The usual characterization of exact solutions of Einstein field equations, including cosmological solutions, is based on the symmetry properties of their corresponding metrics which is obviously mathematically involved. Here we present a physical characterization of the static and stationary perfect fluid solutions of the Einstein field equations by employing the  Usually to have a sound grasp of these kind of classifications and the related spacetime metrics and their characteristics, one needs a somewhat advanced mathematical background. Here we introduce a more physical classification, specifically of static and stationary perfect fluid solutions, which explains more clearly their physical chracteristics. These spacetimes have played a pivotal role in the evolution of the cosmological models, and have been discussed extensively in the exact solution literature2. In these solutions there could be more than one perfect fluid source, each with a different barotropic equation of state (EOS). Employing the quasi-Maxwell form of the Einstein field equations for multi-component perfect fluid sources, here we show how a combination of different choices for the gravitoelectromagnetic (GEM) fields, along with different EOS for different perfect fluid sources, could naturally lead to well-known static and stationary perfect fluid spacetimes as the representative of each class, hence furnishing a physical characterization of these spacetimes. The presence or absence of either or both of the gravitoelectric (GE) and gravitomagnetic (GM) fields could in some cases, not only restrict the minimum number of the perfect fluid sources, but also fix their EOS. We will treat the cosmological term, static single-component perfect fluid solutions of EFE in the non-comoving frames3. Characterizing them in this way, the apparent paradox raised by some authors5 on why there are different static spacetimes with Einstein spaces , and their characterization based on different symmetry groups of either geometric objects, such as Weyl and Ricci tensors, or the energy-momentum tensor of the sourceperfect fluid nature for the cosmological term and assign a 4-velocity to this dark fluid, in order to be able to interpret the directional expansion of the de Sitter-type spacetimes in their dynamical forms3.These findings motivated the idea that one should consider a Here we will show how the static and stationary dark fluid universes could be characterized in terms of their gravitoelectromagnetic fields in a fundamental observer’s frame adapted to the time-like Killing vector field of the corresponding spacetimes.threading formulation of spacetime decomposition, and the quasi-Maxwell form of the Einstein field equations. In four subsections of “The outline of the paper is as follows. In the next section we introduce the Throughout, the Latin indices run from 0 to 3 while the Greek ones run from 1 to 3, and we will use the units in which fundamental observers who are at fixed spatial points in a gravitational field. In other words, these worldlines, sweeping the history of the spatial positions of the fundamental observers, decompose the underlying spacetime into timelike threads6. In stationary asymptotically flat spacetimes, these observers are at rest with respect to the distant observers in the asymptotically flat region. Employing propagation of radar signals between two nearby fundamental observers the spacetime metric could be expressed in the following general form,dl gives the element of spatial distance between any two nearby events. Also, synchronized proper time between any two events. In other words any two simultaneous events have a world-time difference of simultaneous at the point B with the moment 7The mentclass2pt{minim8).Obviously, in the case of static spacetimes , and in accordance with Einstein’s idea that any form of energy gravitates. As an example, the Kerr metric,9.One could also extend the analogy between the quasi-Maxwell part of the EFE and electromagnetism by introducing a gravitational Maxwell-type field tensor whose elements are nothing but the GE and GM fields13.For general time-dependent spacetimes, it is not difficult to show that in the weak field approximation, the standard linearized field equations could be written in terms of the gravitoelectromagnetic 4-potential 7,i-th component of the source fluid as defined in , in, in19),  to Eqs.  and 18)I.VanishiAs the representative solution in each family with the lowest number of parameters, in the case of static spacetimes we consider spherically symmetric solutions whereas in the case of stationary spacetimes we restrict our attention to axially and cylindrically symmetric cases.It is a static spacetime.With Now Eq.  we we\documincoherent radiation. Of course photons as particles of radiation are not timelike and do not satisfy If we take the first case and substitute 0 in Eq. , that wiisfy Eq. . Also itisfy Eq.  which taFrom a physical point of view, that a single-component fluid does not lead to a solution is expected, since any kind of normal matter will produce attractive gravity, and hence leads to a collapsing system with comoving frame (say Einstein static universe in which the relation between the two fluid densities is given by Static universe filled with incoherent radiation in which the relation between the two fluid densities is given by Static universe filled with stiff matter (SM) in which the relation between the two fluid densities is given by In terms of the cosmological constant, the metric of the above three static spherically symmetric spacetimes are given by,Therefore to have a solution we need at least a 2-component fluid which, when plugged into Eqs. –22), le, le22), 3, where it is shown that it leads to a unique characterization of de Sitter and de Sitter-type spacetimes as the only one-component static perfect fluid solutions of Einstein field equations in a non-comoving frame. The well known de sitter spacetime2527. Obviously apart from these 1-parameter solutions there are other solutions of (Starting from Eqs. –19) and and19) a28.The two classes of solutions and their representative spacetimes considered in this section and in the previous one, namely Einstein static Universe and de Sitter model, share the property that both have a vanishing GM field in a fundamental observer’s frame. Born in the same year (1917), the two models of the universe had different fates. Einstein static universe turned to a case for pathological studies after Hubble’s discovery of the expansion of the universe, while the inflationary scenarios and the discovery of the accelerated expansion of the Universe in the present epoch, resurrected interest in the cosmological constant and de Sitter spacetime. Specifically, in the latter case, the cosmological constant is taken as the main candidate driving the repulsive gravity accounting for the unexpected observation. The final fate of a universe with matter and radiation diluted by an ever-expanding feature generated by a cosmological constant, would be a de Sitter universeabsence of the gravitoelectric field requires a constant time-time component of the metric, i.e 29), these observations reduce the general form of the metric  into1,the z-axis. Starting from Eqs.  and andabsenThe one-component perfect fluid of the dark-type with EOS ing Eqs.  and 35)\documentution of  which wiIf on the other hand we insist on having a dark fluid component, as we have done so far, then we should look for a solution of the above equations with two perfect fluid sources namely,negative density dark fluid component , where the corresponding densities satisfy the first equation in , an, an\docue metric , as follThe Gödel spacetime presents a homogeneous rotating model of a universe with a rigidly rotating fluid source, and as such is not a realistic model of our universe. On the other hand it contains very interesting properties, such as the existence of closed timelike curves, which makes it an important pedagogical example in the study of cosmological solutions.stationary axially or cylindrically symmetric spaces. These are equivalent to the stationary, axially or cylindrically symmetric solutions of the Eqs. NUT solution, which in a Schwarzschild-type coordinate system is given by,36. Its gravitoelectromagnetic fields are given by7. This interesting feature is also demonstrated in the scalar invariants of the space, for example in its Kreschtmann invariant which is given byObviously keeping both fields the Eqs. –19) wit wit\docul, also called NUT charge, is mainly interpreted as representing a gravitomagnetic monopole charge which is the gravitational analogue of a Dirac monopole7. Its observational consequences, mainly different gravitational lensing and microlensing patterns, are already discussed in the literature37. Now the inclusion of the cosmological constant along with the NUT charge in the above solution, produces a cosmological solution which could be interpreted as the spacetime of a NUT hole in a de Sitter background, somewhat similar to the interpretation of the Schwarzschild-de Sitter solution as a black hole in the de Sitter background. This is an interesting solution which introduces a stationary cosmological model in the absence of any rotating fluid as a source.The NUT parameter 3.We have shown that the simplest static and stationary single and two component perfect fluid solutions of Einstein field equations, which all include a dark component with EOS olutions  and 32)\document38. By doing so we are ignoring the vital role of the fluid’s 4-velocity in dictating anisotropic expansion in the corresponding de Sitter-type spacetimes3. Finally the above results could be summarized in the following table.To elaborate on this point, it is well know that the cosmological term


CRE isolates are considered a threat to human health. Until recently, the treatment options for CRE were limited. New BL/BLIs, such as ceftazidime-avibactam (CAZ-AVI), meropenem-vaborbactam (MVB), and imipenem-relebactam (IMI-REL), are active against most of these isolates, but these agents display some variation in activity. In this study, we evaluated the activity of these new BL/BLIs and comparator agents against CRE isolates collected in US hospitals.A total of 27,834 Enterobacterales isolates were collected in 74 US hospitals and susceptibility tested by reference broth microdilution methods. CLSI breakpoints were applied. CRE isolates resistant to imipenem or meropenem were submitted to whole genome sequencing and data analysis for the detection of β-lactamases.p=0.06). This increase was noted in isolates from bloodstream infections and among patients hospitalized with pneumonia; a slight decrease was observed among urinary tract infections. Among the CREs, an increase in E. cloacae, K. aerogenes, and less common Enterobacterales species was observed. CRE isolates included 8 species in 2019/2020 and 12 species in 2021. Most CRE isolates produced KPC enzymes, mainly KPC-2 and KPC-3, but a decline was noted over time, from 73.8% in 2019 to 67. 5% in 2020 and 54.1% in 2021. Conversely, MBLs increased from 3.8% in 2019 to 18.4% in 2021. MBLs were mainly NDM-1/-5, but also IMP-27 and IMP-4. OXA-48-like enzymes without NDM were detected among 8 isolates. Overall, CAZ-AVI inhibited 86.6% of CRE isolates. When comparing the activity of 3 new BL/BLIs tested against 2021 isolates, CAZ-AVI was active against 78.6% of the CRE isolates whereas MVB inhibited 76.5% and IMI-REL inhibited 73.3%.CRE comprised 0.9% (261) of the isolates. An increase in CRE prevalence was noted in 2021 (1.1%) compared to 2019 (0.8%) and 2020 (0.9%; An increase in CREs driven by an increase in MBL-producing isolates was noted in 2021. This increase is worrisome since there are no effective therapeutic options against MBL producers. CAZ-AVI was the most active agent against CREs, but the increase in MBLs should be closely monitored. New therapies against these isolates are urgently needed.Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Timothy Doyle, MS, AbbVie: Grant/Research Support Valerie Kantro, BA, AbbVie: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support.


