There is scarce research on the effects of mindfulness in individual therapy. As many practitioners integrate mindfulness exercises into individual therapy, empirical evidence is of high clinical relevance.We investigated the effects of a session‐introducing intervention with mindfulness elements (SIIME) in a randomized, controlled design. The effects of SIIME on therapeutic alliance and symptomatic outcome were compared with progressive muscle relaxation (PMR) and treatment‐as‐usual (TAU) control conditions. The sample comprised 162 patients with anxiety and depression.Multilevel modeling revealed a significant symptom reduction and significant increase of alliance over the course of therapy. There were no significant time–condition interactions on outcome and alliance, indicating the comparable efficiency of all three treatment conditions.We found no advantage of SIIME versus PMR and TAU. Add‐on mindfulness might not improve individual therapy related to alliance and outcome. Participating trainee therapists had predominantly little mindfulness experience and did not have a formal mindfulness qualification . Inclusion criteria for patients were (a) a primary anxiety or depressive disorder diagnosis; (b) age between 18 and 65 years; (c) fluency and literacy in German; and (d) informed consent. Exclusion criteria were (a) psychotic disorders; (b) a major medical illness ; and (c) current suicidal risk. Comorbidities with disorders not on the exclusion list were generally not considered limitations to entering the study, as long as anxiety and depressive disorders were of primary concern. We chose these two mental disorders because of patients being treated at German university therapy‐training centers; approximately 30% suffer from a primary anxiety disorder, and approximately 40% suffer from a primary major depression  were randomly selected and rated by a second diagnostician for reliability information. The agreement between diagnosticians was high .The Structured Clinical Interview (SCID), according to the DSM‐5 criteria  and a good predictive validity of 0.30 ≤ r ≤ 0.72 , and is outcome predictive with r = 0.28, as has been demonstrated in meta‐analyses , a split‐half reliability of r = 0.70, a retest reliability of r = 0.75, and convergent validities of 0.50 ≤ r ≤ 0.61 , a split‐half reliability of r = 0.72, a retest reliability of r = 0.75, and convergent validities of 0.71 ≤ r ≤ 0.89 , and is outcome predictive , intraclass correlations (ICCs) were 0.78 ≤ ICC ≤ 0.82, and cutoff to remission ≤ 6 , ICCs were 0.74 ≤ ICC ≤ 0.96, and cutoff to remission ≤ 7 . In addition, observer adherence was assessed using video‐based analyses. The six items (range: 0–4) assessed whether participants conducted the interventions adequately .Standard measures of the demographic data of patients were assessed at baseline. In addition, the intensity of the patients’ and therapists’ mindfulness and PMR experiences before the study, as well as the current intensity of patients’ and therapists’ mindfulness and PMR exercises conducted outside of the therapy sessions, was assessed. Furthermore, we assessed adherence to the interventions with self‐report and video‐based observer ratings. Patients’ and therapists’ self‐reported adherence was assessed with eight items (range: 0–4). The items assessed whether participants could adequately perform the different components of SIIME or PMR . Internal consistency was excellent with α = 0.97.To test potential allegiance effects related to the session‐introducing interventions, we applied an adapted version of the allegiance scale developed by Falkenström, Markowitz, Jonker, Philips, and Holmqvist . The scaα ≤ 0.86 and a predictive validity of 0.26 ≤ r ≤ 0.68 . Details on the study protocol are published elsewhere . Both workshop programs were offered by an expert with over 20 years of mindfulness and PMR experience. Both programs were based on the following procedure: a first workshop included background information and practice of relevant exercises. Then, a 6‐week home practice interval followed. In the second workshop, home practice experiences were discussed. Ideas on how to integrate the exercises into therapy routine and to continue the home practice were the focus. The workshops were well accepted and participating therapists reported positive experiences in qualitative interviews , spoken by an experienced mindfulness expert. The SIIME is an adapted version of the breathing‐space exercise , spoken by an experienced expert. We chose PMR as the active control group for the following reasons: on the one hand, PMR is a broadly accepted and an easy‐to‐implement relaxation exercise. On the other hand, it does not include the hypothesized specific effective ingredients of the SIIME (observing moment‐to‐moment