Numidia meleagris) production as an alternative source of meat and poultry has shown potential for economic viability. However, there has been little progress in characterizing the transcriptome of the guinea fowl. In this study RNA-sequencing and de novo transcriptome assembly of several Guinea fowl tissues  which play key roles in regulating feed intake, satiety, and immune function was performed using Illumina’s Hi-Seq 2000.Guinea fowl  contains supplementary material, which is available to authorized users. Population projections and high production costs necessitate research to identify and develop alternative sources of meat and poultry. The guinea fowl (GF) is a provocative poultry alternative due to its superior nutritional value and economic potential . Over thTranscriptome analysis of the guinea fowl will provide fundamental data needed to develop species-specific management tools, such as feed and disease prevention regimens. The coupled selection of metabolic and immune function traits is of the utmost importance, as continual selection for single metabolic or growth traits have had a detrimental effect on immune function in poultry . At firsAttaining these goals requires a wealth of genetic information and an in-depth understanding of the role played by each gene involved in the regulation of metabolism, satiety, feed utilization, conversion and metabolism, and immune function. De-novo sequencing and transcriptome assembly of the guinea fowl pancreas, hypothalamus, liver, bone marrow and bursa was performed as a first step to developing this required pool of data unique to the GF. Such information is essential in revealing new metabolic pathways that may be utilized to improve growth and production performance of both traditional and non-traditional poultry such as chickens and guinea fowl, respectively.(i) perform targeted de novo assembly of the guinea fowl transcriptome of the pancreas, liver, hypothalamus, spleen, bursa and bone marrow; (ii) to compile a database of functional annotations for the assembled guinea fowl transcriptome; (iii) to perform comparative analysis of the assembled guinea fowl transcriptome using chicken and turkey protein databases; (iv) to identify guinea fowl transcripts with metabolic and immune function.The aim of this study was to In this study we set out to develop a transcriptome library that would reveal unique gene sequences to aid the understanding of key and unique metabolic and immune processes in the guinea fowl. Application of the data generated in this study will serve to improve the production performance of guinea fowl and other related avian species.As mentioned above, the original 74 million Illumina reads (4.9GB of raw data) was reduced to approximately 53 million reads through trimming and filtering. These remaining reads were assembled into 96,491 contigs  hits after blastp and BlastX searches against the SwissProt section of UniProt and the entire Chicken and Turkey proteomes.A total of 47,079 contigs assembled by Trinity returned statistically significant  limit of 0.01  and turkey  . Following sacrifice by cervical dislocation, liver (approximately 5 g from the mid-portion of the anterior sub-segment of the right lobe), pancreas , tibial bone marrow, and bursa (whole organ) were removed and submerged in an RNA stabilization solution (pH 5.2) containing 0.5 M EDTA, 1 M sodium citrate, and 700 g ammonium sulfate dissolved in ultrapure water overnight at 4°C. Whole heads were flash-frozen in liquid nitrogen. Subsequently, hypothalami were excised by micro-dissection and submerged in an RNA stabilization solution. All tissue samples were stored at −80°C until use. Total RNA was isolated from each tissue using Qiagen’s RNeasy® Mini Kit according to the manufacturer’s protocol.  Total RNA concentrations were determined via NanodropTM Spectrophotometer . Each sample was diluted to 50 ng/μl, separated into 50 μl aliquots and immediately frozen at −80°C. Sample quality was evaluated by visual inspection of a 1% formaldehyde gel ran at 100 volts for 1 hour. Gel images were captured using the Kodak Gel Logic 1500 Imaging System . Experion™ Automated Electrophoresis System  was used to confirm RNA quality according to the manufacture’s guidelines. Sample quality was also confirmed using a BioAnalyzer . The resulting RIN values for the hypothalamus, pancreas, liver and bursa/bone marrow samples were 9.5, 7.9, 5.3 and 5.5 respectively.The pancreas, liver, hypothalamus, bone marrow and bursa were harvested from an eight week old male guinea fowl which was housed under a 12-hour light/dark cycle and fed a diet comprising of 3,340 kcal of metabolizable energy/kg of diet and 23% crude protein. Feed and water were provided for st then 2nd strand cDNA synthesis. The cDNA was end-repaired, size selected and then ligated to adapter sequences. The cDNA libraries were multiplexed and sequenced in one lane using Illumina’s Hi-Seq 2000  single end read sequencing platform. The sequencing run produced approximately 74 million single end reads with average length of 101 bp. The resulting reads were de-multiplex and reported as separate runs and deposited in the National Institutes of Health (NIH) Short Read Archive (http://ncbi.nlm.nih.gov/sra) .The cDNA library construction was conducted at the Vanderbilt University’s Genomic Sciences Resource Center (VUGSR), Nashville, TN (VUGSR). During library construction, mRNA was isolated from 100 ng of total RNA followed by fragmentation, 1Prior to assembly, all reads were run through quality control procedures to ensure that Illumina adapters were removed and that only high quality data was used in the assembly. The FastQC program was used to perform an examination of the reads. Based on those results, tools in the fastx toolkit were used to remove Illumina adapters, performing end trimming of reads, as well as filtering reads out of the dataset that had average quality values < 30 .Assembly and annotation was performed on Blacklight, a SGI UV 1000 cc-NUMA shared-memory system available to U.S. academic researchers through the NSF XSEDE program (http://trinityrnaseq.sourceforge.net/) using the default settings [Transcripts were assembled de novo using Trinity (r2012-08-14) . The Trinotate suite provides for the functional annotation of de novo assembled transcriptomes and makes use of several annotation techniques including blastp/blastx database searches against reference sequence databases, PFAM domain searches, and various signal predictions. Trinotate integrates this initial annotation information into a relational database that includes reference information from Uniprot, and eggNOG/GO Pathways databases. Due to the modular design of the system and the use of an SQL database, the system was modified to include information contained within the complete proteome sequences of both the chicken and turkey.The transcriptome produced was annotated using Trinotate (r2013-08-26) (available at fastannotator.cgu.edu.tw) was used to analyze the distribution of gene ontology terms, develop graphical representations of the data set and for enzyme identification [The assembled transcripts were also submitted to FastAnnotator for comparative annotation and identification of domains and potential enzyme functions. Fast Annotator (available at fication . Gene OnThe data sets supporting the results of this article are included within the article and its supplemental files.


Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45–150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6–8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for <4 hrs. These results indicate that long exposure of calcium AM dyes to the intracellular cytoplasm did not alter the intracellular calcium concentration, the functional range of the dye or viability of the neurons. This data extends our previous work showing that a custom recovery chamber can extend the viability of neuronal tissue, and reliable data for both electrophysiology and imaging can be obtained >24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals. Calcium is one of the most common second messengers in the brain, playing a key role in a variety of intracellular physiological processes, ranging from cell proliferation  to synapCalcium signals occur within cells  and also between cells . Interce2+ signals can be classified as transient , sustained  or oscillatory signals [d)[Cytosolic Caytoplasm). Functioytoplasm). Due to plasm)[d),8,9. Theplasm)[d).in-vitro dye loading of brain slices, the incubation time of the dye is correlated to the age of the animal [One of the limitations of calcium imaging lies within the calcium indicators, which are unable to cross the lipid membrane easily due to their charged nature. However calcium acetoxymethyl (AM) indicators, which were first introduced by Roger Tsien in the early 80’s  can entee animal , occasioin vivo biology, including functional local intercellular circuitry with preserved brain architecture that allows easy access to the studied cells as well as precise control of the extracellular environment. Moreover, slices from the CNS allow direct visualization of the cellular circuit, which enables researchers to locate, identify and easily access the cells being studied. Additionally, it is possible to locally load cells with the dye, which would otherwise be blocked by the blood brain barrier. Calcium imaging in acute brain slices offers a high degree of temporal and spatial resolution [Acute brain slices conserve various properties of solution  and thersolution .Like brain slices, retinal explants conserve whole tissue networks and are by far the most accessible model for CNS function. However calcium-imaging in the retina has never been a commonly used technique for two main reasons: the exquisite sensitivity of this tissue to visible light, and the difficulty loading retinal neurons with calcium dyes. With new optical breakthroughs such as two-photon microscopy  and the We present evidence that calcium dyes loaded into retinal wholemount and brain slices remains in cells for more than 24 hours. Moreover, we show that measuring neurophysiological properties using calcium imaging is possible even 24 hrs after slicing and loading the cells, as no significant alterations were detected either in the total number of cells that were loaded with the dye (loaded cells) or the number of activated cells (evoked cells). This study demonstrates that the functional activity of neurons in acute brain slices can be maintained for long periods (>24 hrs), once the external environment is highly regulated, and that calcium AM dyes remain stably loaded and functional for extended periods. The costs and time spent on preparing such experiments are high, and the use of the Braincubator provides a way to maximise this significant investment, by at least tripling the experimental time. Moreover, animal numbers can be significantly reduced, providing strong ethical reasons for the adoption of such methodology.rd/rd) mice . All animals were healthy and handled with standard conditions of temperature, humidity, 12hr light/ dark cycle, free access to food and water, and without any intended stress stimuli. All experiments were approved and performed in accordance with the University of Western Sydney Animal Care and Ethics committee and according to the animal use and care guidelines .For this study we used 3–30 weeks old Wister rats, C57BL/6 and C3H/HeJArc  were cut with a vibrating microtome (Leica VT1200S) as previously described [Animals were deeply anesthetized by inhalation of isoflurane (5%), decapitated, and their brains quickly removed and placed into ice-cold physiological solution ) containing (in mM): 125 NaCl, 2.5 KCl, 1 MgCl; pH7.4; ). Parasaescribed ,20 and tescribed . Initial3, 3 KCl, 2 CaCl2, 1 MgCl2,10 Glucose and saturated with carbogen , at roo; pH7.4;  was modiTo extend the viability of acute brain slices, we incubated the tissue in a custom-built recovery incubation system, called the Braincubator, as reported