The false codling moth is an indigenous pest of the citrus industry in southern Africa. It is a major threat as it can cause substantial fruit damage through larval feeding. The microorganisms associated with insects are often studied for their potential to kill their host instead of how they could aid them. However, in recent years, this aspect of their interaction has received much attention. The codling moth has been shown to have a close relationship with the naturally occurring yeast found within their digestive tract. These beneficial yeasts help the adult female moths locate a suitable host for egg laying. During their larval phase, these yeasts help them develop faster and increase their chance of survival. These interactions could be manipulated for the purposes of biological control, as one might be able to combine these yeasts with existing biological control agents to improve their effectiveness. These yeasts may also provide additional methods for monitoring field populations of insect pests. In this study, we identified yeasts that occur naturally in the guts of false codling moth larvae and investigated whether any of them are beneficial to their host.Thaumatotibia leucotreta is endemic to southern Africa and is highly significant for various fruit industries, including the South African citrus industry, due to its classification as a phytosanitary pest. Mutualistic associations between C. pomonella, closely related to T. leucotreta, and yeasts have previously been described and reported to reduce larval mortality and enhance larval development. Here, we determined which yeast species occur naturally in the gut of T. leucotreta larvae and investigated whether any of the isolated yeast species affect their behaviour and development. Navel oranges infested with T. leucotreta larvae were collected from geographically distinct provinces in South Africa, and the larvae were processed for analysis of naturally occurring associated yeasts. Six yeast species were isolated and identified from the guts of these T. leucotreta larvae via PCR amplification and sequencing of the ITS region of rDNA and D1/D2 domain of large ribosomal subunit. Larval development and attraction assays were conducted, and T. leucotreta larvae that fed on Navel oranges inoculated with yeast had accelerated developmental periods and reduced mortality rates. Neonate T. leucotreta were also attracted to YPD broth cultures inoculated with yeast for feeding. Oviposition preference assays were conducted with adult T. leucotreta females. Navel oranges inoculated with yeast were shown to influence the oviposition preference of adult females. Yeasts harbour the potential for use in biocontrol, especially when combined with other well-established control methods. This study provides a platform for future research into incorporating yeast with current biological control agents as a novel option for controlling T. leucotreta in the field. Microbes are the most abundant living organisms on earth, and apart from being found within the general environment, they are also known to colonise other living organisms such as insects . The relDrosophila melanogaster (Meigen) (Diptera: Drosophilidae) [Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), commonly known as the codling moth, larvae are closely associated with yeast from the genus Metschnikowia [C. pomonella larvae and reducing mortality. Additionally, the metabolites produced by yeasts contributed towards adult C. pomonella moths recognising and finding their host plant [C. pomonella larvae and yeasts from the genus Metschnikowia has led to the development of novel biocontrol strategies [Unlike their bacterial counterparts, interactions between insects and yeasts have been understudied, despite their importance to insect fitness and behaviour ,19,20. Thilidae) . The prehilidae) ,22,23. Yhilidae) . Yeasts hilidae) . For Cydhnikowia . These yst plant . Identifrategies ,28,29.Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), commonly known as the false codling moth, is a phytophagous insect, endemic to southern Africa [T. leucotreta in South Africa [T. leucotreta [C. pomonella and not for T. leucotreta.n Africa . It is in Africa . The pesn Africa ,33,34. Th Africa . Most noucotreta . It has ucotreta ,28. ThisT. leucotreta larvae from geographically distinct citrus-producing regions across South Africa and to examine whether any isolated yeast species affected the behaviour and development of neonate larvae, as well as the oviposition preference of adult female moths. These yeasts could potentially represent rich resources of new biological agents for the control of T. leucotreta when combined with CrleGV.Herein, we aimed to determine which yeast species occur naturally in the gut of Citrus sinensis L. Osbeck) infested with T. leucotreta larvae were collected from one orchard in each of three geographically distinct citrus-producing regions in South Africa, namely Stellenbosch (Western Cape), Addo (Eastern Cape) and Nelspruit , between March and June of 2018 (Ten Navel oranges ( of 2018 .n = 10) were surface-sterilised by rinsing in 10% NaOCl, followed by a rinse in 70% ethanol solution (v/v) and finally double-distilled H2O (ddH2O). Yeasts were isolated from the digestive tract of T. leucotreta larvae according to Witzgall et al. [2O, filtered through muslin cloth and spread onto a Pen/Strep YPD agar plate. Plates were incubated at 27 °C for 48 h under aerobic conditions, with emerging yeast colonies being selected and discontinuously streaked on Pen/Strep YPD agar plates. This process was conducted twice to obtain pure colonies.The extracted larvae . Genomic DNA (gDNA) was extracted using the YeaStar™ Genomic DNA Kit  per the manufacturer’s instructions. The concentration of gDNA was determined using a NanoDrop 2000 spectrophotometer . The amplicons of the intended targeting region were amplified using 50 ng of gDNA template with the site-specific primers  for the T. leucotreta culture, known as “Mixed Colony”, held at Rhodes University’s Department of Zoology and Entomology, South Africa. The colony was established in 1996 using T. leucotreta collected from Citrusdal (Western Cape Province), Zebediela (Limpopo Province) and the Eastern Cape [Eggs and pupae were obtained from the heterogeneous ern Cape . The larern Cape .T. leucotreta larvae. Navel oranges were collected from orchards in the Sundays River Valley in the Eastern Cape Province of South Africa and stored in a 4 °C cold room to preserve the fruit until use. Oranges were sterilised in a 0.5% bleach solution, rinsed twice in ddH2O and allowed to air dry in a controlled-environment (CE) room at 25 °C overnight. Yeast cultures were grown in YPD medium containing 40 units/mL of Pen and 40 µg/mL of Strep for 20 h at 27 °C while shaking; cell counts were adjusted to 2 × 106 cells mL−1 with ddH2O. Sterilised Navel oranges were dipped three times into a specific yeast culture, placed on a platform and allowed to air-dry in a 25 °C CE room. Five newly hatched T. leucotreta neonates were then placed onto each Navel orange and maintained in a 25 °C CE room for 35 days (d). Cotton wool was placed between the Navel oranges 10 d later to provide emerging 5th instars with a pupation site. Thereafter, the cotton wool was checked daily, and the number of pupated T. leucotreta larvae was recorded. After 35 d, each Navel orange was dissected to check for any remaining T. leucotreta larvae. Assays were conducted on four dates, with nine Navel oranges per treatment.A modified version of the larval development assay described by Witzgall et al.  was usedT. leucotreta yeast feeding and behaviour, adapted from Ljunggren et al. [7 cells mL−1 with ddH2O. Two-choice bioassays were conducted whereby two 50 µL drops of yeast culture and YPD medium were pipetted across from one another, approximately 1 cm from the edge of a plastic petri dish. Red and blue colourants  were used at 1:25 dilution to colour the yeast and YPD medium to distinguish between neonate T. leucotreta that fed on yeast culture, blank medium, or both. Preliminary tests did not show a bias in neonate T. leucotreta attraction to either the red or blue colourants ). Neonate T. leucotreta were starved for 24 to 48 h after hatching before being placed in the centre of a petri dish using a fine paintbrush. A glass lid was used to cover the petri dish to prevent the neonates from escaping and was left for 2 h to allow the larvae to feed. The colouration of the neonate’s gut was then observed under a dissecting microscope. Twenty-two independent replicates with 10 T. leucotreta neonates were performed for each yeast.Two-choice bioassays were used to assess neonate n et al. . Yeast clourants   under light conditions. Trials were conducted in a 25 °C CE room with 30–60% RH under a day/night light cycle of 12 h of light and 12 h of dark. Navel oranges were collected and stored as previously described and removed from cold storage 1 d prior to being used in the ovipositional preference trial. The fruits were checked for T. leucotreta eggs, those with eggs being discarded, before being thoroughly washed in a 0.5% NaOCl solution, rinsed twice in ddH2O and allowed to air dry in a CE room at 25 °C overnight. Yeast cultures were grown as previously described, with counts adjusted to 2 × 106 cells mL−1 with ddH2O. Sterilised Navel oranges were dipped three times into a specific treatment and allowed to air-dry in a 25 °C CE room. Treated Navel oranges were then placed onto 4 cm tall plastic platforms 15 cm apart in a plastic container (60 × 40 × 40 cm) covered with muslin cloth. A single pair of mated adults was then released into the container. The eggs oviposited on the Navel oranges were counted after 48 h. Ten ovipositional preference trials were conducted per yeast species.A modified version of the ovipositional preference trials described by Love et al.  was usedThaumatotibia leucotreta larval development assays were analysed using a Fisher’s exact test [T. leucotreta on yeasts was compared using a Student’s t-test, with the level of significance set to p = 0.05 [t-test, with a significance level of p = 0.05 [All statistical analyses were performed using GraphPad Prism version 9.0.0 . act test . The larp = 0.05 . The ovip = 0.05 .T. leucotreta larvae were screened for yeast, which resulted in the isolation of six yeast species. The isolated yeast species were successfully identified via the gene sequence analysis of the ITS region and D1/D2 domain of the LSU , H. uvarum (p = 0.0092) and S. cerevisiae (p = 0.0024) performed the best out of all the applied yeast treatments, as not only was development significantly faster than that of the control at a 99% probability level, but pupation success was highest for these three species. Although P. kudriavzevii (p = 0.0586), K. marxianus (p = 0586) and C. lusitaniae (p = 0.002) also significantly decreased the larval development period at the same probability level, the number of pupated larvae was similar to that of the control. Additionally, although not recorded, it was noted that the occurrence of mould was lower on yeast-inoculated Navel oranges compared to the untreated control.Navel oranges treated with yeast significantly decreased the larval development period and increased the larval pupation rate compared to sterilised fruit with no yeast treatment . Pichia T. leucotreta larvae was significantly lower on Navel oranges treated with M. guilliermondii (p = 0.0452), P. kluyveri (p < 0.0001), H. uvarum (p = 0.0144) and S. cerevisiae (p = 0.0194) (Clavispora lusitaniae (p > 0.9999), P. kudriavzevii (p = 0.0586) and K. marxianus (p = 0.0586) did not decrease larval mortality. Overall, P. kluyveri provided the most benefit to T. leucotreta larvae, as the yeast decreased larval mortality by 20.55% and 74% of larvae pupated before 25 d.The mortality rate of  0.0194) . ClavispT. leucotreta was influenced by four yeasts, viz. P. kluyveri (p = 0.0043), H. uvarum (p = 0.0037), P. kudriavzevii (p = 0.0001) and K. marxianus (p = 0.0005) (M. guilliermondii (p = 0.0606) and S. cerevisiae (p = 0.2579), had no significant effect on larval feeding. The only time larvae preferred the YPD medium was in the case of C. lusitaniae (p = 0.5907).The feeding preference of neonate  0.0005) . Two yeaM. guilliermondii (p = 0.0090), P. kudriavzevii (p = 0.0471) and H. uvarum (p = 0.0013) compared to the untreated control