There is a continued need to understand varicella-zoster virus (VZV) pathogenesis and to develop more effective antivirals, as it causes chickenpox and zoster. As a human-restricted alphaherpesvirus, the use of human skin in culture and mice is critical in order to reveal the important VZV genes that are required for pathogenesis but that are not necessarily observed in the cell culture. We previously used VZV-expressing firefly luciferase (fLuc), under the control of the constitutively active SV40 promoter (VZV-BAC-Luc), to measure the VZV spread in the same sample. However, the fLuc expression was independent of viral gene expression and viral DNA replication programs. Here, we developed robust reporter VZV viruses by using bacterial artificial chromosome (BAC) technology, expressing luciferase from VZV-specific promoters. We also identified two spurious mutations in VZV-BAC that were corrected for maximum pathogenesis. VZV with fLuc driven by ORF57 showed superior growth in cells, human skin explants, and skin xenografts in mice. The ORF57-driven luciferase activity had a short half-life in the presence of foscarnet. This background was then used to investigate the roles for ORF36 (thymidine kinase (TK)) and ORF13 (thymidylate synthase (TS)) in skin. The studies reveal that VZV-∆TS had increased sensitivity to brivudine and was highly impaired for skin replication. This is the first report of a phenotype that is associated with the loss of TS. Varicella-zoster virus (VZV) is the human neurotrophic alphaherpesvirus that causes varicella (chickenpox) during primary infection, and herpes zoster (shingles) upon the reactivation from latency in the sensory nerve ganglia. Zoster is particularly morbid, as it is frequently complicated by acute and chronic pain. While vaccines for both varicella (targeted to children) and zoster (targeted to adults 50+ years old) have decreased the incidence of the disease in the countries where they are used ,2, VZV dThe human-restricted nature of VZV greatly complicates its study, particularly as there are no effective animal models for the latency, reactivation, or reactivated disease ,10. FurtE. coli required the insertion of a ~10 Kbp replicon into the genome. Two pOka BACs were developed with the insertion of the replicon into the same pOka cosmid set, which was followed by virus derivation and the retransformation of E. coli with circular DNA from virus-infected cells luc exprepression  have reppression . ORF17, pression ,57, althpression . ORF17 cpression . ORF57 ipression . In thisluc is controlled by the SV40 promoter [VZV-BAC-Luc has long been the gold standard reporter virus. Strong luciferase activity enables the bioluminescence imaging of the VZV spread, which has been instrumental in understanding VZV pathogenesis and in evaluating the antiviral compounds in cells, skin, and in vivo ,26,27,44promoter , which iAcyclovir and its derivatives are the first-line antiviral therapy for VZV infections, and they are activated by TK phosphorylation. Mutations in the TK gene are the most common cause of resistance to acyclovir and other nucleoside analogs that lack a phosphonate group ,60,61. NVZV encodes several proteins for nucleic acid metabolism that may impact the mechanism and activity of antiviral nucleoside analogs. VZV ORFs 18 and 19 encode a ribonucleotide reductase that generates deoxyribonucleotides, and the deletion of these genes potentiates the activity of acyclovir . VZV ORFluc gene. VZV-ORF57-Luc appears to be an ideal reporter virus for its maintenance of the virulence in a human skin model and for the responsiveness to inhibition of DNA replication. We used this background to develop isogenic reporter viruses with deletions in ORF36 and ORF13 as additional tools for antiviral research. In cells and skin, we show that TK was dispensable, while TS was found to have an unprecedented requirement for virulence. Furthermore, the loss of TS enhanced the antiviral sensitivity to certain nucleoside analogues. Taken together, this work lays the foundation for the probing of the viral gene contributions to pathogenesis in human skin and for the evaluation of novel antiviral compounds.In summary, we detail a strategy to generate VZV reporter viruses to assess the gene contributions to virulence in cells and skin, as well as to evaluate novel antiviral compounds. The reporter viruses were constructed from a recombinant pOka strain with a T2A ribosome skipping motif between the VZV ORF and the


ObjectiveTo determine whether there is a correlation between pain and the amount of fluid present at the distal insertion of the iliotibial band (ITB) in runners, as measured by USG.MethodOur retrospective cross-sectional study evaluated 100 male and female runners prior to the start of a race. A valid and reliable questionnaire collected demographic, pain, and training data. If a runner reported knee pain, a numeric pain rating scale was used to record the degree of pain. Participants then underwent USG on both knees to determine the presence or absence of fluid at the distal insertion of the ITB.ResultWe found no statistically significant correlations of fluid measurements with pain score, running experience in years, or age. In addition, we found no other differences in fluid measurements between those with and without knee pain or between the sexes.ConclusionsOur findings indicate that the presence or absence of fluid at the distal insertion of the ITB does not correlate with knee pain in runners, regardless of age, running experience, or sex. Iliotibial band syndrome (ITBS) is one of the most common afflictions experienced by runners . It is dGiven that experienced runners may have better running and training techniques than novice runners , it coulFor this retrospective cross-sectional study, 100 male and female participants were recruited during a weekend race event that featured a 5K, 10K, relay, half marathon, full marathon, and ultra-marathon (110 miles). Exclusion criteria were: 1) being under 18 years of age; 2) having previous knee surgery; 3) being a wheelchair user; 4) having received a knee injection within the previous three months, and 5) having already competed in one of the races during the race weekend. Informed consent was obtained from all participants, as stipulated by the Mayo Clinic Institutional Review Board (IRB number 16-010500). With a sample size of 100 participants, there was more than 80% power to detect a correlation coefficient of |0.3| or larger at the 5% significance level (two-sided).Participants were asked to complete a survey asking about their age, BMI, presence of knee pain, an NRS score for knee pain (if present), previous diagnosis of any knee condition, and previous discontinuation of running due to pain. Participants were also asked about the average run duration, modification of training, running experience in years, weekly mileage, training pace, and the number of races in which they had participated.Fluid measurements were taken with the participant at 0° of extension and in the standing position. Sonographic measurements were obtained in the long-axis area of the left (LAA-L) and right (LAA-R) knees, the long-axis circumference of the left (LAC-L) and right (LAC-R) knees, and the short-axis area of the left (SAA-L) and right (SAA-R) knees Figure .This study had multiple hypotheses and aims: 1) Individuals with higher pain scores on the NRS would have more fluid present at the distal insertion of the ITB; 2) More experienced runners would have a less fluid and lower score on the NRS, validating to some extent, the belief that more experienced runners have better running and training techniques than novice runners; 3) Female runners would score higher on the NRS and have a greater amount of fluid present at the distal insertion of the ITB compared to male runners, given that female runners are anatomically predisposed to ITBS due to having wider hips; and 4) Older individuals would have less fluid present at the distal insertion of the ITB as a result of the degeneration of the bursa expected with age.All analyses were performed using SAS, version 9.4 (SAS Institute Inc.). Survey responses and fluid measurements were descriptively summarized for all participants and separated according to sex. Spearman rank correlations (ρ) were used to evaluate the correlation of ITB fluid measurements with pain scores (Aim #1), years of running (Aim #2), and age (Aim #4), along with the correlation of pain scores with years of running (Aim #2). Wilcoxon rank-sum tests were used to compare pain scores and fluid measurements between males and females (Aim #3). As an additional analysis to supplement Aim #1, we compared fluid measurements between those with and without lateral knee pain present at the time of the survey using Wilcoxon rank-sum tests. P-values of 0.05 or less were considered statistically significant without adjustment for multiple testing.Table Fluid measurements and survey responses are summarized by sex in Tables Spearman correlations were used to evaluate correlations of each fluid measurement with pain score, years of running, and age. There were no statistically significant correlations of fluid measurements with pain score , years of running , or age .ITBS is the most common presentation of ITB disease . It is mOur study demonstrated that fluid measurements in and around the distal ITB did not significantly correlate with pain score, sex, years of running, or runner age. There were no other differences in fluid measurements between those with and without knee pain. This information may help to characterize fluid in and around the distal ITB of ITBS sufferers as unrelated and perhaps help with a more thorough definition of sonographic findings that should be considered helpful in diagnosing ITBS. Peridistal ITB fluid is most likely incidental and should not factor into the diagnostic criteria for ITBS.Our study had certain limitations. Early pathologic findings  and more advanced stages (thickening and heterogeneity of the ITB itself) were not considered. However, we felt that the fluid in the distal ITB may not be as related to these changes as it would be to acute activities that inflame or damage the region . Fluid mAdditionally, the ultrasound machine determined the area of fluid being measured using the appropriate lines documented. The machine calculated the area and assumed it to correlate positively with the total fluid volume in the pocket, but this may not necessarily be true. The ultrasound machines used were identical models, tested prior to use, of great quality, and similar to the models used widely by practitioners in the field.Future research should focus on specifying anatomic parameters that may change fluid presence in other regions of the knee and fluid presence associated with runners' knee pain that may be detectable using sonography. Contributors to larger knee circumference  could be individually examined in future studies.There are no statistically significant correlations of fluid measurements with pain score, sex, years of running, or age. There were no other differences in fluid measurements between those with and without knee pain. Therefore, the presence or absence of fluid at the distal insertion of the ITB is likely not a predictor of knee pain in runners, regardless of age, running experience, or sex. Bogginess or swelling noted clinically at this location, especially in runners, in the context of complaints compatible with ITBS, may be irrelevant in this population.