previously . This sy2-5% CO2. Following dye loading, the tissue was washed with aCSF and transferred to the Braincubator until experimental use.Calcium dyes  were dissolved in DMSO (50 μg in 50 μl) and 1% pluronic acid-127 (Molecular Probes), sonicated for 15 min and loaded into the tissue by bath application to a final concentration of 10 μM (brain slices) and 20 μM (retina), as reported by  respectiFor imaging, tissue was placed in a submerged recording chamber under an Olympus BX51W microscope, and perfused with oxygenated aCSF at a flow rate of 4–5 ml/min at room temperature (~22°C). To prevent the tissue from moving in the fluid stream the slices it was held under a ‘‘harp” (made of nylon or gold threads stretched and glued across a U-shaped piece of gold or platinum wire). Image time series were acquired with water-immersion objectives at different rates ranging from 0.3–3 Hz.For ratiometric imaging of Fura-2, the excitation light was filtered through an Ultra High Speed Wavelength Switcher  to provide wavelengths of 340 and 380. Emission light from individual cells was passed through an emission filter of 510 ± 20 nm, and captured by a digital camera (Q-Imaging ROLERA-XR or ANDOR Xion). For a single excitation wavelength of Fluo-4, the excitation light was filtered through a 460–490 nm band pass filter and the emission light passed through a 515–550 nm band pass filter, and captured by a high speed digital camera .ImageJ /FIJI software ((http://fiji.sc/Fiji) [Acquisition protocols consisted of 5–7-minute time-lapse sequences of Fura-2 or Fluo-4 fluorescence. Alterations in fluorescence as a function of time were measured at a single wavelength (Fluo-4) or the wavelength ratio method (Fura-2), as previously described by . All anasc/Fiji) . To visuImageJ—FIJI software (NIH). Stained cells were classified as loaded cells, evoked cells or spontaneously active cells according to the staining pattern. An unpaired two-tailed student t-test was used for comparison between different cell populations or different groups of slices. P-values of less than 0.05 were considered statistically significant.Data is reported as mean ± S.E.M. Analysis of calcium signals was completed using This study aimed to demonstrate the length of time acute brain slices and excised retinae, loaded with calcium indicators, can be maintained to produce reliable and reproducible data about cell function. To increase neuronal tissue viability for more than 24 hours, we incubated the slices in the Braincubator, which provides a highly controlled environment, including pH, temperature, carbogen flow and low bacteria levels as reported previously ,17. To cIn all experiments, the cell bodies of the neurons were monitored individually and simultaneously for their calcium responses. Consistent with previous studies ,28, we hIn brain slices, the average number of cells that were still loaded with the AM dye (Loaded Cells) 24 hrs post loading was comparable to the number of loaded cells in slices that were imaged <4 hrs post loading . This indicates that long term exposure of the intracellular milieu to either fura-2 AM or Fluo-4 AM did not impact the membrane or dye integrity.The average percentage of spontaneously active cells in the field of view was not significantly different between slices incubated for <4 hrs , median and dynamic range of the calcium signals did not change significantly  was comparable between the two groups of slices (27±3% in slices that were imaged <4 hours after slicing (n = 18) and 24±3% in slices that were imaged >24 hours post slicing (dF/F); p>0.5, two-tailed student t-test).To study calcium dynamics following the activation of voltage gated calcium channels, we added a high concentration of KCl to the recording bath . As the ntration , bath apntration , NMDA rentration ). On aveevoked cells that exhibit recovery following short local application of KCl  or glutamate  was comparable between retinae that were measured <4 hrs post slicing (94±3%) and retinae that were incubated for >24 hrs in the Braincubator ; p>0.7), indicating that the dynamic range of the spontaneous calcium signal was not altered following prolonged incubation with the AM dye. Similarly, following KCl application, a fluorescence increase of 0.28±0.01 ; n = 180, >24 hours; p>0.5, n = 2 two-tailed student t-test). These results indicate that calcium dynamics remained intact for a minimum period of 24 hours post slicing.The average frequency of spontaneous calcium events imaged <4 hours post slicing was 1.71±0.2 events per minute , that act together with inositol- 1,4,5-trisphosphate receptor (IP3) to induce further release of Ca2+ from the internal stores. The off mechanisms are activated at high cytoplasmic calcium levels and involve activation of the second messengers system, specifically pumps and exchangers ) to remove calcium ions out of the cytoplasm [Calcium signals, carried by alterations in intracellular calcium concentration, are an essential element in many neurophysiological processes, such as signal transduction, gene expression and neural plasticity , which mectively . The on Calcium concentrations are strongly associated with neuronal electrochemical properties, which impact the calcium influx through transmitter gated  and voltage dependent calcium channels , suggestThe proposed study had two aims: the first was to investigate whether the effectiveness of calcium AM staining persists following long incubation periods post cell loading, and the second aim was to test whether the functional properties of calcium signals in acute brain slices and wholemount retinae altered following extended incubation periods in a tightly controlled environment.Our results indicate that under standard aCSF incubation in the Braincubator at 16°C, both Fura-2 AM and Fluo-4 AM remained within the cells long after loading , cell culture techniques exhibit anatomical reorganisation and altered expression profile of ion channels and receptors, which develop over time (reviewed by ). Hence,Retinal wholemount present an additional challenge for calcium imaging, as the endfeet of Müller cells form a barrier that does not allow the penetration of the dye into the cells. In this paper, we have used a proteolytic enzyme, papain, that digest the membrane endfeet and exposes the cells . This prOne of the advantages of calcium imaging over electrophysiological recording is the spatial resolution. Indeed, voltage sensitive dyes can also support high spatial resolution, however, voltage sensitive dyes are highly toxic to the cells and therefore cannot be incubated for long periods. In this paper we used calcium imaging to show that using highly controlled environment incubation system extends the viability of acute brain slices and retinal wholemount. Moreover, intracellular calcium transients were indistinguishable >24 hours post dissection to freshly prepared tissue, indicating their functional properties are still intact. Furthermore, the long exposure of the AM dye did not alter calcium signals, showing that it was not toxic and still efficient to use. We believe that this method can be applied to many more AM dyes, including selective markers to NO . BeyS1 Video(MP4)Click here for additional data file.S2 Video(MP4)Click here for additional data file.S3 Video(MP4)Click here for additional data file.S4 Video(MP4)Click here for additional data file.


Aims. T2DM often remains undiagnosed for many years because hyperglycemia develops gradually and may not produce any symptoms. As patients with T2DM are at increased risk of microvascular and macrovascular complications, the preclinical diagnosis of the state is the key point of the disease management. Methods. We evaluated parameters such as GAGs/PGs, NAG, and NGAL in urine samples from 43 normoalbuminuric T2DM patients and 31 apparently healthy control subjects. Results. The total urinary GAG excretion showed no significant differences between patients and controls. The electrophoretic analysis evidenced the presence of UTI and its degradation products (LSC and SM-LSC), CS, and HS. We observed modifications of HS and total UTI (including UTI and its degradation products) relative contents in T2DM patients compared with controls whereas no differences in CS percentage were found. NGAL levels were significantly increased in T2DM patients and were positively correlated with both NAG  and the presence of hypertension . Conclusions. These data suggest that the assessed molecules could represent useful markers to detect early renal impairment in patients with T2DM. The chronic noncommunicable diseases represent one of the most difficult challenges for all health care systems, in both industrialized and developing countries, due to their continuous and relentless growth. The most paradigmatic example is certainly represented by diabetes mellitus. Epidemiological evidences suggest that, without effective prevention and control plans, diabetes will likely continue to increase globally . In partThe renal impairment in diabetes mellitus affects ~40% of type 1 and type 2 diabetic patients. Diabetic nephropathy is responsible not only for ESRD, but also for a significant increase in cardiovascular risk in this population. Although CKD is a common comorbidity condition of T2DM, in the early stages it is often unrecognized, especially in the elderly, and, therefore, untreated . BecauseOver the past few years we have described quali-quantitative variations of GAGs/PGs in both normoalbuminuric type 1 and 2 diabetic patients with respect to healthy control subjects, suggesting that both levels and relative abundance of urinary GAGs may be predictive of altered GAG metabolism in diabetes mellitus , 8. FurtIt has been shown that tubular damage occurs early in the course of diabetic nephropathy and that it is not merely secondary to glomerular damage as previously thought . From a Therefore, the aim of the present study is to assess parameters such as urinary GAGs/PGs, NAG, and NGAL to detect alterations of renal function in normoalbuminuric patients with T2DM.43 normoalbuminuric patients with type 2 diabetes mellitus, referred to the Unità Operativa di Diabetologia e Malattie del Ricambio, AOU-Sassari, were enrolled. Exclusion criteria were ketoacidosis, fever, infection, surgery as well as evidences of systemic diseases, renal, cardiac, or hepatic diseases, and malignant tumors. Normoalbuminuria was defined as a urinary albumin excretion rate lower than 30 mg/24 h (mean of three different samples over a period of three months). The mean age of the T2DM patients included (16 men and 27 women) was 64.21 ± 7.18 years and the mean value of known duration of diabetes was 7.02 ± 5.15 years. The patients had no complications at the time of the study, but 27 of them  were hypertensive (systolic blood pressure ≥ 160 mmHg and/or diastolic blood pressure ≥ 90 mmHg) and were under antihypertensive treatment. The control group was composed of 31 apparently healthy subjects matched for age, sex, and BMI with T2DM patients.Informed consent was obtained before enrolment. Institutional Review Board approval was obtained. The study was conducted in accordance with the ethical principles of the current Declaration of Helsinki.Urinary GAG/PG purification and quali-quantitative analysis were performed by a method previously described . BrieflyUrinary NAG was estimated kinetically by using NAG colorimetric kit , whereas NGAL levels were performed with the Lipocalin-2/NGAL Human ELISA Kit . The assays were performed according to the protocols provided by the manufacturers.t-test or the Mann-Whitney rank sum test when data failed the normality test. Association between the examined variables was investigated nonparametrically by Spearman's rank correlation analysis. The statistical significance was set at p < 0.05.Statistical analyses were performed using the software package Sigma Stat 3 (Systat Software). Values are reported as mean (±SD) or median (interquartile range). Differences between groups were assessed by Student's