experiences with an attitude of acceptance and openness). The PMR control condition was developed based on the same iterative procedure as the SIIME.2.4.5In the TAU condition, no standardized session introduction was applied. Participants were free to arrange the start of therapy the way that seemed most consistent for them. Typically, CBT routine introductions  were applied.2.4.6Before conducting the first intervention, a standardized rationale that provided clinical reasons for the relevant condition  was presented by the therapists (wording listed in the Supporting Information Appendix). In addition, all components of the relevant intervention were explained and practiced. Furthermore, in the first therapy sessions, a brief inquiry (brief exploration of experiences during the exercises) was conducted after SIIME and PMR. If required, the inquiry was conducted in later therapy sessions too.2.5f = 0.12) for the interaction between time  and treatment condition  = 0.80, number of groups = 3, number of measurements = 4, pre–post correlation r = 0.5, nonsphericity correction = 1) for the GSI primary outcome resulted in a sample size of 123 patients. Although this should only be seen as a proxy for the power of the reported multilevel models (MLMs), previous research showed that power in MLM is comparable to (or even higher than) power in an ANOVA design  as nested within patients (Level 2), which are nested in therapists (Level 3). For all research questions, model fit of the competing models was compared by means of likelihood ratio tests for nested models and the Akaike Information Criterion . We used Level 1 pseudo ng to Xu  as a meaWe predicted symptom severity ratings obtained at Pre, S5, S15, and S25 from the treatment condition  and time. The condition variable was represented by two dummy variables comparing the mindfulness condition to the PMR condition (SIIMEvsPMR) and the mindfulness condition to the TAU condition (SIIMEvsTAU), respectively. Time was coded as 1, 2, 3, and 4 and log transformed before the analyses, in line with previous research showing a faster initial change in symptom severity followed by a more decelerated change toward the end of therapy  as a dependent variable, and perspective, time (logarithmized session number), and the perspective × time interaction as fixed and random effects predictors . From this model, we extracted person‐specific regression coefficients for each patient for change in alliance from patient and therapist perspective (see Supporting Information Appendix for details). Hence, each patient’s trajectories are described by four parameters . These patient level predictors were then entered into the MLM, predicting symptom severity. We examined the moderating effect of condition for the association between these change parameters and change in symptom severity by testing the condition × change parameter × time (Pre through S25) interactions.α‐level of 0.05 (two‐sided).Finally, on the basis of the guidelines of Jacobson and Truax , we comp33.1d , as well as subjective adherence ratings by patients  and therapists , were high. In addition, the perceived quality of mindfulness implementation reflected in PQ‐M mean scores  and the in‐session therapeutic presence reflected in TPI mean scores  were high, further indicating good subjective adherence to mindfulness. PMR adherence ratings by observers , as well as subjective adherence ratings by patients  and therapists  were high. None of the participants performed exercises or components of exercises from the not assigned condition. None of the participants of the TAU performed SIIME or PMR at the beginning of therapy sessions. This indicates that the interventions were applied according to the protocol.Mindfulness adherence ratings by observers  ≤ 2.12; all ps ≥ 0.124.We found no significant differences across the three study conditions in the subjective ratings of the therapists related to the intensity and frequency of the application of relevant CBT techniques , all 3.2.3SD = 0.66), the TAU+PMR , and the TAU  condition, F  = 0.08; p = 0.926. These results also indicate that the level of therapist competence was satisfactory in all three treatment conditions according to Shaw et al. , TAU+PMR , and TAU , F = 0.47, p = 0.625. However, from the therapist perspective, allegiance to the session‐introducing intervention differed between the three conditions, F = 23.50, p < 0.001. A post hoc Tukey test showed that therapist allegiance was significantly lower in the TAU+PMR condition  than in the TAU+M  and TAU condition , all ps < 0.001. Regarding allegiance to the offered treatment, conditions differed neither from the patient perspective  = 0.61, p = 0.545), nor from the therapist perspective  = 0.01, p = 0.992).From the patient