Curcuma comosa Roxb. (C. comosa) extract demonstrated significant estrogenic activities both in vitro and in vivo. This study aimed to develop and comprehensively evaluate a mucoadhesive vaginal gel for the sustained release of DAs. Different mucoadhesive polymers as gelling agents were investigated. C. comosa ethanolic crude extract was used as a source of DAs. All C. comosa gels were light brown homogeneous with pH within 4.4–4.6. Their flow behaviors were pseudoplastic with a flow behavior index of 0.18–0.38. The viscosity at a low shear rate varied from 6.2 to 335.4 Pa·s. Their mechanical and extrudability properties were associated well with rheological properties. Polycarbophil (PCP):hydroxypropyl methylcellulose (HPMC) blends had a higher mucoadhesiveness to porcine vaginal mucosa than those of PCP-based or HPMC-based gels. All C. comosa gels exhibited a sustained, zero-order DA release pattern over 72 h. Korsmeyer and Peppas equation fitting indicated a non-Fickian, case II transport release mechanism. C. comosa gels had good physical and chemical stability under low-temperature storage for up to 12 months. PCP:HPMC-based mucoadhesive gels could be a proper delivery system for vaginal administration of DAs. Diarylheptanoids (DAs) characterized by a 1,7-diphenylheptane structural skeleton are considered a novel class of phytoestrogens. The DAs available in  Glycine Max isoflavone gels and Pueraria mirifica gels—on the restoration of the symptoms associated with the genitourinary syndrome of menopause have been demonstrated .,36.C. corecorded . The TexC. comosa gels was conducted under sink conditions using the modified Franz diffusion cell with a diffusion area of 2.01 cm2 and receiver volume of 14.0 ± 0.2 mL. The diffusion cells were attached to a water bath  maintained at the temperature of 37.0 ± 0.5 °C. A receiver medium, 50% v/v PEG 400 in pH 5.5 SVF, was loaded into a receiver compartment where its hydrodynamic was preserved by a magnetic stirring . The hydrated polyamide membrane , as the supported membrane, was mounted between the donor and receiver compartment with a clamp. Then, 0.6 g of C. comosa gel was carefully placed on the membrane on the donor side, which was tightly covered with paraffin film. At predetermined times , a 0.8 mL of receiver medium was collected from the receiver compartment and the fresh 50% v/v PEG 400  was replenished after each sampling. The content of DA released was determined by using an HPLC assay.In vitro release of DAs from the Release data analysis: The cumulative amount of DA released per area was computed and plotted against time. The cumulative release results were fitted to zero-order, Higuchi, and Korsmeyer–Peppas models, which are defined by the following equations.tM refers to the amount of DA released at time t. tM/M∞ refers to the fractional DA released at time t. K0, HK, and KPK are the zero-order, Higuchi, and Korsmeyer–Peppas rate constants, respectively. To interpret the release mechanism, the n, denoted as the release exponent, was determined from the slope of the plot between the log value of t, Equation (5) [tion (5) ,38,39.C. comosa gel formulations were investigated according to the ICH guidelines [C. comosa gel was packed in a 20 g collapsible aluminum tube and kept under long-term (5 ± 3 °C) for 12 mo and accelerated (30 ± 2 °C with 75 ± 5% relative humidity (RH)) conditions for 6 mo. After 6 and 12 mo of storage, the aged C. comosa gel was withdrawn and investigated for percentages of DA remaining and the physicochemical characteristics, namely, appearance, pH, and viscosity. The percentage viscosity, which refers to the values of viscosity relative to that of its fresh gel, was calculated.The physical and chemical stabilities of the selected idelines . The fre2 > 0.9999) throughout the determination range of 1–50 μg/mL.The DA amount was quantified using a validated HPLC protocol previously reported by Tunpanich et al. . An Agilp < 0.05.The experiment results are presented as the mean ± S.D. One-way analysis of variance (ANOVA) was used to test the statistical significance of differences among groups. The significant difference in the means was further determined by using Tukey’s post hoc test. The statistical tests were operated using the SPSS program for Windows software . The statistical significance was set at C. comosa ethanolic extract were fabricated using the mucoadhesive polymers—PAA (C980 and PCP), cellulose derivatives (HPMC K15M and HPMC K100M), and a mixture of PAA:cellulose derivatives (2:1 PCP: HPMC)—as a gel-forming and mucoadhesive agent. Because of the hydrophobicity of the DAs (C. comosa extract [C. comosa extract had to be homogeneously blended with the high HLB surfactant (polysorbate 80) as a wetting agent before mixing with other ingredients, to obtain the uniformity mixture with the aqueous polymeric dispersion, especially for the HPMC K15M-based formulations. The C. comosa gels were also composed of glycerol as a humectant and methylparaben as a preservative. Sodium hydroxide solution was used to adjust the gel pH to the range of 4.5 ± 0.1. In the present study, all the C. comosa gels were designed to possess a moderately acidic pH to conform with the normal vaginal pH ranges (3.8–5.0) for women [In this study, the mucoadhesive gels of  the DAs  and othe extract , C. comoor women . It is kC. comosa gels as compared with the C. comosa extract. All the C. comosa mucoadhesive gels were light brown, opaque, homogeneous viscous gels with a characteristic odor of C. comosa extract, whereas the placebo gels—the gels without C. comosa extract—were colorless, almost clear to clear gels (C. comosa gels appeared to be a little more yellow-white than the others. The homogeneity and consistency of C. comosa extract with the gel bases was in the order of PAA (C980 and PCP) > HPMC K100M > HPMC K15M. It should be noted that C. comosa extract can be easily incorporated into PAA-based gels regardless of polysorbate 80 utilization.ear gels . The PCPC. comosa extract, was captured. In the case of PAA-based gels, the jelly-like gels were obtained through neutralization, which induced the ionization of carboxylic acid groups, uncoiling the PAA chains through electrostatic repulsion [C. comosa gels had a pH in the designed range of 4.4–4.6. The actual loading of DA1 and DA2 in C. comosa gels were determined and found to be 0.47–0.49% of each DA, which were corresponding to 94.1 ± 3.3 to 97.4 ± 2.75% and 96.8 ± 2.9 to 99.6 ± 3.6% of theoretical loading of DA1 and DA2, respectively.Different mucoadhesive polymers yielded different viscosity gels, among which HPMC K15M yielded the mucilaginous characteristic. The viscous gel matrix was caused by the continuous polymer network, physically structured through the physical entanglements and/or secondary interactions between polymer chains, whereupon an aqueous phase, as well as epulsion ,30. All C. comosa extract with mucoadhesive polymers as well as gel compositions was assessed using ATR-FTIR. As presented in C. comosa extract comprised the bands at 3333 cm−1 (O-H stretching), at 2800–3100 cm−1 , 1601 cm−1 (C=C stretching), and at 900–700 cm−1 (C-H bending) [−1, a strong band for C=O stretching at 1697 cm−1, bands for C=O and C-O stretching at 1450 and 1166 cm−1, respectively. For HPMC K15M and HPMC K100M, the major absorption bands observed were a band at 3415 cm−1 due to O-H and stretching, at 2900 cm−1 due to C-H stretching, and a strong band at 1051 cm−1 assigned to C-O stretching. The FTIR spectrum of PCP and HPMC K15 physical mixture (2:1 ratio) revealed distinct bands comparable to those of PCP [−1), aliphatic C-H stretching (2931 and 2880 cm−1), and H-O-H bending (1640 cm−1) [−1 (O-H stretching) and a band at 1637 cm−1 (H-O-H bending). It should be noted that the intermolecular interaction between PCP and HPMC was not observed in the FTIR spectra of C. comosa and placebo gels. These could probably be concealed by the strong H-O-H bending band due to glycerol and water molecules. According to the reports published previously, the hydrogen bonding between the –COOH group of PAA polymers (carbomer [The compatibility between bending) ,14,18. C40 cm−1) . The ATRcarbomer  and PCP carbomer ) and –OHcarbomer , could bcarbomer  and PCP:carbomer .C. comosa mucoadhesive gels prepared with C980 and PCP displayed the definite bands corresponding to those of placebo gels and C. comosa extract at approximately the equivalent positions. For C. comosa gels based on C980 and PCP, the characteristic bands of C. comosa extract at around 650–750 and 1400–1550 cm−1 were clearly observed. These indicate the absence of considerable incompatibility among C. comosa extract and the components in the C980-based and PCP-based gel formulations. Interestingly, in the case of HPMC-based and PCP:HPMC-based gels, the disappearance of the FTIR characteristic bands in the 650–750 and 1400–1550 cm−1 regions of C. comosa extract was observed. These suggested the possible interaction between HPMC and the components of C. comosa extract. HPMC is a hydrophilic polymer possessing the ability to form a polymer-type complex [C. comosa extract contains also numerous phytochemical compounds, namely, diterpenes, curcucomosins, flavonoid glycosides, curcucomosides, and sesquiterpines [The FTIR spectra of  complex . Neverthterpines ,46. To pC. comosa gels were assessed using the basic destructive (flow) technique. This destructive method is a commonly used technique to determine the rheological performance of vaginal gels and to provide information regarding in what manner the gels behave under a shear stress circumstance owing to the motions of the vaginal surfaces, and gravitational and capillary flow [C. comosa gels were determined at 37 °C to mimic the rheological performances of these gels in the vaginal cavity. Their rheograms, which illustrated the relation between the shear rate and shear stress, are presented in C. comosa gels exhibited non-Newtonian (pseudoplastic) flow revealing a shear-thinning appearance, and a high viscosity at a low shear rate, which subsequently decreases with shear.Rheological behaviors directly influence the gel performance such as application, spreadability, and retention time in the vaginal cavity. The rheological properties of ary flow . The floτ) and shear rate (γ) with a coefficient of determination of 0.95–0.99. The flow behavior index (n), as well as consistency index (k), differed in each gel formulation depending upon the type and concentration of mucoadhesive polymer. The degree of shear-thinning behavior was determined by the n value [C. comosa gels have n values of less than one, within the range of 0.177–0.376, confirming the shear-thinning phenomena. The closer the n value is to zero, the larger the shear-thinning behavior of C. comosa gels. Blending PCP with HPMC yielded a more pronounced pseudoplastic behavior gel when compared with the neat PCP-based or HPMC-based gels. This might be associated with the intermolecular complexation among PCP and HPMC [n value due to the increase in the attraction bond between adjacent molecules, which enhances the existence of temporary entanglement associations [k value, it was noticed that the higher the k value, the more viscous consistency of the C. comosa gels with a lower n value.Flow behavior in the shear-thinning region was well characterized by the power law, which is a practical equation most utilized for pseudoplastic material expression. As presented in  n value . The C. and HPMC ,43,44. Iciations . With reC. comosa gels differ between formulations