenging to supply remote medical centers with PLT products. Cryopreservation of PLTs in dimethyl sulfoxide (DMSO) and storage at -80°C enables an extended shelf life up to 2 years. Although cryopreserved PLTs have been widely characterized under resting conditions, their ability to undergo agonist-induced activation is yet to be fully explored. STUDY DESIGN AND METHODS: Buffy coat PLTs were cryopreserved at -80°C with 5% to 6% DMSO and sampled before freezing and after thawing. PLTs were analyzed under resting conditions and after agonist stimulation with adenosine diphosphate, collagen, or thrombin receptor-activating peptide-6. The expression of activation markers, microparticle formation, and calcium mobilization were analyzed by flow cytometry. Soluble PLT proteins present in the PLT supernatant were examined by enzyme-linked immunosorbent assay. Protein phosphorylation was investigated with Western blotting. RESULTS: After cryopreservation, PLTs displayed increased surface activation markers and higher basal calcium levels. Cryopreserved PLTs demonstrated diminished aggregation responses. Additionally, cryopreserved PLTs showed a limited ability to become activated (as measured by CD62P and phosphatidylserine exposure and cytokine release) after agonist stimulation. A reduction in the abundance and phosphorylation of key signaling proteins (Akt, Src, Lyn, ERK, and p38) was seen in cryopreserved PLTs. CONCLUSIONS: Cryopreservation of PLTs induces dramatic changes to the basal PLT phenotype and renders them largely nonresponsive to agonist stimulation, likely due to the alterations in signal transduction. Therefore, further efforts are required to understand how cryopreserved PLTs achieve their hemostatic effect once transfused.

Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry.

Cryopreservation of platelets, at - 80 °C with 5-6% DMSO, results in externalisation of phosphatidylserine and the formation of extracellular vesicles (EVs), which may mediate their procoagulant function. The phenotypic features of procoagulant platelets overlap with other platelet subpopulations. The aim of this study was to define the phenotype of in vitro generated platelet subpopulations, and subsequently identify the subpopulations present in cryopreserved components. Fresh platelet components (n = 6 in each group) were either unstimulated as a source of resting platelets; or stimulated with thrombin and collagen to generate a mixture of aggregatory and procoagulant platelets; calcium ionophore (A23187) to generate procoagulant platelets; or ABT-737 to generate apoptotic platelets. Platelet components (n = 6) were cryopreserved with DMSO, thawed and resuspended in a unit of thawed plasma. Multi-colour panels of fluorescent antibodies and dyes were used to identify the features of subpopulations by imaging flow cytometry. A combination of annexin-V (AnnV), CD42b, and either PAC1 or CD62P was able to distinguish the four subpopulations. Cryopreserved platelets contained procoagulant platelets (AnnV+/PAC1-/CD42b+/CD62P+) and a novel population (AnnV+/PAC1-/CD42b+/CD62P-) that did not align with the phenotype of aggregatory (AnnV-/PAC1+/CD42b+/CD62P+) or apoptotic (AnnV+/PAC1-/CD42b-/CD62P-) subpopulations. These data suggests that the enhanced haemostatic potential of cryopreserved platelets may be due to the cryo-induced development of procoagulant platelets, and that additional subpopulations may exist.

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli.

Small proteins (< 50 amino acids) are emerging as ubiquitous and important regulators in organisms ranging from bacteria to humans, where they commonly bind to and regulate larger proteins during stress responses. However, fundamental aspects of small proteins, such as their molecular mechanism of action, downregulation after they are no longer needed, and their evolutionary provenance are poorly understood. Here we show that the MntS small protein involved in manganese (Mn) homeostasis binds and inhibits the MntP Mn transporter. Mn is crucial for bacterial survival in stressful environments, but is toxic in excess. Thus, Mn transport is tightly controlled at multiple levels to maintain optimal Mn levels. The small protein MntS adds a new level of regulation for Mn transporters, beyond the known transcriptional and post-transcriptional control. We also found that MntS binds to itself in the presence of Mn, providing a possible mechanism of downregulating MntS activity to terminate its inhibition of MntP Mn export. MntS is homologous to the signal peptide of SitA, the periplasmic metal-binding subunit of a Mn importer. Remarkably, the homologous signal peptide regions can substitute for MntS, demonstrating a functional relationship between MntS and these signal peptides. Conserved gene-neighborhoods support that MntS evolved from an ancestral SitA, acquiring a life of its own with a distinct function in Mn homeostasis. Significance: This study demonstrates that the MntS small protein binds and inhibits the MntP Mn exporter, adding another layer to the complex regulation of Mn homeostasis. MntS also interacts with itself in cells with Mn, which could prevent it from regulating MntP. We propose that MntS and other small proteins might sense environmental signals and shut off their own regulation via binding to ligands (e.g., metals) or other proteins. We also provide evidence that MntS evolved from the signal peptide region of the Mn importer, SitA. Homologous SitA signal peptides can recapitulate MntS activities, showing that they have a second function beyond protein secretion. Overall, we establish that small proteins can emerge and develop novel functionalities from gene remnants.