fruit during the two-choice tests (Pichia kluyveri (p = 0.3768), C. lusitaniae (p = 0.5729), K. marxianus (p = 0.2838) and S. cerevisiae (p = 0.0517) did not influence the oviposition preference of adult T. leucotreta females.Significantly more eggs were oviposited on Navel oranges inoculated with ce tests . Pichia T. leucotreta.Most interactions between insects and microorganisms have been studied for their potential use as insect pathogens and not for their role as mutualists . Yeasts Meyerozyma guilliermondii, P. kudriavzevii, P. kluyveri and H. uvarum are widely distributed in the environment and often associated with citrus fruits or fermented food [T. leucotreta is not unexpected. Hanseniaspora uvarum is also thought to have a mutualistic relationship with Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), a significant pest of soft summer fruits elsewhere in the world [T. leucotreta larvae by reducing their development periods and mortality. There is growing support that the vast majority of insects feed and benefit from yeast in their diet. Yeast multi-cultures have been shown to improve insect development [Drosophila species, larval diets containing mixed yeast cultures provide more significant development speed and survivability benefits than single yeast cultures [ted food ,46,47,48he world ,50,51,52elopment . The comelopment ,54. Durielopment ,53. In Dcultures . The yeacultures ,53.Kluyveromyces marxianus has been reported to be an effective biological control agent against Penicillium digitatum, a fungus that causes green mould in citrus fruit, in combination with sodium bicarbonate (NaHCO3) [Hanseniaspora uvarum, in combination with phosphatidylcholine, has also shown great potential as a biological control agent against P. digitatum [Meyerozyma guilliermondii has also exhibited potential as a biological control agent against fungi responsible for postharvest spoilage of fruit and vegetables [Thaumatotibia leucotreta-infested fruit often do not rot, or rotting is delayed, particularly when compared to Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (Mediterranean fruit fly) infestations, indicating that T. leucotreta larvae may feed on secondary fungal infections, preventing, or delaying, the fruit’s decay. A similar observation has been made in the case of C. pomonella larval feeding galleries, which rarely become infested with mould [T. leucotreta used to be a Mucor or Rhizopus fungal species-inoculated maise meal diet [Detrimental fungal infections were observed less frequently on Navel oranges treated with yeast than those without, during larval development assays. (NaHCO3) . Hanseniigitatum . Meyerozgetables . Thaumatth mould . This steal diet ,60.T. leucotreta populations [T. leucotreta was influenced by H. uvarum, P. kluyveri, P. kudriavzevii and K. marxianus. Volatiles produced by yeasts are thought to stimulate larval feeding [Spodoptera littoralis   larvae preferred phyllosphere yeasts over yeasts associated with fruit and frugivorous insects [Integrated pest management programmes in South Africa successfully use both semiochemicals and pathogens to manage ulations ,36,61. Culations ,63. The  feeding . Insect  feeding ,64. In l insects . Increas insects ,28,29.Meyerozyma guilliermondii, P. kudriavzevii and H. uvarum were shown to significantly affect adult T. leucotreta females’ oviposition preference. Both H. uvarum and P. kudriavzevii have now been shown to influence T. leucotreta neonate and adult female behaviour. They are known to be associated with citrus; thus, their influence on T. leucotreta is not unexpected. To date, the behavioural influences of these yeasts on lepidopteran moths have not been well documented. However, their influence on other agriculturally important pests has been reported. Hanseniaspora uvarum has been shown to affect the ovipositional behaviour of drosophilid flies, acting as both an attractant and repellant [H. uvarum as a live yeast bait for a selective lure against D. suzukii has been suggested [C. pomonella are mediated by the yeasts’ volatile signatures [epellant ,65,66. Uuggested . The odouggested . Microoruggested . Moreoveuggested . Volatiluggested ,26,41,53gnatures .T. leucotreta larvae collected from geographically distinct citrus-producing regions. Larval feeding and oviposition assays demonstrated that the isolated yeast species elicit behavioural responses in T. leucotreta neonates and adult females. The results generated from this work may help further the development of environmentally safe semiochemicals for population monitoring and control of T. leucotreta in South Africa. Future work arising from this study would involve conducting detached fruit bioassays and semi-field trials to evaluate the efficacy of a combination with biological control agents currently used against T. leucotreta.Six yeast species were successfully isolated and identified from


Acousto-microfluidics uses acoustic waves to manipulate and sense particles and fluids, and its integration into biomedical technologies has grown substantially in recent years. Fluid manipulation and measurement with surface acoustic waves rely on the efficient transmission of acoustic energy from the device to the fluid. Acoustic transmission into the fluid can be reduced significantly by slip at the fluid-solid interface, but, up until now, this phenomenon has been widely neglected during the design of acousto-microfluidic devices. Here our interpretation supports that the slip dynamics at the liquid-solid interface in acousto-microfluidics are highly analogous to the Amontons-Coulomb laws for dry friction between solids. In particular, there is a relationship between the local fluid pressure and shear stress, where we show that pressure-shear stress conditions can be divided into slip and no-slip regions, similar to the cone of friction found in dry friction. This improved understanding of slip will enable more reliable and predictable acousto-microfluidic technologies, thus expanding their use in new applications in biology and medicine. Acoustic waves can be used to manipulate particles and fluids in biomedical applications. The authors show that slip at the fluid-solid interface, characterized by a lower acoustic transmission into the fluid, is similar to Amontons-Coulomb friction, as found between solids.  For example, acoustic resonators, including quartz crystal microbalances (QCM), are used to measure analyte concentrations in fluids by tracking changes in mechanical resonances8. More recently, acoustic waves emitted from a resonant device located within a microchannel have been used to efficiently trap, separate, and sort particles in fluids5. In general, acousto-microfluidic devices leverage the absorption of acoustic energy by the fluid to manipulate the fluid, as well as particles within it, or to detect the properties of the fluid in real-time. The acoustic waves are typically generated with piezoelectric actuators embedded in the substrate that supports the microfluidic network. However, the acousto-fluidic interactions used in these applications are complicated by the presence of slip at the fluid–solid interface, which reduces the absorption of acoustic energy and can dramatically change the behavior of acousto-microfluidic devices.Acousto-microfluidic actuators12, including the role of shear stress15, wall roughness17, and surface tension18. However, the introduction of acoustic waves at the fluid–solid interface dramatically changes the slip dynamics26 and alters the function of devices26. Specifically, the slip from acousto-fluidic friction generates lower-than-expected damping, or attenuation, of the acoustic waves28. The slip coefficient is defined as the difference in damping with and without slip20 and is proportional to the slip length, which is a more physically intuitive parameter for quantifying the slip at the fluid–solid interface22. These parameters have been used to characterize slip in several acousto-fluidic systems, including an electroacoustic delay line for studying slip in non-Newtonian liquids21 and QCMs, where in one example, polymer pillars were fabricated on top of a QCM and the slip between droplets and the pillar array was studied23. Previous work has focused on devices without flow and has not looked at fluids confined by a microfluidic channel, except for one study with a suspended QCM sensor25 where slip was not observed. In addition, the effects of viscosity, density, pressure, shear stress, and device geometry on slip have largely gone unexplored in acousto-microfluidics, which is limiting the design of efficient, reliable, and predictable devices.Slip at the fluid–solid interface without acousto-fluidic interactions has been studied extensively12. In addition, the local acousto-fluidic pressure, shear stress, and sensor contact area with the fluid have been varied. From these detailed experiments, it was found that the slip dynamics in acousto-microfluidics are highly analogous to the Amontons-Coulomb laws for dry friction between solids30. The Amontons-Coulomb friction laws explain that slip between two solids is the result of a coupling effect between the normal and tangential forces at the contact point, with a coupling coefficient that depends on material properties and surface roughness. Although the relationship between local fluid pressure and shear stress is analogous to the normal and tangential forces, respectively, in the Amontons-Coulomb laws, the two factors have never been studied together in microfluidics until now. Our results show a cone of friction that divides the regions of slip and no-slip, which are defined by a relationship between fluid pressure and shear stress. These observations support the use of a cone of friction representation of the slip dynamics that can be used to develop acousto-microfluidic devices that either avoid slip to maximize the acousto-fluidic interaction, as needed for particle trapping and sorting, or leverage slip as a sensing modality .Here we present, for the first time, a set of experiments that show the slip dynamics in acousto-microfluidics under a wide range of operating conditions. We have integrated surface acoustic wave sensors into a microfluidic chip that is used to measure the slip coefficient due to acousto-fluidic friction, where the surface acoustic waves travel at the interface between the vibrating substrate and the fluid. We have investigated two cases, with and without fluid flow in the microchannel, allowing us to observe the complex slip dynamics resulting from acousto-fluidic interactions, which are not well understooddf, as shown in Fig. M1 to M5 are used for the primary results of the paper, which operate at a frequency of 45.6 MHz, and N1 to N5 are used for supportive results, which operate at 38.0 MHz  and 1.1 mm , and are spaced in intervals of 0.1 mm. The contact area between the fluid and the vibrating surface of the delay line is defined as w × df, where w is the channel width. Therefore, the area of M5 (and N5) is 1.6 times greater than M1 (and N1), such that the dependence of slip on the contact area can be investigated. See Methods for fabrication details.The sensors have a surface acoustic wave delay line configuration Fig.  with twoMHz Fig. . The sen.m−3, and the solution’s viscosity from 1 to 10.8 mPa.s. Using these seven solutions, the increase in fluid pressure as a result of the increase in density and the increase in fluid shear stress due to the increase in viscosity can be studied. To assess the effect of local pressure on slip due to the acousto-fluidic friction, we evaluated the slip with and without a pressure drop along the channel length, as described in Fig. D1 and D2  from 44 to 47 MHz for M1 to M5, and from 37 to 39 MHz for N1 to N5 using a vector network analyzer (VNA). The central frequency of the acoustic delay line is 45.6 MHz for M1 to M5 and 38.0 MHz for N1 to N5 as shown in Fig. S21 amplitude of the five sensors in each set (M and N) with each of the seven solutions. By comparing the S21 amplitude at the same acoustic frequency for a given sensor set, we compare the sensors at the same acoustic shear rate generated from the acoustic wave. The two sets generate different acoustic shear rates because their operating frequencies are different. The electrical power measured at the receiver of each sensor depends not only on the amount of acoustic energy transferred into the fluid but