perspective, there were no significant differences in allegiance to the session‐introducing interventions in TAU+M  by adding the condition × time interaction. Model fit did not improve, χ²(2) = 0.09, p = 0.958, suggesting that the change in symptom severity did not differ between the three conditions. Results for BDI and BAI were conceptually identical with symptoms showing a significant decrease over time , which was comparable across conditions as indicated by no improved model fit when adding the condition × time interaction, χ²(2) ≤ 1.24, ps ≥ 0.539. KIMS scores significantly increased over time, b = 0.119, p < 0.001, but again, model fit did not improve when adding the condition × time interaction, χ²(2) = 2.16, p = 0.340. Consequently, the results falsified our assumption that there is a stronger symptom reduction in the mindfulness condition (Hypothesis 1).In the first set of analyses, we examined whether the three groups differed in the change of GSI scores across therapy. To that end, we set up models of increasing complexity. The first model  time (Model 1) yielded a significant increase across therapy, b = 0.143, p < 0.001. Adding all two‐way interactions between perspective, condition, and time improved model fit significantly, χ²(13) = 165.25, p < 0.001 (Model 2). However, none of the five additional fixed effects were significant, p > 0.211 for all, suggesting that this increase in model fit might be attributed to the existence of meaningful interindividual differences in the differential change of alliance rated from the two perspectives. Hence, although on average there were no differences between the rates of change in alliance as rated from the therapist and patient perspective, respectively (indicated by the nonsignificant time × perspective interaction), patients and therapists differed in the degree to which there were differences between these two trajectories. We tested whether these differences could be explained by a treatment condition by including the condition × time × perspective interaction (Model 3). This did, however, not improve model fit, χ²(2) = 0.853, p = 0.653. Consequently, the results falsified our assumption that there is a stronger increase in therapeutic alliance in the mindfulness condition (Hypothesis 2). Figure Fluctuations in therapeutic alliance across the 25 therapeutic sessions were estimated via multivariate MLMs, allowing for simultaneously estimating the trajectories in the ratings from the patient and therapist perspective. Reliabilities of within‐person fluctuations in therapeutic alliance were estimated via McDonald’s ω in a multilevel confirmatory factor analysis  = 26.12, p < 0.001. Inspection of the fixed effects revealed a significant main effect of the intercept: patients with relatively higher intercepts  reported less severe symptoms at pre, b = −0.248, p = 0.004. In addition, there was a significant main effect of a change in alliance and a significant change in the alliance × time interaction: patients who reported stronger increases in alliance across therapy had lower symptom ratings at pre, b = −0.733, p = 0.020, and reported a stronger symptom decrease across therapy, b = −0.480, p = 0.018. Adding the interactions with condition did not improve model fit, χ²(8) = 4.30, p = 0.829, suggesting that condition does not moderate the association between a change in therapeutic alliance (rated from the patient perspective) and a change in symptom severity across therapy. There were also no moderating effects when replacing the change parameters with the respective parameters from the therapist perspective or running the analyses with the BAI and BDI, all χ²(8) < 8.92, all ps > 0.349. Consequently, the results falsified our assumption that the mindfulness condition is a significant moderator of the alliance–outcome association (Hypothesis 3).Furthermore, we examined the potential moderating effects of the mindfulness condition on the alliance–outcome association. More specifically, we investigated whether the association of WAI change trajectories and GSI change trajectories was moderated by the treatment condition. We used the person‐level variables representing a change in therapeutic alliance  as predictors of change in symptoms. Adding the person‐specific change parameters of alliance rated from the patient perspective as main effects and in interaction with time (Pre through S25) improved model fit, 3.6ps ≥ 0.122. Hence, we found no significant moderating variables. This partly falsified our fourth hypothesis: The findings contradicted our expectations that mindfulness‐related variables would show significant moderating effects (Hypothesis 4a), but were in line with our assumption that demographic and diagnostic variables would not be significant moderators (Hypothesis 