The development of precision nanomedicines to direct nanostructure-based reagents into tumour-targeted areas remains a critical challenge in clinics. Chemical reaction-mediated localization in response to tumour environmental perturbations offers promising opportunities for rational design of effective nano-theranostics. Here, we present a unique microenvironment-sensitive strategy for localization of peptide-premodified upconversion nanocrystals (UCNs) within tumour areas. Upon tumour-specific cathepsin protease reactions, the cleavage of peptides induces covalent cross-linking between the exposed cysteine and 2-cyanobenzothiazole on neighbouring particles, thus triggering the accumulation of UCNs into tumour site. Such enzyme-triggered cross-linking of UCNs leads to enhanced upconversion emission upon 808 nm laser irradiation, and in turn amplifies the singlet oxygen generation from the photosensitizers attached on UCNs. Importantly, this design enables remarkable tumour inhibition through either intratumoral UCNs injection or intravenous injection of nanoparticles modified with the targeting ligand. Our strategy may provide a multimodality solution for effective molecular sensing and site-specific tumour treatment. Directing nanomedicines to desired locations - such as tumour sites - is difficult to achieve selectively. Here, the authors develop a method to covalently crosslink peptide-modified upconversion nanocrystals into tumour sites for photodynamic therapy and show in vivo tumour inhibition in mice. This often leads to an inevitable overheating effect because of significant water absorption35Recently, rare-earth doped upconversion nanocrystals (UCNs) have been widely demonstrated for use in biomedical applications. In general, UCN particles offer deep tissue penetration capability for enhanced bioimaging and better tumour treatment arising from their unique non-linear photon upconverting process upon light irradiation at near-infrared (NIR) window212223242526272830313233in vivo covalent localization of particles at the tumour site. Different from the process involving nonspecific tumour targeting, such unique platform can respond to tumour-specific enzyme and undergo cross-linking reaction, which thus enables the selective tumour accumulation. More significantly, compared with the particles that cannot undergo cross-linking reaction, the enzyme-triggered covalent cross-linking of UCNs possess an enhanced light upconverting emission when illuminated at 808 nm. Such enhancement can effectively amplify the production of reactive singlet oxygen  from the photosensitizers loaded on the particle surface, which therefore represents promising nanomedicine for improved photodynamic tumour treatment (PDT) as well as non-invasive fluorescence and photoacoustic imaging in vitro and in vivo.In this work, a tumour microenvironment-sensitive UCNs platform has been designed and prepared to perform 3+ ions for improved pumping efficiency at 808 nm. These Nd3+-doped nanocrystals were further coated with polyacrylic acid (PAA) and polyethylenimine (PEI) to improve biocompatibility and to facilitate subsequent surface modification (fication . Transmification .2) linker on the particle surface. The successful formation of peptide and Ce6-modified UCNs (CRUN) conjugate was confirmed by spectroscopic characterization , with favourable optical properties was chosen and coupled to the PEI/PAA@UCNs. In order to achieve selective tumour localization, an enzyme-responsive peptide, Ac-FKC(StBu)AC(SH)-CBT  containirization . The opt surface . In prinurface 13. During −1) with CtsB (55 nM), transmission electron microscopy and dynamic light scattering analysis indicated significant particle aggregation in solutions  detected in solution after 4 h reaction . General383940Furthermore, we also investigated the underlying reason of the enzyme-responsive luminescence enhancement of CRUN by monitoring the decays of the luminescence lifetime. As shown in 42−1) at 37 °C for 4 h. The different expression levels of CtsB were verified by western blotting and quantitative human CtsB ELISA kit in HT-29 (40.1±4.4 ng mg−1 protein) and NIH/3T3 (2.2±1.4 ng mg−1 protein) cells, respectively  through the standard singlet oxygen sensor green1O2 from the activated photosensitizers after 1 h illumination (1O2 generation (2.9-fold decrease) with 660 nm irradiation, while there was only 0.6-fold decrease in 1O2 generation when illuminated at 808 nm. The higher singlet oxygen production with 808 nm excitation in the presence of pork tissue clearly indicated a better penetration depth as compared with irradiation at 660 nm, which makes UCN-based PDT suitable for effective tumour treatment in living system.We also evaluated the capability of CRUN to generate cytotoxic singlet oxygen (mination . Importain vitro toxicology assay (TOX8). As shown in −1) incubated HT-29 cells, there was higher cytotoxicity (∼53% cell viability) observed in HT-29 cells than that of cells treated with the control NCRUN (∼64% cell viability). In addition, similar photodynamic inactivation was also studied in CRUN-incubated HT-29 cells but with CtsB inhibitor or in the negative control (CtsB-deficient NIH/3T3 cells). Compared with the laser-excited HT-29 cells with CRUN, there was no significant enhancement of cytotoxicity detected in these control experiments. Meanwhile, 808 nm laser irradiation alone did not induce obvious cytotoxicity in both HT-29 and NIH/3T3 cells  light for living cell PDT treatment.The potential photodynamic cytotoxicity of CRUN was further evaluated through an T3 cells , indicatT3 cells , the prein vivo theranostic efficacy via intratumoural injection of CRUN into living female Balb/c nude mice. We implanted HT-29 cells (CtsB overexpressing) into the left and right flanks of nude mice. At 2–3 weeks post-implantation, the tumours grew to 3–5 mm in diameter and the living mice underwent photoacoustic imaging and PDT studies. Typically, one group of xenograft mice bearing two tumours were subjected to CRUN and controlled NCRUN conjugates, respectively , followed by photoacoustic imaging. As compared with the photoacoustic intensity before injection, the mice exhibited a significant increase in photoacoustic signals at 4 h post-injections for both conjugates. Moreover, the photoacoustic signals in the tumour injected with CRUN were found lower (∼30% decrease in intensity) than that subjected to NCRUN , CRUN was injected into the implanted tumour on the left flank, and followed by an 808 nm laser irradiation at the tumours after 4 h injection. As contrast, group 2 (n=8) mice were intratumourally injected with the control NCRUN on the right flank and subsequent light exposure under identical conditions. Tumour-bearing mice with direct injection of CRUN but no NIR light irradiation were assigned as group 3 (n=8). The last group  consisted of mice injected with saline only. The tumour sizes in the different groups were measured to assess the PDT efficacy over a period of 2 weeks. After CRUN-mediated PDT treatment (P=0.038), the tumour growth in group 1 had been significantly inhibited as compared with that observed in group 2 with control NCRUN  and 4, the tumour sizes grew exponentially over the period of time, suggesting that the treatment with 808 nm laser only or CRUN without light irradiation induced minimum inhibition of tumour growth. To further evaluate the therapeutic efficacy, we also performed TUNEL staining on the tissue sections from different groups after 11 days of PDT treatment  spacer containing amino and thiol group at each terminus. Such FA-PEG5000-SH linkers were then coupled to enzyme-responsive CRUN conjugate through NHS-dPEG2-Mal as a linkage moiety to afford final product of FA-PEG@CRUN , to the surface of CRUN for their recognition to folate receptors that overexpress in most tumours. Meanwhile, considering the fact to enhance the biocompatibility of CRUN in living system, the FA was first coupled with polyethene glycol  and 3  were found to be reduced significantly over the therapeutic period, whereas the tumour sizes in control group 1 (n=8) with injection of saline revealed a fast growth rate. In addition, during the treatment, the tumours in group 3 displayed more effective inhibition than those in group 2. These data suggested that the facilitated tumour uptake by FA ligands and effective tumour reduction was mainly from the production of 1O2 from Ce6 anchored on the CRUN surface.Encouraged by the imaging data that revealed the effective tumour uptake of CRUN particles, we continued the light-mediated PDT therapy via intravenous injection of surface-modified CRUN conjugates or saline into six different groups of mice, and P=0.008) laser irradiation of living animals with intravenous injection of FA-PEG@CRUN resulted in comparable PDT efficiency, in which tumour growth has been greatly suppressed , the presence of pork tissue could significantly compromise tumour therapy for the mice treated with FA-PEG@CRUN and 660 nm laser excitation , further demonstrating the benefits of deep tissue penetration in 808 nm laser-mediated tumour treatment.Notably, direct 808 nm (group 3) and 660 nm  than those tumours treatment in control groups  was used to increase the tissue depth; however, the dramatic drop of therapeutic effect was observed in the similar treatment under 660 nm laser excitation  in 1 ml DMEM media which was incubated for 24 h at 37 °C. The seeded cells were subsequently treated with CRUN (50 μg ml−1) and incubated for 4 h. The cell nuclei were stained by DAPI (1 μg ml−1) for 20 min and then the cells were washed with PBS (pH 7.4) for three times. Control experiments were setup by treating the two cell lines with CRUN in the presence of CtsB inhibitor and controlled NCRUN conjugates. For the cell-inhibition assay, the cells were first pretreated with CtsB inhibitor  for 2 h at 37 °C followed by the addition of CRUN and incubated for further 4 h. Fluorescence imaging of the samples were then carried out under confocal microscopy system using a continuous-wave 980 nm laser as excitation source (EINST Technology). The quantification of CtsB enzyme expression in HT-29 and NIH/3T3 cells were performed by Human CtsB ELISA kit according to the standard procedure described by the manufacturer .The human colorectal adenocarcinoma cell line  and mouse embryonic fibroblast cell line  were provided from American type culture collection and checked for mycoplasma contamination. These cells are not listed by ICLAC as misidentified cell lines (3 October 2014). CtsB-overexpressing HT-29 cells and CtsB-deficient NIH/3T3 cells were seeded with a cell density of 1 × 106 HT-29 cells into both flanks or only right side of mouse. When the tumour volume reached a palpable size, the mouse was used for further studies.All animal experimental procedures were performed in accordance with the protocol #120774 approved by the Institutional Animal Care and Use Committee (IACUC). Female Balb/c nude mice (∼6–8-weeks old) were purchased from Charles River Laboratories . Xenograft mice models were established by injecting 0.1 ml of tumour cells suspension  containing 3 × 10in vivo PDT treatment, the tumour-bearing mice were randomized into four groups  and treated by intratumoural injection of particle samples. In group 1, CRUN  was directly injected into implanted tumours, followed by an 808 nm laser irradiation after 4 h injection. In group 2, same amounts of controlled NCRUN conjugates were injected with subsequent laser exposure. Control experiments with injection of CRUN but no NIR light irradiation (group 3) and saline with laser treatment (group 4) were used, respectively. The effective exposure time for each mouse was 45 min  with a power density at 0.4 W cm−2 . A second- and third-dose injection followed by PDT treatment described above was repeated at 4 days and 8 days after the first-dose injection, respectively. Tumour size was measured three times a week using a vernier caliper upon PDT treatment. The tumour volume was calculated using the following equation: tumour volume (V)=length × width2/2. Relative tumour volume was calculated as V/V0 .Xenograft mice models were treated when the tumour volumes approached 3–5 mm in diameter. For in vivo tumour targeting PDT treatment, the tumour-bearing mice had been randomly arranged into six different groups  to perform a series of intravenous injections of saline (group 1), PEG@CRUN (group 2) and FA-PEG@CRUN (group 3–6), respectively . After 4 h of intravenous injection, laser treatment was performed on groups 1–6 by irradiating the tumour region with a continuous 808 or 660 nm laser. The effective exposure time for each mouse was 45 min  with a power density at 0.4 W cm−2 . The tumours in group 4 and 6 were covered by 2-mm thick pork tissue and then irradiated with 808 or 660 nm lasers under the same condition. The PDT treatment with laser irradiation was repeated every 2 days. A second- and third-dose injection of the PDT treatment described above was repeated at 4 days and 8 days after the first-dose injection, respectively. Tumour size was measured and calculated as described above.Xenograft HT-29 tumour model on the right side of female Balb/c mice were first developed as described above. For How to cite this article: Ai, X. et al. In vivo covalent cross-linking of photon-converted rare-earth nanostructures for tumour localization and theranostics. Nat. Commun. 