also on the electrical losses in the transducers and delay line. To isolate the acoustic loss from the electrical losses, we define the acoustic loss in the fluid, L, as the difference between the amplitude of S21 in air and in the fluid  the acoustic resistance in the fluid, R; (2) the acoustic resistance in air, Ra; (3) the acoustic resistance in the piezoelectric substrate, Rc; and (4) the wavelength of the acoustic wave, λ, as described by Eqs. ,,R increa0 in Eq. , the acoL, for the five sensors, M1 to M5, with the seven water-glycerol solutions and no flow. To compare the loss in the different solutions, the solutions are characterized by their Rns values. To highlight the presence of slip, which is indicated by L < Lns, Lns is also plotted in Fig. L, is smaller than Lns for Rns between 1.67 × 106 kg.m−2.s−1 and 2 × 106 kg.m−2.s−1, which corresponds to water-glycerol solutions with a mass fraction of glycerol between 0.2 and 0.5 and a viscosity between 1.76 mPa.s and 6 mPa.s , γSL is the most difficult to measure, but can be calculated using θ and γL33. The interface affecting the acoustic wave is between the silicon oxide surface and the liquid. We measured the contact angle between this surface and the liquid to calculate γSL, as reported in Table .m−2, and for a water-glycerol mixture with wG = 0.6 it is 84 mJ.m−2  and kinetic friction (slip). For dry friction, the slip velocity is constant once the slip occurs. In microfluidics, the slip length, β, is more commonly used and is a function of the slip velocity and the viscous pressure drop , τ, at the fluid–solid interface. When looking at the relationship between P and τ, there is a linear boundary between the no-slip and slip regions defined by τ = µP, where µ is the coefficient of friction and the boundary is called the cone of friction29. This boundary defines the critical shear stress for slip depending on the pressure applied by the solid. To further understand the fluid–solid interaction in this context, we now look at how slip depends on P and τ. Under flow conditions, the increase in flow rate, Q, generates an increase in flow shear stress, τ, as expressed in Eq. , d in Eq.  with chaFrom Eq. , the floors Fig.  changes Q and x, the resulting  values were calculated using Eqs.  and , we measured the acoustic loss and extracted the slip coefficient. Based on this data, in Fig. ’ in blue) when s is over 150 −9 m2.s.kg−1; otherwise, the fluid adheres to the piezoelectric substrate (‘’ in orange). The slip coefficient threshold value between the adhesion and slip regimes is estimated based on the confidence interval of no-slip  values, is shown in the Supplementary Information  and  and Q anP/η, y-axis in Fig. x-axis is represented in terms of the viscosity since the acoustic shear stress is proportional to the viscosity multiplied by the acoustic frequency, which is fixed at 45.6 MHz in the experiments for M1 to M5.Pressure depends on the density and the flow rate. However, here we present the pressure, normalized by the viscosity , and the area where the shear stress surpasses the fluid pressure (slip markers). However, for η = 10.8 mPa.s  does not exist in dry friction. As mentioned previously, the increase in fluid density  is proportional to the resultant of the normal forces (P in our case) and that the effort needed to maintain the slip is smaller than the effort needed to generate the slip . In microchannels, with or without slip, ΔP/η and τ are coupled as expressed in Eq. . This slet Fig. . TypicalFrom our assumption that water-glycerol solutions slip at a fluid-surface acoustic wave interface, we have shown that the slip happens once the shear stress surpasses a critical value. Our experiments confirm that the critical shear stress depends on the pressure applied by the fluid at the interface. When the fluid is static, the slip transition depends on the fluid density and viscosity, and when there is flow, the slip transition depends on the local flow pressure and shear stress. Moreover, we have shown that the critical shear stress does not depend on the area of contact. We have also shown that the acoustic loss, the slip coefficient, and the slip length depend on the combination of the viscous shear stress and the fluid pressure generated by the flow and applied at the interface. This behavior closely resembles the dynamic dry friction described by the Amontons-Coulomb laws, which has not previously been observed in acousto-microfluidic systems. Our experiments show that, in the pressure-shear stress graph, the no-slip area for acousto-fluidic friction is confined in a cone that is analogous to the cone of friction found in dry friction. Furthermore, this phenomenon leads to a slip length that varies along the channel length. This behavior was found to be highly repeatable in our sensors across different sensor geometries. Future work will focus on an analytical description of these complex slip dynamics at the fluid–solid interface.4 using the acoustic radiation force, which depends on the acoustic pressure. In this paper, the slip was studied for ranges of density and viscosity similar to those found in biofluids, including human blood plasma. The natural variance in viscosity for human plasma37, between 1.15 and 1.8 mPa.s, lies between the adhesion and slip regimes for our sensors , 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6. Their properties are reported in Table Glycerol solutions were prepared by weighing glycerol anhydrous for each of the mass fractions, and then the final mass for each solution was reached by adding pure water. The mass fractions, 3) wafers with a diameter of 100 mm and thickness of 500 μm were used to fabricate the surface acoustic wave devices. First, the wafers were cleaned by soaking in acetone for 5 min, then in isopropanol for another 5 min, and finally in deionized (DI) water for at least 5 min. The wafer surface was activated by an argon plasma and an oxygen plasma using a reactive ion etching chamber. These steps improved the adhesion of the lift-off resist on the wafer. The argon plasma activation was done under a pressure of 33.33 kPa (=250 mT), 50 W, and a flow of 15 cm3.min−1 (=15 sccm) during a 20 s period. The oxygen plasma activation was carried out at 26.66 kPa (=200 mT), 200 W, and a flow of 50 cm3.min−1 for a period of 20 s. The lift-off resist was deposited on the wafer using a spin-coater. After spin-coating with lift-off resist, the wafer was placed on a hot plate, at room temperature (~20 °C). The wafer was then heated up to the optimal soft-baked temperature and then cooled down using a ramp of 5 °C/min. Ramping the temperature up and down at these rates prevented the lithium niobate wafer from breaking due to the pyroelectric effect. The photoresist was spin-coated on top of the lift-off resist to get a photoresist thickness of 1.3 μm. A soft bake step was then carried out. Then, the wafer was electrically discharged by short-circuiting its two sides. Using a mask aligner, the interdigitated transducer design was transferred into the photoresist by UV exposure of the wafer through a chrome mask. The photoresist was developed by the photoresist’s developer. The wafer was then rinsed in DI water and blow-dried using nitrogen gas. The wafer surface was activated by using an argon plasma followed by an oxygen plasma, using the same conditions as described above. This surface activation helped the titanium to stick on the lithium niobate. An adhesion layer of titanium (10 nm thick) followed by a layer of gold (90 nm thick) was deposited on top of the lithium niobate using an electron beam evaporator. The resists were then lifted off in a resist remover for 24 h. The wafer was finally cleaned using a three-step process: immersion in acetone for 5 min, then in isopropanol for 5 min, and finally in DI water for at least 5 min. The wafer was then blow-dried with nitrogen gas. A resist was then spin-coated on top of the wafer to protect the wafer from dust. The wafer was diced into 3 cm Piezoelectric 128° XY-cut black lithium niobate  was configured to measure the magnitude in decibels (dB), with a drive power of 1 mW, and the phase between the output and the input. The amplitude ratio, also called the acoustic loss, was measured on a frequency interval from 44 to 47 MHz with a frequency spacing of 1.874 kHz. Fluid was loaded into the channel using a syringe pump. The inlet was connected to the syringe with a solvent compatible and flexible tube. The outlet was connected to a waste container with a solvent compatible and flexible tube. The two tubes for the inlet and outlet had the same length. The loading flow rate was between 10 and 50 μL/min. Once loaded the flow was stopped. The fluid rested for 5 min. Then, using the same configuration as for the reference measurement, the acoustic losses were measured using a VNA. The flow rate was then increased by 50 μL/min using a syringe pump. The measurement was done after waiting between 20 s to 5 min, depending on the fluid viscosity. The rest time allowed the fluid to reach its steady-state. This step was repeated until the flow rate reached 250 μL/min. Then, the flow rate was stopped. The full measurement was repeated two more times after a resting time of 5 min between each measurement. This was done for the 10 sensors on the chip. Then, the inlet and the outlet were exchanged to revert the flow direction. The full measurement was repeated as described above. At the end of the experiment, the fluid was removed with air using a syringe pump.The medium we used as a reference for measuring the Supplementary Information


The purpose of the study was to investigate the short-term response profile after an intravitreal injection (IVI) of anti-vascular endothelial growth factor (VEGF) in patients with neovascular age-related macular degeneration (nAMD) and incomplete response to anti-VEGF.In this monocentric prospective observational study, we recruited patients with incomplete response to anti-VEGF, defined as presence of subretinal fluid (SRF) and/or intraretinal fluid (IRF) on optical coherence tomography (OCT) for at least 6 months despite monthly anti-VEGF treatment. Each patient underwent complete ophthalmic exam and imaging study  the day of their scheduled monthly IVI. Intermediate visits were performed weekly thereafter , until week 4. Fluid metrics were quantified using an artificial intelligence-based algorithm at baseline and at each subsequent weekly visit. Main outcomes were residual fluid volumes of SRF and IRF for each time point, and its relative change after treatment. Particular interest was given to each patients’ nadir point, which was used for association analysis with imaging parameters.A total of 28 eyes of 26 patients were included into the study. The maximal response was reached at 1.93 weeks on average. The relative fluid resolution at nadir point was 66 ± 36.7%, with quartile limits at 49.1%, 83%, and 96.1%, respectively. Mean residual fluid volume was 64.9 ± 128.8 µl at nadir point. Residual fluid was positively correlated with baseline SRF  and larger pigment epithelium detachment . Polypoidal choroidal vasculopathy was associated with larger residual fluid (p = 0.0013).Incomplete anti-VEGF responders in nAMD showed significant mean fluid resolution between injections, typically after 2 weeks. However, complete resolution was the exception, and the amount of residual fluid varied greatly. To understand the role of the unresponsive fluid, further studies are needed. Age-related macular degeneration (AMD) is the leading cause of visual impairment in developed countries . Among pDespite good efficacy of anti-VEGF treatments in nAMD, some cases remain poor responders. Several terms have been used to qualify a suboptimal response to treatment, such as “incomplete anti-VEGF response”, “unresponsive”, “refractory to anti-VEGF”, or “resistant to anti-VEGF” to name a few , 7. AlthThe proportion of eyes with incomplete response to anti-VEGF is not well defined and studies have shown a wide array of results depending on definitions, ranging from 5% up to 53% –11. ExudA recent publication  has suggThe goal of the present study was to evaluate the short-term response profile and its inter-individual variability after anti-VEGF IVI in incomplete responders. In addition, we intended to investigate imaging factors predictive of the inter-IVI response profile.This is a monocentric