p = 0.201). The electrophoretic profiles of urine GAG/PG samples , whereas the median value of NGAL was significantly increased in T2DM patients with p = 0.025  and the presence of hypertension .The total urinary GAG excretion showed no significant differences between patients and controls (3.757 (2.868–5.234) mg of hexuronate/g creatinine versus 4.40 (3.475–5.635) mg of hexuronate/g creatinine,  samples  showed t = 0.025 . The comThe identification of sensitive markers of renal function continues to be of considerable interest. Although UAE is considered the “gold standard” for diagnosing renal dysfunction, other markers, detectable at an earlier stage of the disease, may have diagnostic and/or prognostic value. Moreover, the association between glomerular lesions and MA is less pronounced in T2DM. In this respect, Fioretto et al.  found stSeveral studies have been performed to elucidate the molecular mechanisms underlying glomerular ultrafiltration, suggesting a role of PGs, especially their carbohydrate moieties, in maintaining the glomerular filtration barrier . In thisThe alteration of tubular reabsorption is found almost always in the early stages of kidney disease, including those characterized by predominant glomerular involvement. In this study, we observed an increase of NGAL in normoalbuminuric diabetic patients. Being an iron transporter, NGAL may be expressed by the damaged renal tubule to induce regeneration, since this element is necessary for reepithelialization. Furthermore, it is known that pathological changes in diabetic nephropathy involve accumulation of extracellular matrix, the degradation of which involves mainly MMPs. The activities of these enzymes are dependent on metal ions and are limited by TIMP-1. In particular, it has been reported that a high glucose concentration causes a reduction in the amount of MMPs secreted by the mesangial cells  and an uIn our study, the urinary levels of NGAL show a highly significant relationship with the urinary NAG levels and a positive relationship with the presence of hypertension. We did not find any relationship among these parameters and serum creatinine levels or eGFR, probably because the increase of serum creatinine may not be evident before 50% or more of the nephrons are damaged.Image analysis of GAG/PG profiles also enabled us to detect a significant increase in the relative content of total UTI in T2DM patients.The role of inflammation in the pathogenesis of type 2 diabetes and associated complications is now well established. Multiple mechanisms underlie defective insulin secretion and responses in type 2 diabetes. These include glucotoxicity, lipotoxicity, oxidative stress, and endoplasmic reticulum stress. Interestingly, all of these mechanisms are associated with inflammatory responses .αI family proteins through proteolytic cleavage by neutrophil elastase in the peripheral circulation or at the inflammation site [During inflammation, UTI is released from Iion site , therefoion site –9, 30.In conclusion, we observed an increased level of NGAL, as well as an altered relative content of HS and total UTI in normoalbuminuric T2DM patients with significant correlations among NGAL levels and both NAG excretion and presence of hypertension. It is likely that, rather than a single biomarker, the identification of a panel of strategically selected analytes may represent a useful tool for the accurate prediction and monitoring of the disease. In this perspective, our results, although preliminary, suggest that the assessed markers could represent good candidates to detect early renal alteration in normoalbuminuric patients with T2DM. Further longitudinal studies with a larger sample size are required to clarify these results.


Persistence of antibiotic resistant bacteria, including multidrug resistant (MDR) pseudomonads, is an important environmental health problem associated with drinking water distribution systems (DWDS) worldwide. There is paucity of data on the molecular characteristics of antibiotic resistance genes and their mode of transfer among pseudomonads from DWDS located in resource-challenged areas such as southwestern Nigeria.MDR pseudomonads (n = 22) were selected from a panel of 296 different strains that were isolated from treated and untreated water in six DWDS located across southwest Nigeria. Primarily, the isolated pseudomonads strains were identified by 16S rDNA sequencing and antibiotic-resistance testing was completed using agar breakpoints assays. The final panel of strains of resistant to more than three classes of antibiotics (i.e. MDR), were further characterized by PCR genotyping, Sanger sequencing, and plasmid profiling.tet(A) (31.8 % of isolates), sul1 (31.8 %), blaTEM (40.9 %) and aph(3″)c (36.4 %). Class 1 integron sequences were evident in 27.3 % of isolates and they harbored genes encoding resistance to aminoglycosides , trimethoprim  and sulphonamide (sul1) while the plasmid ranged between 22 and 130 kb.Pseudomonad resistance to gentamicin and streptomycin ranged from 22.7 to 54.6 % while resistance to tetracycline, ceftiofur and sulphamethoxazole ranged from 40.9 to 77.3 %. The most commonly detected antibiotic resistance genes were Pseudomonas spp, isolated from these DWDS possess resistance genes and factors that are of public and environmental health significance. Therefore, has the potential of contributing to the global scourge of resistance genes transfer in human, animals and environments, thereby, useful in the epidemiology of antimicrobial resistance. Pseudomonas spp, is a ubiquitous and diverse genus of Gram-negative bacteria that can be found in soil, water, decaying vegetation and animals. According to Craun et al.  that cause resistant by producing protein that modifies the ribosome.Similarly, tetracycline resistance gene has also not been described in among Pseudomonads from Nigeria. Although our recent report  stated ti et al.  who repol genera . None ofaadA2, aadA1), trimethoprim  and sulphonamide (sul1) resistance.Occurrence of class 1 integron among these bacteria showed their potential ability to shuttle antibiotic resistance between different bacteria species. This is because integrons are mobile DNA element with the ability to integrate and express gene cassettes by site-specific recombination , 31. IntP. aeroginosa. The relationship between plasmid profiles and MDR patterns observed in this study suggests that plasmids may have great role to play in the multidrug resistance of the Pseudomonas. Plasmids usually evolve as an integral part of the bacterial genome, consisting of several extra-chromosomal traits, one of which is their resistance genes, which can be exchanged among bacteria of different origins by conjugation [The size of plasmids observed from our study contradicts previous studies from Nigeria , 33 whicjugation . Global jugation . TherefoPseudomonas harbouring various antibiotic resistance genes, class 1 integron and plasmid in drinking water systems signify that these environmental pseudomonads could significantly contribute to wide-scale transfer of resistance genes in these environments thereby posing health hazard to man and animals that depend on these water sources. This could play a major role in the epidemiology of antibiotic resistance in Nigeria and could also be responsible for prolong treatment of bacterial infection, the emergence of emerging and re-emerging infectious diseases. We hereby recommend integrated antibiotic resistant surveillance system and holistic one health approach as well as promoting prudent use of antibiotics both in clinical and agriculture settings in order to prevent and control multidrug resistant bacteria in southwestern Nigeria.In summary, the occurrence of MDR


The extracts were partitioned into acidic and neutral polyphenolic fractions and following estimation of total phenolics in the crude extract, acidic and neutral fractions and oil, all were analyzed for their DPPH  scavenging potential, ferric reducing ability and chelating power. The highest amount of polyphenols was found in ajwain (8330 ± 107), then in mustard seeds (2844 ± 56.00) and in fenugreek (1130 ± 29.00), and least in poppy seeds (937 ± 18.52). The higher amounts of polyphenols were estimated in neutral fraction compared to acidic (p < 0.05). % Inhibition of DPPH by the crude extract and fractions of all oilseeds was quite significant, being higher for acidic than neutral. The highest % DPPH inhibition was shown by ajwain extract than mustard > fenugreek and least by poppy seed extracts (p < 0.05). The reducing power and the chelating effect of the oilseeds followed the same order as DPPH, but higher % chelation was shown by neutral than acidic fraction (p < 0.05). Though low in polyphenols, the oil fractions were as strong antioxidants as the acidic one. Though oilseeds are used in very small quantity in food, they are potential sources of natural antioxidants and may replace synthetic ones.The polyphenolic extracts and oils were obtained from ajwain, mustard, fenugreek and poppy seeds The foods containing antimutagenic, antibacterial, antiviral and anti-inflammatory phenolic compounds have increasingly been gaining importance since numerous studies have proved strong protective effects of these novel phytochemicals against many diseases ,2,3,4. PPolyphenolic antioxidants do not only help combat with the reactive oxygen species but also minimize rancidity, reduce the formation of toxic oxidation products, maintain nutritional quality and extend shelf life when added to foods ,15. The in vivo and vitro to deal with oxidation stresses but also quite active in stabilizing the edible oils and fatty food against rancidity and oxidative deterioration [Spices and/or oilseeds are an important category of food that has been used since long to enhance the taste and aroma of foods. Besides imparting characteristic flavor and color to foods, they also produce several medicinal effects and hence are used in several indigenous systems of medicines ,17,18,19ioration .Trachyspermum ammi (ajwain), Brassica alba (mustard/rai), Trigonellafoenum graecum(Feenugreek) and Papaver somniferum (poppy seeds/khashkhash) were extracted for polyphenols and oil using Bligh and Dyer method. Polyphenolic extracts were then partitioned into neutral and acidic polyphenolic fractions. The polyphenolic and oil fractions were then compared for their antioxidant potential to seek the possibility of using them either in medicinal preparations or as preservatives for frying edible oil and high fatty foods.The oilseeds The oilseeds were purchased from the local market, washed to remove extraneous matter, dried in open air turning up and down time to time and then stored in portions in polyethylene bags at room temperature. Lichrolut RP-18 SPE (solid phase extraction) cartridges were supplied by Merck and all other reagents and chemicals were of analytical grade supplied either by Merck or BDH.One kg of each oilseed was ground to coarse size using an electric grinder . In order to avoid losses of heat sensitive antioxidants, and to make the process more feasible, the oil was extracted with chloroform-methanol mixture by Bligh and Dyer method  with the3/MeOH  and homogenized well. Subsequent to this, 1 L of CHCl3 was added, homogenized, 1 L of doubled distilled water was added, homogenized again and then finally centrifuged at 1000 rpm for 5 min at room temperature to give a two-phase system  and CHCl3 (bottom). 