7:10432 doi: 10.1038/ncomms10432 (2016).Supplementary Figures 1-26, Supplementary Methods and Supplementary References


Fluid infusion is one of the most common critical care interventions, yet approximately 50 % of all fluid interventions are unnecessary and potentially harmful. An improved approach to identification of fluid responsiveness is of clinical importance. Currently fluid responsiveness is most frequently identified by blood pressure (BP) measurements or a surrogate. However fluid responsiveness is simply the increase in stroke volume (SV) associated with volume expansion, and may not be reflected in BP or BP surrogates. Guyton demonstrated that BP = CO x SVR, and it is know that baroreceptor mediated autonomic nervous system regulation of SV and SVR to preserve BP may mask significant and critical changes in haemodynamics. Dr Pinsky in his recent J Clin Monit Comput Editorial evaluated the relative merits of pulse pressure variability (PPV) methods, a variant on BP measurement, for assessment of fluid responsiveness and promoted the use of physiologic challenges to augment the applicability of PPV. However this guidance is only half right. This letter reminds clinicians of the physiologic limitations of PPV as a measure of fluid responsiveness, even when combined with physiologic challenges, and recommends the replacement of BP with SV measurements. The combination of accurate Doppler measurement of SV and physiologic challenges, as Dr Pinsky recommends, is a physiologically rational and effective approach to identification of fluid responsiveness with established evidence. The direct monitoring of SV and SV changes has the potential to improve a long standing critical care and anaesthetic conundrum; when to give fluid and when to stop. While we have the utmost respect for both the great baseball philosopher Yogi Berra and Michael Pinsky, it would seem that in Pinsky’s recent Editorial “It is amazing what you can see if you look” on fluid responsiveness and physiologic challenges, we have been provided with only half the clues .Dr Pinsky’s central point is that physiologic challenges improve the effectiveness and applicability of PPV monitoring for identifying fluid responsiveness, and this is incontestable. However basic physiology ultimately limits the effectiveness of PPV monitoring for identification of fluid responsiveness as Benes and others have observed , 3.This editorial raises two questions related to physiology. Does PPV approximate SVV, and is the change in PPV–dPPV, equivalent to the change in SV–dSV, the definitive measure of fluid responsiveness as demonstrated by Frank and Starling?While detection of fluid responsiveness by PPV is limited to approximately 2–51 % of ICU patients , Pinsky Despite being a common intervention and clinical conundrum, fluid responsiveness is defined quite simply; fluid responsiveness is a change in SV associated with volume expansion. Yet current methods of assessing fluid responsiveness are predominantly based on surrogates measures of BP—MAP, CVP, IVCd, RAP, PCWP and now PPV. This persistent use of BP and its analogues, such as PPV, to guide fluid therapy, may explain the persistent observation that 50 % or more of all fluid infusions are unnecessary, and potentially harmful .No matter how hard we look at BP or its pressure surrogates, with any number of trials of selected patient cohorts, using any number of devices attached to fingers, or inserted in arteries, and during whatever physiologic challenges, ultimately the baroreceptor modulated Autonomic Nervous System (ANS) ensures that we only see the BP, and not the SV response!Guyton demonstrated that BP = CO × SVR, thus any correlation between BP (PPV) and CO (SVV) is dependent on SVR remaining constant. While PPV may show some correlation with SVV and fluid responsiveness in normal stable patients, the dynamic beat to beat regulation of ventriculo-arterial coupling by the ANS precludes its application in the dynamic circulation of sick patients. BP, the product of CO(SV) and SVR, is effectively preserved in a baroreceptor determined range by the ANS, so even substantial derangements of either CO/SV or SVR/Ea, will be paradoxically regulated by the ANS to preserve BP. Especially in children, such compensation can effectively mask significant underlying abnormalities of CO/SV and or SVR which may require therapy . If BP iThis dynamic modulation of BP complicates the relationship between PPV and SVV, and prevents PPV from accurately tracking CO(SV) and SVR, and thereby identifying cardiovascular abnormalities discrete from BP. This ensures that PPV is both a poor guide for fluid responsiveness, and an ineffective guide for inotropes and vaso-active therapies.From a physiologic stand point it is elementary that the accurate measurement of SV is fundamental for the reliable identification of fluid responsiveness, and that BP measures, including PPV, are therefore only useful insofar as they correlate with SV or SVV, or those in whom ANS regulation is relatively inactive.Whilst Pinsky may challenge the retrospective methodology of Benes’ study, physiology, animal research and other clinical studies support these findings of limited PPV applicability.In 2013 Bouchacourt et al. studied dynamic arterial pressure surrogates, including PPV, to track SVV changes during progressive induced hypovolemia in a rabbit model of hemorrhage while increasing vasomotor tone with phenylephrine . They foThough PPV may have some application in detection of fluid responsiveness in normal patients, the presence of any source of disturbed or dynamic ventriculo-arterial coupling, such as arrhythmia, vaso-active therapies, irregular or complex respiration, and of course diseases with dynamic effects on cardiac and vascular function, such as sepsis, further inhibits the effectiveness and application of PPV , 7. In sThe limits of isolated pressure-based circulatory management have been further highlighted by Asfar et al.  in a recEven if PPV had some applicability for identification of fluid responsiveness, the reliability and reproducibility of methods used for PPV measurements is poor. Pinsky’s own work comparing three different arterial pulse pressure methods  concluded that although they all generally generated approximately the same mean values, they often trended in different directions and could not be used interchangeably . de WildGiven the crucial physiologic, technical and clinical limitations of PPV, why not really open our eyes, as Pinsky exhorts, and observe fluid responsiveness by directly measuring SV, and not the modulated surrogate BP? Well validated Doppler technologies accurately measure both SV and SVV, and are readily available to detect haemodynamic abnormalities in sepsis and guide fluid, inotropes and vasoactive therapies, and are noninvasive.Doppler has an in vitro accuracy of 97 % measured against ultrasonic transit time flow probes , 13. A cTransoesophageal Doppler monitoring has the best outcomes evidence of any technology for the management of perioperative fluid guidance, with a recent meta-analysis by the UK NHS Technical Adoption Centre reporting results from 10 RCTs and 2 nonrandomised outcomes trials that demonstrated improved mortality, morbidity and reduced costs using transoesophageal Doppler (CardioQ) for intra-operative fluid optimisation .Combining accurate SV measures, in place of PPV, with the physiologic challenges recommended by Pinsky, results in a potent solution to the identification of cardiovascular reserve and fluid responsiveness, even in patients in whom PPV is otherwise excluded . Thiel et al. demonstrated a positive predictive value of 91 % for detection of fluid responsiveness in unselected ICU patients using USCOM measured SV changes associated with passive leg raising. Of the 102 patients studied, 61 % were diagnosed with sepsis, where both the SV and SVR can range from high, normal to low, 18 % had cardiac arrhythmias, 56 % were on vasopressors, 34 % were spontaneously breathing (66 % ventilated)  have ideWhile we totally agree with Dr Pinsky, and Yogi Berra, that physiologic challenges are important tests to identify cardiovascular reserve, only when combined with an accurate measure of SV can we truly “see” the circulatory changes they induce. Non-invasive and accurate monitoring of SV at baseline and after intervention is the gold standard for effective and personalised haemodynamic monitoring. This physiologically rational approach is not just effective for the identification of fluid responsiveness, but also for detection of circulatory disease and guidance of inotropes and vasoactive therapies, and can be applied in adults and children across a range of diseases including hypertension, heart failure and sepsis.With regard to the application of PPV for identification of fluid responsiveness, physiology and clinical evidence are in agreement with both recommending we look elsewhere, as Benes and Mahjoub concluded. While we agree with Yogi Berra, it was Sherlock Holmes who gave us the vital next clue for improving fluid management; “Watson, You see, but you do not observe. The distinction is clear” . And it