prospective observational study conducted in the tertiary referral hospital of Jules Gonin, Lausanne, Switzerland. The research methods and analysis plan adhered to the tenets of the Declaration of Helsinki and the research protocol was approved by the Ethical Committee (CER-VD 2017-02175).Patients were prospectively screened during regular AMD clinics for incomplete response to anti-VEGF, defined by the presence of exudative signs on OCT for at least 6 months, namely IRF and/or SRF, despite monthly IVI. Patients had to have undergone anti-VEGF treatment for at least 12 months, following a Treat and Extend regimen or an Observe and Plan regimen . In addiA baseline visit was performed, immediately prior to the next planned monthly IVI. The latter was administered on the same day, or exceptionally within the next 7 days. Follow-up visits were performed weekly after the IVI until week 4 (W1 to W4). At each visit, a complete ophthalmic exam was performed, comprising best corrected visual acuity (BCVA) using an Early Treatment Diabetic Retinopathy Study (ETDRS) chart, intraocular pressure, anterior segment exam, dilated posterior segment exam. Spectral-domain OCT (SD-OCT) and infrared (IR) images were obtained at each visit , applying the inbuilt follow-up mode. In addition, color fundus photography, fundus autofluorescence (FAF) , fluorescein angiography (FA) (HRA Heidelberg), indocyanine green angiography ICGA (HRA Heidelberg), and OCT angiography (OCT-A)  were performed at baseline.Fluid metrics were quantified using an artificial intelligence (AI)-based algorithm , whose performance has been demonstrated previously . To thisIn addition, general patient information was retrieved, including age and gender, the number of previous IVI, treatment duration, the anti-VEGF drug currently used, and the number of previously performed drug switches. Furthermore, multimodal imaging analysis was performed in the following way: baseline OCT was analyzed for the presence or absence of: IRF, SRF, signs of polypoidal choroidal vasculopathy (PCV) (defined as an abrupt pigment epithelium detachment accompanied by double layer sign), reticular pseudodrusen, retinal pigment epithelium (RPE) atrophy , viOutcome parameters included the values and their changes from baseline for BCVA, IRF, SRF, PED, for each time point. The nadir time point for minimal residual fluid volume of IRF plus SRF was determined, and associations with anatomical parameters were evaluated. Maximal response was analyzed at each eye’s individual time point where the response was highest (nadir time point). For eyes with no fluid reduction after the IVI, their relative response was considered to be 0% (no response). These values of the nadir time point were then used for the association analysis.Statistical analyses were performed in JMP software for Windows . Descriptive statistics were performed, and paired changes over time were analyzed using Wilcoxon signed-rank test . Association analyzes were performed using logistic regression, ANOVA test, and Pearson correlation analysis, according to categorical or continuous data.P-values < 0.05 were considered statistically significant.A total of 28 eyes of 26 patients were included. Mean age was 80 ± 6.56 years (range 67 to 96 years). Gender distribution was 22 (79%) females and 6 (21%) males. The mean number of previous IVI was 54.0 ± 24.7 over a mean treatment period of 67.5 ± 31.0 months. Mean number of previous treatment switches was 1.0 (range 0 to 5). At baseline, the mean time from preceding IVI was 30.1 ± 7.7 days. The drug used at the time of the study was Ranibizumab in 11 eyes, Aflibercept in 16 eyes, and Bevacizumab in 1 eye.At baseline of the study, the mean VA was 73.3 ± 12.8.Disease activity at baseline comprised IRF in 15 eyes and SRF in 17 eyes. Multimodal imaging signs of the cohort are summarized in Table OCT metrics, specifically CRT, IRF, SRF, and PED volumes at baseline and at subsequent visits are summarized in Table No statistically significant difference in VA was noticed throughout the follow-up period. Mean VA at baseline was 73.3 ± 12.8 letters. It was 73.4 ± 11.7 letters at W1 (p = 0.96), 73.7 ± 11.6 letters at W2 (p = 0.48), 74.0 ± 10.0 letters at W3 (p = 0.37), and 75.3 ± 9.7 letters at W4 (p = 0.72).At baseline, mean algorithm-computed fluid was 156 ± 211.3 µl. The repartition per compartment was the following: 25 ± 45.5 µl of IRF and 129.4 ± 176.5 µl of SRF. Therefore, 83.8% of the total fluid at baseline was SRF.Referring to individual nadir time points, maximal fluid reduction was reached on average at 1.93 weeks. This nadir time point was reached in 10 (35.7%) eyes at W1, in 9 32.1%) eyes at W2, in 5 (17.9%) eyes at W3, and in 1 (3.6%) eye at W4, while 3 (10.7%) eyes showed no improvement at all after the IVI , with quartile limits at 49.1%, 83%, and 96.1%, respectively. The distribution of relative response is shown on Fig. Residual fluid at the nadir time point was noted in 23 eyes (82.1%). Of the 17 eyes who presented SRF at baseline, 3 eyes showed a complete resolution of SRF at nadir, while 14 eyes (82.3%) still had remaining SRF. Of the 14 eyes with IRF at baseline, 3 eyes showed complete resolution at nadir, while 11 eyes (78.6%) displayed residual IRF. Mean algorithm-computed fluid volume of the eyes with residual fluid was 64.9 ± 128.8 µl. The repartition of residual fluid per compartment of these eyes was: 5.3 ± 6.7 µl IRF and 59.6 ± 128 µl SRF. In other words, 91.9% of residual fluid was SRF. The distribution of residual fluid is shown on Fig. An association analysis for (a) maximal treatment response and (b) residual fluid amount was performed with respect to demographic, imaging or treatment factors. The results are summarized in Table Patients’ demographics such as age and sex were not linked with response or residual fluid patterns, although there was a trend for more fluid resolution in male patients (p = 0.056). Furthermore, no association was found between preceding treatment characteristics, including time from last injection, drug type, time from treatment initiation, and number of past drug switches.Similarly, no statistically significant associations with imaging parameters were found to explain relative treatment response.However, residual fluid (sum of IRF and SRF), was positively correlated with baseline SRF  and larger PED . In other words, the higher SRF or PED volumes were at baseline, the more residual fluid was present.Of note, some imaging parameters were present in insufficient numbers and therefore excluded from the association analysis. These included presence of serous drusen, multifocal choroidal hyperpermeability, and MNV type. Presence of PCV on ICGA also fell into this category (present in two eyes on ICGA), but given its well-known origin of potential refractory fluid, it was kept into the analysis. Indeed, there was one outlier with 598 µl residual fluid at week 2 (nadir) which also presented a PCV lesion on ICGA, which induced a statistically significant association (p = 0.0013) between PCV and residual fluid despite the low numbers.This study showed the response profiles of incomplete anti-VEGF responders in nAMD, in terms of OCT metrics between monthly anti-VEGF IVI. An AI-based algorithm computed IRF, SRF, PED volumes and showed significant fluid reduction between injections, with its maximum typically 2 weeks after injection, and relapse at week 4. However, despite statistically significant resolution, most eyes still showed some residual fluid (IRF + SRF) at the nadir time point, and a proportion of eyes showed only little or no fluid reduction at all. Thus, these patients with incomplete response appeared to be a heterogeneous group varying between non-responding residual fluid and short-term responders.To our knowledge, this is the first study specifically designed to evaluate short-term response on nAMD patients with incomplete response to anti-VEGF, using an automated quantitative approach to measure pathological fluids. Short-term response to IVI treatment in nAMD has previously been studied in terms of CRT . BontzosIn our cohort incomplete anti-VEGF responders only were included. The cohort included a surprisingly high proportion (89%) of type 1 MNV. Type 1 MNV has been reported to be the most frequent type of MNV,  but our During IVI treatment with anti-VEGF, a mechanism of drug tolerance may induce suboptimal response to treatment . SwitchiWhen considering incomplete anti-VEGF response in nAMD, an anatomically special situation (such as PCV) or a masquerading differential diagnosis can be the cause of the suboptimal response. Indeed, PCV has been shown to be potentially resistant to anti-VEGF alone  and a coSome masquerading pathologies could be the origin of unresponsive fluid on OCT, such as central serous chorioretinopathy (CSC), epiretinal membranes, vitreomacular traction syndrome, or inflammatory causes , 25. TheThe main interest of the study focused on the fluid profile in between injections. The results showed significant fluid reduction in all compartments, with its mean maximum after approximately 2 weeks. However, it is interesting to note that complete resolution was infrequent (17.9%), and some eyes (10.7%) did not show any improvement at all. In other words, there was a large variation of the relative response profile, and even in between injections, residual fluid was the most frequent scenario, suggesting a role of other pathogenic pathways than VEGF.However, it is unclear what quantity of residual fluid is clinically relevant. Indeed, the question of fluid tolerance remains open, with no definitive consensus on whether and how much persistent fluid could be accepted without functional consequences. The FLUID study has shown no difference in VA at 24 months if patients with SRF < 200 µm were left untreated. However, the difference in mean number of injections in comparison with the intensive treatment arm was so small that it is difficult to draw conclusions . Other sThere is a general agreement that care should be taken to avoid undertreatment, as it plays a major role in the prognosis of nAMD . Some adIn contrast, the presence of any residual IRF is generally accepted as indicator for treatment need, as its presence is associated with poorer visual outcomes compared to SRF , 33–35.Residual fluid despite maximal anti-VEGF treatment may indicate a VEGF independent source of fluid coexisting with the VEGF pathway in patients with incomplete anti-VEGF response. Therefore, it might be important to recognize its quantity and type, measured at the correct time point between injections, in order to potentially orientate the patient towards adjuvant treatment, if available. In this sense it is interesting to mention previous studies about the development of artificial intelligence-based algorithms to predict the response to anti-VEGF based on OCT scans in nAMD , 37.AMD is a complex disease and its pathophysiology combines many different pathways, including among others, inflammatory molecules, the complement system, and lipoprotein metabolism, in addition to the influence of genetic susceptibility and environmental factors . On thatDemographic and treatment factors did not show any association with the relative response of residual fluids, which was somewhat surprising as we expected to find some biomarkers. However, residual fluid amounts were associated with baseline SRF amount, PED volume, and the presence of PCV on angiography. SRF is the dominating element in the sum of SRF and IRF. Indeed, in terms of volume, SRF represented 83.1% of total fluid (IRF + SRF) volume at baseline, and 91.9% of residual fluid volume. It is therefore not surprising that larger amounts of baseline SRF were associated with larger residual fluid volume.Larger PED volumes at baseline were also associated with more residual fluid. This finding is in accordance with the literature, as the presence of PED has often been linked with more frequent injections need, and with poorer prognosis . HoweverAs mentioned above, due to the selection process of the cohort, only two eyes presented PCV on ICGA. However, its presence was associated with more residual fluid, particularly due to one outlier case. Although the association was statistically significant, conclusions must be handled with care due to the small number.We acknowledge that 28 eyes with nAMD and incomplete response to anti-VEGF is a limited number of participants. However, the prospective design of the study, selecting specifically patients with incomplete response to anti-VEGF, and the detailed imaging documentation are some strengths.In addition, the use of an AI-based algorithm to objectively quantify and categorize fluid compartments, allowed for detailed evaluation of the OCT imaging data with high precision.In conclusion, the study showed that a majority of nAMD patients with incomplete response to anti-VEGF displayed some short-term response to treatment, with a maximum response after approximately 2 weeks and an early relapse at week 4. However, this response was frequently incomplete throughout the weekly analyzed study period, with permanent presence of residual fluid to a variable degree in most patients. A small proportion of patients showed little or no fluid reduction in between injections, with predominantly unresponsive fluid. This highlights the heterogeneity of the group of nAMD patients with incomplete response to anti-VEGF, which suggests that many different factors come into play, including presumably anti-VEGF independent mechanisms.