4b).In the final set of analyses, we examined whether patients with specific characteristics benefit more from the mindfulness condition. Therefore, we additionally tested potential moderator variables. Neither mindfulness‐related variables  nor demographic and diagnostic variables  moderated treatment effects, as indicated by a nonsignificant three‐way interaction with time and condition, all 3.7χ²(2) < 0.32, all ps ≥ 0.852.We assessed response and remission based on observer ratings  and based on self‐ratings (GSI). On the basis of HAM‐D and HAM‐A scores, we identified 56% responders in TAU+M, 53% responders in TAU+PMR, and 53% responders in TAU. We identified 39% remitters in TAU+M, 40% remitters in TAU+PMR, and 41% remitters in TAU. On the basis of SCID ratings, we identified 49% remitters in TAU+M, 52% remitters in TAU+PMR, and 53% remitters in TAU. Finally, we also analyzed response and remission based on the GSI. We identified 53% responders in TAU+M, 57% responders in TAU+PMR, and 51% responders in TAU. We identified 29% remitters in TAU+M, 27% remitters in TAU+PMR, and 31% remitters in TAU. There were no differences in response and remission rates across TAU+M, TAU+PMR, and TAU, all 4In the current study, we investigated whether session‐introducing mindfulness in individual therapy under routine conditions of CBT training has beneficial effects on therapeutic alliance and outcome in a sample of 162 patients with anxiety and depression. We designed an RCT with the three conditions TAU+M versus TAU+PMR versus TAU. Contrary to our expectations, TAU+M did not result in stronger effects on general symptom severity and therapeutic alliance as compared with TAU+PMR and TAU.Contrary to our expectation, the results of our strictly experimental study design showed no stronger symptom reduction in the mindfulness condition (Hypothesis 1). The treatment effects of all three conditions were of similar strengths and comparable with the effectiveness of routine psychotherapy in meta‐analyses . In all three treatment conditions, patients and therapists showed positive alliance ratings that increased across the course of therapy with medium effect sizes. There were no significant differences in the strengths of alliance ratings or in the increase of alliance ratings across the course of therapy. Furthermore, we explored whether the alliance–outcome association was different in the three treatment conditions. Therefore, we conducted an MLM moderator analysis. We did not identify differences in the alliance–outcome association across the three treatment conditions. Consequently, the results falsified our prediction that mindfulness is a significant moderator of the alliance–outcome association (Hypothesis 3). As in most studies (Flückiger, Del Re, Wampold, & Horvath, 2018; Horvath et al., The results of our study are of importance for routine practice, as many practitioners directly integrate mindfulness into their individual therapies without an empirical basis for whether this component actually improves outcomes Crane, . SpecifiA large part of the current therapist sample started mindfulness training with the workshop presented at the beginning of the study. Hence, according to the framework for an MBI teacher training program, the participants were in the first stage of foundational training (Crane, Kuyken, Hastings, Rothwell, & Williams, The current study has several limitations. First, as we investigated routine therapy, treatment was not manualized and several patients suffered from comorbid disorders. This might have resulted in internal validity issues. To address this aspect, we controlled for potential confounding variables in the MLM analyses. In addition, routine care investigations have high external validity and are, thus, relevant for clinical practitioners. Second, we applied only self‐report outcome measures for the repeated assessments. We only assessed observer ratings using SCID, HAM‐A, and HAM‐D at the beginning and end of treatment and did not have the opportunity to apply these ratings at all measuring times. Future studies should apply multiple observer ratings to investigate whether our effects generalize to other outcome rating perspectives. Third, 15% of TAU+M, 22% of TAU+PMR, and 13% of TAU were dropouts, indicating that treatment was not accepted by all patients. According to a meta‐analysis, 19.7% of study participants drop out in RCTs (Swift & Greenberg, Our study delivers first evidence that mindfulness as an add‐on component in individual therapy has similar effects as PMR and TAU on therapeutic alliance and clinical symptoms in anxiety and depression. Add‐on mindfulness might not improve individual therapy related to alliance and outcome.Supporting InformationClick here for additional data file.