PhylOPDb contains 74 003 25-mer probes targeting 2178 genera including Bacteria and Archaea.In recent years, high-throughput molecular tools have led to an exponential growth of available 16S rRNA gene sequences. Incorporating such data, molecular tools based on target-probe hybridization were developed to monitor microbial communities within complex environments. Unfortunately, only a few 16S rRNA gene-targeted probe collections were described. Here, we present PhylOPDb, an online resource for a comprehensive phylogenetic oligonucleotide probe database. PhylOPDb provides a convivial and easy-to-use web interface to browse both regular and explorative 16S rRNA-targeted probes. Such probes set or subset could be used to globally monitor known and unknown prokaryotic communities through various techniques including DNA microarrays, polymerase chain reaction (PCR), fluorescent Database URL:http://g2im.u-clermont1.fr/phylopdb/ Over the past decades, most promising high-throughput approaches were developed including DNA microarrays and next-generation sequencing that can also be coupled to gene capture  ribosomal RNA gene, i.e. 16S rRNA gene, is particularly well adapted to survey prokaryotic communities in complex environments, as it contains highly conserved and variable moieties permitting reliable and detailed bacterial classification. Moreover, the advent of many PCR-based approaches, as well as sequencing projects, has led to the explosion of 16S rRNA gene sequences now available in major specialized sequence repositories, such as Greengenes , SILVA  and RDP Although numerous oligonucleotide probes have been reported, available collections of rRNA-targeted oligonucleotide probes are rare. The oligonucleotide probe database (OPD)  was propBacteria and Archaea.Here, we present PhylOPDb (‘phylogenetic oligonucleotide probe database’), a comprehensive phylogenetic OPD targeting 16S rRNA gene sequences. We used two high-throughput probe design software, PhylGrid and KASpOD, to select both regular and explorative 16S rRNA gene-targeted oligonucleotide probes. PhylOPDb is composed of 74 003 probes of 25 mer targeting 2178 genera including et al.  and environmental (ENV) divisions of the EMBL nucleotide sequence database were used as a reference to build this database. First, 16S rDNA gene sequences were extracted and filtered according to their quality and size. Only sequences that met the following criteria were kept: (i) sequence length is between 1200 and 1600 nucleotides, (ii) sequence is assigned to the genus level in EMBL database, (iii) the percentage of ambiguous nucleotides is <1% and (iv) the maximum number of consecutive unknown bases must not exceed five. Then, extracted sequences were grouped at the genus taxonomic rank according to the NCBI taxonomy database. For each genus, sequence orientation of all sequences was checked using BLASTN  and reduUsing this custom 16S rRNA gene sequences database, a total of 3 553 975 degenerate probes of 25 mer were selected. The probe length of 25 nucleotides offers the balance of highest sensitivity and specificity in the presence of a complex background , 13. CovFive non-overlapping probes showing the best specificity and coverage were then selected for each genus. During this step, some cross-hybridizing probes may have been selected. Nevertheless, the program ensured that the simultaneous analysis of the five selected probes could not cause misleading interpretations of hybridization data. A set of 19 874 25-mer probes corresponding to 10 320 degenerate probes was obtained.k-mer–based software dedicated to the design of group-covering oligonucleotide probes. It allows selecting highly specific and explorative probes based on large data sets , was implemented using PHP, HTML5, CSS3, JavaScript, jQuery, JSON and MySQL. PhylOPDb will be updated annually adding newly designed probes, removing deprecated probes and re-computing coverage and specificity. Moreover, the 16S rDNA sequences databases used for the probe design can be downloaded through the PhylOPDb web interface.To make all the phylogenetic oligonucleotide probes easily available, we developed a web interface  to fetchBased on the EMBL taxonomy, the PhylOPDb web interface provides a hierarchical browse of the database contents. When a taxonomic group is selected, corresponding to a kingdom, phylum, class, order, family or genus, the oligonucleotide probes of this group are then displayed. Selected probes can be downloaded both in CSV and FASTA formats. Furthermore, for each non-degenerate probe, associated information is given: identifier, genus name, sequence, length, corresponding degenerate probe, degeneracy, coverage percentage, position according to the consensus sequence of the genus, putative cross-hybridizations, melting temperature and the probe design tool used.Probes can also be obtained using a rapid search by keywords that can represent a part of the sequence of a probe, a genus name or a design name (PhylGrid or KASpOD). Only probes that match these keywords are then displayed. Probes can also be fetched through an advanced search using multiple criteria .Over the past decades, high-throughput molecular tools have opened an unprecedented opportunity for microbiology by enabling the culture-independent genetic study of complex microbial communities, which were so far largely unknown. Among these tools, environmental microarrays, including POAs, are key technologies that are well adapted to profiling environmental communities , 24–29. PhylOPDb is also well adapted for other molecular tools using primers or probes  with the availability of group-specific signatures. One of the goals of our database is to exhaustively provide the most specific probes or primers at a fine phylogenetic level. When biologists are interested in specific microbial taxa, it is difficult to reveal them using ‘universal’ probes or primers. For such biological applications, we consider that our database will be helpful. Furthermore, thermodynamics of nucleic acids hybridizations is not fully understood, molecular approaches such as FISH, PCR, DNA microarrays and gene capture are still empiric and need biological validations. With our complete database, biologists could test various probes or primers to select the most adapted to answer their biological questions. To reduce the complexity of probes and primers selection, we indicate in the database degenerate signatures from which specific probes and primers are deduced. The problem of probes accessibility is particularly relevant for the FISH approach. However, it is possible to resolve this difficulty using helper oligonucleotides, as previously described . Recentlin silico approaches for predicting the hybridization behaviour of microarray probes are limited: the only solution is to perform an extensive empirical testing of the probes . Thus, additional probes specific to the targeted environments could be defined. Furthermore, specificity tests will be performed against reduced database limiting computational resources needs.Probes provided through the PhylOPDb web interface were designed at the genus level but it would be interesting for some purposes such as PCR-based analyses to define probes at different phylogenetic levels. In addition, the current probe collection of the PhylOPDb will be extended to include 18S rRNA gene-targeted oligonucleotide for investigating fungal species within complex environments, where they may play crucial role.French ‘Direction Générale de l’Armement’ (DGA) and the program Investissements d'avenir AMI 2011 VALTEX. Funding for open access charge: LIMOS, Clermont Université.Auvergne Regional Council, the European Regional Development Fund, the Conflict of interest: None declared.


When one liquid is introduced into another immiscible one, it ultimately fragments due to hydrodynamic instability. In contrast to neck pinch-off without external actuation, the viscous two-fluid system subjected to an oscillatory flow demonstrates higher efficiency in breaking fluid threads. However, the underlying dynamics of this process is less well understood. Here we show that the neck-thinning rate is accelerated by the amplitude of oscillation. By simply evaluating the momentum transfer from external actuation, we derive a dimensionless pre-factor to quantify the accelerated pinch-off. Our data ascribes the acceleration to the non-negligible inner fluid inertia, which neutralizes the inner phase viscous stress that retards the pinch-off. Moreover, we characterize an equivalent neck-thinning behavior between an actuated system and its unactuated counterpart with decreased viscosity ratio. Finally, we demonstrate that oscillation is capable of modulating satellite droplet formation by shifting the pinch-off location. Our study would be useful for manipulating fluids at microscale by external forcing. In many natural systems, coordination between dynamic and geometrical parameters plays a crucial role in retaining the reliability and efficiency for system performance. For example, a beating heart expands and contracts periodically to pump blood through vessels467Rmin obeys scaling laws of α determined by the asymptotic force balances under various circumstancestc is the critical time at neck pinch-off and t is the time. Unlike pinch-off in air, viscous effect of the surrounding liquid is non-negligible in viscous two-fluid systemsη, ρ and γ represent respectively the dynamic viscosity, volumetric density and interfacial tension, and subscript i and o stand for inner and outer phase, respectively. In Stokes regime, capillary effect is counteracted by inner and outer viscous dissipations, while fluid inertia is negligible, which gives a linear scaling of Rmin with α = 11617λ (λ = ηi/ηo) of the two-fluid system161718Rmin < LT20kb and T being Boltzmann’s constant and temperature, respectively. The neck profile is symmetric and the liquid-liquid interface is rough due to the effects of thermal fluctuations. Both experimental20α ≈ 0.42 for thermal-fluctuation regime. However, LT is usually nanometric for simple liquids, where the effects of thermal fluctuations are hardly to be observed at laboratory-scale. As such, Stokes flow is the final asymptotic regime in viscous two-fluid pinch-off, provided the molecular scales are not reached.The dynamics of Newtonian threads rupture without external forcing have been well established for both liquid-in-air and two-fluid systemsRmin still obeys the scaling law C is the proportionality factor), the neck thinning velocity gives C or the exponent α would induce variations in the dynamics of droplet pinch-off, and thus the neck thinning velocity Vr. Nevertheless, knowledge of pinch-off in microfluidics subjected to external forcing is limited, leaving several questions unaddressed like whether the external forcing would affect C or α or both and to what extent, and how it varies satellite droplet formation.Except for the pinch-off of complex liquids in air232528293132336α = 1). However, the neck thinning velocity Vr is accelerated by the oscillation amplitude compared to that without oscillation . In the operation window of Qo and Qi, only dripping was observed, no jetting occurred. A mechanical vibrator  perturbed the inner fluid microtubing  in the gravitational direction with a displacement of L  and thus prevents the external perturbation from growing . Captured images and videos were analyzed by ImageJ.We conducted experiments in a co-flow capillary microfluidic device that was fabricated by aligning one round glass capillary with a taped nozzle (outer diameter of the nozzle e device . The disevice (L  between ηo = 492.875 mPa s), and inner fluids were various glycerol-water mixtures (viscosity: 0.992 mPa s ≤ ηi ≤ 831.07 mPa s). This choice of two-fluid systems enables a wide range of viscosity ratios (10−3 < λ < 10), but narrow variations in interfacial tension (24.2 mN m−1 ≤ γ ≤ 32 mN m−1) and fluid density (0.998 g cm−3 ≤ ρi ≤ 1.261 g cm−3), so as to isolate the effect of viscosity ratio on pinch-off dynamics. Viscosity was measured by a viscometer , interfacial tension by a ring tensiometer , and density by quantifying the volume of a known-mass fluid.Water-in-oil two-phase flow was examined in experiments, where outer fluid was silicone oil , ηi is the viscosity of inner fluid, Pp and Pn are pressures respectively at the perturbing location and the nozzle, and L is the length between the two locations. Based on Qi, we evaluate the steady ejection velocity of the inner fluid as,where Pp into the time-dependent Pp(t),For perturbed