In this work, a numerical investigation was analyzed to exhibit the mixing behaviors of non-Newtonian shear-thinning fluids in Kenics micromixers. The numerical analysis was performed using the computational fluid dynamic (CFD) tool to solve 3D Navier-Stokes equations with the species transport equations. The efficiency of mixing is estimated by the calculation of the mixing index for different cases of Reynolds number. The geometry of micro Kenics collected with a series of six helical elements twisted 180° and arranged alternately to achieve the higher level of chaotic mixing, inside a pipe with a Y-inlet. Under a wide range of Reynolds numbers between 0.1 to 500 and the carboxymethyl cellulose (CMC) solutions with power-law indices among 1 to 0.49, the micro-Kenics proves high mixing Performances at low and high Reynolds number. Moreover the pressure losses of the shear-thinning fluids for different Reynolds numbers was validated and represented. Different applications of micromixers can be found in biomedical, environmental industries and chemical analysis, where they are essential components in the micro-total analysis systems for such applications requiring the rapid and complete mixing of species for a variety of tasks ,2. VarioThere are not many works in the literature for non-Newtonian fluid mixing using Kenics micromixers. Therefore, the idea of the current study is to investigate the performance of a microKenics for mixing shear thinning fluids, trying to attain a high mixing quality and pressure drop. Using CFD code, numerical simulations were carried out at Reynolds numbers ranging from 1 to 500 in order to examine the flow structures and the hydrodynamic mixing performances within the concerned Kenics micromixer. Various CMC concentrations were proposed to investigate the chaotic flow formation and thermal mixing performances within the suggested micromixer. In order to get important homogenization of the fluids’ indices and pressure losses will be appraised.TM 16 CFD software  [−6 of root mean square residual values. Steady conservation equations of incompressible fluid are solved numerically in a laminar regime by using the ANSYS FluentPL, USA) , which i3. The coherence coefficient and the power law indexes of the CMC solutions are indicated in −11 m2/s.A non-Newtonian solution of carboxyméthyl cellulose (CMC) is used as working fluid for the simulation of fluid flows. The density of CMC solutions according to Fellouah et al.  and Pinh3) is the fluid density, P (Pa) is the static pressure, i-th species. i” in the mixing.The 3D governing equations for incompressible and steady flows are continuity, momentum and species mass fraction convection diffusion equation:−1) is the shear rate.For power-law non-Newtonian fluids the apparent viscosity is:g) is defined as [For a shear thinning fluid , the generalized Reynolds number (Refined as :(5)Reg=ρTo measure the efficiency of the Kenics micromixers, mixing rate is defined as follows :(6)MI=1−i, and −1) is the standard deviation at the inlet part.N indicates the number of sampling locations inside the transversal division, The boundary conditions are a condition of adhesion on the walls where the velocities are considered to be zero, uniform velocities are executed at the inlets, the mass fraction of the fluid at the inlet 1 equal to 1 and that of the inlet 2 equal to 0, an atmospheric pressure condition is considered at the exit. All walls are considered adiabatic.The configuration  is based6. In this work, which investigated fluid mixing in laminar flow in which the convergence is limited by the pressure-velocity coupling, a converged and stable solution was obtained using SIMPLE algorithm. The pressure correction under relaxation factor is given at 0.3, which facilitates the acceleration of convergence for the second order upwind scheme. The convergence of iterative calculations was attained when the specified value of the residual quantities are less than 10To choose an adequate mesh; an unstructured mesh has been generated with tetrahedral elements; several grids were tested for the present proposed geometry . NumericA numerical study of the pressure drop in a T-Junction Passive micromixer to verify the accuracy of the CFD with that of C. Kurnia et al. , see TabTo compare the numerical results a quantitative comparison was made for Newtonian fluids in a Kenics micromixer as shown in The mixing indices were compared using the range of Reynolds number between 0.1 to 120. The Kenics micromixer and TLSCC displayed exceptional mixing performance compared to the other two micromixers for Re The mixing index at the exit of Newtonian and non-Newtonian fluid in Kenics was compared with the curved micromixer of Bahiri et al. . For rang = 25 and n = 0.73. The twist of element and the sharp change of angle between blades affected the intensity of the fluid particles’ movement and the mixing performance.The effects of the Reynolds number and the behavior index on the chaotic mixing mechanism were qualitatively analyzed by presenting the contours of the mass fraction at the various transverse planes P1–P7 and the exit. g 0.1 and 50. The flow field enhanced the secondary flow along the micromixer for all cases of Re due to the blade conurbation of the Kenics device.When the Reynolds number increases to 50, the quality of the mixing begins to improve, therefore a homogeneous mixture is obtained at the exit plane in the microKenics for all the values of the behavior index.For low Reynolds numbers (Re = 0.1–5), when the species have more contact time to achieve a perfect mixing with the Kenics, the mixing index loses a part corresponding to nearly 14% of its value. In addition, by increasing the Reynolds number the fluid homonezation augments due to increases of secondary flows and advection, compared with TLCC micromixers for all cases of the power-law index.The Newtonian fluid with n = 1 is independent of shear rate and maintains a constant value of viscosity among different numbers of Reynolds . BesidesTherefore, The pressure drop obtained from CFD simulations was compared with the TLCC micromixer , for theA high mixing performance of the micromixer is generally associated with a high-pressure loss that involves the required energy input for the mixing process. In this work, mixing of CMC non-Newtonian fluids in a microKenics device was numerically investigated for different regimes (Re = 0.1–500), using CFD code. The analyses showed that the mixing performances of the Kenics micromixers consisting of repeating short twisted helical configurations is better than that of other micromixers at low Reynolds numbers.It can be achieved that for fluids with all power-law indices studied  and low Reynolds numbers (less than 8) the micromixer is an excellent one, while for the fluids with


Oncorhynchus mykiss). The ovarian fluid affected significantly spermatozoa performance: it supported high velocity for a longer period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout ovarian fluid induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating medium. The interaction of the spermatozoa with the attracting agents was accompanied by the “turn-and-run” behavior involving asymmetric flagellar beating and Ca2+ concentration bursts in the bent flagellum segment, which are characteristic of the chemotactic response. Ovarian fluid created the optimal environment for rainbow trout spermatozoa performance, and the individual peculiarities of the egg (ovarian fluid)–sperm interaction reflect the specific features of the spawning process in this species.The fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid are likely the major sources of these triggers, which we confirmed here for rainbow trout ( Sexual reproduction is the fertilization of female ova by male spermatozoa. For successful fertilization, the spermatozoa must reach the egg soon after ovulation. Externally fertilizing freshwater fish spermatozoa are activated after direct contact with freshwater, and their motility is sustained for only a very limited period, typically tens of seconds . FreshwaWhat are these mechanisms based on? Generally, ideas vary from the predominance of a male factor, such as the “fair raffle” concept, in which the success of a particular male depends only on the number of spermatozoa it can provide to the “fertilization lottery” , to a spThe first observations of sperm activation and rise in motility traits in the vicinity of the eggs were made in marine invertebrates about 100 years ago , and “egSeveral reports about the chemokinetic reactions of fish male gametes, i.e., changes in spermatozoa velocity and trajectory depending on the presence of ovarian fluid in the medium ,17,18,19Oncorhynchus mykiss), we searched for any evidence of chemotactic behavior to determine the potential mechanism of the gametes’ encounter guidance.In our study, we performed comprehensive testing of the interaction between spermatozoa and ovarian fluid. Using a representative of the freshwater spawning Salmonidae family, the rainbow trout  i), semen was suspended in 4–5 volumes of immobilization solution, i.e., artificial seminal fluid (Morisawa and Morisawa 1988) containing 0.05% Cremophor EL  and 20 μM Fluo-4 AM (Dojindo), and incubated for dye loading at 10 °C for 2 h. Then, the sperm was activated in an observation chamber, and a microinjector with test liquid was introduced. Fluorescence signals emitted by the swimming sperm were captured by a microscope Olympus IX71  equipped with fluorescence illumination and a digital CCD camera QImaging Retiga-EXi  according to the method described by Shiba et al. [To image intracellular calcium ion content  were put into a plastic beaker, covered with test solution , and the sperm was added. Then, the beakers were placed on a shaker (around 100 rpm), and after incubation for 1 min, the eggs were rinsed and transferred to glass Petri dishes. Each point was done in three technical replicates.The dishes were transferred into the tank with baskets for further incubation at 11 °C. The tank had a closed water circuit with aeration, UV treatment, and temperature control. All fertilizations were done in three replicates. The outcome of in vitro fertilization was assessed after 11 days by the number of developing embryos ; simultaneously, the share of albino embryos was counted. The hatched larva was counted as well. The hatching rate (number of hatched larva/number of eggs) did not differ significantly from the number of developing embryos. Thus, only the latter was presented.2+ concentrations and osmolarity. Curvilinear velocity (VCL) and path linearity (LIN) were obtained from the motility measurement from 50–300 spermatozoa per replicate per time point during 10–59 s post-activation with 3 s increment. Then, the motility parameters  were log and logit transformed  to ensure normal data distribution, and an analysis of the interactive effects between variables was performed using repeated measure ANOVA in Statistica v. 13 . Media and post-activation time were considered as within-subjects factors and VCL or LIN were considered as dependent ones. In the case of the calcium ion/osmolarity cross-effect, calcium concentration, medium osmolarity, and time were considered as within-subject factors. Pair-wise