Bevacizumab improves symptoms via reducing the peritumoral edema and/or normalizing blood brain barrier, and occasionally via reducing the tumor size. However, the effect against active cystic components has not been documented yet.Between 2008 and 2018, 139 patients with primary or metastatic brain tumors were treated with bevacizumab (BEV) in our institution. The images and symptoms before and after administration of BEV were examined, and changes in size of cysts were evaluated as follows: CR (complete disappearance), PR (reduction by ≥50%), MR (reduction by ≥25%), SD (size change <25%), PD (increase by ≥25%). The effect of BEV on tumor itself was determined according to Response Assessment in Neuro‐Oncology criteria.P = .0055) and at best response timing . Patients with cysts showed significant improvement in symptoms after the treatment with BEV compared to patients without cysts (P = .0033). However, response rate was not different between patients with or without cysts. Overall survival after starting BEV was not different between glioblastoma patients with or without cysts.Of the 139 patients, 21 (15.1%) had cystic components. The best responses of cysts to BEV treatment were as follows: CR 6, PR 7, MR 4, SD 4. The group of patients with progressively increasing cysts prior to BEV treatment had significant cyst size reduction compared to stable cyst size groups, at initial imaging after BEV  compared to patients without cyst. Bevacizumab is effective against progressively increasing cysts. Although cyst reduction effect and tumor response and/or overall survival are independent, BEV may be effective in patients who are symptomatic due to cystic enlargement. Bevacizumab binds and inactivates all isoforms of VEGF, and works as an anti‐angiogenic agent.Glioblastoma, the most common malignant primary neoplasm of the central nervous system, also expresses VEGF and its receptors. Vascular endothelial growth factor has been investigated as a potent mediator of brain tumor angiogenesis, vascular permeability, and glioma growth. Bevacizumab was approved for the treatment of recurrent glioblastoma in 2009. However, despite its approval, promising preclinical data and early clinical trials, multiple randomized large phase 3 clinical trials have failed to show survival benefit of BEV in patients with glioblastoma.Cystic formation is sometimes observed in glioblastoma and other brain tumors.2Our institutional review board approved this retrospective study (IRB No. 2953/ E‐1585). Between September 2008 to December 2018, 139 patients with recurrent brain tumors were treated with BEV. Recurrence was defined as imaging progression with or without symptomatic progression. Their age ranged from 1 to 94 years , and 79 were male, 60 were female patients. Histological diagnosis was as follows: 65 glioblastoma , 8 anaplastic astrocytoma, 12 anaplastic oligodendroglioma, 4 diffuse astrocytoma, 18 diffuse midline glioma, 5 pilocytic astrocytoma, 3 anaplastic ependymoma, 5 embryonal tumors , 2 diffuse leptomeningeal glioneuronal tumors (DL‐GNT), 1 germ cell tumors, 16 metastatic tumors . Bevacizumab administration schedule was as follows: 10 mg/kg at every 2 weeks, or 15 mg/kg at every 3 weeks. Treatment schedule of BEV was modified in each patient in the event of development of 3+ proteinuria as an adverse effect.All patients underwent magnetic resonance imaging (MRI) study which included non‐enhanced T1‐weighted imaging, T2‐weighted imaging, fluid‐attenuated inversion recovery (FLAIR), diffusion‐weighted imaging, and gadolinium‐enhanced T1‐weighted imaging with slice thickness 7 mm or less. In this study, we defined cystic lesions as high intense lesion with long axis of 1 cm or more on T2‐weighted imaging, with thin wall defined by T2 low band and/or gadolinium‐enhanced T1‐weighted imaging. We excluded necrotizing cysts defined as enhancement inside the enhanced cyst wall on gadolinium enhanced T1WI, or obvious heterogeneity inside the cysts on FLAIR imaging. Overall, in our case series 21 patients were defined to have cystic lesions before BEV treatment. The area of cysts was defined as the product of the long axis and the length of the axis perpendicular to it, also called the short axis.First, we compared the images just before administration of BEV with the images 1‐3 months prior. We defined patients with cysts as follows: active cysts group (cysts size was increase by ≥25%), inactive cysts group (cysts size was increase by <25%). Second, we compared the images just before administration of BEV and images within 2 months after BEV treatment. Third, we compared the images just before administration of BEV and at the points when the size of cysts was smallest (best response point of cysts). Changes in cysts size were evaluated as follows: CR (complete disappearance), PR (reduction by ≥50%), MR (reduction by ≥25%), SD (size change <25%), PD (increase by ≥25%). We also evaluated the effect of BEV on the tumor itself according to (Response Assessment in Neuro‐Oncology) RANO criteria. Best response to BEV was assessed in each patient.Then, we evaluated the BEV effect against