case, we simply assume that the gravitational displacement of ρi and the gravitational acceleration being g. Since the linear pinching time-scale tl , we assume that the Poiseuille’s law is still valid to estimate the fluctuating inner flow rate Qi(t) in the linear pinch-off regime. When Pp is replaced by Pp(t) in Qi(t) as a function of time t,with inner fluid density being Vn discharged from the nozzle is approximated to be Now the oscillatory velocity where Vn, such as shaking and deformation of the microtubing, and fluid acceleration in the microtubing, the simplified form of Qi(t) in Vn in ε0, in association with material properties (ρi and ηi) and geometrical parameters (Riw and L), modulates the ejection velocity. We verify Although we ignore several effects that might influence the transient velocity λ. Keeping silicone oil as the outer fluid, and choosing various glycerol-water mixtures as the inner fluid, we can tune viscosity ratio λ over three magnitudes (10−3 < λ < 10). Fluid system with smaller λ means less viscous inner fluid in the context, and vice versa. Dmin (Dmin = 2Rmin) versus time τ  for different viscosity ratios λ. Fluid system with smaller viscosity ratio pinches off faster , outer radial viscous stress Fλ) under periodic perturbation. When undergoing pinch-off subjected to an oscillatory flow, the droplet initially grows in its size until the neck appears. Afterwards, a sudden suction of the inner fluid, caused by the pulsatile velocity Vn .Now we consider the fluid system with fixed viscosity ratio . As a rinch-off . The necitude ε0 . However≤ 100 Hz , probablDminversus the product of time τ and the dimensionless factor ε.We now quantify the acceleration of perturbed pinch-off by estimating the extra momentum contribution from mechanical vibration. After simply calculating the root mean square of the fluctuation term in Eq. (5), inch-off . The enhε0, but ε, in that larger enhancement of pinch-off is observed for fluid system with the same ε0 but smaller viscosity ratio λ . Therefore, In subsequent tests, we focus on the influence of viscosity ratio on fluid systems with oscillation. In comparison to unactuated case, the oscillation-enhanced pinch-off has a faster neck thinning rate for all fluid systems examined, as shown in as shown in τ > 1 ms  by impacting the transient local force balance.Having quantified the accelerated pinch-off, we now explore the origin of the acceleration in pinch-off with oscillatory flow. For conventional pinch-off scenario, two-fluid Stokes flow dominates near the singularity. However, oscillation modifies the force balance via disturbing the flow velocity, therefore affecting the whole pinch-off dynamics. A recent study proposed that, when pinching in air, fluid thread passes through a plethora of transient regimes before transferring into the final inertial-viscous regimeτ > 1 ms , especiaRmin and length z thins radially with velocity Vr, while fluid flows axially out of the thread with velocity u. Local volume-flux conservation requires u is thus estimated as Vr is accelerated by the oscillation amplitude, u increases with ε, so does the inner axial fluid inertia Re in the neck for perturbed and unperturbed cases, respectively. Having Re versus time τ during neck pinching (λ = 0.0387). Close to pinch-off, a sharp increase of Re occurs for the perturbed neck, while undisturbed Re varies more gently. The increased Re indicates an undoubted increase of inner fluid inertia due to perturbation. It is now probably invalid to ignore the effects of oscillation-enhanced fluid inertia in determining the neck thinning dynamics. Actually, the increased fluid inertia neutralizes the inner fluid viscous dissipation.We hypothesize that oscillation amplifies the effects of axial inner fluid inertia. During pinch-off in Stokes regime, inner fluid thread of radius ηi . Preliminary, an observation . This can be done by calculating λ = 0.157 perturbed by ε0 = 3 mm for example, we find out λ1 = 0.1305. Experiments show that, close to pinch-off, the perturbed system with λ = 0.157 pinches equivalently to the undisturbed one with λ1 = 0.1305  before pinch-off. Two boundaries are emphasized in Z0 bifurcates, pentagon) and pinch-off at the nozzle . The transition of pinch-off location is the result of momentum transport. Because axial velocity u of inner fluid scales as ε. Experimentally, we find that, in the tested range of viscosity ratios, εc = 0.110 and εc = 0.225 .


Chikungunya virus (CHIKV) and o’nyong nyong virus (ONNV) are mosquito-borne alphaviruses endemic in East Africa that cause acute febrile illness and arthritis. The objectives of this study were to measure the seroprevalence of CHIKV and ONNV in coastal Kenya and link it to demographics and other risk factors.Demographic and exposure questionnaires were administered to 1,848 participants recruited from two village clusters  in 2009. Sera were tested for alphavirus exposure using standardized CHIKV IgG ELISA protocols and confirmed with plaque reduction neutralization tests (PRNT). Logistic regression models were used to determine the variables associated with seropositivity. Weighted K test for global clustering of houses with alphavirus positive participants was performed for distance ranges of 50–1,000 meters, and G* statistic and kernel density mapping were used to identify locations of higher seroprevalence.486 (26%) participants were seropositive by IgG ELISA. Of 443 PRNT confirmed positives, 25 samples (6%) were CHIKV+, 250 samples (56%) were ONNV+, and 168 samples (38%) had high titers for both. Age was significantly associated with seropositivity ; however, younger adults were more likely to be seropositive than older adults. Males were less likely to be seropositive . Adults who owned a bicycle  or motor vehicle  were more likely to be seropositive. Spatial analysis demonstrated hotspots of transmission within each village and clustering among local households in Milalani-Nganja, peaking at the 200–500m range.Alphavirus exposure, particularly ONNV exposure, is common in coastal Kenya with ongoing interepidemic transmission of both ONNV and CHIKV. Women and adults were more likely to be seropositive. Household location may be a defining factor for the ecology of alphaviral transmission in this region. Alphaviruses, such as chikungunya and o’nyong nyong viruses, are likely important causes of human disease in endemic regions, but are often misdiagnosed as malaria in the acute care setting. Our objective was to uncover the burden of alphavirus exposure in our study region, rural coastal Kenya. Of 1848 participants tested, 26% were seropositive by screening ELISA, demonstrating intense transmission to humans in this area. Surprisingly, confirmatory PRNT testing revealed that the majority of alphavirus exposures were due to o’nyong nyong virus, rather than chikungunya virus. Both CHIKV and ONNV antibodies were confirmed in young children, demonstrating undocumented and ongoing transmission in this region. Of the examined risk factors, older age and female gender were associated with alphavirus seropositivity. Alphaviruses are endemic to many regions of Kenya; however, due to limited surveillance and documentation during and in between known outbreaks, the alphaviral burden is yet to be fully recognized. O’nyong-nyong virus (ONNV) and chikungunya virus (CHIKV) are closely related alphaviruses in the Semliki Forest antigenic complex . CHIKV wBoth CHIKV and ONNV cause febrile illness in humans. Clinically, the symptoms of CHIKV are difficult to distinguish from those of dengue fever , 8. BecaWhile there is growing research interest in CHIKV as it spreads within Europe and into the Americas, the role of ONNV in endemic regions, especially in sub-Saharan Africa, remains unclear despite its close relationship to CHIKV . A smallAll participants in this study consented to participation in their own language. Written consent was obtained from all adult participants; children provided assent with parental written consent. For the parent study of community infectious burden, IRB protocols were approved by Case Western Reserve University (protocol # 11–07–42) and the Kenya Medical Research Institute (SSC 2611). In addition, for this study, IRB approval was obtained from Children’s Hospital & Research Center Oakland (CHRCO).This study was performed using banked serum specimens from an NIH-funded study on polyparasitism in coastal Kenya , as described by Bustinduy and Bisanzio , 19. In Vero81 cells were infected with CHIKV strain 181/25 at an MOI = 0.01 in a 30 ml volume in T175 flasks and the infection was allowed to progress 48 hours until the monolayer was destroyed. The supernatant was harvested from flasks and centrifuged at 3000 rpm at 4°C for 20 minutes. A 40 ml aliquot of the infected cell supernatant was added to one of four new T175 tissue culture flasks, followed by addition of 1.3 ml binary ethylenimine (BEI) to a final concentration of 3 mM to inactivate the virus [Sera were separated from whole blood specimens and screened for anti-alphaviral IgG antibodies using a direct enzyme-linked immunosorbent assay (ELISA) with CHIKV 181/25 antigen. Briefly, 96-well polystyrene ELISA plates were coated with 1ug/ml of vaccine strain 181/ 25 CHIKV antigen (see above for production), and were held overnight at 4°C. After four washes with phosphate-buffered saline (PBS) with 0.05% Tween-20 (PBS-T), non-specific binding was blocked by adding 5% non-fat dry milk in PBS with 0.5% Tween-20 and plates were incubated for 60 minutes at 37°C. Sample sera were added at a 1:100 dilution and plates were incubated for 60 minutes at 37°C. Each serum sample was run in duplicate. A 1:1,000 dilution of conjugated goat anti-human IgG  was used as the second antibody. Plates were incubated for 60 minutes at 37°C. Presence of CHIKV antibodies was detected by adding alkaline phosphatase substrate (Sigma Aldrich), and the absorbance was read at 405 nm. Positive samples required a mean optical density (OD) value ≥ 0.2 above that of the negative control for each ELISA plate. The range of OD values for positive serum specimens in this study was 0.3–1.5 above that of the negative control. Positive controls were obtained by creating a pool of sera that had tested positive by ELISA and concurrently confirmed positive by plaque reduction neutralization testing (PRNT), as detailed below. Negative controls were obtained by creating a pool of sera that had tested negative for alphavirus exposure by ELISA and PRNT.80) in the number of plaques. Prevalence results reported in this analysis are based on PRNT results. Samples were considered CHIKV PRNT positive if the titer was >20 and the ONNV titer was < four-fold lower than the CHIKV titer. Because there is a unique one-way antigenic cross-reactivity between CHIKV and ONNV [A subset of samples  was subjected to PRNT confirmatory testing to verify ELISA screening results and determine whether seropositivity was due to CHIKV or ONNV infection. Briefly, the samples were heat-inactivated for 30 min at 56°C. Using 96 well cell culture cluster plates  samples were serially diluted two-fold in Dulbecco’s Minimum Essential Medium supplemented with 10% FBS, 100 U/ml of penicillin, and 100 mg/ml of streptomycin  for a final volume of 60 ul per well. A suspension of 50 PFU/60 uL (CHIKV COM125 strain) was then mixed 1:1 with the diluted serum samples and the mixture incubated for 1 h at 37°C. The serum-virus suspension was then transferred onto 12-well cell culture plates  containing a confluent monolayer of Vero cells. The virus/cell mixture was incubated for 1 h at 37°C with the plates agitated every 15 min. Each well was then overlaid with a 0.4% Genepure LE agarose/DMEM medium