analysis was performed to establish significant differences in interaction between media and the time between various activation conditions . The corresponding correction of p-value was done to consider the multiple comparisons; the threshold p-value was set to 0.0005.The assessment of motility parameters in different activation media was conducted in triplicate for 26 males in water, ovarian fluid, and NaCl isotonic saline (performed as a control during motility and chemotactic tests), 16 males in ovarian fluid dilutions, 12 males in media with varying osmolarity based on sodium chloride, and 11 males in MWCO fractions of ovarian fluid. Sperm samples from 8 males were used for experiments with the combined effects of Ca2). The hypothesis for the equality of regression slopes was checked using ANCOVA, and the threshold p-value was set to 0.0001.Then, the data for spermatozoa velocities in several media  were used to obtain linear regression dependencies in GraphPad Prism version 5 for Windows software , and the following parameters were obtained: slope (A), intercepts with x and y axes (B and C), coefficient of determination or the goodness-of-fit (Rp-value was set to 0.0005.A binomial model was used to assess significant differences between the groups in the fertilization test. Three technical replicates for each point were pooled together to increase the statistical power of the analysis. The analysis was done using G*Power 3.1.9.6. software . The folThus, in the frame of one investigation, we performed detailed testing of the features accompanying the gamete encounters in a representative of freshwater spawning fish, rainbow trout. These tests revealed the triggering effect of female fluids on the performance of male gametes, which conformed to the features of spawning behavior in these species. We did not find a specific agent responsible for changes to spermatozoa motility; rather, we supposed a combination of several factors. Nevertheless, finding the exact nature of the active agents and the responsive structures in the male gametes is the prospective goal of future studies.The studying of mechanisms underlying reproductive processes is an important part of evolutionary developmental biology. These mechanisms adapt to the specific conditions of the environment both in the course of evolution and over the lifetime of an individual. These adaptations entail the arising and modification of reproduction strategies and tactics. These are incredibly varied in fish due to the different ecological niches they inhabit. The fact that most fish reproduce externally makes them a good model for performing various experiments in conditions close to in vivo or with a controlled shift of in vivo conditions. This is especially true of freshwater species since, in addition to other factors, their process of fertilization is highly synchronous and limited to several minutes. Thus, the broader inclusion of fish into future studies of gamete encounters will significantly improve the overall understanding of the evolutionary aspects of reproduction, their molecular mechanisms, and their effects on reproductive behavior and vice versa.


Varicella poses an occupational risk and a nosocomial risk for susceptible healthcare personnel and patients, respectively. Patients with varicella are thought to be infectious from 1 to 2 days before rash onset until all lesions are crusted, typically 4–7 days after onset of rash. We searched Medline, Embase, Cochrane Library and CINAHL databases to assess evidence of varicella-zoster virus (VZV) transmission before varicella rash onset. Few articles (7) contributed epidemiologic evidence; no formal studies were found. Published articles reported infectiousness at variable intervals before rash onset, between <1 day to 4 days prior to rash, with 1–2 patients for each interval. Laboratory assessment of transmission before rash was also limited (10 articles). No culture-positive results were reported. VZV DNA was identified by PCR before rash onset in only one study however, PCR does not indicate infectivity of the virus. Based on available medical literature, VZV transmission before rash onset seems unlikely, although the possibility of pre-rash, respiratory transmission cannot be entirely ruled out. Primary infection with varicella-zoster virus (VZV) causes varicella (chickenpox), a highly contagious disease that can result in outbreaks in susceptible populations. Following primary infection, VZV remains latent in the body and may reactivate years or decades later to cause herpes zoster (HZ). VZV transmission poses an occupational risk and a nosocomial risk for healthcare personnel and susceptible patients, respectively –6. CurreAirborne transmission of VZV has been suggested by some epidemiologic evidence indicating varicella in susceptible persons who had no direct contact with a varicella or HZ index patient and no other potential exposures to the virus , 4, 8, 9Implementation of effective infection control measures for varicella depends on understanding routes and timing of VZV transmission. We reviewed the medical literature to assess evidence for transmission of VZV before rash onset.We searched Medline, Embase, Cochrane Library and CINAHL databases for articles published in any language, from database inception through 31 October 2019, using the search terms (‘chickenpox’ or ‘varicella’ or ‘herpes zoster’) and (‘transmission’ or ‘spread’ or ‘epidemic’ or ‘isolation’) and . The complete search strategy is described in Supplementary Table. Two authors independently reviewed each title and abstract to determine if the article likely included information on transmission of VZV before rash onset. For articles that passed this initial screen, we reviewed the full text; the summary of the full text review was reviewed by a second reviewer. We also reviewed references that described transmission of VZV identified in the reference sections of papers retrieved by the database search. Only articles that included original reports of assessment of VZV transmission before rash onset were retained.We describe the evidence by whether it was provided using epidemiologic or laboratory data.We screened 693 articles and identified 59 for full-text review; of these, 16 met inclusion criteria for our review . IncludeOf the seven articles that addressed VZV transmission before rash onset with epidemiologic evidence, four were published during 1896–1940 and three during 1989–2003. Three contained data from reports of varicella case series during outbreaks in hospitals, three included institutional outbreak investigations (two in schools and one in a jail) and one was a case report . Each reThe authors reported that patients with varicella were infectious at various intervals before the rash appeared: <1 day (9 h) before (one report) , at leasOf the 10 articles that addressed VZV transmission before rash onset with laboratory evidence, five were published during 1966–1989; they reported results of virologic techniques (culture) to identify virus in the oropharynx , 24–26. None of the studies that used culture to identify VZV in oropharyngeal specimens reported detection of the virus during the varicella incubation period. While these studies included a limited number of children who were tested before developing the characteristic varicella rash (between 23 and 27), the number of oropharyngeal specimens collected during the entire incubation period was ~160; ~47 were collected during the 3 days before rash onset . These i3 copies) did not detect viral DNA in this study. Conversely, 90% of throat specimens (n = 39) collected during the first 3 days after rash onset were PCR positive . Asano reported VZV DNA in throat specimens of one child of 13 tested (susceptible), with the specimens collected 4 h after recognising onset of varicella in a contact who had only a few maculopapular lesions [Positivity of oral specimens collected before rash onset was reported when VZV PCR was used for testing, although in rare instances . On a li lesions . This re lesions . The VZV lesions , 21.This review confirms the scarcity of evidence in the medical literature on transmission of VZV before varicella rash onset and substantiates limitations on reaching definitive conclusions about VZV transmission before appearance of skin lesions.Few reports contributed epidemiologic evidence; no formal studies have been published, and most information was anecdotal and reported decades ago. The epidemiologic investigations reported that patients with varicella were infectious at various intervals before rash onset, ranging from <1 day to 4 days before, with only 1–2 patients identified for each interval. One study that included more cases concluded that transmission during the incubation period occurred based on person-time analysis of in-class transmission, where exposure was defined as the day before a classmate stayed home with varicella . The epiLaboratory evidence supporting transmission of VZV before rash onset is also very limited. No culture-positive results were reported. PCR was a major advance for investigating disease caused by VZV, proving much easier, cheaper and more sensitive than culture of VZV. VZV DNA was found in the skin, saliva and respiratory tract of patients with varicella and HZ when rash was present , 29. VZVVaricella is a highly contagious viral disease. Many viral infections are spread by the respiratory route and virus can be readily identified in respiratory secretions of infected patients. Nevertheless, in patients with varicella, isolation of VZV in the respiratory tract has rarely been reported. Coughing and sneezing, associated with respiratory transmission of infectious diseases, are not characteristic of varicella. On the other hand, the pruritic vesicles characteristic of varicella contain high levels of well-formed, highly infectious virions  and itchBased on the available medical literature, VZV transmission seems unlikely prior to rash onset; however, it is impossible to prove it never happens. We did not find conclusive evidence to alter existing infection control guidelines. Providers should continue to implement appropriate infection control measures since the possibility of pre-rash, respiratory transmission of VZV cannot be entirely ruled out and sufficiently early identification of the rash, when only a few lesions are present, might be missed. Considering the scarcity and age of the existing evidence, additional evidence, using current laboratory assays would be beneficial. These studies can be undertaken in regions where varicella infections are still high; varicella rates are now low in the United States due to widespread use of the varicella vaccine  and situ


Dear Editor,The ubiquitin and ubiquitin‐like protein modifications contribute to functional changes of substrate proteins and thus regulate a range of biological processes including cancer.To further explore the SCNA patterns of UFMylation genes, we calculated the high‐level alteration of SCNAs and found that 11.08% of TCGA samples have high‐level copy number alterations in at least one of the eight genes. Noteworthy, the high‐level amplifications and deletions of UFMylation genes appeared to be occurred in a pattern of mutually exclusive Figure , suggestAlthough most UFMylation genes generally have low mutation frequencies in cancers, we found that UFSP2 is frequently mutated in colon adenocarcinoma and uterine corpus endometrial carcinoma. The high recurrent copy number loss and frequent somatic mutations of UFSP2 suggest that UFSP2 may function as a tumour suppressor. It has been previously demonstrated that knockdown UFSP2 promoted breast cancer cell growth and tumour formation, suggesting that UFSP2 is a tumour suppressor in breast cancer.In summary, we found that UFMylation family genes have a high frequency of SCNAs. Especially, UFSP2 is recurrently and focally deletion in a total of 14 human common cancers with the highest alteration score. We further demonstrated that depleted UFSP2 expression significantly promotes the growth of tumour cells both in vitro and in vivo, suggesting that UFSP2 may function as a tumour suppressor in colon cancers. Our integrated genomic analysis and functional studies provide insights in understanding this new post‐translational modifier in cancer and potential therapeutic opportunities.Supporting informationClick here for additional data file.


