Combined effect of hierarchical topography and fluorination supplied to the surfaces omniphobic and icephobic properties. The study of the stability of the Cassie wetting state is reported. High apparent contact angles were accompanied with the low contact angle hysteresis and high stability of the Cassie air trapping wetting state. Time delay of the ice crystallization as high as Omniphobic and icephobic twin-scale surfaces based on the “urchin”-like fluorinated Al The problem of creating of ice-phobic surfaces attracted the attention of researchers in the last decade9. The notion of “icephobicity” is ambiguous3. There exist are at least three very different approaches to the interpretation of icephobicity, as discussed in Ref.3. First, icephobicity implies relatively low energy of adhesion (as estimated with the Young–Dupre equation12) between ice crystals and a supporting surface. It was demonstrated in Ref.11 that the ice adhesion strength correlates with the work of adhesion required to remove a liquid water drop from each smooth surface. The authors of Ref.11 studied flat strongly hydrophobic surfaces and concluded that reduction in ice adhesion strength will require developments of specially textured surfaces, as no known materials exhibit receding water contact angles on smooth surfaces that are above those reported in Ref.11. And this is an approach suggested in the present paper in which icephobic properties of the textured urchin-like surfaces are addressed. The reasonable question is: how the ice adhesion strength should be quantified? The most widespread approach is based on the measurement of the critical shear stress necessary for de-icing16, although the normal stress is used as well3.Icing is a widespread phenomenon in subzero climates where water and/or moisture are present and it crucially influences the quality of life and industry including transportation systems, power transmission, infrastructures and energy supply systems18. Icephobic should be, therefore, a surface with a minimum ice nucleation temperature. Such surfaces can be successfully operated even at a few degrees above this temperature, allowing an extended freezing delay time18. The optimal texture developed on such surface should have multitier structuring, designed also to account for heterogeneous nucleation thermodynamics, to simultaneously reduce and optimize the liquid–solid contact area (lower nucleation temperature) and to improve the droplet mobility, namely to reduce contact time, and to prevent the Cassie–Wenzel wetting transition19. And it was concluded that such surfaces should be designed for the specific environmental conditions18. Obviously, design of the tailor-made surfaces preventing ice formation calls for profound understanding of water nucleation on the micro- and nano-rough interfaces21. It should be emphasized that superhydrophobic surfaces, with very high receding contact angle may have strong adhesion to ice22. It was demonstrated recently that there exists the critical confinement length scale enabling catching of liquid water (as opposed to ice) in between roughness features and thus reducing the strength of ice adhesion by over a factor of twenty-seven compared to traditional hydrophobic surfaces17. The reduction in ice adhesion is due to the meta-stability of liquid water; as ambient ice cleaves from the textured surface, liquid water leaves confinement and freezes—a process which takes the system from a local energy minimum to a global energy minimum17. This phase transition generates a detachment force that actively propels ambient ice from the surface17. Finally, the third, alternative approach to the design of icephobic interfaces was suggested, implying bouncing of incoming small droplets at the temperatures below the freezing point25. Generally, icephobic surfaces should: prevent freezing of water condensing on the surface, prevent freezing of incoming water if ice formed, and they should have as weak adhesion strength with the solid as possible25. In spite of a much effort invested into the development and manufacturing of icephobic surfaces the interfaces, which repel efficiently ice, remain scarce27. We conclude that the development of the effective anti-icing surfaces remains a challenging scientific and engineering problem. We report the robust ice-repelling surface based on the urchin-like Al2O3 particles.The alternative approach defines icephobicity as the ability to prevent ice formation on the surface2O3 were synthesized by the hydrothermal method described in detail in Ref.28. SEM images of the particles are depicted in Fig. Epoxy oligomer , 1H,1H,2H,2H-perfluorodecyltrichlorosilane (FDTS)) and 3-aminopropyltriethoxysilane (abbreviated APTES) were supplied by Alfa Aesar, GB. Aluminum sulfate, Aluminum nitrate, Dimethyl sulfoxide ≥ 99.5% (DMSO), Ethyl Alcohol 96.0–97.2%, were supplied by Sigma-Aldrich, 2-propanol 98%, Urea 99% were supplied by Acros Organics. Urchin-like spherule particles of Al2(SO4)3:Al(NO3)3 = 80:20 ml%/mol%. The molar ratios of the components of the synthesis were Al3+/CO(NH2)2/H2O/i-PrOH = 1:2:100:5, the synthesis scheme is described in detail in28.Urchin-like particles were obtained by the method of hydrothermal synthesis, from an aqueous-alcoholic solution of aluminum salts taken at a ratio of Al2O3 were introduced into the solution under mixing concentration 7.5%. The suspension was homogenized with the ultrasonic disperser Bandelin Sonopuls HD 3200 (modulus KE 76) during 2 min. After this Al2O3 particles were dried under 2O3 particles were used for manufacturing of the icephobic, omniphobic coating.Particles were hydrophobized as follows: at the first stage of the process 0.4 g of FDTS were introduced into 50 ml of hexane. 4.0 g particles of Al2O3 were introduced and homogenized with ultrasound during 30 s. The total energy necessary for homogenization was estimated as 0.8 kJ. The obtained suspension was deposited on the solid substrate by spraying or dip coating. Al and Ti plates with the thickness of 0.1 and 1 mm, cleansed by ultrasound, ethanol and acetone, were used as the solid substrates. Solid substrates coated with the omniphobic coating were exposed to the thermal annealing under the temperature of 150 °C during 1 h. Thermal annealing was followed by dipping of the solidified coating into 0.1% wt. hexane solution of FDTS. The coating was dried under 20 °C during 5 min, and finally annealed under the temperature of 150 °C during 1 h. SEM images of the eventual coating are supplied in Fig. The icephobic coating was manufactured according to the following protocol: 1 g of APTES was mixed with 1.5 g of TEOS and 0.3 g of the epoxy oligomer. Mixing was performed with the magnetic stirrer during 10 min. In the course of the mixing 0.4 g of Al2O3 particles manufactured as discussed in “Topography and morphology of the urchin-like AlWetting characteristics of the coating (apparent contact angles and the contact angle hysteresis) were measured with the precise goniometric system (Kruss DSA-100). 8 θA beyond which the triple line started to move. The threshold contact angle θA was registered as the advancing contact angle10. When a droplet was deflated with a syringe its volume was decreased to a certain limiting value; in parallel the contact angle decreases till a threshold value θR, registered as the receding contact angle, denoted θ = θR, the triple line suddenly moved. Both θA and θR are equilibrium, although metastable contact angles10. The difference between θA and θR, namely 10.Contact angle hysteresis was established with the needle-syringe method. 5 μl droplet was placed on the surface and inflated with a syringe; the triple (contact) line was pinned to the substrate up to a certain volume of the droplet. When the triple line was pinned, the contact angle increased till a certain threshold value In parallel, sliding angles were established with the inclined (tilted) plane method. The minimal angle at which 5–10 µl water droplets started to slide along the studied surface was registered. The establishment of the contact angle hysteresis and the sliding angle were performed at the ambient conditions  which is 10 nm. The needles are fluorinated as evidenced by the EDS spectroscopy, illustrated with Fig. 2O3 particles demonstrated pronounced omniphobic properties, namely APCA values as high as Address first the topography of interfaces, manufactured as described in the “35. Various experimental techniques were implemented for the study of the stability of the Cassie wetting state19. We tested the stability of the Cassie air trapping wetting with the technique enabling establishment of the threshold  value of the surface tension of liquid , which are This finding evidences the pronounced omniphobicity of the reported surfaces, displayed in Fig. 41 with its fluorination, illustrated with Fig. 43. It is noteworthy, that the established critical value of surface tension was markedly smaller than that reported in Ref.43, namely The surface tension of DMSO 22. Highly developed superhydrophobic surfaces sometimes promote heterogeneous nucleation of ice, as mentioned in Refs.21. Contrastingly, the reported surfaces demonstrate the pronounced icephobic properties. Consider first water crystallization, which took place on the cooled flat aluminum surfaces. Crystallization was accompanied with the change in the transparency of droplets: this change, evidencing formation of ice was registered at t = − 7 ℃, and it finally was registered after 90 s of cooling under the APCA 2O3 particles was frozen after 3 min of cooling. It was impossible to remove droplets from the surface with an air jet.It should be emphasized that hydrophobic and even omniphobic properties of the interface do not ensure its icephobicity, as discussed in Refs.t = − 6 ℃. The rate of this decrease is approximately t = − 6 ℃ the APCA was stabilized at the high level of 44.Now address water crystallization observed on the fluorinated “urchin”-like surfaces and illustrated with the Fig. Interfacial crystallization was followed by the bulk crystallization, which started with an essential delay to be addressed below in detail. Bulk crystallization of water within a cooled droplet was accompanied with the displacement of the crystallization front. The propagation of the crystallization front within the droplet, which started from the contact area, is illustrated with the Supplementary Video R is the radius of the droplet and α is the thermal diffusivity of water at t = 0 °C45) yields The characteristic time of the movement of the front of crystallization within a droplet, depicted schematically with Fig. mentclass2pt{minim t = − 6 ± 0.5 °C, the rate of crystallization was markedly slowed; cooling procedure which started at t = − 6 ± 0.5 °C and continued for t = − 15 ± 0.5 °C. Water within a droplet was completely crystallized after t = − 15 ± 0.5 °C. Thus, the pronounced total time delay of the bulk ice crystallization (i.e. freezing delay time span) as high as At the temperaturementclass2pt{minim46, namely The delay of the bulk ice crystallization was markedly larger than the maximal time span of the delay reported in Ref.46. The high values of the APCA under crystallization evidence that the crystallization occurs within the Cassie wetting regime. This assumption is supported by the fact that the crystallized droplets are easily blown out of the surface by the air stream with the velocity The delay in crystallization is reasonably related to the temperature increase within the droplet due to the release of latent heat of fusion, as suggested in Ref.entclass1pt{minima44. The reasonable explanation of this effect was proposed in Ref.46; namely, it was suggested that the supersaturation of water vapor in the proximity of the triple line influences the solid–vapor and solid–liquid surface tensions. Consequently, the surface appears more hydrophilic at decreasing surface temperatures. The increase in contact area between the cooled droplet and the surrounding vapor occurs despite the increase in the surface tension of water in a course of cooling47. The contact area was stabilized within the range of temperatures of ∆t = − 6 – (– 10) °C, as shown in Fig. 2O3 surfaces demonstrate the pronounced icephobic properties when compared with flat and non-fluorinated ones. Crystallized ice is easily removed from the surfaces. Heated surfaces restored their omniphobic and icephobic surfaces.Crystallization was accompanied with the change in the area of contact area and the shape of the cooled droplet, as shown in Fig. 2O3 particles. The highly developed hierarchical surface demonstrates two well separated spatial scales, namely the large scale “urchins” which is ca40.We report twin-scaled fluorinated surfaces built of “urchin”-like fluorinated Al48.The surface demonstrated distinct omniphobicity. The onset of the Cassie–Wenzel wetting transitions corresponds to the critical value of liquid ca 88 min was registered when compared to the ice formation on the flat aluminum and non-fluorinated “urchin”-like surfaces. Crystallization of ice started from the water/solid interface44; the velocity of propagation of the crystallization front was established experimentally as 12. Heated “urchin”-like surfaces completely restored their omniphobic and icephobic surfaces after thawing, which is extremely important for the industrial applications of the reported surfaces. The pronounced omniphobic and icephobic properties of the hierarchical “urchin”-like surfaces emerge from the combination of the twin-scale topography and fluorination of the interface, decreasing its specific surface energy13. In our future investigations we plan to study condensation of droplets on the reported “urchin”-like fluorinated surfaces46.The reported urchin-like hierarchical fluorinated surfaces also demonstrate pronounced icephobicity. The essential time delay of the bulk ice crystallization as high as Supplementary Figure S1.Supplementary Video Legends.Supplementary Video 1.Supplementary Video 2.