ranging from approximately 1 to 237 Pa. The extent of yield stress indicates the intensity as well as the strength of the closed pack between adjacent molecules of the gel structure [Yield stress can be defined as the minimum stress that has to be placed to start the flow. As listed in tructure . Yield structure . The higC. comosa gels was caused by the change in polymeric structure upon shear stress exposure. The investigated gels might contain a highly entangled polymer in a relaxed state, which, as a whole and/or polymer chain segments, would disentangle and then align themselves in the direction of shear. The solvent located in the coil structure was released. The number of entanglements between polymer chain segments and side chains was decreasing with shear stress. Under high shear stress, polymer chains were becoming highly mobile and started to slide past one another due to the inter- and intra-molecular bond destruction, offering low resistance to flow, and the apparent viscosity was decreased [The nonlinear response to shear stress of ecreased ,47.Pseudoplastic behavior is considered a preferable characteristic for vaginal gels. A gel exhibits considerable viscosity at low shear representing the initial physical stability and a high potential to retain at the applied location, while a decrease in an apparent viscosity under high shear stress provides the ease of administration as well as spreadability on the application.C. comosa gels as a function of shear rate is presented in C. comosa gels at low shear rates (5 s−1) was also determined, their viscosity varies from 6.2 to 335.4 Pa·s. It should be noted that the presence of 3% w/w C. comosa extract did not affect the flow behavior of the gels; however, it slightly increased the viscosity values for all formulations when compared with placebo gels. The viscosity at a low shear rate could be used to indicate the viscosity during resting in the vaginal cavity. Similar to the yield stress values, the viscosity of the gels increased with polymer concentration. C980 yielded a higher viscous gel as compared with PCP. The viscosity of 3% PCP:HPMC K100M was found to be the highest, while the HPMC K15M-based gels showed the lowest viscosity among all the formulations. The low viscosity may result in the prompt loss of C. comosa gel from the application site, while the high viscous gels might exhibit poor spreadability on the vaginal mucosa after application.The shear viscosity at 37 °C of the C. comosa gels were investigated as per their mechanical properties using a texture profile analyzer. Texture profile analysis contributes the details regarding the response to the external compressional force and the ability to generate the gel structure deformations, either reversible or irreversible [C. comosa gels. The hardness of C. comosa gels ranged from 0.021 to 0.310 N, depending on the type and concentration of mucoadhesive polymers. For the gels prepared from the same concentration of neat mucoadhesive polymer, the hardness of the gels was in the order of HPMC K15M < HPMC K100M < PCP < C980 (p < 0.05). Blending HPMC K15M with PCP significantly increased the gel hardness (p < 0.05). Increasing the concentration of mucoadhesive polymer enhanced dramatically the gel hardness values (p < 0.05). This was linked to the denser polymeric network in the gels when the higher polymer concentration was used. The compressibility of C. comosa gels ranged from 0.098 ± 0.002 to 1.448 ± 0.051 N·s. Analogous to the hardness values, the compressibility was determined by the type, molecular mass, and concentration of mucoadhesive polymers. These textural properties were associated well with the rheological properties of the gels. This agrees with the previous reports investigating the compression behaviors of various polymers—carbomers, hydroxyethylcellulose, polyvinylpyrrolidone, and polycarbophil [Vaginal gels shall possess suitable mechanical characteristics, namely, low hardness, ease of application, mucoadhesive, and retention prolongation at the application site. Therefore, versible ,35. TablC. comosa gels exhibited good cohesiveness ranging from 0.914 ± 0.037 to 1.236 ± 0.052 of which the HPMC-based gels had the highest cohesiveness (p < 0.05). The PAA-based gels exhibited comparable cohesiveness, which was not influenced by the polymer concentration or the presence of HPMC in the systems (p > 0.05).The C. comosa gels from a container as well as the applicability to the target site, producing a homogeneous layer and providing the product comfort. Low hardness and compressibility values signify the ease of gel removal from the container and its spreadability over the application site. Cohesiveness refers to the proportion of the area under the force–time curve generated during the second compression over that formed during the first compression cycles [Hardness, the so-called firmness, refers to the maximum compression force required to reach a given deformation, whereas compressibility refers to the work needed to deform the gels during the first compression. Hardness and compressibility manifest the work or stress needed to withdraw the n cycles . Cohesivn cycles ,35.C. comosa gels exhibited comparable elasticity with values ranging from 0.999 ± 0.007 to 1.019 ± 0.009 mm (data not shown). These indicate that the rate at which the deformed gels recovered to their original structure of C. comosa gels was not affected by the type, molecular mass, and concentration of mucoadhesive polymers. Additionally, it was found that the textural properties of C. comosa gels were comparable to those of placebo gels, indicating that the incorporation of 3% C. comosa extract did not affect the mechanical behaviors of the investigated mucoadhesive gels.It should be noted that all P < HPMC K100M < C980 (p < 0.05). Blending PCP with HPMC dramatically enhanced the extrudability values as compared to the gels prepared with neat polymers (p < 0.05). The extremely high extrudability of 3% PCP:HPMC K100M might be unfavorable for the practical administration of gels with a syringe. Interestingly, increasing the polymer concentration from 2% to 3% had no significant effect on the extrudability of PCP-based gels, whereas raised the extrudability value of PCP:HPMC K15M for about 1.5 folds. This extrudability parameter indicates the ease of administering the C. comosa gels with an applicator or syringe. It has been claimed that the work to discharge the semisolid formulations through an applicator  to periodontal pockets sites should be within 20–380 N·mm for injectable application via syringe systems [Extrudability was investigated and the results are listed in  systems . NeverthmaxF) and work of adhesion (adW), of the C. comosa gels to the porcine vaginal mucosa was therefore conducted. As presented in C. comosa gels was found, with their maxF and adW ranging from 0.060 to 0.237 N and 0.029 to 0.131 N·mm, respectively. The ranking of mucoadhesiveness of C. comosa gels prepared with 2% mucoadhesive polymer was HPMC K15M ~ C980 < HPMC K100M < PCP (p < 0.05). Blending of PCP with HPMC K15M yielded a significant increase in mucoadhesiveness than those of HPMC K15M gels. An increase in the PCP concentration from 2 to 3% decreased the mucoadhesiveness, while the mucoadhesiveness of PCP:HPMC K15M increased with the polymer concentration increment.The mucoadhesive ability of a vaginal gel formulation is considered an essential variable to achieve prolonged retention and release duration at the application site. The adhesive strength, in terms of maximum detachment force (3 or –CH2CH(CH3)OH functional groups, whereas C980 and PCP are the high molecular weight PAA possesses numerous –COOH in their structure [It is known that mucoadhesion, the adhesion between mucoadhesive formulations and mucin glycoprotein in the mucus layer, arises after intimate contact between these two surfaces. The swelling of the gels upon contact with the mucosal membrane induces the relaxation of the entangled or twisted polymeric chains, enhancing the chain mobility for interdiffusion/interpenetration with a biological substrate. The physical entanglement followed by the secondary non-covalent interactions—hydrophobic, hydrogen, and van der Waals—between substrates occurs and strengthens the mucoadhesion ,50. Conctructure ,28,50. Ttructure ,43,44. Ttructure . Additiotructure ,27.C. comosa gels were therefore examined in vitro via a static diffusion cell. Because DA compounds are practically insoluble in the aqueous medium (v/v PEG 400 was incorporated into SVF pH 5.5 to obtain the sink condition. The solubility of DA1 and DA2 in SVF pH 5.5 (at 37 °C) was found to be 4.3 ± 1.8 and 9.2 ± 2.9 μg/mL, respectively. The presence of 50% PEG 400 in the SVF yielded the DA solubility enhancement of approximately 63- and 84-fold for DA1 and DA2, respectively, compared to those in SVF. PEG 400 has been proven as an efficient cosolvent to enhance the solubility of DAs in the receiver medium [C. comosa gels exhibited the DAs release over a period of 72 h in a sustained release pattern. It can be seen that the HPMC-base gels provided the highest DA release compared to the other C. comosa gels. The rate and extent of DA release from the C. comosa gels were found to be retarded by the presence of PAA. It is obvious that the release of DA2 was significantly higher than that of DA1 (p < 0.05), irrespective of the type and concentrations of the mucoadhesive polymer in the formulations. This phenomenon was associated with the higher solubility of DA2 in the medium (p < 0.05). This is in agreement with the previous report regarding the DA release from C. comosa mucoadhesive vaginal tablets [The release behavior of active compounds from a gel matrix is a prerequisite for mucosal permeation. The DA release patterns from the s medium , 50% v/vr medium ,16,51. T tablets .C. comosa gels, the release data were fitted through the typically utilized kinetic equation models, zero-order and Higuchi. As presented in C. comosa gels was best explained by the zero-order of which their coefficient of determination values was ≥0.99, whereas the coefficient of determination values obtained from the Higuchi model was ≥0.97. These suggest that the rate of DA release from the C. comosa gels was constant and did not depend upon the DA concentration. The rates of DA1 release, K0, from 2% HPMC K15M and 2% HPMC K100M were comparable, whereas 2% HPMC K100M yielded a significantly lower rate of DA2 release than that from 2% HPMC K15M. This might be associated with the lower solubility of DA1 than DA2, which caused the less sensitivity of DA1 release to the higher molecular weight HPMC three-dimensional structure. It is interesting to note that the release of DA2 from 2% HPMC K15M was almost complete with the Q72h of 3 mg (0 of both DAs from C980-based and PCP-based C. comosa gels was comparable regardless of the polymer concentration. A combination of PCP with HPMC could significantly retard the DA release when compared with neat HPMC-based gels (p < 0.05). The DA release rate was decreased with the PCP:HPMC concentration (p < 0.05).To assess the pattern of DA release from the  of 3 mg  or approC. comosa gels, the release data were fitted as per the Korsmeyer–Peppas model, the so-called power law, which is a semi-empirical equation useful for characterizing the controlled release of polymeric systems including hydrogels, especially when the release mechanisms are engaged with more than one phenomenon [C. comosa gel release data also showed high linearity with the coefficient of determination values