95% of the bottom phase were recovered with the assistance of separating funnel while the remaining 5% were collected following addition of 100–200 mL of CHCl3, vigorously shaking and collecting from bottom. Oil fraction was recovered from the chloroform phase by evaporating CHCl3 on rotary evaporator at 40 °C while for polyphenols; methanolic phase was concentrated to remove alcohol and resolubilized in water. To retain the stability of oil portion, moisture was removed by adding anhydrous sodium sulfate. The clear dry oil was then collected by decantation and stored at 4 °C till further study. Yield of oil in percent and total extractable solids from aqueous methanol were calculated for each oilseed.One kg of the ground sample was added to 3 L of CHClPolyphenolic extract of each oilseed was fractionated into acidic and neutral polyphenols by using the procedure of Oszmianski and Lee . Two polet al. [et al. [n-hexane (1:2 w/v). The dissolved oil was mixed with MeOH/water  in the ratio of 1:5 (w/v), vortexed for 2–3 min and then centrifuged at 3000 rpm for 10 min. The aqueous MeOH was separated from lipid phase, concentrated and set aside. The lipid residue was redissolved in MeOH/water  and the extraction was repeated. The aqueous MeOH extracts so obtained were combined, redissolved in acetonitrile and washed with n-hexane. The polyphenols recovered in acetonitrile were concentrated under vacuum at 30 °C, dissolved in methanol and then analyzed for total phenols. Crude extracts were also analyzed for total flavonoids by the method of Zhishen et al. [The total phenolics in the crude polyphenolic extracts, acidic, neutral and oil fractions were determined by the method of Jayaprakasha et al. , however [et al. . The oiln et al. .The antioxidant potential of the polyphenolics extract, fractions and oil was determined by three different methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, reducing power assay and Fe(II) chelating activity. Butylated hydroxyl anisole (BHA) was used as a positive control in these assays.et al. [et al., (2002) [The DPPH scavenging activity was evaluated according to Negi et al.  with litet al. . The oil, (2002) .et al. [3 (0.1%) were added and the absorbance was noted at 700 nm. The reducing power of the samples was determined by observing increase in absorbance.Ferric reducing power was determined by the method of Jayaprakasha et al. . To the 2+) was measured by 2,2ʹ-bipyridyl assay [4 solution (1 mM) was added 0.25 mL of extract or fraction (50–250 µg/mL in ethanol), 1 mL of Tris-HCl buffer (pH 7.4), 1 mL 2,2ʹ-bipyridyl solution (0.1% in 0.2 M HCl) and 2.5 mL of ethanol. The absorbance was measured at 522 nm and used to evaluate Fe2+ chelating activity using Na2EDTA as a standard.Chelating activity (Feyl assay . To 0.25p < 0.05) analysis of variance (ANOVA) and Tukey’s tests were performed.For the statistical analysis, three replicates of each sample were used and the data were expressed as mean ± S.D. Pearson correlation test was conducted to determine the correlation between total phenolics and antioxidant activity. To identify significant differences among the mean values (at p < 0.05)  .p < 0.05)  .i.e., ajwain > mustard > fenugreek > poppy seeds but unlike DPPH, neutral fraction > acidic fraction (p < 0.05)  .% yield of the oil obtained from the oilseeds has been depicted in o-dihydroxy group) structures in the aromatic ring of the phenolic compounds like caffeic and gallic acids enable them to form intramolecular hydrogen bonds with the free radicals and enhancing high antioxidant activity of these molecules in the extracts predominantly containing these species [% DPPH inhibition indicated that polyphenolics extracts of oilseeds have substantial potential to scavenge the free radicals. The higher % inhibition of DPPH by acidic fraction showed relatively higher efficiency of phenolic acids to scavenge the free radicals. Though the total phenols were higher in neutral fraction than acidic yet the activity of the acidic fraction was higher than neutral. This indicated that the antioxidant activity is not only based the total phenolics but also on the nature of the phenolic compounds ,34. The  species . Accordi species , the preR2 = 0.25–0.36) was found between the total phenolics in crude extract, neutral and acidic fractions and the % DPPH activity of the oilseeds indicating that there are some other factors responsible for antioxidant activity in the oilseeds besides the polyphenols. Significant antioxidant activity of acetone extract of ajwain in linoleic acid system, owing to the presence of compounds like thymol (39.1%), has already been demonstrated by Singh et al. [i.e., mustard > fenugreek > poppy seeds however, compared to the EC50 values determined in our study  the EC50 values reported by Chan et al. [At a concentration of 100–250 µg/100 µL, the activity of ajwain (74%–95%) was comparable to BHA (78%–97%) which means that use of synthetic antioxidants like BHA, BHT could be avoided by replacing them with the natural ones that not only effectively scavenge the free radicals but also preclude the side effects like liver damage caused by these synthetic antioxidants. A low correlation .The higher the antioxidant potential, the higher is the conversion of Feoperties . In addi% Chelating effect shown by the oilseeds extracts and fractions demonstrate that they not only have the ability to combat oxidation reactions directly but can also decelerate the oxidation reactions by chelating prooxidant metals. Transition metals like copper, iron and nickel are prooxidant and thus promote oxidation by acting as catalyst. Therefore sequestering of these metals ions by antioxidants decrease their prooxidant potential by stabilizing their oxidized form . The % cet al. [In general the oil fraction was found to be more powerful radical scavenger and reducing agent than the corresponding neutral fraction; however the chelating power of the oil fractions was least. Total polyphenols were very low in oil fraction as expected; however, the oil fraction was as active as the acidic one with respect to the antioxidative potential. This may be justified on the basis of explanation given by Ramadan et al. . Accordiet al. .The results of the study show that, though oilseeds are used in very small quantity in food, they are potential sources of antioxidants. The study also suggests that the natural antioxidants may preferably be used for food preservation and securing health effects, as they are equally good in strength to synthetic ones.


Optimizing resuscitation is important to prevent morbidity and mortality from perinatal asphyxia. The metabolism of cells and tissues is severely disturbed during asphyxia and resuscitation, and metabolomic analyses provide a snapshot of many small molecular weight metabolites in body fluids or tissues. In this study metabolomics profiles were studied in newborn pigs that were asphyxiated and resuscitated using different protocols to identify biomarkers for subject characterization, intervention effects and possibly prognosis.2 0.21 or 1.0, different duration of ventilation before initiation of chest compressions (CC), and different CC to ventilation ratios. Plasma and urine samples were obtained at baseline, and 2 h and 4 h after return of spontaneous circulation . Metabolomics profiles of the samples were analyzed by nuclear magnetic resonance spectroscopy.A total of 125 newborn Noroc pigs were anesthetized, mechanically ventilated and inflicted progressive asphyxia until asystole. Pigs were randomized to resuscitation with a FiOPlasma and urine showed severe metabolic alterations consistent with hypoxia and acidosis 2 h and 4 h after ROSC. Baseline plasma hypoxanthine and lipoprotein concentrations were inversely correlated to the duration of hypoxia sustained before asystole occurred, but there was no evidence for a differential metabolic response to the different resuscitation protocols or in terms of survival.Metabolic profiles of asphyxiated newborn pigs showed severe metabolic alterations. Consistent with previously published reports, we found no evidence of differences between established and alternative resuscitation protocols. Lactate and pyruvate may have a prognostic value, but have to be independently confirmed. Globally, perinatal asphyxia is one of the leading causes of morbidity and mortality in the neonatal period  and promMetabolomics is an approach to quantitate a large number of small metabolites with molecular weight typically below 1000 Da within cells, tissues or biological fluids. The metabolites comprising the metabolome are end products of cellular activities and give a snapshot of physiological and pathophysiological processes. During asphyxia and resuscitation normal homeostasis is severely disturbed with profound effects on metabolism . PreviouThe aim of this study was to analyze the impact of asphyxia and the different CPR protocols described in –5 on theThe experimental protocol has been described in detail elsewhere –5. The eBriefly, 125 healthy newborn Noroc pigs, aged 14–36 h (1.8–2.7 kg), were anesthetized with 5% Sevoflurane gas prior to a bolus of intravenous midazolam, fentanyl and pentobarbital. The animals were tracheotomized, placed on a mechanical ventilator, and intravenous access in the left external jugular vein was established. Each piglet was allowed to recover/stabilize for one hour prior to asphyxiation, and heart rate, arterial blood pressure, and transcutaneous oxygen saturation were monitored continuously. Clinical assessment with regards to e.g. pain and seizures was performed continuously. There were no unexpected deaths. The only deaths prior to the study endpoint at 4 hours after ROSC were caused by unsuccessful resuscitation, which was expected as per protocol. Even though the protocol dictated euthanasia if persistent signs of pain despite bolus analgesia or clinical seizures, no animals had to be euthanized before the study endpoint according to these criteria. This was not a survival study, but an acute experiment, meaning that the animals were euthanized with 150 mg/kg pentobarbital at a predetermined time point (4 hours after ROSC). This decision was partly made based on an expectation that having the animals surviving the experiment would be unethical as the animals would have suffered large neurological injury.2 to inspired air and reducing FiO2 to 0.08. The ventilator rate was reduced by 10 min–1 every 10 min until asystole occurred. After 20 s of asystole, the animals were resuscitated using six different, randomly assigned protocols [Baseline urine and blood samples were collected after stabilization, immediately before initiation of asphyxiation (t1). Then progressive asphyxia was introduced by adding COrotocols   as a frequency and concentration reference.Plasma and urine samples were thawed at room temperature. Plasma samples were centrifuged at 2,000 g and 4°C for 10 min to precipitate debris that had not been completely removed immediately after sample collection. Two hundred and fifty μL of each sample were then mixed directly in 5 mm tubes  with 250 μL NaH2PO4/KOH at pH 7.4 in pure D20, containing NaN3 and TSP, and centrifuged at 13,400 g and 4°C for 5 min, before 500 μL were transferred to 5 mm tubes.For urine, 550 μL of each sample were buffered with 55 μL KH1H nuclear magnetic resonance (NMR) spectra with water presaturation were recorded for all samples on a Bruker AV 600 spectrometer  as previously described [One-dimensional escribed .All spectra were preprocessed with an in-house program written in GNU Octave , which f2 and Q2 [The urine and plasma spectra, respectively, were scaled to unit variance and subjected to principal component analysis  to obse2 and Q2 . In addi2 and Q2 . The loa2 and Q2 .Known identifiable metabolites were quantified by comparing the area under their spectral signals with that of the TSP reference. A number of consistent but unidentified signals were also measured, but their concentrations are consequently only reported in arbitrary units due to the unknown numbers of contributing protons per signal.Metabolite concentrations were log-transformed and used for analysis of variance (ANOVA) and subsequent pairwise t-tests with respect to the classification of intervention groups. The metabolite concentrations at the different time points are presented as (back-transformed) medians and interquartile ranges. Change over time was assessed using pairwise t-tests on the metabolites’ log differences and presented as fold change (FC). Pearson correlations were calculated