Cleavage at S1/S2 is important for efficient viral entry into target cells. The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous cleavage motif was present at S1/S2 of the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV. We show that a crucial third arginine at the left middle position, comprising a motif RRxR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage. Further, we describe a surprising finding that the two serines at the edges of the insert SPRRAR↓SV can be efficiently phosphorylated by proline-directed and basophilic protein kinases. Both phosphorylations switch off furin’s ability to cleave the site. Although phospho-regulation of secreted proteins is still poorly understood, further studies, supported by a recent report of ten in vivo phosphorylated sites in the Spike protein of SARS-CoV2, could potentially uncover important novel regulatory mechanisms for SARS-CoV2.The Spike protein of the novel coronavirus SARS-CoV2 contains an insertion  One of the hypotheses that emerged immediately after the release of the SARS-CoV2 sequence in January3 was that a unique insertion in its Spike protein, predicted to carry a crucial cleavage site for furin protease, could be the key for enhancing zoonotic transmission and infectivity6.The novel SARS-CoV2 coronavirus that has caused hundreds of thousands of deaths worldwide in 2020 has puzzled scientists with its high infectivity and severe consequences for infected organs7. It is composed of two subunits  are easily accessible for enzymes and other protein signaling modules, and often encode short linear motifs (SLiMs) that act as recognition sequences for such binding partners. Intriguingly, besides the RxxR motif, the sequence of the loop SPRRAR↓SV contains two serines that match the consensus of proline-directed kinases (SP), and basophilic protein kinases (RxxS), the two largest subfamilies of mammalian kinases22. However, the Spike protein, except for its short C-terminal tail, is considered to be facing the endoplasmic reticulum (ER) or the Golgi lumen during the viral replication cycle23 and is not present in cytoplasm or the nucleus, where most of the kinase signaling is taking place. Nevertheless, despite still being a poorly studied field, it is well known that protein phosphorylation does not only occur on cytoplasmic and nuclear proteins, but also takes place on secreted proteins in ER and Golgi lumen, and also in the extracellular space27. Strikingly, supporting evidence for the idea of Spike phospho-regulation arises from a recent report that confirmed more than ten in vivo phosphorylated sites in the Spike protein of SARS-CoV228. Furthermore, phosphorylation has been shown to regulate fibroblast growth factor 23 targeting by furin29, indicating that kinases can regulate the secreted proteins as well as furin targeting.Recent Cryo-EM structures of the SARS-CoV2 Spike protein revealed that the insertion with the furin-like cleavage site emerges as a disordered loop on the side of the proteinSPRRAR↓SV can be phosphorylated in vitro by different protein kinases and both phosphorylations affect the ability of furin to cleave the site. Finally, we discuss the possible novel regulatory mechanisms that such interplay of three different and mutually inter-dependent enzymatic modifications within the insert may present for SARS-CoV2 function.In this study, we analyzed the furin cleavage site specificity of the SARS-CoV2 Spike using biochemical assays with substrate constructs based on the S1/S2 cleavage site sequence. We discovered that a motif RRxR, with a crucial P3 arginine , was required for cleavage of the S1/S2 site substrate constructs in vitro, while the motif in common with MERS-CoV (RxxR) was not sufficient for furin cleavage. Further, we describe a surprising finding that the two serines flanking the insert 20  , that changes the furin core motif RxxR↓x to QxxR↓x, also completely inhibits furin activity towards the reporter protein  that greatly increases the furin cleavage efficiency of MERS-CoV S1/S2 also enhances its phosphorylation by CDK  as a representative for proline-directed kinases and the protein kinase A (PKA) catalytic subunit as a representative for basophilic kinases. To examine the phosphorylation of different S1/S2-GB1 reporter proteins, we stopped the in vitro phosphorylation reactions at specific time points and analyzed the phosphorylation efficiency using Phos-tag SDS-PAGE to separate the phosphorylated protein from the non-phosphorylated substrate. The SARS-CoV2 S1/S2-GB1 reporter was fully phosphorylated by both CDK and PKA by the 60-min time point Fig. c. The S1Next, we analyzed how phosphorylation at these sites affects furin cleavage. For this, the S1/S2-GB1 reporter proteins were first phosphorylated by either CDK or PKA for 60 min, resulting in the phosphorylation of the majority of the substrate, followed by the addition of furin. Phosphorylation of either site adjacent to the core furin motif, S680 and S686, significantly inhibited the furin cleavage Fig. a,b. To cNext, we tested the effect of mutation of the phosphorylation sites to aspartic acid, often used to mimic phosphorylation. The S680D mutation decreased furin cleavage efficiency to the same extent as phosphorylation. However, S686D had a smaller effect on furin activity, similar to S686A mutation Fig. a. Thus, A similar phospho-inhibitory effect was seen with MERS-CoV(RRVR)-GB1 Fig. d,e, alth18, others have argued against this due to an off-target effect of the furin inhibitor dec-RVKR-CMK19. Our finding suggests that SARS-CoV2 may have acquired a true furin cleavage specificity in contrast to the coronaviruses causing the two previous outbreaks. In addition, the discovered possibility of tight phospho-regulation at two serines creates an interesting complexity where a disordered loop carrying a SLiM facilitates specific regulatory inputs for three different modifying enzymes. The in vivo functionality of this SLiM could emerge as one of the key functional elements in the SARS-CoV2 Spike protein, given that proteolytic processing on viral glycoproteins has been found to be a key virulence factor. Indeed, previous studies have shown that highly pathogenic influenza virus forms have been found to harbor optimal polybasic furin processing sites, whereas forms with low pathogenicity have monobasic cleavage sites36. The glycoprotein cleavage specificity also determines tissue tropism, as furin is ubiquitously expressed, whereas the proteases that process monobasic cleavage sites, like TMPRSS2, are expressed mainly in the aerodigestive tract38.Our results confirm that a four amino acid insertion to the S1/S2 site of the SARS-CoV2 Spike protein introduces a furin cleavage site. However, the P3 arginine, not present in analogous site in MERS-CoV, is crucial for furin-dependent in vitro cleavage of a substrate construct carrying the sequence of the S1/S2 site. While some previous reports have noted furin-mediated activation of MERS-CoV Spike40. More recent reports, however, have defined a core motif of 8 amino acids . The proteins were eluted in buffer containing 25 mM Hepes–KOH (pH 7.4), 300 mM NaCl, 10% glycerol, 200 mM imidazole.Constructs containing a 20 amino acid region from the S1/S2 linker were fused via a linker with the sequence ELQGGGGG to the GB1 domain  and a C-terminal 6xHis tag. The pET28a vectors were transformed to 47. The phosphorylation reactions were carried out in buffer consisting of 50 mM Hepes–KOH (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 50 mM imidazole, 2.5% glycerol, 0.2 mg/ml bovine serum albumin, 0.15 mM EGTA, 1 mM β-mercaptoethanol, and 500 μM ATP. The kinase reactions were performed in 30 µl containing 2.5 µg of S1/S2-GB1-6xHis substrate (7.6 µM). The concentration of PKA was 375 nM and cyclin B-Cdk1 5 nM. The reactions were carried out at room temperature and were stopped at 10 s (0 min), 5 min, 20 min, and 60 min by pipetting 5.5 µl of the reaction mixture to 8 µl of 2 × Laemmli SDS-PAGE sample buffer containing 1 mM MnCl2.The phosphorylation of the S1/S2 linker constructs was examined in vitro using purified cyclin B-Cdk1 (Millipore 14-450) and PKA (murine cAMP-dependent protein kinase), purified as described in2. The electrophoresis was carried out at 15 mA until the bromophenol blue dye front reached the bottom of the gel. Following electrophoresis, the gels were soaked in fixation solution  with gentle agitation for 15 min. The proteins were stained with colloidal Coomassie Blue G-25048.The stopped samples were incubated at 72 °C for 5 min and were loaded on Phos-tag SDS-PAGE gels containing 12.5% acrylamide, 100 µM Phos-tag, 200 µM MnCl2, 16 mM imidazole, 2% glycerol, 0.2 mg/ml bovine serum albumin, 0.15 mM EGTA, 500 μM ATP, 0.5% Triton-X100, 2 mM CaCl2, 2 mM β-mercaptoethanol. In case the effect of phosphorylation on furin cleavage was studied, the S1/S2-GB1-6xHis substrate was first phosphorylated for 60 min as described above, followed by addition of furin. The reactions were carried out at room temperature and were stopped at 0, 5, 20 and 60 min by pipetting 6 µl of the reaction mixture to 2 × Laemmli SDS-PAGE sample buffer.The furin cleavage specificity was assayed by incubating 2.5 µg (7.6 µM) S1/S2-GB1-6xHis substrate with 2 U furin  in 30 µl reactions. The reaction buffer contained 50 mM Hepes–KOH (pH 7.4), 150 mM NaCl, 5 mM MgClThe stopped reactions were heated at 72 °C for 5 min and loaded to 15% acrylamide SDS-PAGE. Following electrophoresis, the gels were immersed in fixation solution for 15 min and stained with colloidal Coomassie Blue G-250.49.Furin cleavage reactions were set up as described above, except that BSA was not added to the reactions. The reaction mixtures were incubated for 60 min at room temperature and were stopped by adding EGTA to 10 mM concentration to inhibit furin. Reactions where 10 mM EGTA was present at the time of furin addition were carried out for controls. The peptides arising from furin cleavage were purified using C18 StageTips. The peptides were separated using Agilent 1200 series nanoflow system (Agilent Technologies) and sprayed into an LTQ Orbitrap mass spectrometer (Thermo Electron) with a nanoelectrospray ion source (Proxeon). The data was analyzed using MaxQuantSupplementary Figures.Supplementary Tables.


Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), are responsible for high mortality and morbidity worldwide. Resistant lineages were previously confined to hospitals but are now also causing infections among healthy individuals in the community. It is therefore imperative to explore therapeutic avenues that are less prone to raise drug resistance compared with today’s antibiotics. An opportunity to achieve this ambitious goal could be provided by targeted antimicrobial photodynamic therapy (aPDT), which relies on the combination of a bacteria-specific targeting agent and light-induced generation of ROS by an appropriate photosensitizer. Here, we conjugated the near-infrared photosensitizer IRDye700DX to a fully human mAb, specific for the invariantly expressed staphylococcal antigen immunodominant staphylococcal antigen A (IsaA). The resulting immunoconjugate 1D9-700DX was characterized biochemically and in preclinical infection models. As demonstrated in vitro, in vivo, and in a human postmortem orthopedic implant infection model, targeted aPDT with 1D9-700DX is highly effective. Importantly, combined with the nontoxic aPDT-enhancing agent potassium iodide, 1D9-700DX overcomes the antioxidant properties of human plasma and fully eradicates high titers of MRSA. We show that the developed immunoconjugate 1D9-700DX targets MRSA and kills it upon illumination with red light, without causing collateral damage to human cells.Infections caused by multidrug-resistant   An immunoconjugate for targeted photodynamic therapy of Staphylococcus aureus infections kills MRSA with high efficacy upon illumination with red light. Staphylococcus aureus is a Gram-positive bacterium, part of the normal human microbiota, but also a leading cause of bacteremia, endocarditis, osteomyelitis, and skin and soft tissue infections (S. aureus (MRSA) (fections . Such ins (MRSA) , 3 and ps (MRSA) . The stas (MRSA) . Therefo1O2), that kill the infecting organism. Moreover, bacterial resistance to photosensitizers has not yet been reported. An ideal photosensitizer should be selectively toxic to the pathogen with minimal damage to the host. In addition, the photosensitizer should have a large absorption coefficient in the visible spectrum, especially in the long wavelength (near-infrared) region, to allow effective penetration of light into the infected tissue  , an emerd tissue . Recentld tissue –12. HoweS. aureus isolates tested so far , the 1D9-700DX immunoconjugate can fully overcome potent antioxidant defenses of the human body, allowing the complete eradication of high titers of MRSA.Building on the observation that 1D9 can be applied for targeted infection imaging and image-guided surgery, we envisaged that this mAb could be applicable as a targeting agent for aPDT to treat spectrum . Furtherspectrum –26. The N-hydroxysuccinimide (NHS) ester, to obtain 1D9-700DX, was achieved through reaction of the NHS ester with the primary amines of lysine residues in the mAb. Judged by the absorbance values at 280 nm (protein) and 690 nm (IRDye700DX), this resulted in approximately 1–3 conjugated dye molecules per mAb molecule. MALDI-TOF mass spectrometry was used to determine the exact mass of the mAb 1D9 — 148,503 m/z — and to visualize the shift toward a higher m/z of the 1D9-700DX immunoconjugate — 152,110 m/z — corresponding to approximately 3 dye molecules per mAb (https://doi.org/10.1172/jci.insight.139512DS1). Furthermore, the covalent association between the mAb and IRDye700DX was evidenced by LDS-PAGE of 1D9-700DX under reducing conditions, where the separated heavy (~50 kDa) and light (~25 kDa) chains were both shown to fluoresce  , an established 1O2 scavenger  . Of notecavenger  29). 1D. 1D1O2. aureus. The mAb 1D9 binds IsaA with high affinity, but like any human IgG1, it also binds to the 2 immunoglobulin-binding proteins SpA and Sbi of S. aureus (S. aureus strains (SH1000 and Newman), and the respective isogenic deletion mutants lacking IsaA (MS001) or SpA and Sbi  were incubated with 1D9 that had been labeled with Alexa Fluor 555 (1D9-Alexa555). The 1D9-Alexa555 immunoconjugate readily detected all the strains, but the detectable binding was notably reduced in the IsaA-deficient strain  (–2 (P+L+ DAB+). These experiments, therefore, highlight the ability of 1D9-700DX to produce 1O2 at the bacterial cell surface, underlining its applicability for targeted aPDT.To visualize the localization and photoactivity of 1D9-700DX upon binding to py (TEM) , 33. In aureus by photoactivated 1D9-700DX. To study the in vitro antimicrobial activity of 1D9-700DX, we first investigated the survival of WT or IsaA-deficient S. aureus incubated with or without the immunoconjugate in the absence or presence of red light  Xen36 strain and the MRSA strain AH4807 with different concentrations of the immunoconjugate in the presence or absence of red light. The advantage of using these strains is that they have been genetically engineered to become bioluminescent through the expression of modified lux genes derived from Photorhabdus luminescens  and other peroxides in biological samples by luminescence. As shown in 2O2 in the irradiated sample groups compared with the ones that were kept in the dark. Of note, exposure to red light alone at 30 J.cm–2 is not toxic for the bacteria, as presented in 1O2, are also produced.Lastly, the production of ROS after aPDT with 1D9-700DX against S. aureus NCTC8325-4 were grown on coverslips and subjected to aPDT. Subsequently, LIVE/DEAD staining was performed with the dyes Syto9 and propidium iodide to, respectively, visualize live (green) and dead (red) bacteria by confocal laser scanning microscopy. Of note, some parts of the biofilm contain thicker layers, which translates into a brighter fluorescent signal and different biofilm phenotypes. Importantly, Z-stacks  or hospital-acquired (HA) MRSA isolates of the USA300 lineage . The survival rate of the larvae was assessed 72 hours after irradiation. The highest dose for which no larval death, melanization , or other indicators of toxic effects  were observed was reached after 45 seconds of irradiation at 100 mW.cm–2 (4.5 J.cm–2)  . Additioe larvae .–1 can be applied. Indeed, aPDT with 1D9-700DX was able to rescue CA- or HA-MRSA–infected larvae, with the best survival rates at 72 hours after irradiation being observed at a dose of 40 mg.kg–1 (–1 of 1D9-700DX and irradiation at 4.5 J.cm–2) to carefully compare larval survival upon infection with midexponential or early-stationary phase CA- or HA-MRSA inocula, or with inocula of the engineered CA-MRSA strain AH4807 that had been used for the above-described in vitro studies. Indeed, aPDT with 1D9-700DX significantly prolonged larval survival at 24, 48, and 72 hours after irradiation for all 3 MRSA strains grown to midexponential and early-stationary phase  in the larval hemolymph were quantified at 24, 48 and 72 hours after irradiation, using surviving larvae that had, respectively, been infected with the 3 different MRSA strains. As shown in Since a significantly increased survival of G. mellonella larvae, we wanted to evaluate possible cyto- or phototoxic effects of this immunoconjugate toward human cells. To this end, HeLa cells were incubated with 1D9-700DX for 30 minutes; thereafter, the cells were exposed to red light at 30 J.cm–2 either directly or after removal of unbound immunoconjugate by washing with PBS. To assess possible cytotoxic effects of 1D9-700DX, control experiments were done where HeLa cells were mock treated with PBS or with 1% SDS and kept in the dark. Cell viability was determined 24 hours after treatment using the colorimetric assay based on the reduction of the yellow MTT by mitochondrial dehydrogenase activity to a purple formazan precipitate. Hence, the reduction of MTT is directly correlated with the metabolic activity of living cells (Since no toxicity of (photoactivated) 1D9-700DX was observed in ng cells . No signng cells . In partng cells , which cAs a nontargeted control, the above-mentioned IRDye700DX carboxylate was incubated with the HeLa cells at the same concentration of 3.3 μM that was used for 1D9-700DX. This resulted in ~95% of cell mortality, comparable to 1% SDS control, even after washing with PBS . This hi2DCFDA), immediately after red light exposure (30 J.cm–2). Indeed, no significant ROS production was detectable, neither in the 1D9-700DX– and red light–treated PBS-washed HeLa cells, the unwashed HeLa cells, or the respective negative controls . Importantly, placement of the nitrocellulose membrane under the skin and subcutis did not negatively influence the efficiency of the treatment (3.19 log10 [CFU/mL] after aPDT). In contrast, no decrease in the CFU counts was observed for S. epidermidis (6.26 log10 [CFU/mL]), as expected, due to its lack of 1D9-binding sites. Together, these observations provide proof-of-principle that 1D9-700DX is applicable for use in the treatment of subdermal S. aureus infections in humans.The decision to use IRDye700DX as a photosensitizer for the present studies was based on the fact that it absorbs light in the near-infrared range, which has higher penetration in human tissue (~1 cm) than white light . To tested above . In paraS. aureus infections. Therefore, the antioxidant effect of human plasma was investigated ex vivo by treating the CA-MRSA strain AH4807 with 3.3 μM of 1D9-700DX and red light at 30 J.cm–2 upon suspension in plasma, a dose that kills 50% of 2 × 107 CFU/mL of the bacteria in PBS. Consistent with its presumed antioxidant activity, the plasma fully blocked the aPDT effect of 1D9-700DX  with high specificity for S. aureus  at 72 hours after treatment. The latter must be attributed to the larval innate immune defenses. Together, these findings show that, at least in the larvae of G. mellonella, aPDT with 1D9-700DX reduces the bacterial burden to such an extent that the host’s immune responses can overcome infections caused by multidrug resistant S. aureus. Of note, the G. mellonella infection model adheres to the principles of replacement, reduction, and refinement (known as 3Rs) and can potentially reduce the number of vertebrates used for experimental infection studies.ndidates . In the To enable the future clinical application of 1D9-700DX aPDT, possible photo- and cytotoxic effects must be verified. The immunoconjugate displayed no cytotoxicity