layer  and plates were incubated at 37°C for 48h for CHIKV and 72h for ONNV. The agarose layer was removed and the wells were covered with a fixative/staining solution  for 10 min. Plates were then rinsed with water. Plaques were counted and titers were calculated and expressed as the reciprocal of serum dilution yielding a >80% reduction .Fingerprick specimens for all subjects were tested for Descriptive analyses on the demographic and household variables were conducted on the sample across the villages and for different age groups. Children were defined as those being less than 16 years of age, while adults were those individuals that were 16 years of age or older. Analyses included means, measures of variability, proportions and confidence intervals. Human alphavirus seropositivity was the principal outcome measure for the study. Initial exploratory assessment of differences in categorical measures with alphaviral seropositivity was done using chi–square or Fisher’s exact test. We used Student’s t-test for comparisons of continuous measures. Logistic regression models were used as the primary analyses with the binary outcome of alphavirus infection (seropositive vs seronegative). These models adjusted for the differences between villages . A significance level of 0.05 was used for all statistical tests. Subset analysis was performed for the children (under 16 years) and adults. The statistical software package SAS v 9.3  was used to analyze the data. The unweighted and weighted K test  and kernel density mapping were performed for distance ranges of 50–1,000 meters using ArcMap 10.1 . Getis G* statistic testing, with correction for multiple comparisons, was performed using ClusterSeer .Of the 1864 samples initially tested for anti-alphavirus IgG antibodies by ELISA, 413 (22%) were positive, 134 (7%) were equivocal and 1317 (70%) were negative. Ninety percent (371/413) of ELISA positives, eighty seven percent (117/134) of ELISA equivocal and 4.5 percent (59/1317) of ELISA negatives were PRNT tested. In the 83 cases in which the PRNT result differed from the ELISA result, the PRNT result was used as the final result and the sixteen equivocal samples that were not PRNT tested were removed from the final analysis. Eighty four percent (313/371) of ELISA positive, 89% (105/117) of ELISA equivocal and 42% (25/59) of ELISA negative were confirmed positive by PRNT. In our final analysis, 486 out of 1848  participants were positive for anti-alphavirus IgG antibodies by ELISA and 443 were confirmed positive by PRNT . PRNT daTwo hundred seventy (56%) alphavirus-exposed participants were from Milalani-Nganja. Using analysis by location, village of residence was not significantly associated with seropositivity (P = 0.458). 25.4% (95% CI 22.5%–28.5%) of Vuga participants were seropositive vs. 27.0% (95% CI 24.3–29.9%) in Milalani-Nganja. Three hundred fifty-three (72%) of seropositives were adults. Age was significantly associated with seropositivity . Mean age of alphavirus-exposed subjects was 27 years (range 1–79 y). Males were less likely to be seropositive . Adults who owned a bicycle  or motor vehicle  were more likely to be seropositive . In multIn children , the only variable significantly associated with seropositivity was age (p<0.001), with older children being more likely to be seropositive. Among adults (N = 1139), female gender was associated with seropositivity . Age was also associated with seropositivity within the adult subgroup, however in this case it was younger adults who were more likely to be seropositive than older adults .Of 443 PRNT confirmed positives, 250 samples  were positive for ONNV, 25 samples  were positive for CHIKV, and 168 samples  had high PRNT titers for both CHIKV and ONNV. A positive CHIKV test was more likely to be from Vuga , whereas a positive ONNV test was more likely to be from Mililani-Nganja , though this was not statistically significant. If a more conservative 4-fold titer cutoff was used instead of 2-fold cutoff to designate ONNV antibody positivity, 37% and 57% would be considered ONNV and undetermined alphavirus antibody positive, respectively. PRNT titer to CHIKV or ONNV did not drop over time until after age 70 years .Anopheles spp. mosquitoes.For those who were ONNV antibody positive (N = 250), mean age was 28.4 years (range 2–75y) and the majority (75%) were adults. Fifty-eight percent of ONNV-exposed participants came from Milalani-Nganja, and 64% were female. For those who were CHIKV antibody positive (N = 25), mean age was 21.7 years (range 7–54y) and the majority (75%) were adults. Only 24% of CHIKV-exposed participants came from Milalani-Nganja and 70% were female. The mean age of the undetermined group was 27.9 years (range 1–79y). We did not see a significant association between current malaria and ONNV exposure in our study subjects despite being likely transmitted by the same Geographic Information Systems (GIS)-based spatial analysis Figs.  and 5 waAlphavirus exposure, particularly ONNV exposure, was found to be common in coastal Kenya, despite little previous public health attention or research. Evidence of high transmission rates for ONNV was unexpected, given that the last known outbreak in this region was in 1961. The fact that multiple children and young adults were exposed shows that ONNV transmission has occurred in the past five years and remains undetected or poorly reported within the clinical setting. Not surprisingly, children were less likely to be seropositive than adults, likely due to fewer years of mosquito exposure. This suggests that there is endemic circulation with additive risk for exposure over time. Interestingly, younger adults were more likely to be exposed than older adults in this study; however, we did not find evidence that older adults were losing their titers over time . These tAedes are blood seeking, while men are in the fields, tending to animal herds, or away from home earning livelihoods in more urban areas. There was no significant difference in seroprevalence between those who slept under a bed net and those who did not, even though the majority of alphaviral infections we identified were ONNV which, unlike CHIKV, is spread by night-feeding Anopheles spp. Although ONNV is transmitted by the malaria vector, Anopheles gambiae [prior ONNV exposure via IgG antibody testing, whereas active P. falciparum infection was documented by circulating antigen testing and this may explain the lack of association.Women were more likely to be exposed to alphaviruses in this study. The epidemic and urban vectors for CHIKV are highly anthropophilic and are daytime or crepuscular feeders. It may be that because of cultural roles in this region, women in this community are more likely to stay around the homestead where  gambiae , we did Aedes and Anopheles habitats and insect abundance. Although we did not include common daytime gathering points in our local cluster analysis, our spatial analysis suggests that household location is one of the defining factors for CHIKV and ONNV transmission in these villages. Data from Vuga showed local but not global clustering, suggesting that the household factor in Vuga might be less influential than exposure in a shared, more central location. Adults who owned a bicycle or motor vehicle were more likely to be seropositive and this may signal that greater mobility adds a greater level of alphaviral exposure, probably beyond the household/village landscape that was assessed. Individual risk for alphaviral exposure likely includes both household and daytime exposures.Spatial analysis demonstrated global clustering of positive homesteads in the larger Milalani-Nganja complex of villages, but not in the smaller Vuga community. Local clustering analysis further identified ‘hotspots’ and ‘coldspots’ of exposure within each village. Where observed, global clustering was most prominent at the 250–400m ranges, further suggesting that there are local factors that influence Many of our samples had high titer (≥20) to both ONNV and CHIKV by PRNT. The presence of antibody titers against both viruses could be due to several factors: 1) both viruses could be co-circulating in the region, 2) continued evolution of ONNV has made it antigenically more similar to CHIKV than previously reported resulting in greater cross neutralization (21), 3) another alphavirus that induces antibodies that cross-neutralize both CHIKV and ONNV could be circulating in the area. Individuals who had high titer to both CHIKV and ONNV were relatively young (mean age 28 y), which makes double exposure to both viruses less likely and suggests circulation of another cross-reactive alphavirus or an evolved ONNV lineage. Determination of the exact cause of comparable titers against both viruses requires additional studies with isolation of virus for biological characterization.Our study has several limitations. We used biobanked sera taken as part of a separate study on parasites; therefore we do not have information on mosquito avoidance or exposures in our participants. We were unable to trap vectors for viral testing in this study and so cannot associate vector type abundance or mosquito infection rates with human data. Due to cost, time, and labor considerations, PRNT could not be performed on the entire study sample set (547 of 1862 samples were tested). As expected, the screening ELISA had several false positives and false negatives detected by PRNT testing, so there may have been misclassification of some participants in our overall survey sample, which would bias our results to underestimation of prevalence: if the 42% false negative rate was applied to our total sample set, our alphaviral prevalence estimates would become 52% (966/1864). Nonspecific false positives from sticky serum can occur, but should have been corrected by PRNT testing. Also some ELISA positive individuals who were PRNT negative may not truly be false positives but may have been exposed but not mounted neutralizing antibodies responses. However, because of the robust size of our sample, we feel that our conclusions are well supported. Finally, a large subset of PRNT positives (38%) could not distinguish between CHIKV and ONNV exposure, which raises the question whether another cross-reacting alphavirus might be co-circulating .Acute disease due to CHIKV or ONNV is very infrequently diagnosed in this region of Kenya, as most febrile illness is thought to be due to malaria (9). Given that our data suggests at least 26% (486/1848) of the population is exposed to CHIKV or ONNV, it is likely that a substantial proportion of febrile disease due to alphaviruses is being missed in this clinical setting. CHIKV infected patients have been shown to be more likely to receive inappropriate antibacterial or antimalarial therapy than other febrile patients (10). This has important implications for individual disease management and prevention, and for the emergence of antibacterial or antimalarial drug resistance.In conclusion, CHIKV and ONNV infections were found to be common in coastal Kenya. It is likely that both CHIKV and ONNV infections are causing human disease that is going undiagnosed in this area. Unrecognized ONNV transmission, in particular, has been ongoing and underappreciated in this region. Among local residents, women and adults were found to be more likely to have been exposed. Finally, household location may be one of the defining factors for the ecology of alphaviral transmission in this region, given our spatial analysis results, and the fact that anophelines transmit ONNV and are associated with biting in homes.S1 Fig(DOC)Click here for additional data file.S1 Data set(XLS)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.