of ≥0.98. The exponent of release, n, values derived from the slope of the plot calculated based on the Korsmeyer–Peppas equation indicate that the release mechanisms of DA1 and DA2 from C. comosa gels follow the non-Fickian transport, an approximate Case II (n ~ 1), which provide release characteristics approaching zero-order. These suggest that DA release from C. comosa gels is controlled by swelling and involves polymer relaxation and chain disentanglement [It is generally acknowledged that the principal mechanisms determining the release from the hydrogel-based drug delivery systems are diffusion, swelling, polymer relaxation and disentanglement, and/or erosion of the polymeric matrix. Diffusion of active ingredients from the polymeric matrix may happen on a molecular level—the passage between polymer chains—or on a macroscale level—the diffusion pores in a polymer matrix. A three-dimensional macromolecular mesh networks made of reticular polymers behave as a diffusion layer proving a sustained release capability. The release behaviors could be caused by explicit or sophisticated mechanisms depending on the physicochemical properties of both the active ingredient and the polymeric matrix, as well as active ingredient-matrix interaction ,52,53. Benomenon . The pownglement ,54.n > 1), an intense form of non-Fickian transport, is developed. For Super Case II transport, the solvent diffusion occurs at a faster rate, accelerating the solvent penetration. The tension generated during the movement of the gel–vitreous interface causes polymer breakage [A non-Fickian mechanism transport generally occurs with the glassy (vitreous) polymeric system having the glass transition temperature beyond the experimental temperature. In this situation, the mobility of polymer chains is insufficient to allow the penetration of water molecules into the system core ,54,55. Cbreakage ,55.C. comosa gels may be associated with the difference in the rate of polymer relaxation and chain disentanglement caused by the molecular structure of the polymer. HPMC K15M and HPMC K100M are soluble methyl cellulose ethers with molecular weights of 750 and 1150 kDa, respectively [2–4 × 106 and 3.5 × 106 kDa, respectively [The differences in the release rate and extent between HPMC-based and PAA-based ectively . C980 anectively . The enoectively .It is noteworthy that the rapid DA release from HPMC-based gels, as well as the comparable release of DAs from PCP:HPMC-based as compared with PCP-based gels, indicate the lack of the DA and HPMC interaction .C. comosa gels were maintained throughout the stability experiment; however, a slight color fading was observed. Physical properties, namely, pH and percentage viscosity of aged C. comosa gels versus the fresh gels are presented in p > 0.05), the 12 mo long-term-aged gels and 6 mo accelerated-aged gels had significantly higher pH than the fresh gels (p < 0.05). The viscosity of aged gels was likely to be decreased upon storage (p > 0.05), especially the PCP-based gels stored under long-term condition for 12 mo during which their viscosity decreased to approximately 10% of the initial value. It becomes apparent that the gel viscosity diminishes with aging, which is caused by the elastic contraction of polymer chains [It should be noted that the visual appearance and consistency of the investigated r chains .C. comosa gels after storage are presented in p > 0.05), ranging from 99.1 to 100.6% and 95.5 to 100.1% for 6 and 12 mo storage. For C. comosa gels storage under accelerated condition, the percentages remaining of DA1 were in the range of 42.1 to 66.2% of which 2% PCP ~3% PCP < 2% PCP:HPMC K15M < 3% PCP:HPMC K15M (p < 0.05). Interestingly, the percentages remaining of DA2 in all aged C. comosa gels insignificantly increased irrespective of the storage condition and time (p > 0.05). The higher remaining content of DA1 and comparable DA2 content of PCP:HPMC K15M-based than those of PCP-based C. comosa gels suggest the absence of incompatibility between DAs and HPMC (ATR-FTIR result, The percentages remaining of DAs in C. comosa mucoadhesive tablets based on HPMC K100M [C. comosa crude extract was not only a time—but also a temperature-dependent process. After 6 mo storage under 40 °C, the percentage remaining of DA1 was 13.3 ± 0.1%. The proposed degradation processes of DA1 and DA2 were oxidation and hydrogenation or dehydration. The increase in DA2 content upon storage under high temperatures, e.g., 40 °C, was associated with the degradation of some other components contained in C. comosa extract into DA2 [C. comosa extract was a product intended to be stored in a refrigerator or under low temperatures.The significant decrease in DA1 content upon storage under accelerated condition is in line with the previous report that investigated the MC K100M . It has into DA2 . This stC. comosa. The C. comosa mucoadhesive gels could be successfully formulated with the investigated mucoadhesive polymers, namely, C980, PCP, HPMC K15M, HPMC K100M, and 2:1 PCP:HPMC blends. Because of the large and hydrophobic molecular structure of DAs, their release from C. comosa gels could be sustained, in the zero-order release manner after a predetermined lag time, from the polymeric network of the investigated mucoadhesive polymers. The Korsmeyer–Peppas model fitting suggested that the release mechanisms of DA from C. comosa gels follow the non-Fickian transport, an approximate Case II transport, involving the polymeric swelling—relaxation and disentanglement of the polymer chains. The stability investigation indicates that C. comosa gels possessed excellent physical and chemical stability for up to 12 mo under a refrigerator or low-temperature storage. Considering the rheological, mechanical, mucoadhesiveness, in vitro release, and stability of DAs, the PCP:HPMCK15M-based gels were considered as a favorable mucoadhesive vaginal gel system for C. comosa crude extract. Nevertheless, it should be also noted that the high mucoadhesiveness formulations can give rise to local pain and tissue damage. Future investigations regarding the local irritation and clinical efficiency of the C. comosa mucoadhesive vaginal gels for the treatment of vaginal dryness and/or the symptoms associated with the genitourinary syndrome of menopause shall be performed.This study was designed to investigate the mucoadhesive vaginal gels for sustained release delivery of phytoestrogen DAs from


Aseptic cell sorting is challenging, especially when a flow‐cytometric cell sorter is not operated in a sterile environment. The sheath fluid system of a cell sorter may be contaminated with germs such as bacteria, yeasts, viruses, or fungi. Thus, a regular chemical cleaning procedure is required to prepare a sorter for aseptic cell sorting by flushing the fluidic system. However, this procedure is time consuming, and most importantly, the researcher can never be sure that the cleaning process was successful. Here we present a method in which the sheath fluid of a cell sorter was decontaminated by irradiation with UV‐C light using a flow‐through principle. Using this principle, we were able to achieve a 5 log reduction of bacteria in the sheath fluid. LBlysogeny brothLEDlight‐emitting diodeP. aeruginosaPseudomonas aeruginosaPIpropidium iodideUVultraviolet1Flow‐cytometric cell sorting is a key technology in bio‐medical research laboratories used to separate cells based on their biological and physical properties . The conFlow‐cytometric cell sorters typically do not operate within a sterile environment, making contamination with microorganisms very likely, for example, when the sheath fluid reservoir is opened for refilling. Thus, a chemical cleaning procedure based on sodium hypochlorite or ethanol is required to prepare a cell sorter for aseptic cell sorting . Such a In many cell sorting laboratories, antibiotics are added to the collection medium of the sorted cells to prevent the proliferation of bacteria entering through the sheath fluid. This, in turn, may lead to an unwanted change in gene expression and regulatory level of the cultured cells and affect the subsequent results . FurtherThe lethal effect of UV light  is well The antimicrobial effect of UV‐C light is based on the absorption of photons in the wavelength range of 200–280 nm by the DNA, resulting in the formation of pyrimidine dimers, which inhibit DNA replication and consequently block transcription into RNA . UV‐C liHere we present a method based on a UV‐C unit for flow‐through irradiation of sheath fluid to enable aseptic cell sorting. In a proof‐of‐principle study, the UV‐C unit was placed between the sheath fluid tank and the nozzle in a BD Influx™ cell sorter  consists of a meandering stainless steel fluid path with a total volume of 60 ml in which the sheath fluid is irradiated by six UV‐C LEDs  with an emission wavelength of 275 nm. The LEDs are located at each channel turning point Figure . The totThe sheath fluid of a flow‐cytometric cell sorter can potentially be contaminated with proliferating microorganisms. Our approach uses UV‐C light emitting diodes (LEDs) to irradiate the sheath fluid of a cell sorter in process. This procedure prevents the effects of bacterial contamination on the cell culture of the sorted cells.The unit is equipped with an active Peltier cooling element Figure  to preveP. aeruginosa at a concentration of 1.1 × 105 bacteria/ml.The efficiency of the UV‐C unit to inactivate microorganisms was characterized using flow cytometry, solid culture medium (LB agar), and the cultivability of Jurkat cells sorted in contaminated and irradiated sheath fluid. To this end, the sheath fluid of a BD Influx™ cell sorter was contaminated with 2 (Figure P. aeruginosa is increasingly damaged with increasing UV‐C dose [The results of the flow‐cytometric analysis using the nucleic acid dyes Syto9 and PI are shown in Figure 2 Figure . However2 Figure . This suV‐C dose , 11, 12 P. aeruginosa could be achieved for all applied doses which meets the effectiveness of the reduction levels of the chemical washing protocols suggested by the cytometer manufacturer [The flow‐cytometric results were confirmed by plating the irradiated and non‐irradiated sheath fluid on LB agar. For all doses applied, no bacterial colonies were detected after 24 h of cultivation. Consequently, using the UV‐C LED unit in the described configuration, a 5 log10 reduction of P. aeruginosa to assess the impact of UV‐C irradiation of sheath fluid on cell cultivability. After sorting, the cells could be cultured for at least 72 h even when applying the lowest UV‐C dose of 1.4 J/cm2. However, if the sheath fluid is not irradiated by UV‐C light, the cell culture was overgrown with bacteria within 24 h of incubation. This confirmed the results determined by plating experiments on LB agar where the lowest UV‐C dose of 1.4 J/cm2 was sufficient to prevent proliferation even if the membrane was not completely degraded as indicated by nucleic acids staining (Figure Jurkat cells were sorted in sheath fluid contaminated with 32, which is about 5400 times above the required dose to achieve a 4 log10 reduction of P. aeruginosa. Thus, even if the applied UV‐C dose is lower than the calculated dose, it is very likely that other microorganisms, such as yeasts, fungi or viruses [Following the calculated dose estimates, the UV‐C unit enables a maximum dose of 42 J/cm viruses , 11 can K.v.V and B.G. are employees of APE. All other authors declare no commercial or financial conflict of interest.SUPPORTING INFORMATIONClick here for additional data file.