against HeLa cells in the absence of activation with red light. Nonetheless, when 1D9-700DX was photoactivated, it did affect the viability of HeLa cells if the unbound immunoconjugate was not removed, since the production of ROS in the proximity of tumor cells causes their killing. Notably, however, the HeLa cell viability was not affected when unbound 1D9-700DX was washed away with PBS, which mimics the in vivo situation where the blood circulation will “wash” away any unbound immunoconjugate from a site of infection, with the exception of the causative bacterial agent that is targeted by the immunoconjugate. Importantly, the nontargeted IRDye700DX carboxylate strongly affected HeLa cell viability, despite a washing step with PBS. This argues in favor of using targeted agents for aPDT of bacterial infections, as this will minimize adverse effects. In addition, our present findings show that 1D9-700DX is not easily taken up by mammalian cells and, therefore, does not cause intracellular ROS production. Instead, this immunoconjugate is highly specific for IsaA-expressing bacteria.S. aureus. Of note, the postmortem set-up lacks blood circulation, and the overall conditions for aPDT are therefore different from the actual clinical setting. Furthermore, it is important to bear in mind that the maximum penetration depth for red light is about 1 cm  and Newman ΔspA, Δsbi  was cross-linked to the human mAb 1D9 via activated NHS ester chemistry. Cells were incubated with 3 μg.mL–1 of the 1D9-Alexa555 immunoconjugate for 30 minutes in PBS. To remove unbound antibody, the cells were washed with PBS and, thereafter, fixed in 4% paraformaldehyde in PBS for 10 minutes. Last, the bacterial DNA was stained with DAPI (Roche) followed by 1 PBS wash. Cells were spotted on a glass slide for microscopy. Image acquisition was performed with a Leica confocal laser scanning microscope . The recorded images were processed using ImageJ software (NIH). Intensity surface plots were created using FIRE LUT and the surface plot tool in ImageJ.pA, Δsbi , were grS. aureus strain Newman was cultured overnight in TSB at 37°C. A fresh subculture was grown to exponential phase (OD = ~0.7) and then harvested and washed with PBS by centrifuging for 2 minutes at 16,000g at room temperature. The bacterial pellet was resuspended with 1D9-700DX (1.3 μM) and incubated for 30 minutes at room temperature (RT). After incubation with the drug, 1 mg.mL–1 of DAB in sodium cacodylate buffer, pH 7.4, was added, and the bacterial suspension was irradiated with red light at a radiant exposure of 60 J.cm–2 . A control sample consisting of bacteria exposed to light and DAB only (P–L+ DAB+) was included. The sample preparation for TEM was performed as described previously (The WT eviously . BrieflyS. aureus strains (Xen36 and AH4807) were used in this study together with the laboratory strains SH1000 and MS001  and then harvested and washed with PBS by centrifugation for 2 minutes at 16,000g at room temperature. The bacteria were 10-fold diluted, and 50 μL aliquots (~2 × 107 CFU/mL) were incubated with different concentrations of 1D9-700DX (0.7–9.8 μM) or PBS in a 96-well plate at RT for 30 minutes in the dark, and then kept in the dark or exposed to 30 J.cm–2 of red light. For the time-response study, bacteria were incubated with 4.9 μM of 1D9-700DX and subjected to red light at a radiant exposure that ranged between 0 and 30 J.cm–2. After treatment, bacteria were serially diluted in PBS, plated on blood-agar (BA) plates, and then incubated aerobically for 16 hours at 37°C for CFU counting. Visualization of bioluminescence and fluorescence with the IVIS Lumina II (PerkinElmer) was additionally performed with a higher bacterial burden (~1 × 108 CFU/mL) to allow proper visualization.Two bioluminescent nd MS001 . The MSS plasmid . The MRSS. aureus Xen36 and AH4807, grown as described above, were incubated with 6 μM of 1D9-700DX and subjected to red light at a radiant exposure of 30 J.cm–2. H2O2 was detected with 10 μM of an AquaSpark Peroxide Probe (Biosynth Carbosynth), which is a dioxetane-based substrate for luminescence detection of H2O2 and other peroxides in biological samples. Visualization of bioluminescence was performed with the IVIS Lumina II, and the total flux (photons/second) was determined by region of interest quantification with the Living Image 4.5.5 software (PerkinElmer).S. aureus NCTC 8325-4 WT overnight culture was diluted 1:50 in TSB supplemented with 5% glucose and 4% sodium chloride, and 500 μL aliquots were used to inoculate 24-well plates containing 13 mm chemically resistant borosilicate glass coverslips. Upon 24-hour incubation at 37°C, planktonic bacteria on top of the coverslips were washed away with PBS. The biofilms were then incubated with 1D9-700DX (15.5 μM) for 30 minutes at RT. After washing with PBS to remove the unbound immunoconjugate, biofilms were treated with red light at a radiant exposure of 30 J.cm–2 and stained with the LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Fisher Scientific). Briefly, this kit contains 2 dyes: the Syto9 dye penetrates both viable and nonviable bacteria, while the propidium iodide penetrates bacteria with damaged membranes and quenches the fluorescence of Syto9. LIVE/DEAD-stained biofilms were examined using a Leica confocal laser scanning microscope . The recorded images were processed using ImageJ software.For biofilm formation, a G. mellonella larvae were performed as detailed in the Supplemental Materials. Ninety minutes after infection, 10 μL of 40 mg.kg–1 of 1D9-700DX or PBS were administered to the larvae, and after 30 minutes of incubation, the aPDT group (P+L+) was treated with red light at a radiant exposure of 4.5 J.cm–2. Viability was scored according to the G. mellonella Health Index Scoring System  plates for CFU counting. All experiments were repeated at least twice, and representative experiments are shown.The number of bacterial cells in the eviously . Briefly2. A total of 0.25% Trypsin-EDTA (Thermo Fisher Scientific) was used to detach adherent cells for subculturing. Cells were seeded into 96-well cell culture plates at a density of 3 × 104 cells/well. On the following day, cells were treated with 1D9-700DX or IRDye700DX carboxylate in the dark, for 30 minutes. One group was washed with PBS, and fresh DMEM medium was added, while the other group remained with the drug in PBS. Irradiation with 30 J.cm–2 of red light was subsequently performed. Cell metabolic activity at 24 hours after aPDT was determined with the MTT assay, which measures the ability of HeLa cells to reduce MTT (MilliporeSigma) to colored formazan crystals. The formation of formazan was quantified with a microplate spectrophotometer  by measuring the absorbance at 570 nm, using 620 nm as the background wavelength. The percentage of absorbance for each treated sample was normalized to each untreated control.The human cervical cancer HeLa cell line (ATCC) was cultured in DMEM-GlutaMAX medium (Thermo Fisher Scientific) supplemented with 10% of FBS, at 37°C and 5% CO2DCFDA (Invitrogen), a chemically reduced form of fluorescein used as an indicator for intracellular ROS generation for 1 hour at 37°C in the dark. After incubation with H2DCFDA, cells were washed with PBS and mechanically scraped and resuspended in 1% (m/v) SDS solution in PBS. Subsequently, the fluorescence was measured using a Synergy HT microtiter plate reader with excitation and emission filters of 485/20 nm and 540/25 nm. Protein concentrations were determined using the Pierce BCA Protein Assay Kit.After aPDT, HeLa cells were incubated with 5 μM of HS. aureus clinical isolate or S. epidermidis ATCC 38984 were treated with 3.3 μM of 1D9-700DX and spotted on nitrocellulose membranes. The membranes were subsequently implanted on the lateral side of the proximal tibia of a human postmortem leg, which was subsequently irradiated with 30 J.cm–2 of red light. Nonimplanted control membranes were either irradiated or kept in the dark. Subsequently, the membranes were spotted onto BA plates for CFU counting.Dilution series of overnight cultures of an 600 = 0.5), and they were then harvested and washed with PBS by centrifugation for 2 minutes at 16,000g at room temperature. The bacteria were 10-fold diluted and 50 μL aliquots (~2 × 107 CFU/mL) were incubated with 50 μL of a mixture of 3.3 μM of 1D9-700DX or PBS and/or 50 mM of KI (MilliporeSigma) or PBS and/or plasma in a 96-well plate at RT for 30 minutes in the dark. The mixtures with bacteria were then either kept in the dark or exposed to 30 J.cm–2 of red light. After light exposure, the bacteria were serially diluted in PBS, plated on BA plates, and then incubated aerobically for 16 hours at 37°C for CFU counting. The generation of iodine after irradiation was monitored by reading the absorbance of 96-well plates at 340 nm with a microplate spectrophotometer (Synergy HT).CA-MRSA AH4807 was grown to exponential phase  were assessed using 2-way ANOVA tests and subsequently by the Šidák multiple-comparison test. The statistical significance of differences in the killing of G. mellonella larvae by S. aureus was assessed by Gehan-Breslow-Wilcoxon test. P < 0.05 was considered significant.The results are presented as mean ± SEM. All statistical analyses were performed with GraphPad Prism 8.0.1. Statistical significance among 2 unpaired groups was performed with the Mann-Whitney All postmortem experiments were conducted according to institutional guidelines with prior approval from the scientific review committee of the Skills Center of the University Medical Center Groningen, the Netherlands, and according to the applicable law . All individuals involved in the human postmortem studies have provided informed written consent for the use of their bodies for scientific research and teaching. Blood donations from healthy volunteers were collected with approval of the medical ethics committee of the UMCG  and after written informed consent, in accordance with the Declaration of Helsinki guidelines and local regulations.MB and JMVD conceived and designed the experiments. MB, AAS, SS, EJMR, MLA, MH, and WS performed experiments and analyzed the data. FRP, ARH, and KPF contributed strains and reagents. GB, GMVD, MVO, and JMVD supervised the project. MB and JMVD wrote the manuscript. All authors have read and approved the manuscript.