Perioperative hypovolemia and fluid overload have effects on both complications following surgery and on patient survival. Therefore, the administration of intravenous fluids before, during, and after surgery at the right time and in the right amounts is of great importance. This review aims to analyze the literature concerning perioperative fluid therapy in abdominal surgery and to provide evidence-based recommendations for clinical practice.Preoperative oral or intravenous administration of carbohydrate containing fluids has been shown to improve postoperative well-being and muscular strength and to reduce insulin resistance. Hence, the intake of fluid (preferably containing carbohydrates) should be encouraged up to 2 h prior to surgery in order to avoid dehydration. Excessive intravenous fluid administration adds to tissue inflammation and edema formation, thereby compromising tissue healing.During major abdominal surgery a “zero-balance” intraoperative fluid strategy aims at avoiding fluid overload  as well as goal-directed fluid therapy (GDT). Both proved to significantly reduce postoperative complications when compared to “standard fluid therapy”. Trials comparing “restrictive” or zero-balance and GDT have shown equal results, as long as fluid overload is avoided in the GDT group as well .It is possible that high-risk surgical patients, such as those undergoing acute surgery, may benefit from the continuous monitoring of circulatory status that the GDT provides. Data on this group of patients is not available at present, but trials are ongoing.In elective surgery, the zero-balance approach has shown to reduce postoperative complications and is easily applied for most patients. It is less expensive and simpler than the zero-balance GDT approach and therefore recommended in this review. In outpatient surgery, 1–2 L of balanced crystalloids reduces postoperative nausea and vomiting and improves well-being. Intravenous fluid therapy is an integrated and lifesaving part of the treatment of patients undergoing surgery. Hypovolemia leads to insufficient circulation with decreased oxygen delivery to organs and peripheral tissues causing organ dysfunction and shock. Fluid overload, on the other hand, leads to interstitial edema and local inflammation and impairs the regeneration of collagen, thereby weakening the tissue healing with increased risk of postoperative wound infections, wound rupture, and anastomotic leakage. Moreover, it causes impaired cardiopulmonary function –14. It iMaintain or correct fluid balance Maintain or correct plasma constitution (electrolytes)Secure sufficient circulation (in combination with vasoactive and/or cardioactive substances)Secure sufficient oxygen delivery to organs (in combination with oxygen therapy)The goal of perioperative intravenous fluid therapy is to maintain or restore circulation with an adequate fluid and electrolyte balance, thereby creating the preconditions for a favorable outcome for the patient. Hence, the goals of perioperative fluid therapy can be summarized as follows:In daily clinical practice, fluid therapy is guided by knowledge of basic physiological needs and simple cardiovascular measurements as well as the monitoring of the renal function by urine output. However, parameters such as mean arterial pressure (MAP), heart rate (HR), and diuresis are affected by variables not related to the circulatory status, including pain, body temperature, and physiological and psychological stress, as well as anesthetic and analgesic drugs, etc. These parameters are therefore imprecise in the measurement of intravascular status. The blood volume has to decrease by approximately 20 % before hypovolemia is detected, and fluid overload does not change blood pressure or HR at all in patients without heart failure. Therefore, using these parameters, fluid overload is invisible for the treating clinician giving intravenous fluid therapy to surgical patients, and its deleterious effects only become apparent in clinical trials avoiding fluid overload.Consequently, the use of central cardiovascular measurements such as stroke volume or functional parameters  is recommended for the monitoring of circulatory status  to secure sufficient circulation and avoid fluid overload, with or without the simultaneous use of “zero-balance” or “restricted” fluid therapy.The aim of this review is to analyze the literature concerning perioperative fluid therapy in abdominal surgery and to provide evidence-based recommendations for clinical practice.Fasting prior to surgery is mandatory to avoid aspiration of stomach content to the lungs. Six hours fasting from food and 2 h from liquids is generally recommended, and the patient should be encouraged to minimize the fasting period, thus avoiding dehydration .Carbohydrates given orally or intravenously have been shown to improve postoperative well-being and muscular strength and to attenuate insulin resistance, the latter being correlated to prolonged length of hospital stay –20. For Jacob et al. show that a prolonged fasting period is unlikely to affect cardiopulmonary function and cause hypovolemia in healthy patients . Thus, fMechanical bowel preparation prior to surgery has been argued to reduce postoperative leakage and infection. However, the benefit of the procedure has not been shown despite systematic review of the literature in a Cochrane review . In addiLoss of fluid and electrolytes occurs continuously and has to be replaced to maintain homoeostasis. However, replacement regimes vary considerably within studies and unclear categorizations of perioperative fluid therapy as restrictive, conventional, or liberal creates confusion. In the earliest original papers testing the so-called restricted fluid therapy, the fluid regimen was in fact aiming at zero-balance measured as zero body-weight gain, thus, avoiding fluid overload. Therefore, the more descriptive term zero-balance is used in more recent papers from the same authors, as well as in this article. To ensure an optimal and adequate fluid replacement therapy, knowledge of physiological fluid turnover is fundamental.insensible perspiration (evaporation from the skin and the airways—the only loss of pure water from the body) in different circumstances. In 1977, Lamke et al. used a special chamber to measure the water content in the air layer immediately adjacent to the skin in four different zones of the body of adult healthy volunteers. They found insensible perspiration to be approximately 0.3 mL/kg/h [Several investigators have measured  mL/kg/h . Reithne mL/kg/h –27. Duri mL/kg/h . HoweverSensible perspiration is visible sweat consisting of salt and water. The volume varies considerably depending on the surrounding temperature and physiological stress. Lamke et al. estimated visible sweat in patients with a rectal temperature above 39.5 °C to account for 600 mL/day (0.3 mL/kg/h). However, fever and sweating were occasional and only present for 6 h/day [ 6 h/day . In a clDiuresis is affected by a variety of factors including blood pressure, fluid intake, stress response , surgical trauma, and anesthesia. Thus, diuresis reflects many other things than the renal ability to secrete fluid and osmotic components. Urinary output is therefore unreliable as a marker for intravascular fluid status and does not show the adequacy of the fluid therapy in the perioperative setting [ setting .The expected diuresis for postsurgical patients varies in different countries, but a diuresis of 0.5–1.0 mL/kg/h is generally recommended. In several studies, the allowance of perioperative diuresis of 0.5 mL/kg/h in combination with a judicious fluid therapy has been shown to reduce postoperative morbidity .When healthy individuals experience considerable thirst, the kidneys can concentrate urine to approximately 1200 mOsm/L and sodium in an amount of 300 mmol/L urine . The clievaporation from the surgical wound. They used a chamber to cover the wound and the exteriorized viscera and found an evaporative loss correlating to the size of incision ranging from 2.1 g/h in minor wounds with slightly exposed viscera, up to 32 g/h in major wounds with completely exposed viscera [Lamke et al. have measured the  viscera . An addi viscera .The evaporative fluid loss during laparoscopic surgery is considered small, yet dry air is insufflated into the abdomen with an unknown turnover. At present, evaporative loss during laparoscopic surgery is completely unknown.It has been argued that surgical trauma leads to a shift of fluid volume between the fluid compartments of the body, creating a loss of extracellular fluid to a nonanatomical compartment named “the third space”.This has led to the recommendation of giving up to 15 mL/kg/h the first hour of surgery and thereafter declining amounts of fluid in accordance with algorithms.However, having reviewed the literature, this hypothesis is based on few studies using one specific but flawed method of measurement of the extracellular volume. More recent studies using sounder methods cannot demonstrate any such fluid loss. The entire concept of a loss to the third space should therefore be abandoned , 36.Surgical trauma, however, does create an edema in the traumatized tissue as demonstrated by Chan et al. in 1983. They showed that the formation of a small bowel anastomosis in rabbits caused an increase in tissue weight of 5–10 %, due to fluid accumulation. Supplementary intravenous crystalloid infusion of 5 mL/kg/h doubled the edema and destabilized the anastomosis .Transferring these findings to a clinical setting, a hypothetic manipulation of the entire colon (approximately 3 kg) results in water accumulation in the tissue of about 150–300 mL. Substituting this volume, additional edema formation appears, compromising the healing of anastomosis and increasing the risk of leakage . MoreoveNoblett et al. randomized 108 patients undergoing colorectal resection to intraoperative GDT compared to standard fluid therapy (3638 mL vs. 3834 mL) and showed that GDT significantly reduced interleukin 6 levels. This indicates that through securing splanchnic circulation by GDT, a reduction of the systemic inflammatory response due to surgical trauma was achieved . In addiThe balance between sustaining intravascular volume and avoiding extravascular fluid accumulation is delicate. Lobo et al. infused 1 L saline and demonstrated that 68 % had escaped from the intravascular space 1 h after the infusion, compared to 16 % after the infusion of 1 L colloid . LikewisAcetated or lactated Ringers solutions are originally developed from the plasma of amphibians, but are closer to the composition of human plasma than saline. It contains less chloride than saline (100 mmol vs. 154 mmol) but still 140 mmol of sodium. Even though chloride causes hyperchloremic acidosis if given in excess amounts, the importance of sodium in the development of postoperative edema is unknown.The use of colloids for stroke volume optimizing regimes has been shown to reduce postoperative complications , 40–42. Interstitial edema following intravenous fluid administration is formed and sustained as a result of osmotic forces and caused by the diffusion of osmotic active components, primarily excessive sodium and chloride infusion. This means that interstitial edema is not caused by excess of water (hydra = water) but “excess of salt” and should be treated as such. It is important to keep in mind that excess sodium is excreted slower than water , 32.Oxygenation of organs is essential to preserve tissue function and avoid negative implications for wound healing and further complications. The correlation between oxygenation and sufficient circulation is subtle, since fluid optimization causes hemodilution and increases interstitial oedema, thereby compromising oxygen supply , 46. ManBelief in the existence of a third space loss and the fear of hypovolemia has led to a perioperative fluid practice of giving a large volume of intravenous fluid. However, observational studies show that a postoperative weight gain had deleterious side effects , 50, 51 In