Syn-1 and Syn-4 both correlated with VEGFR-1  whereas Hyaluronan correlated with ET-1  and Ang-2 . There was no correlation between Pro-ANP and any of the EG biomarkers. Distinct patterns of association between biomarkers of EG shedding and endothelial cell activation were observed among patients undergoing resuscitation for sepsis. No relationship between IV fluid volume and Pro-ANP or any of the other biomarkers was observed.Endothelial glycocalyx (EG) shedding is associated with septic shock and described following intravenous (IV) fluid administration. To investigate the possible impact of IV fluids on the pathobiology of septic shock we investigated associations between biomarkers of EG shedding and endothelial cell activation, and relationships with IV fluid volume. Serum samples were obtained on admission (T0) and at 24 h (T24) in patients undergoing haemodynamic resuscitation for suspected septic shock in the emergency department. Biomarkers of EG shedding—Syndecan-1 (Syn-1), Syndecan-4 (Syn-4), Hyaluronan, endothelial activation—Endothelin-1 (ET-1), Angiopoeitin-2 (Ang-2), Vascular Endothelial Growth Factor Receptor-1(VEGF-1) and leucocyte activation/inflammation—Resistin, Neutrophil Gelatinase Associated Lipocalin (NGAL) and a marker of cardiac stretch—Pro-Atrial Natriuretic Peptide (Pro-ANP) were compared to the total IV fluid volume administered using Tobit regression. Data on 86 patients  with a mean age of 60 (SD 18) years were included. The mean fluid volume administered to T24 was 4038 ml (SD 2507 ml). No significant association between fluid volume and Pro-ANP or any of the biomarkers were observed. Syn-1 and Syn-4 were significantly correlated with each other (Spearman Rho 0.43,  There is emerging evidence of harm associated with excess fluid balance in sepsis patients4 and clinical trials in some populations have demonstrated better patient outcomes with a fluid-restricted approach6. Consequently, there is clinical uncertainty about the optimal dosing of IV fluid and the associated timing of vasopressor initiation7, and there is substantial variation in practice8.Intravenous (IV) fluid resuscitation is the first line treatment for the haemodynamic resuscitation of patients with septic shock, with international consensus guidelines recommending initial resuscitation with 30 ml/kg of isotonic crystalloid over the first three hours10. The EG is a 0.5 μm thick gel-like layer of proteoglycans anchored to the luminal endothelial surface, with glycosaminoglycan (GAG) molecules incorporated throughout the structure. Syndecans are membrane-bound proteoglycans on the apical surface of endothelial cells to which several different GAGs such as Heparan Sulfate and Chondroitin Sulfate attach covalently to form the EG layer. In contrast, the GAG Hyaluronan is not attached to Syndecan but to cell surface CD44 or other GAG molecules13. In settings such as inflammation, cleavage of the Syndecan ectodomain by sheddases such as matrix metalloproteinases (MMPs) delivers the soluble component to the circulation14.One suggested mechanism for harm with IV fluids is shedding of the endothelial glycocalyx (EG) layer due to hypervolaemia and/or haemodilution15. EG shedding occurs in response to both acute and chronic inflammation and elevation of blood Syndecan-1 (Syn-1) and Hyaluronan concentration has been described in patients with sepsis17. It has been suggested that EG shedding leads to fluid extravasation and propagation of the systemic inflammatory response in sepsis with resultant worsening organ dysfunction and shock20. However, recent preclinical studies have challenged whether EG shedding results in increased vascular permeability22. Additionally, it is unclear whether exogenous fluids have a direct effect on the EG, or whether this is mediated via MMPs activated by natriuretic peptides released in response to cardiac chamber dilatation24.The EG is a key determinant of fluid homeostasis as well as maintaining inflammatory and haemostatic quiescence in health26. Further, studies in healthy subjects and perioperative settings may not be relevant in settings such as sepsis where EG shedding is already established27. The question of the incremental effect of exogenously administered fluids on an already injured EG is unresolved.While it is well established that increased biomarkers of EG degradation are associated with illness severity and mortality in sepsis, data from patients undergoing fluid resuscitation have shown variable associations between IV fluid volume and concentrations of individual EG biomarkersWhile clinical uncertainty remains, it is important to test the hypothesis that IV fluid volume is detrimental in sepsis via the effect of EG shedding. The objective of this study was to examine the relationship between the volume of IV fluid administered during the first 24 h from commencement of treatment in the emergency department (ED) and soluble biomarkers of EG shedding, endothelial activation and inflammation in adult patients with suspected septic shock. The secondary objective was to explore associations between EG biomarkers and biomarkers of endothelial cell activation and inflammation after fluid resuscitation.29. The inclusion criteria were (1) the presence two or more systemic inflammatory response (SIRS) criteria and (2) a clinical decision to administer IV antibiotics for suspected infection and (3) a systolic blood pressure of < 90 mmHg despite at least 1 L of IV fluid along with either (a) blood lactate > 2 mmol/L or (b) a requirement for vasopressors to maintain perfusion targets. These criteria reflect an operational definition for septic shock. Recruitment took place according to the Sepsis 2 criteria which were based upon SIRS. The Sepsis 3 criteria introduced in 2016 placed greater importance on lactate and vasopressor requirement to define septic shock. All of these patients were undergoing resuscitation for hypotension/hypoperfusion related to infection in the ED and thus represent a clinically relevant population in terms of the research question.We undertook secondary analysis of a convenience sample of adult (age 18 years or older) ED patients undergoing haemodynamic resuscitation for suspected septic shock recruited into two observational studies one in Perth, Australia between 2010 and 2015, the other in Boston, USA between 2012 and 2018. The methodologies of each study have previously been describedBlood samples were collected on recruitment in the ED (T0), and subsequently 24 h later (T24). Samples were processed within 2 h of collection, with serum collected by centrifugation at 3000 rpm for 10 min, followed by storage of 0.5 ml aliquots at − 80 °C. Clinical data included baseline demographics, vital signs, laboratory results, suspected source of infection, Sequential Organ Failure Assessment (SOFA) scores, Charlson Comorbidity Score (CCS), admission to the intensive care unit (ICU), length of hospital stay and in-hospital mortality. Fluid volumes were calculated as the total administered IV, prior to each time point , including prehospital fluids, but excluding drug infusions or flushes.32. All analytes except ET-1 were assayed using R&D ‘DuoSet’ Enzyme Linked Immunosorbent Assays (ELISA), . Each kit was individually optimised prior to use, to achieve average intraplate coefficient of variations (CVs) as follows: Syn-1 5.14%, Syn-4 4.39%, Hyaluronan 8.59%, Ang-2 5.40%, VEGFR-1 8.31%, Pro-ANP 5.68%, Resistin 5.09% and NGAL 4.58%. ET-1 was assayed using a fully validated Quantikine ELISA kit  in accordance with manufacturer’s instructions. All biomarker analyses were undertaken in the laboratory of the Centre for Clinical Research in Emergency Medicine in Perth, Australia.Biomarker analyses were determined by enzyme-linked immunosorbent assay (ELISA). Selected EG biomarkers were Syndecan-1 (Syn-1), Syndecan-4 (Syn-4) and Hyaluronan. Selected endothelial cell activation biomarkers measured were Endothelin-1 (ET-1), Angiopoietin-2 (Ang-2) and Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1). We also measured a biomarker of cardiac chamber stretch, Pro-Atrial Natriuretic Peptide (Pro-ANP), and the leucocyte activation biomarkers Resistin and Neutrophil Gelatinase-Associated Lipocalin (NGAL). These biomarkers were selected based upon previous work by our groupsWe measured the relationship between total cumulative fluid volume and biomarkers at T0 and T24 and the change in value of each biomarker between T0 and T24, and with the cumulative total volume at T24. The relationship between pairs of biomarker concentrations at T24 was analysed by Spearman correlation. Because of censoring of some biomarker results both below and above the limit of assay detection, repeated measures Tobit regression was employed with an interaction of time and fluids to test for an association between cumulative fluid volume at T24 and the change over time in the biomarker. For biomarker variables with no censored values, random effects linear mixed models were used. Regression analyses are dependent on assumptions, but these are specific to each regression not to the data set as a whole. All biomarker concentrations were log transformed for multiple reasons. The first is that none of the biomarkers were normally distributed  and while this is not an assumption of regression, but rather normality of the residuals, the latter generally occurs if the former is true. Secondly the log transformation can often solve the problem of heteroscedasticity if present. When the transformation did not successfully address these issues, bootstrapping was employed. This resampling technique produces standard errors and p values that are robust to the violation of assumptions.Associations between the cumulative fluid volume and biomarker concentrations were examined for possible non-linearity using scatterplots with a Lowess fit as well as multivariable regression spline models. Where non-linearity was detected, piecewise Tobit or linear regressions were used to estimate multiple slopes in the one model. All regression models were adjusted for age, sex, baseline mean arterial pressure, baseline lactate, SOFA score, CCS, suspected source of infection  and recruitment site. Where violations of assumptions were detected, regression analyses were boot-strapped to produce robust standard errors and p values. The analyses were performed using Stata v15 . Given the exploratory nature of the study p values were not adjust for multiple testing.The study was approved by the Human Research Ethics Committee of Royal Perth Hospital and the Institutional Review Board of Beth Israel Deaconess Medical Center. Informed consent was obtained from the participant or their next of kin, or under a waiver of consent for low-risk observational research in accordance with local laws.Of 86 adult participants recruited, 11 (13%) died within 30 days. The characteristics of the participants are described in Table The mean cumulative total fluid volume administered prior to T0 was 2330 ml (SD 1698 ml) and to T24 was 4073 ml (SD 2507 ml). A vasopressor infusion was commenced in 55 (64%) participants within 24 h. The predominant fluid administered was isotonic crystalloid. Additionally, fourteen patients received colloid  and one received blood. Two participants received more than 10L of IV fluid (12L and 14L respectively) within the first 24 h.The median concentration of each biomarker at the two sampling time points are shown in Table No significant relationship was observed between the biomarkers and the cumulative fluid volumes at T0 or at T24. No significant interaction between fluid volume at T24 and the change over time was found for any of the biomarkers. Figure Table In this observational study of adult patients with clinically suspected septic shock undergoing fluid resuscitation in two emergency departments, no relationship between volume of intravenous fluid and concentrations of biomarkers of EG shedding or endothelial activation was found. We did observe two distinct patterns of potential EG shedding; a significant association between Syn-1/Syn-4 and VEGFR-1; and between Hyaluronan and Ang-2, ET-1, Resistin and NGAL. There was no observed correlation between Syn1/Syn4 and Hyaluronan, nor between Pro-ANP and any of other EG biomarkers.27. Moreover, soluble biomarkers are a surrogate measure of EG shedding and their utility as an accurate quantitative measure of this process has been questioned in a recent literature review27. The impact upon the expression of EG from vasopressors is unclear. In mitigation of this, the SOFA score does capture the use of vasopressors, and we adjusted for SOFA in the regression analyses. Due to the retrospective nature of this analysis, we were unable to perform direct estimation of EG thickness  which would have provided a more comprehensive assessment33. Finally, the small number of participants precluded any exploration of potentially important differences in the effect of fluid types on the EG 15 and patient subgroups. Given these factors, the lack of any demonstrated positive associations between fluid volume and EG shedding does not necessarily imply that such