This seemingly anomalous result is what might be expected if GPX1 is a substrate for the active protease, leading to its fragmentation. We show that the GPX1 active site sequence is substantially similar to a known Mpro cleavage site, and is identified as a potential cysteine protease site by the Procleave algorithm. Proteolytic knockdown of GPX1 is highly consistent with previously documented effects of recombinant SARS-CoV Mpro in transfected cells, including increased reactive oxygen species and NF-κB activation. Because NF-κB in turn activates many pro-inflammatory cytokines, this mechanism could contribute to increased inflammation and cytokine storms observed in COVID-19. Using web-based protease cleavage site prediction tools, we show that Mpro may be targeting not only GPX1, but several other selenoproteins including SELENOF and thioredoxin reductase 1, as well as glutamate-cysteine ligase, the rate-limiting enzyme for glutathione synthesis. This hypothesized proteolytic knockdown of components of both the thioredoxin and glutaredoxin systems is consistent with a viral strategy to inhibit DNA synthesis, to increase the pool of ribonucleotides for RNA synthesis, thereby enhancing virion production. The resulting “collateral damage” of increased oxidative stress and inflammation would be exacerbated by dietary deficiencies of selenium and glutathione precursors.Glutathione peroxidases (GPX), a family of antioxidant selenoenzymes, functionally link selenium and glutathione, which both show correlations with clinical outcomes in COVID-19. Thus, it is highly significant that cytosolic GPX1 has been shown to interact with an inactive C145A mutant of M Several independent studies have now established a significant association between the outcome of COVID-19 and previously documented regional selenium (Se) status in Chinese cities . These oMost  of the biological roles of Se, both as selenocysteine in selenoproteins, and as redox-active Se-containing metabolites, involve interactions with cysteine thiols and disulfides in proteins and peptides, and their various oxidized forms. Like Se, the essential antioxidant and free radical scavenger glutathione (GSH), a tripeptide thiol, has also proven to be an important factor in various viral infections, particularly in HIV/AIDS, as reviewed in section 2.1.2. of Taylor , and mosboth host Se and GSH status provide important context to a related observation emerging from a proteomics-based study of possible cellular targets of SARS-CoV-2 (SCoV2) proteins. Using affinity-purification mass spectrometry, Gordon et al. identified high-confidence protein-protein interactions between 26 of the SCoV2 proteins and human proteins . The study also included a catalytically inactive C145A Mpro mutant (lacking the active site cysteine), which was also used as a bait protein, in order to discriminate false positives that might bind non-specifically to the Mpro active site cysteine via disulfide bond formation. One of the interactions they identified involved the cytosolic form of the selenoprotein glutathione peroxidase, GPX1, which bound strongly to the inactive C145A Mpro mutant. However, this interaction with GPX1 was not observed with the wild type Mpro. This is precisely what one might expect to see if GPX1 is a protease substrate, because its cleavage would produce two fragments that would necessarily have reduced affinity to the enzyme relative to GPX1.Correlations between COVID-19 clinical outcomes and only to the inactive Mpro C145A mutant, and concluded it was a likely Mpro substrate, because they were able to identify a putative Mpro cleavage site in the TRMT1 protein sequence (PRLQ/ANFT), where the slash (/) represents the cleavage site. Thus, the only piece of evidence lacking to draw a similar conclusion for GPX1 is a candidate Mprocleavage site, which most algorithms will fail to find, e.g., if they are highly stringent about requiring a Q in the P1 position . The downstream sequence past P1′ is highly variable.As shown in due span . This mepro consensus core target sequence combination LQ/G in 2 of 3 positions. Furthermore, as shown in pro cleavage site at the nsp13/14 junction, NVATLQ/A, is remarkably similar to that of the active site sequence of GPx1, NVASLU/G, where the selenocysteine (U) lines up with a glutamine (Q) in the viral sequence. These two amino acids (U and Q) are not highly similar, but are both midrange in size, and polar in nature, because the selenol residue is predominantly ionized at physiological pH. The other two “mismatches” in the important positions P3 and P1′, i.e., S vs. T and G vs. A, both differ only slightly, by the size of a methyl group, and in any case, having a glycine (G) in the P1′ position is a permitted residue (pro cleavage site at the nsp5/6 junction (indicated by * in procleave.erc.monash.edu.au)  .pro.For several reaction intermediates in the GPX1 mechanism, Se is attached to either oxygen, or to nitrogen, as a selenenylamide. The latter is quite stable, and may accumulate during GSH depletion, because GSH is needed to regenerate the selenolate . Selenenpro, it is almost certainly because the virus has evolved to target the host for some reason, rather than the converse. The most parsimonious hypothesis is simply that this is a case of a viral protease targeting a cellular gene for cleavage, for which there are abundant precedents, including the proposed Mpro targeting of TRMT1 and HDAC2  NetCorona (cbs.dtu.dk/services/NetCorona) , (2) PROSPER, the Protease Specificity Prediction Server (prosper.erc.monash.edu.au)  , the onl.edu.au) , which w.edu.au) , and (3)pro sites identified computationally . The most remarkable instance is in selenoprotein F (SELENOF), an ER protein involved in glycoprotein folding quality control. Over the 8 residues spanning P4 to P4′, it has 7 of 8 identical to the Mpro site at the nsp12/nsp13 junction, with the only mismatch at the highly variable P3′ position , making the enzyme incapable of regenerating reduced thioredoxin.Equally important is the hit on the selenoprotein thioredoxin reductase 1 (TXNRD1), which also was highly scored by both NetCorona and PROSPER . Note thA predicted site in selenoprotein P SELENOP,  is at popro itself, which have the highest catalytic efficiency for cleavage of any of the viral Mpro targets (100 and 41%). This suggests that Mpro might be particularly efficient at disrupting GSH synthesis, as compared to action at some of the other target sites predicted here, thereby contributing to the clinical findings in COVID-19 , the rate limiting enzyme for GSH synthesis. The first of these, GCLC-a , would cCOVID-19 .Finally , we inclAlong with GSH reductase, GSH and glutaredoxin comprise the glutaredoxin system, one of two cellular redox systems involved in maintaining reduced thiol states, protein folding and repair, and providing electrons to ribonucleotide reductase (RNR). The other redox system with similar roles is the thioredoxin system, comprised of thioredoxin and thioredoxin reductase .only be synthesized via the reduction of ribonucleotides by RNR, a process which is unsustainable without the participation of one or both of the thioredoxin and glutaredoxin systems. As a consequence of this basic fact of biochemistry, one should expect that RNA viruses might exploit various mechanisms to interfere with components of the thioredoxin and glutaredoxin systems, in order to minimize the diversion of ribonucleotides into DNA synthesis. This is simply the inverse of a strategy used by some large DNA viruses to maximize DNA production, by encoding their own thioredoxin-like proteins, glutaredoxins and even entire RNR genes (both of the essential redox systems required to sustain DNA synthesis, and thereby conserve ribonucleotides to enhance RNA synthesis for virus production.Because DNA is an “add-on” to RNA biosynthesis, deoxyribonucleotides can NR genes . ConsistVitamin D3-mediated activation of both TXNRD1 and GSH biosynthesis could substantially counteract the proteolytic knockdown of TXNRD1 and GCLC predicted by our hypothesis, which, if validated, would thus represent an important mechanism contributing to a role for vitamin D3 in moderating the severity of COVID-19. However, the therapeutic potential of D3 in COVID-19 is the subject of ongoing debate (the effective upregulation of TXNRD1 by vitamin D3 requires the presence of an adequate level of Se (In light of accruing evidence that vitamin D3 status is inversely correlated with severity of COVID-19 –26, it ig debate . Perhapsel of Se . So the pro substrate. Of the other sites we propose, some are more likely than others, but those in TXNRD1 and SELENOF are particularly convincing, and all of the predicted sites map to surface accessible regions of the targeted proteins (these consequences of virus-mediated proteolysis would be taking place in everyone who is actively infected, regardless of their Se status, and would thus be consistent with results suggesting that Se intakes up to several times the minimal dietary requirement are associated with an increase in cure rate from COVID-19 (Altogether, given the protein interaction and functional data , 16, it proteins . If valiproteins , 18. Becproteins , this coCOVID-19  and the These results are not unprecedented—our lab has shown that some RNA viruses target host mRNAs encoding isoforms of thioredoxin reductase via RNA:RNA antisense interactions, which, like proteolysis, would likely also result in host selenoprotein knockdown, but by a different mechanism , 33. If Understanding why RNA viruses may have developed such strategies presents an interesting challenge for future research.All datasets generated for this study are included in the article/This study was conceived and designed by ET, who wrote the initial draft. ET and WR worked on the data analysis and revisions of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Occipitocervical fusion (OCF) is an effective treatment for instability of occipitocervical junction (OCJ). The occipital condyle screw serves as a novel surgical technique for occipitocervical fixation. However, the intraoperative procedures for the occipital condyle screw technique have relied on surgeons’ experience, so the pool of surgeons who are able to perform this surgery safely is limited. The present study aims to evaluate the feasibility and safety of the occipital condyle screw technique using human cadavers and to provide image anatomy for clinical application basis.The scientific study comprised 10 fresh‐frozen cadaveric specimens from the anatomy department of Qingdao University. Placement of the occipital condyle screws (3.5 mm diameter and 20.0 mm length) was performed in the 10 fresh‐frozen cadaveric specimens with intact occipitocervical junctions, respectively. Occipitocervical CT was performed for all specimens and the DICOM data was obtained. Occipitocervical CT three‐dimensional (3D) reconstruction was performed for the cadavers. Morphometric analysis was performed on the bilateral occipitocervical junction of 10 cadaveric specimens based on the 3D reconstruction CT images. Detailed morphometric measurements of the 20 occipital condyles screws were conducted including the average length of the screw trajectory, inside and upper tilting angles of screws, distance to the hypoglossal canal, and to the medial wall of occipital condyle.t = 1.306, P > 0.05). The upper tilting angle of bilateral screws was, respectively, 11.24° ± 0.74° (left) and 11.11° ± 0.64° (right) . The inside tilting angle of bilateral screws was, respectively, 31.00° ± 1.32° (left) and 30.85° ± 1.27° (right) . The screw's distance to the bilateral hypoglossal canal was, respectively, 4.84 ± 0.54 mm (left) and 4.70 ± 0.54 mm (right) . The screw's distance to the medial wall of the bilateral occipital condyle was, respectively, 5.13 ± 0.77 mm (left) and 5.04 ± 0.71 mm (right) .Placement of the occipital condyle screws into the 20 occipital condyles of the 10 cadaveric specimens was performed successfully and the trajectory of implantation was satisfactory according to 3D CT reconstruction images, respectively. There was no obvious injury to the spinal cord, nerve root, and vertebral artery. The length of the bilateral screw trajectory was, respectively, 20.96 ± 0.91 mm (left) and 20.59 ± 0.77 mm (right) (The occipital condyle screw technique