a study of 141 patients undergoing colorectal surgery, Brandstrup et al. showed a beneficial effect of a more restrictive vs. a standard  fluid regimen (2740 mL vs. 5388 mL), reducing overall, major and minor postoperative complications and confirming that fluid overload caused poor tissue healing and cardiopulmonary complications . SeveralHowever, the fear of occult hypovolemia caused by a too restrictive fluid regimen  and the A common factor to the trials on fluid therapy is that blinding is difficult since edema and diuresis is evident for all parts of treating patients. In addition, in all research concerning the surgical patient, many variables affect outcome and are difficult to standardize. Small sample sizes in the presented GDT trials challenge the results potentially affected by confounders. Furthermore, primary outcomes are dominated by LOS, which is a weak parameter influenced by local traditions and doctor and patient preferences and expectations.Lopes et al. randomized 33 patients undergoing high-risk surgery to GDT vs. standard care (4618 mL vs. 1694 mL), perceiving the benefit of GDT with significant reduction in LOS, fewer patients developing complications, and shorter duration of mechanical ventilation . In a stHowever, not all trials showed a benefit –62. In aIt is important to note that diverse methodology, different patient categories, and the use of supplemental crystalloids in both the GDT and the reference group challenge comparison between studies and might explain the heterogeneity in results. Overall beneficial outcomes to GDT appear to be related to patient risk stratification, being more beneficial in groups with higher mortality rates and more comorbidities .The GDT approach is usually applied in addition to “the standard fluid therapy”, compromising the ability of GDT to limit excessive fluid administration, allowing continuous intravenous crystalloid infusion alongside GDT optimization. Crystalloid infusion seems to have an insignificant effect on GDT measurements. This is in accordance with the findings by Lobo et al. and McIlroy and Kharasch, who showed a lower effect of crystalloids on the circulating volume and cardiac output. Hence, “the standard fluid therapy” or “maintenance regimen” should only replace physiological fluid turnover and pathological fluid losses with fluids resembling the loss in quantity as well as quality. The physiological loss is no more than 1–1.5 mL/kg/h substituting diuresis and insensible perspiration and is more than replaced by the fluid given with the different anesthetic and antibiotic medication.Interestingly, recent studies comparing restrictive or zero-balance fluid therapy with GDT based on a zero-balance maintenance regime  have shown no difference in outcome between the two approaches  and showed no difference in mortality and postoperative complications, despite a significant increase in SV in the GDT group. Likewise, Srinivasa et al. randomized 85 patients undergoing colectomy to GDT vs. a restrictive regime (1994 mL vs. 1614 mL) and found superior cardiac indices in the GDT group, but no difference in surgical recovery, LOS, and complications per patient. In the same way, Phan et al. showed improved stroke volume index but no difference in LOS in a study of 100 patients randomized to GDT vs. restrictive therapy (1500 mL vs. 1400 mL). Thus, zero-balance or restrictive fluid approach seems equal compared to the zero-balance GDT approach during elective abdominal surgery with a RR, 1.06   for patients undergoing laparoscopic cholecystectomy. Patients showed an improvement in postoperative nausea and vomiting, performance on a treadmill, and balance test in the group receiving the most fluid . HoweverOral fluid intake should be encouraged up to 2 h prior to surgery, thereby minimizing the need for intravenous compensation. Preferably, carbohydrate-containing fluids should be given due to patients’ proven reduction of postoperative insulin resistance and improved well-being.Perioperative fluid turnover accounts for no more than 1–1.5 mL/kg/h consisting of diuresis, insensible perspiration, evaporation from the wound, and accumulation in the traumatized tissue and should be compensated by carbohydrate-containing (hypotonic) fluids unless counter indications are present. Sensible perspiration varies considerably and is recommended replaced by balanced crystalloids. The assumption that elective surgery causes a fluid loss to the third space is based on flawed methodology and the replacement of a “loss to third space” worsens the postoperative outcome, due to derived fluid overload. Hence, this practice should be abandoned. A delicately balanced fluid therapy is recommended to avoid adverse effects of unnecessary excessive fluid administration as edema, inflammation, and compromised tissue healing.The intraoperative zero-balance fluid approach based on measurement of lost blood and fluid and postoperatively on body weight is easily implemented and has been shown to reduce postoperative major and minor complications. Therefore, a zero-balance fluid approach is recommended in the elective perioperative setting. A GDT approach likewise has shown to improve postoperative outcome, and guidelines recommending GDT seem well supported. However, the GDT practice is not documented to be superior to the zero-balance fluid approach. Nevertheless, high-risk surgery with multimorbid patients might benefit from the dynamic GDT approach. Evidence regarding urgent surgery is lacking, leaving a gap for future studies to explore.In relation to outpatient surgery, 1–2 L balanced crystalloids reduces PONV and improves well-being.


Fluid therapy is still the mainstay of acute care in patients with shock or cardiovascular compromise. However, our understanding of the critically ill pathophysiology has evolved significantly in recent years. The revelation of the glycocalyx layer and subsequent research has redefined the basics of fluids behavior in the circulation. Using less invasive hemodynamic monitoring tools enables us to assess the cardiovascular function in a dynamic perspective. This allows pinpointing even distinct changes induced by treatment, by postural changes, or by interorgan interactions in real time and enables individualized patient management. Regarding fluids as drugs of any other kind led to the need for precise indication, way of administration, and also assessment of side effects. We possess now the evidence that patient centered outcomes may be altered when incorrect time, dose, or type of fluids are administered. In this review, three major features of fluid therapy are discussed: the prediction of fluid responsiveness, potential harms induced by overzealous fluid administration, and finally the problem of protocol-led treatments and their timing. However, in reality both features may contribute in part to what makes adequate fluid administration even more important. This is in contrast with the rather benevolent and uncoordinated use of fluids by clinicians worldwide as demonstrated in the recent FENICE trial [In patients with acute circulatory failure, the primary goal of volume expansion is to increase cardiac output, hence oxygen delivery to the tissues. However, this effect is inconstant : in manyCE trial . In thisThe risks associated with improper fluid administration led to development of several strategies to assess “fluid responsiveness” before performing volume expansion. Although conventional parameters of preload have been used for decades for testing fluid responsiveness, their unreliability has been demonstrated by several studies . TherefoIt is today clearly established that static markers of cardiac preload, such as the central venous pressure or pulmonary artery occlusion pressure, are unable to predict what effect will fluid administration have on cardiac output , 8. The During mechanical ventilation, insufflation increases the intrathoracic pressure, increases the right atrial pressure, and hence decreases the pressure gradient of venous return. If the right ventricle is preload-dependent, this will inevitably reduce the right ventricular outflow. Increase in right ventricular afterload induced by increased lung volume contributes to this reduction of right ventricular outflow during inspiration. As certain time is needed for the transit of blood through the pulmonary vasculature, this will reduce left ventricular preload. During conventional ventilation, this should occur at expiration. If the left ventricle is preload-dependent, the left ventricular stroke volume transiently decreases at expiration. Hence, a cyclic variation of stroke volume under mechanical ventilation indicates preload-dependence of both ventricles .Several surrogates or estimates of stroke volume have been used in order to quantify these respiratory variations. The first one was the systemic arterial pulse pressure , which iAlongside PPV, other estimates of stroke volume have been used to predict preload responsiveness through their respiratory variations: stroke volume estimated by pulse contour analysis, blood flow of the left ventricular outflow tract measured by echocardiography, aortic blood flow assessed by esophageal Doppler, and the amplitude of the plethysmography signal recorded by pulse oximetry .The respiratory variation of stroke volume as a marker of preload responsiveness is not valid under some conditions. First, in case of spontaneous breathing activity, stroke volume variations relate more to the respiratory irregularity compared to preload dependence . Second,Mechanical ventilation could induce some changes in the diameter of venae cavae. Due to their high compliance, the changes are more likely to be observed in case of hypovolemia than in case of normo- or hypervolemia. The respiratory variation of the diameter of the inferior vena cava at the point where it enters the thorax was demonstrated to reliably predict fluid responsiveness . This waAs stated above, during the mechanical ventilatory cycle cardiac preload is reduced in inspiration. Stopping mechanical ventilation at end-expiration for a few seconds interrupts this cyclic decrease, meaning that end-expiratory occlusion (EEO) induces a transient increase in cardiac preload. This increase allows testing preload dependence. If the right ventricle is preload-dependent, the EEO will lead to an increased right ventricular output. If the duration of EEO is long enough for the transmission of this increased output toward the left cardiac cavities through the pulmonary circulation, left ventricular preload will increase. If the left ventricle is preload-dependent, EEO will eventually provoke an increase in cardiac output . Some stBeyond its simplicity, an advantage of the EEO test is that it can be used in case of cardiac arrhythmias since it exerts its effects on a period of time (15 sec) that covers several cardiac cycles  Figure . The EEO2O or 13 cmH2O [The EEO test remains valid whatever the level of positive end-expiratory pressure. One study reported that the prediction of fluid responsiveness by the EEO test was reliable and similar if the positive end-expiratory pressure was either 5 cmH13 cmH2O .The most intuitive way to test fluid responsiveness is to administer a small volume of fluid, observe its effects on cardiac output, and expect that a subsequent larger volume expansion will exert similar effects . The queA “mini fluid challenge” has been described as an alternative . In an iIn a patient lying in the semirecumbent position, elevating the inferior limbs at 45° and lowering the trunk induces a transfer of venous blood from the lower part of the body toward the cardiac cavities. PLR increases right and left cardiac preload and acts like a transient and reversible “self-volume challenge”  Figure . The relSince its hemodynamic effects occur over a long period of time, PLR remains valid in case of cardiac arrhythmias and spontaneous breathing activity. The hemodynamic effects of PLR are independent from mechanical ventilation, which explains that the PLR test can be used in some conditions in which