associations do not exist.Strengths of the study include recruitment during the initial resuscitation phase of suspected septic shock. Limitations include its observational design which, despite controlling for variables that would be expected to influence the volume of fluid administered, residual confounding cannot be discounted. The large number of censored values for Hyaluronan (22/172 censored observations at T24) and VEGFR-1 (71/166 censored observations at T24) reduced the power to detect significant differences for these biomarkers. Measurements were limited to two time points at T0 and T24 and therefore would not capture the biomarker profiles during the first 6 h of resuscitation when the largest volumes of fluid are typically administered. The nature of real-world clinical research among acutely sick patients in the emergency setting is that many patients had treatment initiated prior to enrolment, even pre-hospital. The analyses chosen take account of this by describing the fluid administered prior to T0. The biomarkers selected were not a comprehensive suite of all molecules released by EG shedding and endothelial cell activity34. In patients with sepsis in the ED, Smart et al.described a positive association between fluid volume and hyaluronan during the first 3 h of resuscitation however no such relationship was seen for Syn-1 or Syn-417. Hippensteel et al.reported an association between the volume of IV fluid and the expression of Heparan Sulfate (measured by mass spectroscopy) after 6 h of resuscitation in patients with sepsis25. The timing of sampling in our study prevents direct comparison with these studies.We did not observe any association between IV fluid volume, Pro-ANP or any biomarker of EG shedding, endothelial activation of inflammation. These clinical observations are in contrast to experimental and preclinical studies which have found evidence of EG shedding associated with administration of exogenous fluids, with some studies also showing a concurrent rise in Pro-ANP26. In a multicentre ICU study of 619 patients there was no relationship between fluid volume in the first day of admission and Syn-1 among patients with sepsis, although Syn-1 was associated with illness severity and mortality35. Similarly, in a prospective study of 175 patients with sepsis, Puskarich et al.found that high Syn-1 levels were associated with increased renal injury and mortality but there was no significant difference in the volume of IV fluids administered between two groups categorised according to the level of Syn-136. In contrast, Soraya et al. found that Syn-1 measured at the time of admission to the ICU was associated with subsequent fluid requirements, as well as with organ failure and mortality37. This raises the question of whether an association between elevated EG biomarkers and fluid volume simply represents larger fluid resuscitation requirements in patients with a leaky vasculature rather than fluid induced EG shedding. Further complicating the issue are recent preclinical experimental studies which have challenged whether there is any relationship between EG shedding and increased vascular permeability38.In a randomised trial comparing a fluid restricted resuscitation strategy versus a liberal fluid regimen for resuscitation among patients with septic shock, Soaraya et al.found Syn-1 levels at 6 h to be lower with the limited fluid regimen (geometric mean ratio of 0.82 (95% CI 0.66–1.02), p = 0.07)n = 66) which demonstrated lasting changes in the perfusion boundary region  following IV volume loading in sepsis patients39. There was a transient increased in PBR in elective surgical patients which resolved. A similar effect was seen in septic shock patients, however this persisted at least 120 min after the infusion. Whether this is a direct effect or mediated by the effect of ANP on MMPs is not clear9. Our results are consistent with those of Hippensteel et al.who found no significant relationship between ANP and Heparan sulfate, interleukin-6 levels (a marker of acute inflammation) or mortality25. This, however, may be explained by timing of sampling in different studies. Belavic et al. demonstrated an early rise in ANP along with Syn-1 after administration of IV fluids in surgical patients40. Possible explanations for these discordant results could be due to insufficient volume administered to generate cardiac stretch to increase expression. Alternatively, unlike euvolemic perioperative patients, sepsis patients may have absolute or relative hypovolaemia at baseline.The hypothesis of a relationship between fluid volume and EG degradation is supported by a study by Pouska et al. (41. These distinct mechanisms may explain the unexpected finding of a lack of correlation between the Syndecans and Hyaluronan in this study. Replication of this discordance in further studies is an important first step.Of interest is the potential discordant patterns of EG shedding and associations with the endothelial activation biomarkers. Release of Hyaluronan occurs via incompletely understood mechanisms but includes cleavage of the CD44 molecule by MMP-15, as well as by Hyaluronidase and reactive oxygen species27. The conditions of sepsis may upregulate endothelial Syn-1 and Syn-4 expression, which may contribute to circulating concentrations42. Further, serum Hyaluronan concentration may be influenced by interstitial washout of tissue Hyaluronan secondary to fluid loading, which may confound the temporal pattern of EG shedding of Hyaluronan. Others have found that Hyaluronan peaks within the first 3–6 h of treatment43, and therefore this would not be captured at the time points studied. The lack of increase in Hyaluronan at 24 h suggests that an earlier peak may have been missed. These differences may be due to various mechanisms of shedding, sources of EG biomarkers and temporal patterns and explain why relationships between individual EG biomarkers cannot be established at isolated time points.There are other reasons for EG biomarkers to increase in circulation that is not specifically related to EG shedding44. In contrast Hyaluronan was not associated with VEGRF-1 but was associated with ET-1 and Ang-2. This finding supports recent data which suggests Ang-2 plays a role in Hyaluronan shedding41. It was also interesting to note the positive correlation between Hyaluronan and inflammatory biomarkers; fragmented Hyaluronan is known to be pro-inflammatory and may have driven this association45. When examining associations between biomarkers reported in this study, it is important to note that similar associations were identified at T0 and T24. Glycocalyx shedding and endothelial activation is likely on a continuum prior to, and during, treatment of septic shock, and that, despite attempts to adjust for confounding, this conceals any modification by the volume of resuscitation.There was an association between both Syndecan types and VEGFR-1. This molecule, also known as sFlt-1, has previously been found to be associated with organ failure and an accurate predictor of mortality among ED patients with sepsis46. Another study found no difference in Syn-4 concentrations between critically ill ICU patients and healthy controls47. The correlation between Syn-1 and Syn-4 is consistent with increased endothelial expression of these glycosaminoglycans in response to circulating cytokines in systemic inflammation in the setting of critical illness14. The discordant patterns between T0 and T24 may reflect downregulation of Syn-4 by Syn-1, as has been demonstrated in vitro48.Syn-4 has been studied less than Syn-1 as a biomarker of EG shedding. In one study of patients with pneumonia, Syn-4 levels trended downwards over time, as observed in the present study, and had a negative correlation with illness severity20. The question of the optimal approach to haemodynamic resuscitation in septic shock is important due to the high morbidity and mortality of this condition. A clinical trial in African children with sepsis and hypoperfusion found that fluid resuscitation increased mortality due to shock49. In a preclinical ovine sepsis model, resuscitation with intravenous fluids resulted in a larger subsequent dose of vasopressor to maintain a target blood pressure18. EG shedding is one of several mechanisms by which exogenous fluid resuscitation may be detrimental in septic shock. While emerging evidence favours a fluid-sparing or de-resuscitation fluid strategy among ICU patients with sepsis4, there is uncertainty about the optimal approach to initial haemodynamic resuscitation. Our findings do not support the hypothesis that IV fluid resuscitation exacerbates EG shedding in sepsis. Indeed, recent clinical observational studies have suggested that early intravenous fluid resuscitation is associated with reduced odds of mortality among ED patients with septic shock51. Thus, an overly restrictive fluid strategy in sepsis resuscitation due to fears of causing EG injury may be unwarranted. This is the subject of several active clinical trials54.The clinical importance of this study relates to concerns about a possible detrimental effect of exogenously administered fluids, particularly in the setting of septic shock55. While the results of clinical trials are awaited, a judicious approach to fluid administration which aims to protect the EG remains prudent15.In addition to the impact of IV fluids, further research is required to confirm the apparent finding of different patterns of EG shedding and the implications for clinical practice. The significant heterogeneity of sepsis may make a straightforward answer elusive. Indeed, it is possible that the inability to reproduce the findings of preclinical and healthy volunteer studies in the clinical setting likely reflects the fact that EG shedding is already established in critical illness. Novel therapies directed at restoring the shed glycocalyx is one avenue of current investigationIn conclusion, distinct patterns of association between biomarkers of EG shedding and endothelial cell activation were observed among patients undergoing resuscitation for sepsis. We did not find evidence to support the hypothesis that degradation of the EG is exacerbated by higher resuscitation fluids volumes in patients with septic shock.Supplementary Information.


Candida yeast cells revealed thatdespite their high structural similarity, each of the drug probeshad a unique subcellular distribution pattern. Fluorescent CSF, whichis the least potent of the three echinocandins, accumulated in Candida vacuoles; fluorescent ANF localized in theextracellular environment and on the yeast cell surface where thetarget GS resides; and fluorescent RZF was partitioned between thesurface and the vacuole over time. Recovery of fluorescent CSF from Candida cells revealed substantial degradation overtime; functional vacuoles were necessary for this degradation. Underthe same conditions, fluorescent ANF was not degraded. This studysupports the “target-oriented drug subcellular localization”principle. In the case of echinocandins, localization to the cellsurface can contribute to improved potency and accumulation in vacuolesinduces degradation leading to drug deactivation.Echinocandin antifungaldrugs have a broad spectrum of activitiesand excellent safety profiles. These agents noncompetitively inhibitthe formation of the major polysaccharide component of the fungalcell wall, a reaction catalyzed by the membrane-bound β-glucansynthase (GS) protein complex. We have developed fluorescent probesof three echinocandin drugs: caspofungin (CSF), anidulafungin (ANF),and rezafungin (RZF). Fluorescent echinocandins had the same spectrumof activities as the parent echinocandins, supporting the fact thatconjugation of the dye did not alter their mode of action. Of thethree echinocandins, ANF has the most potent in vitro activity. Investigationof the subcellular distribution of the fluorescent echinocandins inlive  These differences did not alwayscorrelate with the differences in MIC values, suggesting that additionalparameters contribute to differences in antifungal activities of thesedrugs.Comparisonof minimal inhibitory concentration (MIC) values ofCSF to those of ANF indicates that the latter has more potent antifungalactivity in vitro.40 In this study, we applied a similar strategy and designed and synthesizedfluorescent probes of CSF, ANF, and RZF and studied their time-dependentsubcellular distribution and stability in live yeast cells of thegenus Candida.In search of design guidelines for the next generationof echinocandindrugs, we have evaluated how specific molecular characteristics ofthree members of the echinocandin class of antifungals affect theirsubcellular distribution patterns and antifungal efficacies. We havepreviously reported on the development of fluorescent probes for studyingthe effects of antifungal azoles and antifungal cationic amphiphilesagainst cells of pathogenic fungi.To facilitatetracking of the echinocandins in live yeast cells,each drug was conjugated to two different fluorescent dyes that differsignificantly in their molecular weight and formula: tetramethylrhodamine