can serve as a feasible and safe treatment for instability of the occipitocervical junction with meticulous preoperative planning of the screw entry point and direction based on individual differences. Morphometric trajectory analysis is also an effective way to evaluate the surgical procedure. Posterior internal fixation with instrumentation in OCF is still the treatment of choice for occipital cervical instabilityVarious factors can result in occipitocervical instability, leading to local pain, spinal nerve compression symptoms, and even death, in a multitude of conditions including infection, trauma, tumor, and other degenerative conditionsDespite improved surgical techniques, the halo immobilization was still essential because of the lack of internal fixation strength in the 1970s. Furthermore, the prolonged external immobilization could vastly affect the short‐term prognosis as well as the quality of life of postoperative patientsMany studies have demonstrated that OC screws are a feasible option for treatment of occipitocervical instability, with no apparent neurovascular complicationsTherefore, we analyzed the anatomy of the OC in detail. The anatomic relationships between the OC screw and the vertebral artery, the medulla oblongata, and the foramen hypoglossal were also studied. CT‐based morphometric measurements of the craniovertebral junction for OC screw placement were performed, including screw insertion length, the upper tilting angle and inside tilting angles, and other parameters relating to the screws.1–C2 complex; (ii) allow surgeons to evaluate the safety and feasibility of the occipital condyle screw; and (iii) provide spine surgeons with a relevant theoretical basis for clinical application.The aim of the present study was to: (i) enable surgeons to further study the complex anatomy of the occipital‐CTen fresh‐frozen cadaveric specimens with intact OCJ were obtained from the department of anatomy at Qingdao University. Screws  and other surgical instruments were provided by Stryker (USA). We used a 64‐slice spiral CT machine manufactured by Siemens. This study was conducted in accordance with the Declaration of Helsinki and with approval from the Ethics Committee of Qingdao University.Ten cadaver specimens were enrolled in the study. They were all obtained from the department of anatomy, Qingdao University. The cadaver specimens all had intact OCJ. All the cadaver specimens had undergone no previous cervical surgery. Ten cadaver specimens had no obvious deformity or vertebrae fractures at OCJ.All surgical procedures were performed by three experienced surgeons. All specimens underwent thin‐slice CT scanning, respectively. According to the screw entry point and screw trajectory, based on the measurements of CT images, screws were inserted into the OC of specimens.3–C4 level. The skin and subcutaneous fascia were dissected in turn. Muscles were laterally retracted to expose the posterior arch of the atlas. The horizontal segment of the vertebral artery was identified by dissecting laterally in a subperiosteal fashion. Following the exposure of the occipital bone, C1 and C2, the posterior atlantooccipital membrane was then dissected laterally from the foramen magnum to the medial margin of the OC. The soft tissues around the screw entry points were dissected and removed using the operating scalpel and nerve dissector to identify the bony landmark. The placement of the screws was performed at the bilateral entry points according to the measurements.All specimens were placed on the operation table, with the head in a cephalostat in the prone position. A nuchal midline incision was made and it was extended from the external occipital protuberance to the CA 64‐slice spiral CT scan with a slice thickness of 1 mm was performed. The 3D reconstruction images of the sagittal and axial OCJ views along the long axis of the OC were made by using a GE 3D CT image processing workstation , the upper tilting angle (UTA), the inside tilting angle (ITA), the distance to hypoglossal canal (DHC), and the distance to the medial wall of the OC (DMWOC) of the OC screws. We also examined the relationships between the screws and the adjacent structures using the 3D CT images Fig. .The LST was the length of the screw trajectory within the bone. It can be measured based on CT images to help surgeons understand appropriate depth of screw insertion during an operation Fig. .The UTA was the head tilting angle of the screw insertion in the sagittal plane. The sagittal CT images can show the screw insertion angle to help surgeons identify the appropriate head titling angle of the screw insertion to avoid damage to vessels and nerve injury.The ITA is the inside‐and‐outside tilting angle of the screw insertion in the horizontal plane. To avoid damage to vessels and nerve injury, the ITA can be measured from horizontal CT images to help surgeons identify the inside‐and‐outside tilting angle of the screw insertion.The DHC was the inside distance between the occipital condyle screw and the hypoglossal canal in the sagittal plane. The hypoglossal canal is the passageway for the hypoglossal nerves. The DHC can be measured in the sagittal CT images.The DMWOC was the inside distance between the occipital condyle screw and the medial wall of the occipital condyle in the horizontal plane. The DMWOC can be measured in the horizontal CT images.t‐tests to analyze the LST, UTA, ITA, DHC, and DMWOC. The data were presented as mean ± SD. P < 0.05 was regarded as statistically significant.The data was analyzed using the software package SPSS 25.0 . Statistical analysis was performed using paired samples t = 1.306, P > 0.05).The measurements of LST on both sides of the 10 cadavers were obtained based on sagittal CT images. The LST was 20.96 ± 0.91 mm on the left side and 20.59 ± 0.77 mm on right side. There was no statistically significant difference in the LST on either side .The measurements of UTA on both sides of the 10 cadavers were obtained. The UTA was 11.24° ± 0.74° on the left side and 11.11° ± 0.64° on the right side. No statistically significant differences in UTA were shown for either side .The measurements of ITA on both sides of the 10 cadavers were obtained. The ITA was 31.00° ± 1.32° on the left side and 30.85° ± 1.27° on the right side. There was no statistically significant difference in the ITA on either side .The measurements of DHC on both sides of the 10 cadavers were obtained. The DHC was 4.84 ± 0.54 mm on the left side and 4.70 ± 0.54 mm on the right side. No statistically significant differences in DHC were shown for either side   Fig. , Table 1Numerous factors can lead to instability at the OCJ, including trauma, inflammation, neoplasm, and congenital and iatrogenic causes. OCF is an effective treatment for OCJ instability. However, meticulous preoperative assessment is needed because of the unique anatomical location.et al.Several OCF methods have been applied since Foerster first described the approach in 1927et al.et al.With the development of lateral mass screw techniques, plate systems began to be widely used in occipitocervical fusion in the early of 1990s. A long‐term follow‐up study carried out by Sasso The most common surgery requires integrity of the posterior part of the occipital bone, and the thickness and strength can impact bone fusion. The limitations of the occipital bone sclerosis associated with lateral occipital fixation pose the biggest problem for the occipitocervical fusionet al.et al., to avoid vertebral arterial injury, fully revealing the vertebral arteries should be done extremely carefully after exposing the atlantooccipital joint. Therefore, adequate skill is necessary to avoid serious postoperative complications. They made a standard posterior median incision from the external occipital protuberance to the spinous process of C3 or C4. To reveal the horizontal segments of the vertebral artery, dissection along the bilateral periosteum was performed after exposing the arcus posterior atlantis. Full exposure of the occipital bone,Uribe 1 and C2 was achieved and then the membrane of C1 was dissected from the foramen magnum to the occipital condyle using a small sized curet. The dissection was then performed outwards from the foramen condyloideum to the emissary vein along the bone surface to prevent injuring the vertebral artery. The entry point was located on the junction of the occipital condyle and the occipital bone. The angle of the screw trajectory from medial to lateral and the tilt angle to the head were 12°–22° and 5°, respectively. The length of the screw trajectory was 20–24 mm. The author found that the occurrence of complications can be reduced and screwing safety can be significantly improved by certain careful anatomical operations. Cadaveric studies, aiming to evaluate the feasibility of occipital condyle screws, can verify the safe placement for screw insertion on the basis of thorough knowledge of the OC junction anatomy.Cet al.et al.et al.et al.et al.Naderi In our study, the 20 OC were all successfully inserted with the screws, and no spinal cord, nerve root, vertebral artery or hypoglossal nerve was injured. We measured the LST, UTA, ITA, DHC, and DMWOC of the OC screws, and all the results were satisfactory. We used 24‐mm multi‐axis screws for insertion and the screwing entry points were 4–5 mm to the lateral side of the interior edge of the OC; namely, at the junction of the OC and the occipital bone. The DHC and the DMWOC, or the insertion points at the condylar process, were approximately 5 mm. The ideal OC screw channel should pass through the cross‐sectional center of the OC and be parallel to the long axis of the OC, but the screwing angle and an suitable screwing point cannot be achieved in actual operationsIn our study, all 20 cases had differences in the screw angles and entry points. However, the 3D reconstruction showed that the placed screws maintained adequate distances from the surrounding structures, so we believe that screws can be safely inserted with no damage caused to surrounding structures if the differences in the UTA and ITA are kept within 2°, and the screw entry points are kept 3–6 mm away from the lateral side of the posterior OC edge. Due to differences among races, the OC vary largely, but studies that anatomically measure the Chinese occipital condyles are limited.We studied the 3D anatomies of the OC screws in these cadaver specimens and the relationships between OC screws and surrounding tissue structures, and further confirmed that OC screwing with a 30° interior inclination angle and a 10° upper inclination angle is also applicable for Chinese people, and will not damage the peripheral nerve and vascular structures. The screws can maintain a safe distance from the surrounding important structures: 20–24‐mm length screws are safe for use in Chinese and Asian people. We believe that preoperative 3D CT reconstruction can better and more safely guide the screwing procedure based on measuring the OC anatomical morphologies in the reconstructed images, thus improving the screwing safety. As an important structure at the occipitocervical site, the atlantoaxial joint bears 40%–50% of the activity of the cervical spine; the rest is borne by the lower cervical spine. Therefore, appropriately applying the OC screwing technology can maximally retain the cervical activity and achieve better therapeutic effects.Of course, there are still issues with OC screws, including lack of uniform standards, and no specific screw can be selected for the OC. The bones of specimens were not the first option for biomechanical measurements. If OC screws were to be widely used in clinical practice, further biomechanical measurements should be performed to improve the technology. In addition, minimally invasive and precision insertion of OC screws could be performed using a navigation system and robots in the future.Due to unfamiliarity with the anatomical structures around the OC and the serious potential complications caused during the screwing process, this technology is limited in clinical practice. However, the anatomical measurements of the OC have confirmed that it could be a more stable and reliable internal fixation method than traditional OCF methods. Therefore, the OC screw technique could improve on or supplement traditional OCF methods.


Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) compete with each other for nutrients and survival in natural environments, and have been extensively studied because of their intermicrobial interactions in the human microbiome. These are the principal microbes infecting immunocompromised patients and persons with cystic fibrosis, particularly the airways. These intermicrobial studies have largely been conducted in liquid medium or on agar, and thus focus on soluble or diffusible microbial products. Several key inhibitory molecules were defined in such studies. Methods: in the present report, we examine several methodologies which can be conveniently used to study the interaction of microbial volatiles, including capture methods and kinetics. Results: Pa volatiles inhibit Af, and the inhibitory mechanism appears to be the incorporation of the inhibitory molecules into the substrate nourishing the Af, rather than directly onto Af structures. We define by mass spectroscopy some specific volatile Pa products that can inhibit Af. Some of these molecules are selected for interest by the study of gene deletion mutants, producing a few Pa strains that were impaired in inhibition. We presumed the volatiles of these latter strains could be excluded from the search for inhibitors. Conclusion: the Pa inhibition of Af via a gaseous phase could be critical components in their competition, particularly in airways, where more direct contact may not be extensive.Background:  Pseudomonas and Aspergillus are ubiquitous micro-organisms inhabiting soil, water, plants, and the human microbiota [P. aeruginosa (Pa) and A. fumigatus (Af) have the most devastating consequences of any bacterium and fungus, respectively [crobiota ,2. Clinicrobiota ,7,8,9,10ectively ,18,19,20ectively ,32,33,34ectively . For thiectively ,45,46,47ectively ,54,55,56All the chemicals were purchased from Sigma-Aldrich . All the commercial agars and RPMI-1640 medium were purchased from Beckton Dickinson . The Bacto agar used for growth studies was obtained from Carolina Biological Supply Co., Burlington, NC. The RPMI agar was prepared; briefly, 7.5 gm of Bacto agar in 100 mL of distilled water was autoclaved and mixed with 400 mL of prewarmed RPMI-1640 medium. The agar (20 mL) was poured into petri dishes. Fe-depleted agar was prepared by formulating potato dextrose agar (PDA) in the presence of ascorbate and ferrozine, as we previously described . For allA. flavus (CIMR 10-103). The Af strain CBS 144-89 was kindly donated by the investigators from the Institute Pasteur [In determining the activity of volatiles with Af as a target, we desired to have confirmation by demonstrations with four Aspergillus wild types: AF 293 (ATCC MYA-4609), 10AF (ATCC 90240), CBS 144-89, and  Pasteur . Clinica Pasteur .pvdD-, lasR-/rhlR-, pqsA-, coaB, and the PAO728 mutant were kindly provided by P. R. Secor, Departments of Microbiology and Medicine, University of Washington, Seattle, WA, USA [PA14 (genome published) , PAO1 A. The mut WA, USA .The use of all the micro-organisms in our laboratory was approved by the CIMR Biological Use Committee .9/mL) suspension was inoculated in one plate and spread on the surface of the plate using a sterile plastic loop. Three sterile paper discs  were put on the second plate, and 10 µL of Af suspension (5 × 104 conidia/mL)  was inoculated on each disc. The lids of the plates were discarded, and the bottom parts of the plates were sealed to each other with parafilm and tape , and the sealed plates were incubated for an additional 48 h. In a variation of this method, 4 cm agar plates were inverted, 10 mm filter disks were placed in the lids, and they were inoculated with 100 µL of volatile substances or controls. The agar plates were exposed at room temperature for 90 min. The lids were replaced, one new sterile filter disk was placed on each exposed agar, they were inoculated with 10 µL of Af suspension (5 × 104 conidia/mL), and the sealed plates were incubated at 37 °C for 48 h.This method was also used to capture Pa-derived volatiles in agar. An uninoculated agar plate was incubated with a plate inoculated with Pa, as described above, for 48 h. Following incubation, the Pa-carrying plates were removed, filter disks were placed on the exposed agar and inoculated with 10 µL of Af suspension (5 × 109/mL) was inoculated on one part and spread on the surface of the agar using a sterile plastic loop. One sterile paper disc was placed on the second part, and 10 µL of Af suspension (5 × 104 conidia/mL) was inoculated on the disc. Pa was not inoculated on the control plates  was inoculated on each disc. A 5 mL vial was attached to the lid of a jar using a drop of hot silicone glue. The test compounds were placed into the glass vials. With the top of the jar lid resting on a firm surface, the jars with the agar and the lid were screwed together, kept inverted, and sealed with tape. Additionally, the lids were sealed with parafilm  in RPMI or TSB was added to the wells of the outer two columns and rows of 96-well plates. Af suspension  in RPMI was placed into the inner wells of the plates. The plates were sealed and incubated at 37 °C for 40 h. After incubation, an XTT assay was performed to measure the Af metabolic activity as described previously [The 96-well plate method: 75 µL of Pa suspension (2.5 × 104 conidia/mL) on paper discs on TSA.Syringe method: After incubation, the air was aspirated from the double plates by piercing the parafilm wrap with a needle. Each air sample was injected into rubber-stopper sealed glass vials, which contained an Af inoculum (10 µL from 5 × 109/mL) was inoculated on two TSA agar plates, and the plates were combined as described. As controls, TSA plate combinations with no Pa inoculation and double plate setups with no TSA or Pa were used. The double plates were sealed and incubated at 37 °C for 48 h. After incubation, the air in the double plates was transferred into vials by using a one-way air pump. The pump was set up using common laboratory materials such as clamps, tubes, electrical tape, vinyl tubing, and syringes. Silicon glue was heated and applied externally to any possible leakage sites. In this model, the volatiles in the double plate were aspirated and transferred into the glass vial directly. More specifics about the operational details are given in the figure legends and are illustrated.Air pump method: An air pump model was designed that transferred volatiles from double plates to glass vials. The Pa suspension  were exposed to Pa in double plates, and the trapping materials then exposed to Af plates in a second double plate step, as will be described.9/mL) was inoculated on TSA agar and combined with a petri dish with 15 mL of sterile deionized water (test article). As double plate controls, uninoculated TSA combined with 15 mL of sterile deionized water or 15 mL of sterile deionized water with an empty plate were used. The double plates were sealed and incubated at 37 °C for 72 h. After incubation, the water was aspirated and subjected to mass spectrometry.Water samples exposed to Pa volatiles: Three double plates were used. The Pa suspension (40 µL from 109/mL) were inoculated onto the agars. The vials were closed with rubber stoppers and sealed with parafilm. As controls, vials were prepared with TSA without Pa inoculation and vials were prepared with no TSA and Pa. The vials were incubated at 37 °C for 48 h, and the volatiles from these vials were directly used for mass spectroscopy.Live cell samples: After the autoclave sterilization of TSA, 5 mL of agar was put in mass spectroscopy vials. Following solidification, the Pa suspensions  model 6890/5973 was used for the headspace gas chromatography (GC)-mass spectrometry (MS), as per the manufacturer’s instructions and as detailed elsewhere . In briet-test if the two groups were compared and a 1-way analysis of variance (ANOVA) combined with Tukey’s post-test for multiple comparisons. All the data are expressed as the mean ± the standard deviation. The data reported as percentages of the control value were compared with a Student’s t-test after the arcsin transformation of the proportions; these data are presented as percentages. Assays used 4–8 replicate wells for each group studied for statistical purposes, and the number of replicates in each experiment is specified in the figures. The representative experiments shown are the most complete of a series of experiments; the conclusions and statistics for the corroborating experiments are given.The results were analyzed with a Student’s We performed experiments to determine the abilities of the different methods to demonstrate the inhibitory effects of Pa volatiles on Af (10AF). In five experiments , we showed that the double plate method was more sensitive for detecting inhibition than the single plate method . The jarp < 0.001, comparison with another plate that omitted the Pa). Thus, interested investigators have four methods by which the phenomenon of Pa inhibiting Af with no direct contact between the microbes can be manipulated for further studies, which was the purpose of these initial studies.The 96-well plate method is a miniature version for testing the ability of Pa on agar to produce volatiles affecting Af on agar . It enables multiple wells of Pa and Af to be prepared, examined, and scored on the same plate. The inhibition of Af was clear , we found that it made no difference if the Pa plate was on the top and the Af (10 AF) plate was on the bottom, or vice versa. Thus, the inhibitory vapors diffuse freely up or down, as studied in the double plate environment.We tested in five experiments TSA, SDA, PDA, and RPMI agar in all possible combinations (the 16 possible combinations of these four agars) as the substrates for plating Pa (Pa10) or Af (10AF) in the double plate method. We found that the Af inhibition was negligible if the Pa was plated on RPMI agar, regardless of the four agars tested for the Af, possibly related to the apparently inferior Pa growth on RPMI agar. The Pa on PDA agar had similar results to those just described on RPMI, but to a lesser extent and less consistently. In contrast, any combinations of TSA or SDA for the Pa or Af did not affect the inhibition of Af by Pa, nor the use of RPMI agar or PDA for only the Af plate of the pair.In two experiments, we found that the Pa volatile production measured by Af inhibition was inferior when TSB was substituted for TSA, even though the Pa growth on both was robust and quantitatively similar. Removing the iron  from TSAIt was necessary to prove that the plastic plates with agar themselves emitted no Af-inhibitory volatiles . In fourTo assess whether inhibition could be related to only the micro-organisms studied in detail above, we compared  the Pa clinical strain Pa10 and the Pa laboratory strains PAO1, PA14, or PAK with the Af strain 10AF or the reference strain AF293, and found no differences in any combination of Pa-Af in the inhibition we described above.3 to 109 CFU/mL were plated on TSA, the inhibition of 10AF on TSA was dose-responsive from 103 to 106/mL, and there was no Af growth with >106/mL Pa. This was confirmed in a second experiment using TSB for Pa growth.When 40 μL of 10-fold dilutions of Pa10 inocula 10In the first of these experiments, a Pa plate was present only for the first 24 h, then was