Seeking to improve upon limitations of other probes, we have developed a family of fluorescent probes based on a 2-amino-3′-dialkylaminobiphenyl core. This core condenses with N2O3 to form benzo[c]cinnoline structures, incorporating the analyte into the newly formed fluorophore, which results in product fluorescence with virtually no background contribution from the initial probe. We varied the substituents in the core in order to optimize both the reactivity of the probes with N2O3 and their cinnoline products' fluorescence wavelengths and brightness. The top candidates were then applied to cultured cells to verify that they could respond to NO within cellular milieus, and the top performer, NO530, was compared with a “gold standard” commercial probe, DAF-FM, in a macrophage-derived cell line, RAW 264.7, stimulated to produce NO. NO530 demonstrated similar or better sensitivity and higher selectivity for NO than DAF, making it an attractive potential alternative for NO tracking in various applications.Fluorescent probes for nitric oxide (NO), or more frequently for its oxidized surrogate dinitrogen trioxide (N e.g. N2O3, compared to the original NO550.A thorough structure-property relationship study has produced much-improved fluorescence probes for nitric oxide surrogate,  In addition, high oxidation-state metals can be reduced by NO, with concomitant or swiftly ensuing nitrosation of a proximal nucleophile, such as a thiol.64–66 If present, superoxide will react with NO in a diffusion-controlled rate. It produces the peroxynitrite anion, a potent oxidizer responsible for DNA mutations.67 When triplet oxygen reacts with four equivalents of NO, it forms two equivalents of the potent nitrosating dinitrogen trioxide  radical. Reaction with another NO radical produces the asymmetric N2O3, rather than the symmetric nitrous anhydride.Nitric oxide synthases (NOSs) produce the free radical NO when they convert de N2O3, . The dirii), as exemplified by Lippard's Cu(FL) probes.68–74 After NO complexation to Cu(ii), nucleophilic attack by the ligand amine reduces the metal to Cu(i). Metal reduction and N-nitrosation lower the affinity of the ligand for the metal, to the extent that the metal dissociates and no longer quenches ligand fluorescence  quenching of fluorescence. Nitrosation of either of the amines, followed by condensation of the other amine upon the N-nitrosamine, produces a triazole that can no longer efficiently PET quench fluorescence. In this category lies DAF-FM, one of the most cited probes for NO imaging. It has also been suggested that the nitrosation occurs via peroxynitrite, which is formed due to a reaction between superoxide and NO,80 however the probes cannot differentiate the source of nitrosation.The second category consists of the cts N2O3 .75–79 Thortho-diaminobenzene-based probes is limited both by chelation to alkali-earth metals and reaction with other bioavailable dielectrophiles, such as dehydroascorbic acid (DHA) and methyl glyoxal, resulting in the attenuation of PET quenching and thus a non-NO-selective signal.81–84Both sensing strategies produce an enhancement, not a wavelength-shift, of emission as the signal of reaction with NO or its oxidized surrogate. Incomplete quenching of fluorescence in the unreacted probes can result in significant background signal. With no change in wavelength, this background degrades the detection limits of the reaction with NO. Transition metal-based probes are most affected by this incomplete quenching, with relatively small increases (3–40×) in fluorescence due to NO addition when compared to the non-metal-based probes (40–1500×). In addition, the performance of 550  is significantly lower than that for fluorescein-based DAF-FM triazole , due both to AZO550's lower molar absorptivity (4000 M−1 cm−1versus 73 000 M−1 cm−1) and fluorescence quantum yield (0.11 versus 0.81).76 Ghebremariam and co-workers found that not even at 75 μM loading concentration did NO550 match the intensity of only 1 μM DAF-FM in the same endothelial cells at the same time point.86 Loading with higher concentrations of NO550 was also hampered by poor water solubility and cytotoxicity. Herein, we describe the systematic substitution of the 2-aminobiphenyl scaffold, the effect of this substitution on reactivity and fluorescence, and the response of the best candidates to exogenous and endogenous NO in cells, and compare these results to those obtained for DAF-FM.The excitation and emission wavelengths of AZO550's limitations, the results were sufficiently promising to study the 2-aminobiphenyl core more thoroughly, with the goals of increasing brightness, quantum yield, and red-shifting the fluorescence, all while maintaining the selectivity for NO and the spectral resolution of probe fluorescence versus that of the corresponding cinnoline product. 2O3 is dielectrophilic, these probes are dinucleophilic, and positions X and Y on rings A and B, respectively, have the greatest influence on the probes' nucleophilicity. We incorporated ring C to impart bathochromic shifts. Substituent Z on ring C is not only commercially and synthetically convenient but also distant from  the N2O3 reaction sites. It is therefore an attractive attachment point for hydrophobic, hydrophilic, cell-compartment directing, or tethering functionalities. We first evaluated the brightness and fluorescence properties (i.e. azo) products  accessed from the reaction of nitrite with acid , hydroxy , and N,N-dimethylamino  groups. By far, the hydroxyl variant surpassed the others in brightness due to AZO-2's much higher quantum yield of 67%. Unfortunately, this brightness showed a dependence upon the protonation state of the phenol around physiological pH. For fluorescein-based probes, the pKa of the phenol in the excited state normally lies around 6.7,87 meaning that these probes may non-selectively detect (by quenching of fluorescence) pH in cells. We performed fluorescence pH titrations on AZO-2 and AZO-3, establishing their pKa's as 7.8 and 4.5, respectively . The Y substituent was fixed as a methyl group while varying the X substituent on ring A. Since ring A attacks the trapped ectively . Protona2 to CN would result in a bathochromic shift of fluorescence wavelengths. The X = NMe2 and Y = CN cinnoline variant (AZO-4) had excitation and emission maxima 35–40 nm longer than AZO-3; however, the quantum yield of AZO-4 was determined to be less than 1%, even lower than AZO-3's quantum yield (2%).We next evaluated the influence of position Y by changing it from methyl to cyano, postulating that the greater conjugation from NMe3 and AZO550's low quantum yield, we speculated that the excitation energy was lost to non-radiative relaxation through rotation about the ring A aryl-NMe2 bond.88,89 This bond rotation decreases significantly when the N-alkyl groups are fused to the aryl ring, so we synthesized N-methyl-tetrahydroquinoline (5) and julolidine (6) variants on ring A, with methyl at Y on ring B. The ring fusion in both variants resulted in a remarkable increase in quantum yield, from 0.02 and 0.11 for non-fused AZO-3 and AZO550 to 0.36 for AZO-6 and 0.62 for AZO-5. In addition, the molar absorptivity for AZO-5 also increased, making it the brightest of the biphenyl series – nearly twenty times brighter than AZO550, albeit at shorter wavelengths. The julolidine variant did produce the longest fluorescence wavelengths of the group, confirming the expected greater planarity of the dialkyl-N to aryl bond. However, a steric clash between the aromatic proton and the julolidine benzylic protons, as depicted in 6's lower quantum yield and lower molar absorptivity than AZO-5.When considering AZO-5 proved to be the brightest candidate, but AZO-5's excitation and emission maxima (425 and 508 nm) fall short of the ideal wavelengths for the universal FITC filters. We decided to add ring C with the hypothesis that benzannulation would extend fluorescence wavelengths to those more suitable for the FITC filter set. For a thorough comparison, we studied the N,N-dimethylamino, N-methyltetrahydroquinoline, and julolidine variants at position X on ring A. For each of these variants, H occupied position Y and we derivatized position Z with H, N-acetamide (NHAc), and CN – a total of nine variants.Probe 7–15) show that both NMe2 (at X) and CN (at Z) substitutions are detrimental to cinnoline brightness. For the same Z substituent, the NMe2 series members had lower molar absorptivities and quantum yields than their corresponding N-methyl tetrahydroquinoline or julolidine congeners, i.e., cinnolines 7versus10 and 13, 8versus11 and 14, and 9versus12 and 15. This trend is readily explained by the greater non-radiative relaxation rate caused by rotation about the N-aryl bond for NMe2 variants versus the singly fused N-methyl tetrahydroquinoline and doubly fused julolidine variants. Likewise, the Z = CN cinnoline series exhibited lower molar absorptivities than the corresponding Z = NHAc or H series (for the same X substituent). We speculate that the Franck–Condon excited state has significant quinoid character in the Z = CN series, with less quinoid character in the ground state, such that the poor orbital overlap between the two states results in lower probability of photon absorption.Focusing on brightness first, the results listed in 12 and AZO-15), the ground state incorporates greater donation of the aligned nitrogen lone pair into the polyaromatic system than from the more freely-rotating X = NMe2 variant (AZO550), leading to better orbital overlap of the ground state with the excited state, as demonstrated by their higher absorptivities. For