using a click reaction to form fluorescent CSF probes 1 and 2 . The MIC values for all strains,determined using the broth double-dilution method, are summarizedin Table S2.To confirm that, similar to the parent echinocandins, the fluorescentprobes S,4S-dihydroxy-l-homotyrosine phenol. The antifungalactivity of the TMR-labeled CSF probe 1 was superiorto that of the NBD-labeled CSF probe 2, whereas the activityof the NBD-labeled RZF probe 6 was superior to that ofthe corresponding TMR-labeled RZF probe 5. These resultsindicate that the optimal choice of fluorescent dye depends on theindividual echinocandin and that the molecular weight of the fluorescentdye cannot serve as a general guideline for the design of fluorescentechinocandin probes with optimal antifungal activity.In general, labeling of ANF andRZF with NBD resulted in slightlysuperior antifungal activities relative to the corresponding TMR-labeledprobes. In contrast, the antifungal activity of the TMR-labeled CSFprobe was slightly superior to that of the corresponding NBD-labeledprobe. For the conjugation of the fluorescent dye, CSF and RZF probeswere functionalized at the same position, the 3FKS1 and/or FKS2 genes of the GS complex, were resistant to these fluorescent probes,and echinocandin-susceptible strains were susceptible. This supportsthe hypothesis that similar to the echinocandins from which they werederived, probes 1–6 inhibit the activityof the GS complex.Importantly,all of the fluorescent echinocandin probes had thesame spectra of activity as their corresponding parent echinocandindrugs. The echinocandin-resistant strains, in which resistance isdue to mutations in the Candida strains, C. albicans SC5314 and C. glabrata strain ATCC66032, over 60 min of incubation. Comparison of subcellular distributionsof TMR-labeled CSF, ANF, and RZF probes  and of NBD-labeled CSF, ANF,and RZF probes  over time revealed three different and distinguishablepatterns (S9–S11).The subcellular distribution patterns of the fluorescent echinocandinprobes were investigated in cells of two representative echinocandin-susceptible 1 and NBD-based 2, localized to the vacuolesof the yeast cells.32 Vacuolar accumulationintensified and reached a maximum at approximately 60 min. In contrast,TMR-based and NBD-based ANF probes 3 and 4, respectively, did not permeate the yeast cells and remained predominantlyin the extracellular environment and on the yeast cell surface forthe entire 60 min duration of the experiment. The RZF probes 5 and 6 appeared in the extracellular environmentand on the yeast cell surface after 15 min; however, after 60 min,both probes localized mainly to the vacuoles. Consistent with this,TMR-labeled probes  co-localized with the vacuole-specific fluorescent dye CellTrackerBlue 7-amino-4-chloromethylcoumarin (CMAC)41 after 60 min of incubation in C. albicans SC5314, with Pearson correlation values42 of 0.73 ± 0.06, 0.03 ± 0.04, and 0.47 ± 0.04, respectively.Similarly, the NBD-labeled echinocandins  co-localized with CMAC after 60 min of incubationin C. albicans SC5314, with Pearsoncorrelation values of 0.40 ± 0.08, 0.15 ± 0.05, and 0.28± 0.02, respectively.Within 15 min after introduction, the CSF probes, TMR-based 1, 3, and 5 were similar to those of the correspondingNBD-based probes 2, 4, and 6 (S9–S11). The fact that the TMR-labeledand the NBD-labeled echinocandins had similar subcellular distributionpatterns, taken together with the large differences in chemical propertiesof the two dyes, supports the hypothesis that the drug scaffolds,and not the conjugated fluorescent dyes, determined the subcellulardistribution patterns of the fluorescent echinocandin probes.If the fluorescent dyes determinedthe subcellular distributionof the fluorescently labeled echinocandins, all three echinocandinswould share the same subcellular localization. However, fluorescentlylabeled CSF probes accumulated in vacuoles of the yeast cells, fluorescentlylabeled ANF probes localized predominantly to the extracellular environmentand the yeast cell surface, and fluorescently labeled RZF probes appearedin the extracellular environment and on the yeast cell surface shortlyafter their introduction and were detected in vacuoles after a longerincubation period. Of note, the time-dependent subcellular distributionpatterns of the TMR-based echinocandin probes D) values, defined as the ratio of concentrations of ionized to neutralspecies of the compound at the physiological pH of 7.4. CSF probes 1 and 2 were the most hydrophilic of the probeswith logD values of −7.70 and −6.95,respectively, and ANF probes 3 and 4 werethe most hydrophobic with log D values of −0.85and −0.10, respectively. The calculated log D values of RZF probes 5 and 6 were −5.08and −4.32, respectively. The fluorescent probes of the mosthydrophobic echinocandin, ANF, accumulated on the cell surface wherethe target GS complex resides, and those of the most hydrophilic echinocandin,CSF, were cell-permeable. With intermediate log D values, RZF probes concentrated on the surface of the yeast cellsshortly after introduction and then slowly permeated the cell andaccumulated in the vacuoles.In an attempt to rationalize the distinctively different subcellulardistributions of the three echinocandins, our attention was drawnto differences in hydrophobicity/hydrophilicity ratios as evidentfrom their calculated distribution coefficient . Through a time-dependent investigation of the subcellular distributionof fluorescent CSF, we have previously shown that echinocandins causecell lysis under conditions that enable rapid yeast cell growth butdo not cause lysis under conditions that lead to quiescent cell maintenance.32 Thus, growth in the nutrient-rich growth media,which promotes cell lysis and vacuole disassembly, resulted in lessdegradation of the CSF probe than when the cells were maintained ina nutrient-free buffer. As controls, probes were incubated for a veryshort period (5 min) with the cells in the PBS or were incubated withthe lysate of C. albicans SC5314 inthe buffer for 24 h . In bothcases, ∼80% of CSF probe 1 was intact. These resultsindicate that significant CSF degradation requires prolonged incubationin cells with intact functional vacuoles.Due to the superior stability and spectral properties of TMR, weused TMR-based CSF and ANF probes ter 24 h 5A. Of no3 was incubated with cells or lysatesof C. albicans strain SC5314, no significantdecomposition was detected over the 24 h duration of the experimentregardless of the media used (S12). Given the structuralsimilarities between ANF and CSF, these results may be rationalizedby the difference in the subcellular distribution between the twoechinocandins: The CSF probe 1 enters the yeast cellsvia endocytosis32 and accumulates in thevacuoles where it is degraded, whereas the ANF probe 3 localizes mainly to the yeast cell surface and remains intact overthe course of the experiment.When ANF probe Candida cells incubatedwith the fluorescent echinocandins provided visible evidence for differencesin cell permeability and subcellular distribution of these drugs.CSF probes accumulated in vacuoles of the yeast cells, ANF probeslocalized predominantly to the extracellular environment and the yeastcell surface, and RZF probes appeared in the extracellular environmentand on the yeast cell surface shortly after their introduction andwere detected in vacuoles after a longer incubation period. Imagesof yeast cells that were co-incubated with both fluorescent CSF andANF probes revealed that vacuolar uptake of fluorescent CSF was notprevented by the presence of the extracellular-residing fluorescentANF. Comparison of the HPLC chromatograms of extracts of C. albicans cells that were pre-incubated with fluorescentCSF or fluorescent ANF revealed extensive degradation of the CSF probe,whereas the cell-surface-residing fluorescent ANF remained intact.This degradation required intact functional vacuoles.Toenable exploration of the subcellular distribution of differentdrugs belonging to the echinocandin class of antifungals, we synthesizedfluorescent probes of three drugs in this class; CSF, ANF, and RZF.Imaging of live Accumulationof the drug on the fungal cell surface increases itslocal concentration around the target and may therefore contribute,at least in part, to its efficacy as an inhibitor of the GS complex.Uptake by the vacuole results in decomposition of the echinocandinand, as a consequence, can contribute to a reduction in its efficacyover time. The results of this study therefore suggest that designingechinocandins that localize predominantly to the cell surface of thefungal pathogen should contribute to the metabolic stability and potencyof these drugs.1–6 were dissolved in anhydrous dimethylsulfoxide (DMSO) to 5 mg/mL or to 1 mM. CellTracker Blue CMAC waspurchased from Thermo Fisher and was dissolved in DMSO to 10 mM.The antifungaldrug CSF (purchased from S.L. Moran Ltd.), ANF , RZF, and compounds Table S1. Two FKS mutants derived from C. albicans SC5314and two FKS mutants from C. glabrata EF1620 were used in this study.Thelaboratory and clinical isolates and ATCC strains used in this studyare listed in Candida strains werestreaked from glycerol stock onto YPAD agar platesand grown for 24 h at 30 °C. Colonies were suspended in 1 mLof PBS and diluted to an optical density of 2 × 10–4 at 600 nm (OD600) in flat-bottom 96-well microplates(Corning) with YPAD broth containing dilutions of each tested compoundat concentrations ranging from 64 to 0.008 μg/mL in twofolddilutions. Control wells with yeast cells but no drug and blank wellsthat contained only YPAD were prepared. MIC values (Table S2) were determined after 24 h at 30 °C by measuringthe OD600 using a plate reader .MIC values were defined as the point at which the OD600 was reduced by ≥75% compared to the no-drug wells. Each concentrationwas tested in triplicate, and the results were confirmed by two independentsets of experiments.Candida strains were streaked from glycerol stocksonto YPAD agar platesand grown for 24 h at 30 °C. Colonies were grown in tubes in3 mL of YPAD broth for 24 h at 30 °C with shaking. Cultures werediluted 1:100 and incubated in YPAD broth for 2 h at 30 °C withshaking until log-phase growth was observed. Cultures were then centrifuged,washed with PBS buffer, and resuspended in PBS buffer. Cells werestained with CellTracker Blue CMAC (10 μM) and incubated withTMR-based probes  at a final concentration of 1 μM or/and with NBD-based probes at a final concentrationof 5 μM at 30 °C with shaking in the dark over a 2 h timecourse. To optimize the concentrations of the probes, we investigatedthe effect of different concentrations of each probe. After washing with PBS, 2 μL aliquots of Candida cell samples were placed on glass slidesand covered with glass coverslips. Cells were imaged using a NikonTi microscope equipped with a Plan Apo VC 100× Oil objectiveand a Zyla 5.5 sCMOS camera (Andor) using NIS elements Ar software.The bandpass filter sets used to image TMR-based probes  had an excitation wavelengthof 560/20 nm and an emission wavelength of 629.5/37.5 nm. To imageNBD-based probes , the excitation wavelength was 470/20 nm, and the emission wavelengthwas 525/25 nm. For CellTracker Blue CMAC, an excitation wavelengthof 350/25 nm and an emission wavelength of 460/25 nm were used. Imageswere processed using ImageJ. Each probe was tested in at least threeindependent sets of experiments, and at least 1500 cells were capturedin each experiment. The Pearson correlation values were determinedusing co-localization analysis with ImageJ.C. albicans SC5314 cells were streakedfrom glycerol stocks onto YPAD agar platesand grown for 24 h at 30 °C. Colonies were grown in tubes in3 mL of YPAD broth for 24 h at 30 °C with shaking. Cultures werediluted 1:50 and incubated in 5 mL of YPAD broth for 3 h at 30 °Cwith shaking until log-phase growth was observed. Cultures were thencentrifuged , washed with PBS buffer, and resuspendedin 1 mL of PBS buffer or YPAD. Cells were incubated with TMR-basedprobes (1 or 3) at a final concentrationof 10 μM at 30 °C with shaking in the dark for 24 h. Afterwashing three times with PBS, cells were resuspended in 75 μLof TNE buffer  in a 1.5 mL Eppendorf tube, and 0.5 mm diameter beadswere added to the yeast solution. The mixture was agitated on a vortexat maximum speed for 5 min. Next, 200 μL of 2% Triton X-100in TNE buffer (v/v) was added to the agitated solution, and then themixture was sonicated for 15 min. To clarify the yeast lysate, themixture was centrifuged twice , and the supernatant was removed and filtered using a 0.22 μmsyringe filter. The samples were analyzed using analytical reverse-phaseHPLC at a 550 nm wavelength. HPLC conditions: mobile phase: acetonitrilein H2O (containing 0.1% trifluoroacetic acid (v/v)), gradientfrom 10 to 90%; flow rate: 1 mL/min; absorbance wavelength: 550 nm.Each sample was tested in two independent sets of experiments. Thepercentages of intact echinocandin were determined using the areacalculation function available in the Hitachi Primaide Software andare presented as means (two replicates) ± standard deviation(SD).