between time to asystole or time to ROSC and the log-transformed metabolite concentrations at all time points. Concentrations at baseline (t1) were compared between surviving and non-surviving piglets using unpaired t-tests.The pairwise FC rates and t-test p-values derived from the plasma profiles at t1 and t2, i.e. the changes between baseline and 2 h after ROSC, were used as input in an Ingenuity IPA Core Analysis , mapping the observed changes onto known pathways, functions and diseases.To further investigate the relationship between the plasma and urine samples, and between metabolites within the samples, Pearson correlations were calculated for all pairs of log-transformed concentration variables at all sampling time points. The correlations were presented as a clustered matrix with rows and columns arranged such that correlated variables are close to each other. This analysis was also repeated separately for the three time points. However, in these matrices the clustering was performed independently for the plasma and urine variables. Additionally, the lower right half of the (symmetric) matrices was filtered to only show strong correlations where the absolute value of r was >0.5.2 values, indicating a weak association between resuscitation mode and the metabolite profile, but no classification was possible at either time point. This was supported by ANOVA of the log-transformed concentrations, where none of the measured compounds, known or unknown, showed significant differences between the resuscitation groups.PLS-DA classification of plasma NMR spectra with respect to the different resuscitation protocols yielded almost entirely insignificant models at baseline (t1) and 2 h (t2) and 4 h (t3) after ROSC. Only the group resuscitated with a C:V ratio of 15:2 (group 6) produced positive Q2 values were positive, i.e. the cross-validated classification results were not correlated with the resuscitation protocols, and none of the log-transformed concentrations became significantly different in ANOVA at any of the time points both for TSP and creatinine-normalized spectra.The urine spectra invariably produced insignificant PLS-DA models with respect to the resuscitation groups. No QSince there were no consistent differences between the metabolite profiles from the six different resuscitation groups, they were pooled and considered as equal in the following analyses in order to explore the association between the metabolite profile and the severity of asphyxia and outcome, irrespective of resuscitation protocol.Separate PLS regression at the three time points found no spectral features of either the plasma or the urine samples to be associated with the response to CPR in terms of the time to ROSC in seconds.There was a weak Pearson correlation between plasma sample constituents at baseline (t1) and the subsequent duration of hypoxia needed to induce cardiac arrest. The lower the levels of hypoxanthine , the longer the piglet would endure before suffering asystole. There was also an effect of the width of the TSP signal, as a broader TSP peak was correlated with a shorter duration of hypoxia . These findings were not recapitulated in the urine samples.Finally, neither plasma nor urine sample constituents at baseline could predict the intervention outcome, as no identifiable markers differed between survivors and non-survivors.There was no difference in plasma spectra between groups of different resuscitation approaches. All samples at baseline (t1) and after ROSC at 2 h (t2) and 4 h (t3) were therefore pooled. PCA of the plasma spectra  showed t2 of 66%) and valid . The median NMC of 22 (95% CI: 22–24) was significantly lower than that of permutated class labels (107 (93–128), p < 5e-20). A loading plot .PLS-DA of plasma spectra between t2 and t3 did not reveal a significant difference between the spectra  and the median NMC of 13 (12–16) was substantially smaller than random NMC of 97 (82–114), p < 2e-23, indicating that the metabolic profiles had not returned to baseline 4 h after ROSC. At t3, NMR signals associated with lipids were weaker and choline stronger than at baseline. Lactate and alanine concentrations were also higher, but much more ambiguous than at t1 and t2. A univariate characterization of a selection of metabolite concentrations, along with their changes between the time points, is presented in Lastly, the comparison of plasma spectra at time points t3 vs. t1 with PLS-DA yielded a significant model , and better than random NMC of 89 (74–108), p < 2e-9), based on changes in urine lactate, alanine, BCAA, glucose, and hypoxanthine concentrations.There was no difference in urine spectra between groups of different resuscitation protocols. All samples at the three time points were therefore pooled. PCA  and PLS-2 of 18%, Q2 0%; NMC not different from random, p = 0.96), while the comparison of t3 and t1 yielded an even better model  compared with random NMC 52 (45–62), p < 6e-8) than baseline vs. t2 above. The BCAA, especially valine, but also lactate, alanine, glucose, and choline concentrations in urine were clearly higher than at baseline. Selected urinary metabolite concentrations are shown in As with plasma, there was no significant difference between t2 and t3 by PLS-DA . In this model we previously examined differences with clinical and more established biochemical methods including Enzyme-linked immunosorbent assay (ELISA) and Real-Time Polymerase Chain Reaction (RT-PCR) and found few differences between resuscitation protocols ,28. We set. al. [et al. [A profiling study of less asphyxiated pigs by Solberg et. al.  also fou [et al.  found di2 consumption [Independently of resuscitation protocol, we observed a 7.7- and 4.9-fold increase in plasma succinate and fumarate concentrations, respectively, 2 h after ROSC (t2) compared with baseline (t1), whereas the changes in urine at the same time points were more modest and only 1.9 and 1.2-fold. The urine concentrations of the metabolites 4 h after ROSC (t3) were almost back to baseline, and almost in parallel with the reduction of plasma concentrations. The near normalization of urine succinate and fumarate at t3 is in contrast to the concentration changes of other metabolites such as lactate and hypoxanthine, which increased markedly at t2 followed by sustained high urine concentrations at t3, in contrast to the fall in plasma concentrations. Some of the differences can be explained by differences in renal handling of the metabolites. Succinate is actively reabsorbed in the proximal tubules by an apical dicarboxylate transporter, which is activated by lowering extracellular pH as in asphyxia . Lactatesumption . During et al. [A study by Atzori et al.  reportedet al. [Solberg et al.  reportedIn terms of the general characterization of the metabolic trajectories, our plasma data show an increase and subsequent decrease of many profiled substances, most notably lactate, succinate, fumarate, malate and alanine, which could be useful in diagnosis and estimation of prognosis following asphyxia. Choline increased without a subsequent decrease, while the low-density lipoprotein (LDL) lipid signals did the opposite. Choline concentrations in umbilical cord blood was also increased and almost doubled in infants with perinatal asphyxia compared with controls .et al. [The role of these metabolites and how they interact in metabolic pathways has not been clarified. Lipids play a role in critical illness, and in particular high-density lipoproteins (HDL) have been shown to have a protective effect in sepsis.  Lipid meet al. There are, owing to the severe nature of this study, no corresponding studies in humans with which to compare our findings. More generally, though, human studies of newborn asphyxia and hypoxic ischemic encephalopathy (HIE) also found differences in the metabolic profile of healthy compared with asphyxiated newborns, involving many of the substances we have found altered in our study. For example, asphyctic infants were characterized by increased urine lactate and glucose, together with threonine and 3-hydroxyisovalerate, as well as decreased dimethylglycine, dimethylamine, creatine, succinate, formate, urea and aconitate, and succinate . AnotherThe change of the plasma and urinary profiles between the time points before and after asphyxia and resuscitation was analyzed in terms of metabolic pathways and gene networks. The change was found to be consistent with a dramatic reaction of the organism to such a catastrophic event as cardiac arrest and CPR. Not surprisingly, the metabolism switches into an emergency, catabolic state, and there is clear evidence of hypoxemia and acidosis characterized by increased plasma lactate, hypoxanthine and glycerol concentrations, activation of amino acid metabolism and increased plasma creatinine indicating reduced kidney function.We could directly compare plasma and urine profiles and found them at best loosely correlated. Compared to plasma, the urine metabolite profile represents the final result of glomerular plasma filtration, and tubular reabsorption and excretion during the period from the last emptying of the bladder. The concentration of urine metabolites depends not only on the plasma concentrations of the metabolites, but also on the renal handling of the individual metabolites and sampling frequency. For real-time monitoring of acute conditions such as hypoxia, plasma is superior to urine samples. In fact, one of the important findings of this study may be the poor correlation between plasma and urine samples. This will be useful for those attempting to develop clinical markers for hypoxic injury. Urine samples are delayed and are dependent on renal function. In addition individual metabolites have different clearance patterns. The data support the use of caution when using urine as a marker for metabolite alterations.The present study has some limitations. The samples are from a study primarily designed to compare the effects of several different resuscitation protocols. There were too few pigs in each group to make reliable comparisons of metabolite profiles between the different protocols. The pigs were so severely asphyxiated in this study, with a heart rate of zero evaluated by electrocardiogram, that more subtle metabolic differences between resuscitation protocols may have been subdued. As there was no difference in outcome between the protocols, metabolite concentrations in plasma and urine during asphyxia and resuscitation were pooled for all resuscitation protocols. The study presents metabolite profiles in plasma and urine at baseline, and 2 h and 4 h after asphyxia, resuscitation and ROSC. It provides a list of metabolites that alone or in combination may be tested as biomarkers of susceptibility to hypoxia, effect of resuscitation protocols and time to ROSC. However, with a limited number of time points we may miss important metabolite changes occurring before 2 h and after 4 h of rest after ROSC.et al. [2 > 95%). Other analytical techniques may be used for validation and quantification of biomarkers such as RT-PCR to quantify candidate microRNAs in blood and urine [In this study we present results from NMR spectroscopic analysis which can be validated by mass spectrometry analyses or specific metabolite assays. In Sachse et al.  both urind urine . ELISA and urine  are analThe study demonstrates the severe metabolic alterations in plasma and urine caused by asphyxia-induced cardiac arrest and CPR. NMR-based profiling did not find evidence for a differential metabolic response to different resuscitation protocols. Lipoprotein concentration appeared inversely correlated to the tolerance of hypoxia, but no biomarkers were identified that were associated with a faster or slower recovery from cardiac arrest. The metabolic profiles could potentially be useful in predicting outcome after hypoxic ischemic insults in newborn pigs and thereby infants and of future guidance for resuscitation strategy.S1 Supplement(PDF)Click here for additional data file.