the Z = H and NHAc variants, both substituents are less electron withdrawing than the quinoid-inducing CN, meaning that their ground and excited states are more similar and their absorptivities higher.90,91 The Z = CN series also produced lower quantum yields. Here we posit that the excited state relaxes through internal charge transfer to the nitrile π* orbital, with subsequent non-radiative relaxation through the increased hydrogen-bonding of this negatively-charged complex with buffer. N-methyl tetrahydroquinoline variants proved brighter than their A1,3-strained julolidine counterparts, as discussed for AZO-6. Interestingly, for AZO-13 the greater electron-withdrawing nature of the NHAc Z-substituent (acceptor) acting upon the dialkyl amine (donor) lone pair electrons countered the A1,3 strain to promote greater planarity and therefore a higher quantum yield, as compared to Z = H for AZO-14.Within the Z = CN series, for the two alicyclic amine variants at X  induced the greatest shift, but this substitution prohibitively decreased brightness. Variants substituted with NHAc at Z  showed only slight shifts (3–7 nm) to longer wavelengths compared to their corresponding H  substituted ones. Varying X in the benzannulated series from NMe2  to N-methyltetrahydroquinoline  to julolidine  continued the aforementioned pattern in the biphenyl series of red-shifting fluorescence wavelengths with greater ring fusion. Benzannulated julolidine variants therefore produced the longest wavelength probes; unfortunately, julolidine substitution also decreased brightness, as proposed above, due to A1,3 strain.As anticipated, adding ring C to the biphenyl probes bathochromically shifted fluorescence wavelengths. While benzannulation of fluorones to seminaphthofluorones shifts wavelengths by more than 100 nm, we found only 15–30 nm shift for the cinnolines.5, 10, 11, and 13 as the brightest in the group. Importantly, as with first generation NO550 and AZO550, the maximum excitation and emission spectra are well resolved from each other for both the probes and their cinnoline products, obviating homo-Förster resonance energy transfer (FRET) that occurs for high intracellular concentrations of ortho-diaminophenyl-type probes.82 Furthermore, in the case of the four brightest probes, the absorption spectra for the probes minimally overlap with the absorption spectra of their corresponding cinnolines .The spectroscopic data reveal probes s Fig. S1. Consequ15,95,96 up to five equivalents of NO, were added to an air-equilibrated 50 μM solution of each probe variant in 4 : 1 50 mM PBS/DMSO at pH 7.4 , OH (2), and NMe2 (3), their Hammett σ+ substituent constants of −0.78, −0.92, and −1.7, respectively.97 Variant 1 did not produce sufficient cinnoline product to be detected by fluorescence  to readily promote nucleophilic attack by ring A. Subsequently, we set X as NMe2 and changed the weakly donating methyl (σ+ = −0.311) at Y to a strongly-withdrawing cyano group, (σ+ = 0.659).98 With no cinnoline fluorescence detected, we concluded that the nitrile group inhibits the nucleophilicity of the p-amine to such an extent that it could not N-nitrosate.We next looked at fluorescence titrations with NO of the probes in aerobic buffer. Incremental aliquots of a saturated NO solution (1.9 mM),g. S22O3 , a portirescence , whereas2O3  for the most part required less than five minutes to nearly fully react. The high level of fluorescence from species 10, 11, 13, and 14 makes these species the most promising of the group we examined  was previously determined to be 30 nM;85 for the brighter 10, the detection limit is 2 nM, comparable to DAF-FM's detection limit of 3 nM.76With the high reactivity of these probes and with the creation of signal with virtually no background, excellent detection limits were obtained. We defined the lower detection limit (LDL) as the concentration of NO that produces signal equal to three times the experimental setup noise. The LDL of 100 However, with our probes two factors moderate this benefit of ratiometry. First, the probe excitation band is in a shorter, cell-damaging near-UV range than that for the cinnoline products. Second, the shorter excitation results in little cinnoline emission, instead primarily unreacted probe background fluorescence. The later phenomenon however does not affect one of the most significant attributes of our 2-aminobiphenyl system: i.e. that exciting at the cinnoline absorbance maximum results in virtually no background contribution from the probe.The resolution of the probe and cinnoline emission spectra enables ratiometry when the probes and their cinnoline products share an excitation band . Ratiome550 for NO over other common biological substrates.85 Indeed this anticipation proved correct; as one example, 10 in the presence of NO versus that in the presence of various oxidants. Even at one hundred equivalents of peroxynitrite to probe, at most a five-fold increase of fluorescence was observed, as compared with a greater than 1300-fold increase for merely one equivalent of NO. It is important to note that we used purified peroxynitrite for this study. When this is generated in cellulo it involves both super oxide and NO which could potentially activate the fluorescent probe through a free radical mechanism.By keeping the 2-aminobiphenyl core, we expected to maintain in our newer probes the selectivity of NO10 (henceforth named NO530), and 13 (NO562) and to compare them to the first-generation probe 9 (NO550) and commercial probe DAF-FM. In the current studies, we examined the response of these probes to NO in an NIH-3T3 murine fibroblast cell line and a RAW 264.7 murine macrophage cell line because, while NO530 and NO562 readily reacted with NO in solution, most important is their performance in live cells. We first pre-incubated cells with the probes and then exposed cells to either exogenous NO-donor S-nitrosopenicillamine (SNAP) or to a NO solution. Next, we compared the specificity of NO530, NO562, and DAF-FM probes for the detection of endogenous NO produced in RAW 264.7 cells. Finally, we evaluated the cytotoxicity of NO530.Taking into consideration the probes' spectral properties and response to NO, we decided to focus on probes 550, NO530, and NO562, and exposed to 1 mM and 200 μM SNAP solutions, respectively  channel  with appropriate Cy3 filters (data not shown). In contrast, NO530 generated somewhat more SNAP-dependent signal than NO550 in NIH-3T3 cells, although it provided a several-fold lower increase relative to unstimulated cells (28-fold vs. 104-fold). In RAW 264.7 cells, NO530 and NO550 stimulated cell fluorescence was not significantly different, although NO530 provided somewhat higher fractional increase in fluorescence as a result of lower background . All three probes yielded similar signal in RAW 264.7 cells; NO530 provided the best fractional change in signal because of substantially lower background before addition of NO. Taken together, these findings suggest that NO530 was the most promising probe to investigate endogenous production of NO in RAW 264.7 cells.In addition, cells loaded with each probe were exposed to 200 μM NO (NIH-3T3 cells) or 320 μM NO (RAW 264.7 cells) by diluting 1.9 mM saturated NO solution into the cell medium . In NIH-l-arginine. We tested NO530 response in both quiescent and LPS/IFN-activated RAW 264.7 cells, and compared results to those obtained using DAF-FM DA. Data were obtained using cell imaging instrument INCA 2200. Cells were exposed to l-arginine in the presence of NO probes as described in the ESI.530, quiescent RAW 264.7 cells produced low fluorescence signal that did not measurably change after treatment with arginine. In the absence of exogenous arginine, LPS/IFN-activated RAW 264.7 cells produced approximately two-fold higher fluorescence signal than un-activated cells; cellular fluorescence further increased in response to arginine in a concentration-dependent manner , an enzyme that can be stimulated by a combination of lipopolysaccharide (LPS) and interferon-γ (IFN-γ) to produce NO in the presence of NO-synthase substrate e signal . The cur530 was the best performer in terms of fluorescence ratio in NO-dosing studies, we evaluated its cytotoxicity in RAW 264.7 cells at various concentrations with a Calcein Blue/propidium iodide viability assay. For 1.25, 2.5, 5, 10, 20, and 40 μM loading concentrations of NO530, greater than 93% of the cells stained a viable blue  stands as a promising candidate for live cell imaging. Due to its greater response to stimuli, higher selectivity for NO, and the ability to image both reacted and unreacted probe, NO530 provides a attractive option as a nitric oxide probe and a potentially valuable alternative to DAF-FM in various applications.The 2-aminobiphenyl core selectively creates a longer-wavelength fluorophore when it reacts with oxidized NO. We found that attaching electron-donating groups, primarily dialkyl amines, on the nucleophilic aryl group, and avoiding the conjugation of electron withdrawing groups to the 2-amino group, renders the probes sufficiently nucleophilic to readily scavenge NO in acellular media. Furthermore, fusion of the 4′-aminoalkyl groups to the nucleophilic aryl group increased both fluorescence wavelengths and the fluorescence quantum yield of the cinnoline product. Benzannulation of the 2-amino aryl group also provoked a bathochromic shift in fluorescence and opened a functionalizable position, removed from the non-alkylated amine, for attachment of groups with desired properties DYF and AVK are employees of Creative Scientist, Inc.SC-011-C9SC04304G-s001