The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (Rel Ef ) and the small alarmone synthetase (SAS) RelQ Ef . Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQ Ef synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQ Ef also efficiently utilized GMP to form GMP 3=-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQ Ef was activated only by ppGpp. Furthermore, enzymatic activity of RelQ Ef is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCEAccumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQ Ef ), we found that, in addition to (p)ppGpp, RelQ Ef is an efficient producer of pGpp (GMP 3=-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQ Ef and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones. In order to survive under adverse environmental conditions, such as nutrient starvation, bacteria have evolved complex, interconnected regulatory networks that sense and integrate internal and external metabolic cues to activate cellular responses that enhance bacterial survival. One critical and highly conserved mechanism employed by bacteria to cope with nutritional as well as a variety of environmental and chemical stresses is the stringent response (SR). The SR is mediated by the accumulation of two guanine analog...

The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5′-monophosphate-3′-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species. Both ppGpp and pppGpp interact with proteins involved in inhibition of purine nucleotide biosynthesis and with GTPases that control ribosome assembly or activity. By contrast, pGpp interacts with purine biosynthesis proteins but not with the GTPases. In addition, we show that hydrolase NahA (also known as YvcI) efficiently produces pGpp by hydrolyzing (p)ppGpp, thus modulating alarmone composition and function. Deletion of nahA leads to reduction of pGpp levels, increased (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive fitness. Our results support the existence and physiological relevance of pGpp as a third alarmone, with functions that can be distinct from those of (p)ppGpp.

Breast cancer survivors may experience long-term treatment complications, must live with the risk of cancer recurrence, and often experience psychosocial complications that require supportive care services. In low- and middle-income settings, supportive care services are frequently limited, and program development for survivorship care and long-term follow-up has not been well addressed. As part of the 5th Breast Health Global Initiative (BHGI) Global Summit, an expert panel identified nine key resources recommended for appropriate survivorship care, and developed resource-stratified recommendations to illustrate how health systems can provide supportive care services for breast cancer survivors after curative treatment, using available resources. Key recommendations include health professional education that focuses on the management of physical and psychosocial long-term treatment complications. Patient education can help survivors transition from a provider-intense cancer treatment program to a post-treatment provider partnership and self-management program, and should include: education on recognizing disease recurrence or metastases; management of treatment-related sequelae, and psychosocial complications; and the importance of maintaining a healthy lifestyle. Increasing community awareness of survivorship issues was also identified as an important part of supportive care programs. Other recommendations include screening and management of psychosocial distress; management of long-term treatment-related complications including lymphedema, fatigue, insomnia, pain, and women's health issues; and monitoring survivors for recurrences or development of second primary malignancies. Where possible, breast cancer survivors should implement healthy lifestyle modifications, including physical activity, and maintain a healthy weight. Health professionals should provide well-documented patient care records that can follow a patient as they transition from active treatment to follow-up care.

BackgroundHealth has improved markedly in Mesoamerica, the region consisting of southern Mexico and Central America, over the past decade. Despite this progress, there remain substantial inequalities in health outcomes, access, and quality of medical care between and within countries. Poor, indigenous, and rural populations have considerably worse health indicators than national or regional averages. In an effort to address these health inequalities, the Salud Mesoamérica 2015 Initiative (SM2015), a results-based financing initiative, was established.MethodsFor each of the eight participating countries, health targets were set to measure the progress of improvements in maternal and child health produced by the Initiative. To establish a baseline, we conducted censuses of 90,000 households, completed 20,225 household interviews, and surveyed 479 health facilities in the poorest areas of Mesoamerica. Pairing health facility and household surveys allows us to link barriers to care and health outcomes with health system infrastructure components and quality of health services.ResultsIndicators varied significantly within and between countries. Anemia was most prevalent in Panama and least prevalent in Honduras. Anemia varied by age, with the highest levels observed among children aged 0 to 11 months in all settings. Belize had the highest proportion of institutional deliveries (99%), while Guatemala had the lowest (24%). The proportion of women with four antenatal care visits with a skilled attendant was highest in El Salvador (90%) and the lowest in Guatemala (20%). Availability of contraceptives also varied. The availability of condoms ranged from 83% in Nicaragua to 97% in Honduras. Oral contraceptive pills and injectable contraceptives were available in just 75% of facilities in Panama. IUDs were observed in only 21.5% of facilities surveyed in El Salvador.ConclusionsThese data provide a baseline of much-needed information for evidence-based action on health throughout Mesoamerica. Our baseline estimates reflect large disparities in health indicators within and between countries and will facilitate the evaluation of interventions and investments deployed in the region over the next three to five years. SM2015’s innovative monitoring and evaluation framework will allow health officials with limited resources to identify and target areas of greatest need.Electronic supplementary materialThe online version of this article (doi:10.1186/s12963-015-0034-4) contains supplementary material, which is available to authorized users.

The alarmone (p)ppGpp regulates diverse targets, yet its target specificity and evolution remain poorly understood. Here, we elucidate the mechanism by which basal (p)ppGpp inhibits the purine salvage enzyme HPRT by sharing a conserved motif with its substrate PRPP. Intriguingly, HPRT regulation by (p)ppGpp varies across organisms and correlates with HPRT oligomeric forms. (p)ppGpp-sensitive HPRT exists as a PRPP-bound dimer or an apo- and (p)ppGpp-bound tetramer, where a dimer-dimer interface triggers allosteric structural rearrangements to enhance (p)ppGpp inhibition. Loss of this oligomeric interface results in weakened (p)ppGpp regulation. Our results reveal an evolutionary principle whereby protein oligomerization allows evolutionary change to accumulate away from a conserved binding pocket to allosterically alter specificity of ligand interaction. This principle also explains how another (p)ppGpp target GMK is variably regulated across species. Since most ligands bind near protein interfaces, we propose that this principle extends to many other protein–ligand interactions.

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Bacterial stress-signaling alarmones are important components of a protective network against diverse stresses such as nutrient starvation and antibiotic assault. pppGpp and ppGpp, collectively (p)ppGpp, have well-documented regulatory roles in gene expression and protein translation. Recent work has highlighted another key function of (p)ppGpp: inducing rapid and coordinated changes in cellular metabolism by regulating enzymatic activities, especially those involved in purine nucleotide synthesis. Failure of metabolic regulation by (p)ppGpp results in the loss of coordination between metabolic and macromolecular processes, leading to cellular toxicity. In this review, we document how (p)ppGpp and newly characterized nucleotides pGpp and (p)ppApp directly regulate these enzymatic targets for metabolic remodeling. We examine targets’ common determinants for alarmone interaction as well as their evolutionary diversification. We highlight classical and emerging themes in nucleotide signaling, including oligomerization and allostery along with metabolic interconversion and crosstalk, illustrating how they allow optimized bacterial adaptation to their environmental niches.

The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacterial species. Xanthine phosphoribosyltransferase (XPRT) is a purine salvage 3 enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical and genetic analyses, we show that pppGpp and ppGpp, as well as a third putative alarmone pGpp, all directly interact with XPRT and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine salvage enzyme HPRT, suggesting evolutionary conservation in different enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. This results in two distinct regulatory features. First, XPRT cooperatively binds (p)ppGpp with a Hill coefficient of 2. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in Bacillus subtilis, suggesting that regulation of XPRT is key for regulating the purine salvage pathway.