substituted by an uninoculated TSA plate, or the Pa plate was substituted for an uninoculated TSA plate at 24 h. Those two conditions were compared to a Pa plate present for a full 96 h (positive control), or only a 96 h exposure to a TSA plate that was not inoculated with Pa (negative control) . The posIn a second experiment with identical controls but incubation for 144 h, the effect of introducing the Pa plate only after 24 h had the identical total inhibition effect and this lasted until 144 h; when a Pa plate was introduced only after 48 h, there was limited Af growth for the next 24 h but then no further growth.Thus, in summary, the inhibition by Pa volatiles is proportional to the time of exposure; the inhibition is reversible (suggesting a fungistatic effect). Moreover, the inhibition can start even after the Af has started growing  in the absence of an inhibiting Pa plate.p < 0.001) Af growth >50% compared to air extracted from either of the controls.In this experiment, a variant of the double plate method was used. We co-incubated pairs of plates in each of these three combinations: Pa on TSA, TSA with TSA, or two empty plates (each of these three groups in quadruplicate). At 48 h and 37 °C, the air was aspirated from each set of combined plates by piercing the parafilm wrap with a needle connected to a 20 mL syringe, then each air sample was injected into rubber-stoppered sealed glass vials which contained an Af inoculum on paper discs on TSA a. After In a second double plate experiment, a single Pa-inoculated plate, a single uninoculated TSA plate, and a blank plate were each combined with a blank plate, and at 48 h the air content was pumped directly b from eaUsing the double plate method, a TSA plate with Pa10 or an uninoculated TSA plate were incubated below uninoculated TSA plates for 72 h. At the conclusion of the incubation, the upper plates were removed and inoculated with Af (10AF). The results show that the uninoculated plate that had been incubated with Pa was now inhibitory to Af , indicat7 of 10AF conidia in PBS was exposed to a TSA plate inoculated with PA14 or to an uninoculated TSA plate (control). After 72 h, the conidia were harvested and serial dilutions of conidial suspensions were re-quantitated as CFU by inoculation onto PDA plates; also, undiluted conidia were inoculated onto RPMI agar and the growth areas were measured. The incubation with Pa volatiles neither affected the conidial number measured as CFU on PDA, nor the growth area resulting from the conidial inocula. This suggests that the volatiles do not directly affect Af.This conclusion about mechanism was supported by another experiment where, in a double plate method, 5 × 10lasR-/rhlR- (no inhibition), lasR-, hcnA-, and pvdD-/pchE-  of PAO1 were as inhibitory as the parent.In previous studies ,52,53, PD-/pchE- . The pvd(lasR-/rhlR-) with unimpaired growth on TSA but lacking inhibitory volatiles was an important development, in that it created a control for the later analysis of the volatiles from inhibitory strains.This discovery of a Pa mutant strain In a previous epidemiologic study, we found that Pa isolates varied significantly in their inhibition of Af in liquid, with non-mucoid CF isolates > mucoid CF isolates > non-CF isolates . To compAspergillus growth is not restricted to A. fumigatus. When A. flavus was similarly studied, the fungal growth was also inhibited . Then, the Pa plate was quickly replaced with a plate with a 10AF inoculum (exposure phase) and the double plate was incubated a. All thThe exposure period of 72 h with trapped volatiles had resulted in less Af inhibition than 48 h of exposure . To assess this, in another experiment, during the exposure phase, a fresh Petri plate of water that had just trapped volatiles for 72 h from Pa on TSA was put under the Af-TSA plate and replaced by another such fresh plate at 24 and at 48 h of exposure, and the effect was compared to water containing trapped volatiles that had been in the double plate for the whole 72 h exposure phase. With the replacements by the fresh captured volatiles, there was no waning effect when the Af growth was then assessed, indicating that the introduction of freshly trapped volatiles every 24 h during Af exposure could maintain the level of Af inhibition without waning c.To assess the optimal duration of trapping, a plate with water was combined in the double plate method with a plate of Pa for 24, 48, or 72 h, and each water plate was then tested in exposure to Af. The optimal trapping duration was 72 h d. This wIn these studies, we discovered that the trapping of volatiles led to a net alkalinization in the trapping materials, a possible clue to their trapped content. The pH of the deionized water was 5.5; the water in a plate combined with uninoculated TSA in the double plate method for 72 h had a pH of 5.5, whereas the water in a plate combined with Pa10 on TSA in the double plate method for 72 h had a pH of 7.5.The double plate method would not directly allow the analysis of the volatile components. We have described above, in studies showing that the continuous production of volatiles is not necessary for inhibition, that the air between double plates can be aspirated or that the air could be pumped and inhibit Af in vials. This could also be a method to capture volatiles that are thus assuredly inhibitory for spectral analysis. To explore trapping volatiles in larger volumes for subsequent analysis and possibly more simply, we also assessed pumping air from a Pa-inoculated TSA plate through tubing and bubbling it through de-ionized water. The volatiles from the collected water were then inhibitory to the Af growth a; the asHaving studied the variables involved in trapping volatiles, it was necessary to ascertain the preservation of inhibitory activity, with a view toward storing and transporting the volatiles for mass spectroscopy. A series of five experiments was performed which examined the volatiles trapped in de-ionized water for 72 h and then stored either at room temperature; in the refrigerator (4 °C); or frozen (−20°C) for 24, 48, or 72 h. The materials stored in the refrigerator or frozen were brought to room temperature before testing. This study was an attempt to mimic the shipping conditions that might be necessary.The TSA plates were inoculated with Pa10, or uninoculated (the control for comparison), incubated for 72 h in the double plate method with the waters, and the waters then tested for the volatile release of inhibitory activity against 10AF after storage under the several conditions just described, in a second double plate method.p < 0.001) in the inhibitory activity from this water, comparing the Af growth areas after exposure to a comparably prepared unheated preparation.In summary, no significant loss of activity of the trapped volatiles was noted under any of the three conditions, when compared with freshly generated volatiles trapped in water for 72 h. The volatiles from all the preparations inhibited the Af growth ~25% compared to the controls (plates with no water exposure). These results speak to the stability of the volatiles under the several temperature conditions described, as well as after freeze-thawing. However, when the water with trapped volatiles was instead heated in a sealed container at 58 °C for 30 min, there was a 13% deterioration  In these two studies, spectroscopy peaks appearing only in the test samples, and not in the controls or water exposed to the mutant, were considered to be potentially inhibitory volatiles of interest. It was considered that only water-soluble inhibitory volatiles would be captured and identified in these studies.lasR-/rhlR- mutant on TSA, two uninoculated TSA plates, or two empty plates. At the end of the 48 h of incubation, the air that had collected in each pair was aspirated via a needle and one-way air pump into mass spectroscopy vials. An example of a spectroscopy tracing from this study is shown in the In the third study, a similar double plate method was used, but instead of the second plate of water, both plates of the pair were identical (to augment the collection of volatiles). These double plates were combinations of two plates: PA14 on TSA, the lasR-/rhlR- mutant , or a TSA-coated vial or an empty vial was given 40 μL of saline. All the vials were incubated as described above. With the live cells still present in the Pa vials, the air content of all the vials was sampled for mass spectroscopy.In the fourth study, the vials were coated with 5 mL of TSA and inoculated with either PA14 or the In the latter two studies, the peaks present in the Pa vials but not in the three controls in each experiment were considered the inhibitory volatiles of interest, which now included the volatiles that were not water-soluble.From the spectrographs resulting from these four studies, tables were prepared from the peaks identified: In the next series of 15 experiments, we studied those A. fumigatus. 1-undecanol and 2-dodecanone produced only a slight inhibitory effect. These results are shown in 12-HSL), and the Pa siderophore, pyoverdin, in the same way, and they also had no inhibitory effect as volatiles on A. fumigatus.We found 2-heptanone, 2-undecanone, and methylthiobutyrate from In a previous study , we founWe anticipated that the combinations of inhibitory molecules could be synergistic . HoweverPa and Af are the most important bacterium and fungus in the microbiota in many clinical settings, particularly in the lungs of immunocompromised persons and those with CF. Advanced technology has been used to detect the volatiles of Pa and other microbes ,73,74,75It was notable that the volatiles of Pa isolates from CF patients, particularly non-mucoid Pa isolates, were more inhibitory than others (as was reported for the inhibition of Af in liquid medium) . The latOne concern in studies in closed systems, such as those previously described  or our dlasR-/rhlR- mutant was noninhibitory, the lasR- mutant was only somewhat inhibitory, and the rhlR- mutant was fully inhibitory, opens the possibility of now defining which downstream pathways in Pa are responsible for these differences.Microbial volatiles are typically small lipophilic organic molecules . The mecAf also produces volatiles ,69,70 (aThere are several shortcomings of this study. We have not pinpointed all the Pa molecules that could be involved in Af inhibition, as some candidates that were identified by chromatography studies could not be obtained for separate study. A larger issue is how inhibitory molecules interact with each other; it was highlighted by our observations that commonly the sum of the parts was less than expected from the results of the individual components, and that noninhibitory molecules could affect the results of inhibitory molecules. It is also possible that the net Af-inhibitory effect we have described is a result of the interactions of volatiles produced by Af and Pa, an interaction that may be of a synergistic or antagonistic nature; or that one actor may modify the molecules produced by the other ; or thatMany of the volatiles of interest to us as potential Pa inhibitors  have beePseudomonas, may prove useful against fungi that are plant pathogens [Another important potential application of our findings is that specific volatiles, from micro-organisms such as athogens .