Weight change in control group participants in behavioural weight loss interventions: a systematic review and meta-regression study.

BACKGROUND: Unanticipated control group improvements have been observed in intervention trials targeting various health behaviours. This phenomenon has not been studied in the context of behavioural weight loss intervention trials. The purpose of this study is to conduct a systematic review and meta-regression of behavioural weight loss interventions to quantify control group weight change, and relate the size of this effect to specific trial and sample characteristics. METHODS: Database searches identified reports of intervention trials meeting the inclusion criteria. Data on control group weight change and possible explanatory factors were abstracted and analysed descriptively and quantitatively. RESULTS: 85 trials were reviewed and 72 were included in the meta-regression. While there was no change in control group weight, control groups receiving usual care lost 1 kg more than control groups that received no intervention, beyond measurement. CONCLUSIONS: There are several possible explanations why control group changes occur in intervention trials targeting other behaviours, but not for weight loss. Control group participation may prevent weight gain, although more research is needed to confirm this hypothesis.

Regulation of Bacterial Manganese Homeostasis and Usage During Stress Responses and Pathogenesis.

Manganese (Mn) plays a multifaceted role in the survival of pathogenic and symbiotic bacteria in eukaryotic hosts, and it is also important for free-living bacteria to grow in stressful environments. Previous research has uncovered components of the bacterial Mn homeostasis systems that control intracellular Mn levels, many of which are important for virulence. Multiple studies have also identified proteins that use Mn once it is inside the cell, including Mn-specific enzymes and enzymes transiently loaded with Mn for protection during oxidative stress. Emerging evidence continues to reveal proteins involved in maintaining Mn homeostasis, as well as enzymes that can bind Mn. For some of these enzymes, Mn serves as an essential cofactor. For other enzymes, mismetallation with Mn can lead to inactivation or poor activity. Some enzymes may even potentially be regulated by differential metallation with Mn or zinc (Zn). This review focuses on new developments in regulatory mechanisms that affect Mn homeostasis and usage, additional players in Mn import that increase bacterial survival during pathogenesis, and the interplay between Mn and other metals during Mn-responsive physiological processes. Lastly, we highlight lessons learned from fundamental research that are now being applied to bacterial interactions within larger microbial communities or eukaryotic hosts.

The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis.

Manganese is a critical micronutrient for cells, serving as an enzyme cofactor and protecting against oxidative stress. Yet, manganese is toxic in excess and little is known about its distribution in cells. Bacteria control intracellular manganese levels by the transcription regulator MntR. When this work began, the only Escherichia coli K-12 gene known to respond to manganese via MntR repression was mntH, which encodes a manganese importer. We show that mntS (formerly the small RNA gene rybA) is repressed by manganese through MntR and encodes an unannotated 42-amino-acid protein. Overproduction of MntS causes manganese sensitivity, while a lack of MntS perturbs proper manganese-dependent repression of mntH. We also provide evidence that mntP (formerly yebN), which encodes a putative efflux pump, is positively regulated by MntR. Deletion of mntP leads to profound manganese sensitivity and to elevated intracellular manganese levels. This work thus defines two new proteins involved in manganese homeostasis and suggests mechanisms for their action.

The impact of behavioural screening on intervention outcomes in a randomised, controlled multiple behaviour intervention trial.

BACKGROUND: With an increasing research focus on multiple health behaviour change interventions, a methodological issue requiring further investigation is whether or not to employ pre-trial behavioural screening to exclude participants who are achieving a pre-specified level of one or more behaviours. Behavioural screening can be used to direct limited resources to participants most in need of a behaviour change intervention; but may reduce the representativeness of the sample and limit comparability with trials that do not employ pre-trial behavioural screening. Furthermore, the impact of this type of screening on intervention participation and intervention effects is unknown. METHODS: Data for this study come from the Logan Healthy Living Program, a randomised, controlled telephone counselling lifestyle intervention trial which did not employ behavioural screening prior to randomisation. Screening for physical activity, diet or the combination was simulated using baseline trial data. To examine the impact of behavioural screening on intervention participation (in terms of participant characteristics, intervention dose received and retention), characteristics of participants included an excluded under the various screening scenarios were compared. To examine the impact of behavioural screening on intervention effects, results from the main trial analysis were compared with results obtained from the same analyses performed separately for each of the screened groups. RESULTS: Simulated pre-trial behavioural screening impacted minimally on intervention dose received and trial retention rate. Beyond the anticipated effect of reducing baseline levels of the behaviours being screened for, behavioural screening affected baseline levels of behaviours not targeted by screening, and participants' demographic and health-related characteristics. Behavioural screening impacted on intervention effects in ways that were anticipated and positive, but also unexpected and detrimental. Physical activity screening (alone or in combination with diet) resulted in improved intervention effects for physical activity, while fruit and vegetable screening had no impact on intervention effects for these outcomes. All three types of screening impacted detrimentally on intervention effects for behaviours not being targeted by screening. CONCLUSIONS: Behavioural screening may have desirable and undesirable consequences in the context of multiple behaviour intervention trials, and thus its potential merits and pitfalls should be carefully considered.