Aim. To test the hypothesis that walking in a park has a greater positive effect on coronary artery disease (CAD) patients' hemodynamic parameters than walking in an urban environment. Methods. Twenty stable CAD patients were randomized into two groups: 30-minute walk on 7 consecutive days in either a city park or busy urban street. Wilcoxon signed-rank test was employed to study short-term (30 min) and cumulative changes (following 7 consecutive days of exposure) in resting hemodynamic parameters in different environments. Results. There were no statistically significant differences in the baseline and peak exercise systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), exercise duration, or HR recovery in urban versus park exposure groups. Seven days of walking slightly improved all hemodynamic parameters in both groups. Compared to baseline, the city park group exhibited statistically significantly greater reductions in HR and DBP and increases in exercise duration and HR recovery. The SBP and DBP changes in the urban exposed group were lower than in the park exposed group. Conclusions. Walking in a park had a greater positive effect on CAD patients' cardiac function than walking in an urban environment, suggesting that rehabilitation through walking in green environments after coronary events should be encouraged. There is some evidence that green environments are associated with better self-reported health , lower bThe underlying mechanisms for health benefits of green spaces are not fully understood. Recent studies have reported that green space, such as city parks, can reduce noise and air pollution , 22, enhThere is some evidence that walking in a natural environment compared to an urban environment has benefits in terms of psychological and physical restoration in young subjects , 29 and The study was conducted in Kaunas, Lithuania. Twenty male and female Kaunas city residents (62.3 ± 12.6 years of age) with CAD  I-II chronic heart failure) participated in the study. The patients were treated at the Cardiologic Clinic of the Hospital of Lithuanian University of Health Sciences because of MI or unstable angina pectoris and were consecutively selected from the patients register. The mean duration since the last period of CAD hospitalization and cardiac rehabilitation was 1.03 ± 0.5 years. Inclusion criteria were as follows: 45–75 years of age, men or women, who survived MI or unstable angina pectoris, and signed informed consent to take part in the study. Exclusion criteria were as follows: unstable angina pectoris, cardiomyopathy, idiopathic or organic valvular disease, hypertension with SBP > 160/110 mm Hg, diabetes mellitus type 2, electrocardiostimulation, neurological diseases, and limited capacity  .The study was performed under the regulations of the Lithuanian Bioethics Committee and in accordance with the Declaration of Helsinki.n = 10) was a busy street behind the Cardiology Clinic . The green exposure (n = 10) was a pine park located within a 5 min walk of the Cardiology Clinic, accessed through clinic park . Patients' normal medication regimens were not changed during the study. We used standardised protocols for environmental exposure and measurement of physiological responses. Both groups were similar, clinically and in terms of their residential environmental characteristics. Physical activity, eating, and drinking were controlled during the study periods. Data collection took place at the clinic between 12:00 and 15:00, May–September 2013. To minimise the social interaction effects during the environmental exposure, the same trained researcher supervised all subjects, their walking intensity, and their social interaction during the 30 min walk. Exercise capacity testing using a spiroergometer on a treadmill and with ECG monitoring was performed at baseline and day 7. The test provides an accurate assessment of maximal and functional aerobic capacity. Walking intensity was estimated to be 10% lower of the capacity determined during spiroergometry. Patients walked for 30 min each day in their allocated environment, for 7 consecutive days. We studied the short-term  and cumulative 7-day effects of walking alone in the urban or park environment and 7-day changes in specific exercise capacity parameters. Changes in hemodynamic parameters at rest and at peak exercise  were assessed. Before and after 7 days of walking in different environments, we compared changes in hemodynamic parameters between those walking in the urban and the park environments including resting data before walking, 1 min after walking, 30 min after walking, and 3 hours after walking.Patients were randomly assigned to either green or urban exposure groups. The urban exposure .We used the exact Fisher tests to compare the personal characteristics of the urban and park exposed patients. Quantitative variables are reported as means and standard error. Quantitative clinical and environmental variables in both groups were compared using the Mann-WhitneyThere were no statistically significant differences in the demographic or clinical characteristics between the two exposure groups .2 concentration of patients exposed to urban environment was 18.5 ± 5.4 μg/m3 and that exposed to park environment was 20.1 ± 5.3 μg/m3 (P = 0.37), while the residential proximity to the nearest city park was 321.7 ± 251 m and 490 ± 356 m, respectively (P = 0.114). There were significant differences in the characteristics of the urban versus park environments, with higher levels of air pollution  and noise (19.03 dBA higher) compared with the park environment.The mean age, body mass index (BMI), duration of CAD anamnesis, and time since last CAD hospitalisation were all similar for patients in the urban and park groups. Residential environmental characteristics were also similar: the mean residential NOThe two groups did not differ significantly in terms of mean SBP, DBP, and HR before exposure .P = 0.205, resp.), work load, or postexercise HR recovery. Pulse wave velocity was also similar. After 7 days of walking there were no statistically significant changes between the urban and park groups in terms of hemodynamic parameters. However, work load was slightly higher , and SBP and DBP were slightly lower in park group. Both groups demonstrated slight decreases in HR and increases in exercise capacity test duration and HR recovery as a consequence of regular walking. There were no statistically significant changes between the groups in pulse wave velocity (P = 0.475).Baseline exercise capacity testing, which provides an accurate assessment of maximal and functional aerobic capacity, showed that there was no significant difference between the urban and park exposure groups for exercise duration (where longer duration indicates greater capacity) .Analysis of short-term (1 min and 30 min after exercise) changes in hemodynamic parameters on days 1 and 7 revealed statistically significant differences in hemodynamic indices at 1 min after walking compared with baseline . On day P = 0.049 and P = 0.014, resp.). Significant increases in exercise duration  and HR recovery  were also observed in the park group, while in urban environment exposed patients, changes in these parameters were not statistically significant.The difference in resting hemodynamic parameters measured at baseline of days 1 and 7 is presented in The present study aimed to use objective measures to assess the physiological effects of controlled walking in urban and park environments in CAD patients. Data showed that regular 30 min walks of moderate intensity in a park environment performed on 7 consecutive days led to greater favorable changes in resting SBP and DBP, improvements in exercise tolerance, and increases in exercise duration, compared with equivalent walks in an urban environment. Walking in the park also increased patients' HR recovery after everyday physical exercise. Because HR recovery  is treated as an indicator of autonomic function , 16, 17,To our knowledge, no previous studies have compared the effects of controlled walking in urban and park environments on hemodynamic parameters in CAD patients. Our results are consistent with evidence from healthy young adults. The comparison of physiological effects of 15 min of walking in forest and urban environment in 12 Japanese students revealed significantly lower SBP, DBP, and HR and higher HR variability in subjects exposed to a forest environment showing suppressed sympathetic nervous activity and enhanced parasympathetic nervous activity in the forest area . The greIn our study improvements observed in exercise tolerance and increased HR recovery after 7 days of 30 min walks in a park environment may be explained by the positive influence of forest-related activities on cardiovascular relaxation and recovery of homeostasis in CAD patients. This mechanism may be partially confirmed by the findings of young Japanese adult males, indicating that walking in the forest environment can facilitate homeostasis . PhysiolOur findings are in accordance with the results of epidemiological studies, which show positive relationships between the physical activity in natural environment and cardiovascular health. A Kaunas cohort study that investigated associations between the accessibility and use of urban city parks and cardiovascular health showed that the prevalence of cardiovascular risk factors was statistically significantly lower among park users than among nonusers. Men living further away from parks and rarely using them had a higher risk of nonfatal and fatal CVD combined, compared with those living nearby; that is, regular use of green space in a city setting was linked to reduced risk of heart disease . An obse2 was higher by 3.84 μg/m3, PM2.5 by 6.41 μg/m3, and noise level by 19.03 dBA (compared with the park environment). Such differences may have impact on psychophysiological stress, homeostasis, and hemodynamic parameters. Previously published data from Kaunas [2 pollution, noise level, and PM2.5 pollution may increase the risk of hypertension and that this, through increase in SBP and DBP, may promote atherosclerosis and CAD. Short-term increases in exposure to ambient PM2.5 are associated with acute increases in blood pressure in adults [In our study, differences in measured characteristics of the two environments may also partially explain our findings. During walking in the urban street, NOm Kaunas , 40 and m Kaunas  indicaten adults . The parn adults , 45, andn adults –48. ThesThe study results will have direct practical applications for the use of natural environments in cardiac rehabilitation. However, some limitations are recognised. First, the sample size was relatively small, albeit large enough to detect some significant effects. Second, we are unable to identify which specific characteristics of the natural and urban environments were responsible for the observed effects. During walking, patients were affected not only by the traffic emissions but also by the view of trees planted in front of the houses and that may have impact on the decrease of psychophysiological stress level and obtained results.These limitations notwithstanding, this study appears to be the first to analyse the relationship between the controlled physical activity in different environments and CAD patient's hemodynamic parameters, further adding to the growing support for the therapeutic potential of natural environments. Natural environments should be considered for inclusion in physical rehabilitation after CAD, but further research with larger samples is required to draw generalized scientific conclusions on the impact of natural environmental quality on CAD patients.