PPV is not valid, such as low tidal volume, low lung compliance, and very high respiratory rate . The posAnother important point regards the method that must be used for assessing the PLR-induced hemodynamic changes . FirstlyIntra-abdominal hypertension could reduce the validity of the PLR test. It has been suggested that intra-abdominal hypertension could create an obstacle to the transfer of blood from the lower limbs toward the cardiac chambers through the inferior vena cava . One stuThe prediction of fluid responsiveness should be considered differently upon the clinical setting. First, one must remember that preload dependence is a physiological condition. Thus, positive predictors of fluid responsiveness should lead to volume expansion only in case of circulatory failure. Second, in case of an obvious hypovolemia, detecting preload dependence is useless since fluid responsiveness is constant. This is the case at the very initial phase of septic shock, when no fluid has been already administered, in case of hemorrhagic shock or in case of hypovolemic shock due to diarrhea, vomiting, or ketoacidosis, for instance.The operating theatre might be particularly adapted for the respiratory variation of stroke volume or surrogates in anesthetized patients, except if low tidal volumes are used for mechanical ventilation. In addition, such indices can be assessed by means of a simple arterial catheter or by noninvasive hemodynamic monitoring devices, which are suitable for this setting. The EEO test might also be useful, provided that the ventilator allows interrupting ventilation at end-expiration for 15 sec.In critically ill patients, the frequent presence of cardiac arrhythmias, the low lung compliance and ventilation with low tidal volumes associated with ARDS, and the presence of some spontaneous breathing and of low lung compliance often prevent use of PPV and the related indices. The respiratory variation of vena cava can be used as an alternative in case of cardiac arrhythmias. The EEO and PLR tests are often suitable provided that their conditions of application are fulfilled.The utility of predictors of fluid responsiveness may also depend upon the context where they are used. In the perioperative setting, prediction of fluid responsiveness might be part of the preemptive, individualized hemodynamic treatment that has been shown to reduce the rate of postoperative complications and the hospital length-of-stay in different categories of surgical patients –41. In t primum nil nocere principle.Today, we possess clear evidence of how detrimental unjustified and unbalanced fluid administration might be. Similar to several therapeutic interventions, fluid administration is obviously a life-saving intervention in severe hypovolemia and dehydration; however, it can also exert a number of adverse and potentially life-threatening effects . The effA number of guidelines recommend an aggressive and early “rescue” fluid resuscitation, particularly in severe sepsis, hemorrhagic shock, and necrotizing pancreatitis –49. The The primary aim of the fluid load is to increase cardiac output and oxygen delivery in patients with compromised oxygen transport. Thus, the dynamic parameters and functional hemodynamic tests can be of great value in determining the clinically effective volume of fluid load. However, in distributive shock associated with severe GIPS targeting “normal” preload and cardiac index can result in life-threatening complications. In severe ARDS, burns, or shock associated with necrotizing pancreatitis even restrictive fluid load can be accompanied by intense fluid accumulation in the tissues, particularly in the lungs, leading to increase in extravascular lung water (EVLW), hypoxemia, and pulmonary edema. This dilemma exerts a “therapeutic conflict” that forces us to modify the goal-directed intervention and consider “permissive hypovolemia” . WithoutIn the literature, we can find wide range of terms describing fluid therapy in both ICU and perioperative settings including “liberal,” “conservative,” and “restrictive.” In the review of Bundgaard-Nielsen et al.  merging In the ICU, an excessive fluid load in the settings of GIPS, systemic inflammatory response syndrome, and multiple organ dysfunction, particularly, in ARDS and acute kidney injury (AKI), can be devastating . The RENDuring the perioperative period, the liberal fluid therapy is commonly justified by presumption of perioperative dehydration and losses into hypothetical “third space.” Current evidence shows that in many patients these factors can hardly be assumed as an important reason for perioperative hemodynamic distress and should not be considered as a prerequisite for the aggressive intraoperative or preoperative fluid load . Thus, ivolume and rate of fluid administration (deliberate or excessive fluid load),reperfusion phenomenon and microcirculatory recruitment,fluid-specific complications (AKI for hydroxyethyl starches (HES) or dilutional acidosis for unbalanced crystalloids).Therefore, the risks of fluid resuscitation can mainly be related to the following factors:The choice of type of fluid does probably not primarily affect the clinically important outcomes related to the adverse effects of hyperhydration. However, at later stages the effects of type of fluid become unequivocal. According to the range of the current guidelines the use of albumin and semisynthetic colloids does not carry any obvious benefits over crystalloids and can be harmful in particular subgroups of patients like traumatic brain injury . The risInitial fluid resuscitation should routinely be started with crystalloid solutions. However, according to Marik , hyperhyIt is important to note that the goal-directed fluid therapy aiming to central venous pressure, as still required by Surviving Sepsis Campaign , in manyIn previous chapters, we have demonstrated several measures to differentiate between patients who would or would not benefit from fluid administration or in fact require fluid removal. Consequences of inadequate or overzealous fluid administration were also discussed. From this point of view, it sounds rational to use strategies enabling individual titration of fluid balance which should be associated with better outcomes in critically ill patients. However, it seems that our praxis is very divergent , 67–69. Even in the perioperative setting where numbers of single and multicenter trials have proved significant benefit of advanced hemodynamic monitoring guided management on morbidity, contradictory results have also been published recently. The largest study so far on this topic, the OPTIMISE trial , failed It has been established in various scenarios that timing is crucial: immediate commencement of resuscitation and of the time within the target end-points reached is of utmost importance in critical care medicine. As recently pointed out by some most renowned authors , 80 fourThe Rescue phase encompasses the very first minutes to hours aiming at salvage of the patient. Naturally, only those readily available tools and minimalistic targets may be considered. In some cases, overtreatment may even be associated with harm as, for instance, overresuscitation to higher perfusion pressure with fluids in multiple trauma victims . EvidencIn the Optimization phase, the therapeutic goal should be to reach the optimal perfusion of peripheral tissue and, according to some authors, to repay the oxygen debt incurred through the previous course of acute illness . It is n2) are comparable to consultant-led treatment alone. Similarly, there is no evidence supporting the early use of advanced hemodynamic monitoring. With widespread use of critical care echocardiography many information on the heart performance may be gathered with the use of a transthoracic probe and with standard monitoring . More invasive tools of adequate reliability may help to manage difficult patients and also help less experienced physicians to understand the underlying physiology and follow treatment goals. In this view of low evidence, only general recommendation may be derived for the everyday care  in trauma victims , 89. In day care .Perioperative goal-directed therapy (pGDT) has a special place in this topic of the protocol-guided therapy in the Optimization phase. In contrast to other critical care scenarios, both time and severity of the insult are well defined. Furthermore, the population of patients undergoing surgery is usually more homogenous from many aspects. Numerous studies and meta-analyses suggested that pGDT reduces the risk of postoperative complications , 90. Sti2, high lactate, and increased central venous-arterial CO2-gap. Adequate bedside tools and targets of regional tissue perfusion capable of monitoring microcirculation are, however, still undetermined.Nevertheless, surgical patients undergoing general anesthesia with controlled mechanical ventilation are ideal candidates for using dynamic predictors of fluid responsiveness (mostly based on heart-lung interactions) to rationalize intraoperative fluid therapy. Using stroke/pulse pressure volume variation or its surrogates was shown to be effective in reaching better outcomes and reducing postoperative complications in a recently published multicenter trial  as well 2, can be helpful but may not occur in every patient. However, it is important to acknowledge that none of these indices have a high enough sensitivity on their own to be used as a target in every patient; therefore, the so-called “multimodal” approach, meaning to take all that into account, may be necessary. At any case, if the optimization phase lasts longer than 24 hours after the initial insult, it is not associated with improved outcomes.There is limited data regarding protocolized care in Stabilization and Deescalation. Aggressive initial treatment followed by a restrictive approach was demonstrated to be beneficial by Murphy et al. . In ARDSSimilar to the rather blurred borderline between Optimization and Stabilization phases the optimal moment for the initiation of Deescalation is also unresolved. In many patients the fluid “Deescalation” is a naturally occurring process as a result of the spontaneous healing, during which forcing fluid removal is unnecessary. However, in some cases either the positive fluid balance is too large or the ability of the patient to mobilize the edema is diminished by the disease and active intervention is necessary. For this reason, a “diuretic responsiveness test” was recently introduced , which mCardiovascular insufficiency due to critical illness is a very heterogeneous entity. As pointed by Vincent et al. , patientSeveral tools are available today to assess volume responsiveness using dynamic procedures. These tools enable us to administer fluid with the assurance that it will lead to the expected increase in cardiac output. In particular, this should be included into the protocols guiding the hemodynamic treatment in the operating room setting. In the intensive care unit, these tools may be particularly useful in order to refrain from volume expansion that should reduce the risk of overzealous fluid administration. Further studies elucidating the kinetics of glycocalyx injury/regeneration and the role of vascular permeability during the goal-directed, individualized approach to the fluid resuscitation are warranted. The type and volume of the fluid should be thoroughly selected considering the phase of shock, risk of impending organ dysfunction, and individual comorbidity.Failure to use the time-patient-tool-protocol adequate approach may lead to worse outcomes and false conclusions. Use of protocols without proper individualization will always offer simplistic solution and can never lead to improvement of care in all patients coming from some global population of different disease states and severity. In other words, protocols of care can never replace the well-educated and critically thinking physician, who is able choose the appropriate diagnostic tools, put all relevant data into context, and tailor treatment to the patients' individualized needs.