We show that in response to reduced skeletal mineralization at lower pH, corals increase their skeletal macroporosity (features >10 μm) in order to maintain constant linear extension rate, an important criterion for reproductive output. At the nanoscale, the coral skeleton's structural features are not altered. However, higher skeletal porosity, and reduced bulk density and stiffness may contribute to reduce population density and increase damage susceptibility under low pH conditions. Based on these observations, the almost universally employed measure of coral biomineralization, the rate of linear extension, might not be a reliable metric for assessing coral health and resilience in a warming and acidifying ocean.Ocean acidification is predicted to impact ecosystems reliant on calcifying organisms, potentially reducing the socioeconomic benefits these habitats provide. Here we investigate the acclimation potential of stony corals living along a pH gradient caused by a Mediterranean CO Global warming and ocean acidification impact coral ecosystems. Here, the authors show higher skeletal porosity and reduced bulk density at lower pH in corals living along a natural pH gradient in the Mediterranean, which may contribute to reduce population density and increase damage susceptibility. Rising atmospheric carbon dioxide partial pressure pCO2 due to ca. 0.1 U2 emissions continue at their current rates, the average sea surface pH is predicted to drop to 7.8 by the year 2100  sea surface pH predictions associated with different atmospheric CO2 emission scenarios for the end of the centuryNear Panarea Island, off the southwestern coast of Italy, lies a series of active volcanic vents in the seabed releasing COBalanophyllia europaea living along the pH gradient. We studied 74 corals of similar age (mean age of 12 years) that had spent their lives at the CO2-pH gradient. Using a combination of scanning electron microscopy (SEM), atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), micro computed tomography (μCT), nanoindentation, hydrostatic weight measurement and time-domain nuclear magnetic resonance (TD-NMR), we document the skeletal mass, bulk volume, pore volume, porosity, biomineral density, bulk density, hardness, stiffness (ratio between elastic stress and strain), biometry data, net calcification rate and linear extension rate for each coral. Weight measurements combined with TD-NMR data represent a non-destructive technique for quantifying skeletal porosity over length scales spanning from tens of nanometres to tens of micrometres14The present study investigates the effects of environmental pH on skeletal structures and growth at multiple length scales in the solitary scleractinian coral We show that in response to depressed calcification at lower pH, corals increase their skeletal porosity maintaining constant linear extension rate, which is important for reaching critical size at sexual maturity. However, higher skeletal porosity and reduced bulk density and stiffness may contribute to reduced mechanical strength, increasing damage susceptibility, which could result in increased mortality in an acidic environment.TS) of 8.1, equivalent to the average surface pH of modern oceans. S2's average pHTS of 7.9 aligns with IPCC's predictions of a conservative CO2 emissions scenario (Representative Concentration Pathway (RCP6.0)), and the average pHTS of 7.7 for S3 fits the predictions of the ‘business-as-usual' CO2 emissions scenario (RCP8.5). Since no corals were found at S4 , only S1–3, which had growing coral populations and pHTS values ranging from 8.1 to 7.7, were included in the present study. Of the measured parameters at the three sites along the pH gradient , only pHTS differed significantly across sites . The changing pH was accompanied by significant shifts in carbonate–bicarbonate equilibria, with aragonite saturation (Ωarag) decreasing by >30% from S1 to S3  did not vary among sites  and skeletal bulk density  significantly declined , linear extension rate , SEM and AFM showed that the organization of the aragonite fibre bundles  and the The principal component analysis identified three major components: growth, skeletal porosity and biomineral density . Only thB. europaea did not change significantly with decreasing pH, despite a clear reduction in net calcification rates. This reduction in net calcification rate was accompanied by an increase in skeletal porosity  and a consequent decrease in skeletal bulk density and stiffness.In summary, skeletal nano and microstructural features, linear extension rate, interseptal volume fraction and corallite biometry of B. europaea at this same vent site, which revealed no changes in skeletal calcium carbonate polymorph, organic matrix content, aragonite fibre thickness and skeletal hardness in corals growing along the pH gradientResults of the present study complement previous research on B. europaea. At the macroscale, increasing acidity was associated with a reduction in net calcification rate and a parallel increase in skeletal porosity, coupled with a decrease in skeletal bulk density. Linear extension rate and corallite shape  did not depend on pH, probably as a result of the compensation of reduced net calcification rate by increased skeletal porosity. At the micro/macroscale, the declining skeletal stiffness with decreasing pH could be coupled to an increased volume fraction of pores having a size comparable to the indentation area . At the nanoscale, porosity, biomineral hardness and density were not significantly affected by pH. These results, bolstered by qualitative SEM and AFM analyses, suggest that the ‘building blocks' produced by the biomineralization process are substantially unaffected, but the increase in skeletal porosity is both a gain and a loss for the coral. In fact, in an acidic environment, where the net calcification is depressed, enhanced macroporosity keeps linear extension rate constant, potentially meeting functional reproductive needs ; however, it also reduces the mechanical strength of the skeletons, increasing damage susceptibility, which could result in increased mortality and the observed population density declineB. europaea may not be representative of the generalized response of all coral species to OA, they are consistent with field observations made on other reef-building scleractinians. For example, while maintaining constant skeletal linear extension, decreased rates of calcification and losses in bulk skeletal density as a function of reduced aragonite saturation have been observed in Montasraea annularisPorites astreoides17While the results reported here for B. europaea16202122Our findings, together with the well-described detrimental effects of heat stress on the scleractinian zooxanthellate coral 2 vents. The main vent, a crater measuring 20 m × 14 m and ∼10-m deep, generates a persistent column of CO2 bubbles that rise from the seabed to the sea surface. In this hydrothermally stable setting with ambient temperature, CO2 emissions establish a pH gradient that extends ∼34 m from the centre of this crater to its peripheryd) from the main crater centre and mean pHTS values of the three sites from which corals were collected are: S1 (the control site), d=34 m, pHTS=8.07; S2, d=13 m, pHTS=7.87; S3, d=9 m, pHTS=7.74. Water depth varies from 11.6 m within the crater to 8.8 m at the crater edge (S2) to 9.2 m at the outer margins (S1), where the local pH matches that of the surrounding seawater. The study site has stable hydrothermal–chemical propertiesOff the southwestern coast of Italy, near the island of Panarea, an area delimited by the islets of Dattilo, Bottaro, Lisca Nera and Lisca Bianca  is charaB. europaea  between November 2010 and May 2013. This sample size was chosen to limit damage on the natural population, which significantly diminishes in the most acidic sites13europaea  is a solCoral skeletons were submerged in 1% solution of sodium hypochlorite for 3 days to dissolve polyp tissue. After washing with deionized water and drying at 50 °C for 3–4 days, each coral was examined under a binocular microscope to remove fragments of sediment, rock and encrusting organismsVB), the bulk density , the biomineral density  and the apparent porosity VA) to VB). This method is based on the Archimedean principle applied to a specimen after full saturation with the same fluid in which it is submerged (water in our case). It is worth to underline that the pore volume (VA) measured by the buoyant method is only the volume of the pores that can be saturated with water, that is, connected with the external surface. Pores inside the biomineral that are not connected to the external surface (occluded pores) are not measured. An estimate of the occluded porosity gave a negligible maximum value of ∼3% (in porosity units)  , which wm) and then placed inside a dryer chamber and evacuated with a rotary mechanical pump down to a vacuum of 10−2 mbar. After 6 hours, water was gently introduced to fully saturate the samples. The pump was switched off, the chamber was vented to the ambient atmosphere, and the masses of the fully water-saturated samples (ms) determined. With a hydrostatic balance, the masses of saturated samples fully immersed in water (mh) were determined. The skeletal parameters were calculated /ρw; VB=(ms−mh)/ρw; PA=VA/VB=(ms−m)/(ms−mh); db=m/VB; dr=m/(VB−VA).The skeletons were weighed with a precision balance to determine the dry mass  and larger (longer T2) pores. The ratio of the area under the distribution for T2 larger than the cutoff to the total area under the distribution, called ‘macroscale pore volume fraction'  for automatic pulse sequence transmission and data acquisition. The transverse relaxation data were acquired by using the Carr–Purcell–Meiboom–Gill sequenceA homebuilt relaxometre based on a 0.2 T permanent magnet operating at 8 MHz was used for acquisition of the transverse relaxation curve of the To investigate the meso and macroscale organization of the aragonite fibre bundles, SEM analyses were performed. The cleaned coral skeletons were mounted (uncoated) to conductive carbon tape and examined with a Tescan Vega3 GMU (Czech Republic) variable pressure scanning electron microscope. Three corals per site were analysed.The age of each coral was determined based on growth band analysis via computerized tomography2221To compute the corallite interseptal volume fraction , μCT analyses were acquired and the convex hull approach was followed. μCT scans of coral skeletons were performed with a GE phoenix X-ray Nanotom S. The voxel sizes in the scans varied from 2.91 to 13.25 μm depending on the actual size of the investigated coral sample. All image processing steps in the analysis were performed with the open source software FIJITo analyse mechanical properties of the coral skeletons, skeletal stiffness (Young's modulus) and hardness were determined by Oliver–Pharr analysis of load-depth curvesTo obtain information on skeletal porosity at the nanoscale, SAXS analyses  were perTo study the morphology and packing of skeletal mineral grains at the nanoscale, AFM images were acquired. Samples were lightly polished using diamond paste, cleaned with milli-Q water and observed using a Veeco AFM Dimension 3100 Nanoscope III, Plainview, NY. The probe consisted of a cantilever with an integrated silicon nitride tip. Samples were imaged at room temperature and in air using tapping mode phase contrast imaging.χ2) tests were run. Non-parametric tests were performed for data that did not assume normal distributions. Multivariate regression analyses were performed to investigate the relationships between one dependent and one or several independent variables, using ordinary least squares robust to outliers. The model is described by the Statistical analyses  were performed using the Statistical Package STATA 9.0 (StataCorp LP). To test the significance of the differences among sites, parametric (F) or non-parametric  are the best-fit parameters, to be determined by the best fit. Quantile analysis was performed to study the previous relationships for homogeneous groups of values of the dependent variable. This analysis was used to give a more comprehensive picture of the effect of the independent variable (pH) on the dependent variables of where the index How to cite this article: Fantazzini, P. et al. Gains and losses of coral skeletal porosity changes with ocean acidification acclimation. Nat. Commun. 6:7785 doi: 10.1038/ncomms8785 (2015).Supplementary Figures 1-10, Supplementary Tables 1-9, Supplementary Notes 1-4 and Supplementary References.Balanophyllia europaea close to the crater


Streptococcus pneumoniae is an invasive organism that causes a wide range of common diseases, including sinusitis, acute otitis media, and pneumonia. Splenic abscesses and purpura fulminans (PF) are rare complications of pneumococcal disease. Splenic abscesses caused by S pneumoniae have only been reported in the adult literature. PF has been described in the pediatric population as a rare complication in patients with invasive pneumococcal disease (IPD) with and without underlying immunological disorders such as asplenia. Here, we report a patient with IPD complicated by splenic abscesses and PF. Our patient initially presented with bacteremia, septic shock, and disseminated intravascular coagulation. She subsequently developed PF and splenic abscesses. She survived her illness after receiving a total of 8 weeks of antibiotic therapy. This case highlights 2 rare complications of IPD and demonstrates the need to keep pneumococcal disease in the differential diagnosis even in children whose vaccination status is up to date. S pneumoniae from a normally sterile site, such as blood, cerebrospinal ﬂuid (CSF), pleural ﬂuid, joint aspirate, pericardial ﬂuid, or peritoneal ﬂuid.1 Splenic abscesses are rare in the pediatric population.2 Early recognition and intervention are critical because of the high mortality rate associated with delayed diagnosis.3 Splenic abscesses and purpura fulminans (PF) are rare complications of IPD. Splenic abscesses caused by IPD have been reported in adults.4 We report a case of IPD confirmed by blood culture and serotyping in a fully vaccinated 15-month-old girl whose clinical course was complicated by splenic abscesses and PF. To our knowledge, this is the first case of splenic abscesses caused by Streptococcus pneumoniae described in the pediatric population.Invasive pneumococcal disease (IPD) is deﬁned as the recovery of an isolate of This patient was a 15-month-old girl who presented to a local emergency department with fever and cough for 1 day. She was diagnosed with a viral upper-respiratory infection and discharged home. She subsequently became lethargic with poor oral intake over the next day, which prompted her parents to return to the emergency department. She was healthy prior to this illness. Her childhood vaccinations were up to date, including 4 doses of the 13-valent pneumococcal conjugate vaccine (PCV-13). Her family history was negative for immunodeficiencies and sickle cell disease.2 of 35 mm Hg; PO2 of 52 mm Hg; HCO3 of 11 mg/dL; prothrombin time of 37.8 s; international normalized ratio of 3.8; activated partial prothrombin time of 101.4 s; total leukocyte count of 1900/mm3, with 10% banded neutrophils and 25% segmented neutrophils; hemoglobin of 10.8 g/dL; and platelet count of 67 000/mm3. Lumbar puncture was performed and revealed a white blood cell count of 1 per mm3, red blood cell count of 3 per mm3, glucose 108 mg/dL, and protein 83 mg/dL. Blood, urine, and CSF cultures were sent, and she was started on broad spectrum antibiotics with vancomycin and ceftriaxone. She was admitted to the pediatric intensive care unit for management of septic shock, disseminated intravascular coagulopathy, and multiple organ dysfunction syndrome. The blood culture from admission became positive after 8 hours of growth, which was identified as S pneumoniae that was sensitive to penicillin and ceftriaxone, with a minimum inhibitory concentration of less than 0.03 µg/mL. An immunological workup revealed a normal antibody response to childhood vaccinations, including diphtheria, tetanus, and pneumococcus; IgM was 82 mg/dL, IgG was 986 mg/dL, and IgA was 106 mg/dL; total complement (CH50) was 63 U/mL, and alternative complement pathway (AH50) was 65%, all of which were within normal limits. S pneumoniae IgG to 14 serotypes from ARUP laboratories demonstrated >1.3 µg/mL for 10 of the 14 serotypes, which is consistent with an adequate vaccination response. Newborn screen was negative for sickle cell disease, and there was no family history of sickle cell disease. Vancomycin was discontinued after reviewing the sensitivities and documentation of sterile cerebrospinal fluid. She was initially hypotensive despite being placed on vasoactive medications for treatment of refractory shock. On hospital day 4, she developed dry gangrene of the distal phalanges  than those with Gram-positive coccus (31%). Three cases of splenic abscesses caused by S pneumoniae were identified in the adult population.7 One patient had a history of alcoholism and was diagnosed with “Austrian syndrome,” which consists of the triad of endocarditis, meningitis, and pneumonia caused by S pneumoniae.8 Risk factors were not described for 2 of the patients with splenic abscesses.4S pneumoniae was isolated on admission via blood culture after 8 hours of growth, suggesting that our patient’s splenic abscesses were likely a result of direct contiguous spread from bacteremia. A weakness of this case report is that determination of the causative agent of the splenic abscesses was based on a positive blood culture result and not from abscess fluid culture, because the fluid collections were not aspirated and sent for culture. Therefore, the possibility of second bacterial infection or coinfection with a pneumococcal strain that was not 22A/F could not be entirely excluded. However, bacteremia is a known risk factor for developing splenic abscesses, so it was presumed that the 2 processes were related. The fluid collections improved in size while on antibiotic therapy and were no longer rim enhancing at discharge.According to a review of 67 cases of splenic abscesses by Chang et al,2 We elected to treat with prolonged antibiotic therapy to avoid the possibility of these infections. In addition, the presence of multiple abscesses made needle aspiration difficult because this would likely require multiple interventions, increasing the risk of complications such as bleeding or seeding infection into the pleural space. Instead, our patient completed 14 days of intravenous ceftriaxone for treatment of S pneumoniae bacteremia and 6 weeks of intravenous penicillin G for the initial treatment of her splenic abscesses. Clindamycin was chosen to complete the patient’s treatment course because the patient developed an allergy to both penicillins and first-generation cephalosporins. Also, sensitivity of S pneumoniae to clindamycin has been well documented in the literature among non-19A strains.9Splenic-preserving therapies such as partial splenectomy or interventional radiological guide needle aspiration are now preferred over splenectomy in the pediatric age group to avoid overwhelming postsplenectomy infections.1012 Most cases of PF caused by S pneumoniae infections occur in adults and in patients with asplenia,10 though cases have been described with no predisposing risk factors.12 Our patient initially presented with bacteremia, septic shock, disseminated intravascular coagulation, and multiple organ dysfunction syndrome and was later found to have developed digital infarctions as a result of PF and splenic abscesses during her hospitalization. She remained profoundly hypotensive despite vasoconstrictive medications for 10 hours during her initial hospitalization, which would have contributed to her digital infarctions. Our patient did not have any predisposing conditions that would have made her more likely to develop PF, such as sickle cell disease, asplenia, or complement deficiency. She received adequate vaccination against pneumococcus with the 13-valent pneumococcal vaccine, although this does not contain strain 22A/F.Symmetrical peripheral gangrene, also known as PF has been described in the pediatric population as a complication of IPD.In conclusion, splenic abscesses are a potential complication in patients with IPD and persistent fevers despite adequate antibiotic coverage. PF is a potential complication of pneumococcal sepsis and should be considered even in children with no underlying immunological disorder. IPD should not be excluded in the differential diagnosis if the child’s vaccination status is up to date because there are many serotypes not covered in the PCV-13.