symptoms of patients. Karnofsky performance status (KPS) before and after 2 courses of BEV administration was evaluated. Effect of BEV against symptom was defined as follows: improved (increase KPS by at least 10), no change, worsened (decrease KPS by at least 10). Seven patients were administrated only one course because of progressive disease (1), adverse events (3), patient refusal (2) or switching regimen (1), and evaluated KPS after one course of BEV. We also analyzed the prognosis in recurrent glioblastoma patients after BEV administration.U test and Fisher's exact text were used. Symptom changes between patients with cysts and without cysts were evaluated by Fisher's exact text.Statistical analyses were performed with PRISM version 5.0 (GraphPad Software Inc). Cysts increment rate/reduction rate were calculated, and for statistical analysis Mann‐Whitney 3P = .0033, Fisher exact test). However, response rate of solid part of tumor was almost same between tumor with cysts group and without cysts group .Among 139 patients, 21 patients  had cystic components. Characteristics of patients with cysts and without cysts are summarized in Table Histological diagnosis of tumors with cysts was as follows: 7 glioblastoma, 1 anaplastic astrocytoma, 2 anaplastic oligodendroglioma, 3 diffuse midline glioma, 3 pilocytic astrocytoma, 1 ETMR, 1 DL‐GNT, 3 metastatic tumors. The characteristics of primary or metastatic brain tumor with cysts, symptoms, and response to BEV are summarized in Table P = .0251, .0058, respectively, Fisher's exact test).Before administration of BEV, cysts size condition was as follows: PD 14, SD 7. We subdivided patients with cysts as follows: active cysts group (PD), N = 14, inactive cysts group (SD), N = 7. On the first imaging following administration of BEV, the response of cysts was as follows: CR 2, PR 7, MR 7, SD 5. The best response of cysts to BEV treatment were as follows: CR 6, PR 7, MR 4, SD 4. No cyst showed increase in size (PD). We then sub‐divided patients with cysts as responder (MR/PR/CR) and non‐responder (SD), and statistically evaluated the difference between active and inactive cysts group. Responders were more frequently observed in active cysts group compared to inactive cysts group both at initial image after administration of BEV and best response point (P = .0055) and at best response timing .We subsequently compared the actual change in cysts size between active and inactive cysts group. The group of patients with active cysts responded to BEV and showed significant cysts size reduction compared to inactive cysts group, at initial imaging after BEV  or message of the manuscript;None declared.Writing—original draft: FY; writing—review and editing; FY, MK, MT, UY, IN, NI, AT, SO, VJA. YT, YN, KK, KS; conceptualization: FY, MT; data curation: FY, MK, MT, UY, IN, NI, AT, SO, VJA; formal analysis: FY; supervision: YT, YN, KK.


A cobalt sulfide/PVDF(Polyvinylidene Fluoride) composite was prepared by a simple blending method, and the microstructure of the composite was investigated through X-ray diffraction, scanning electron microscopy, and field emission scanning electron microscopy. Increased absorption properties at frequencies ranging from 2 GHz to 18 GHz were studied, and the mechanical properties were investigated via tensile tests and finite element method simulations. The results indicated that the cobalt sulfide/PVDF composites exhibited strong microwave absorption intensities (−43 dB at 6.6 GHz) with a low filler loading (5.0 wt%). The elastic modulus increased with the cobalt sulfide mass fraction. Cobalt sulfide could improve the mechanical properties of the composite, especially in the lengthwise direction. In recent years, porous semiconductor nanomaterials, as the reinforced phase of composite, have widely intrigued researchers due to their good microwave absorption properties, light weight, thin thickness, and good compatibility6. Good strength is required in practical applications; therefore, the mechanical properties must also be investigated8.As new functional materials, microwave absorbing materials have been broadly applied in the fields of industry, commerce, and military13. Liu et al.14 prepared a SiC fiber/paraffin wax composite with microwave absorption properties. Its minimum reflection loss (RL) was −28.47 dB at 12 GHz. Meng et al.15 found that SiC microtubes have a minimum RL of −23.9 dB at 17.5 GHz. Meanwhile, a polypyrrole-reduced graphene oxide–Co3O4 nanocomposite with maximum RL of −33.5 dB at 15.8 GHz was reported by Liu et al.16. According to Xu et al.17, smart composite absorbers comprising silicone rubber, multi-walled carbon nanotubes (MWCNTs), and flaky carbonyl iron particles can absorb microwave ranging from approximately 4 GHz to 11 GHz. Many researchers have also investigated PVDF/GIC composites18, RGO/CuS/PVDF composites19, PVDF/RGO composites20, ZnO/PVDF composites21, and CuS/PVDF composites24 in terms of their absorption properties and obtained excellent results. One such finding is that a synergistic effect exists between PVDF and nanofillers26, and it can improve the wave absorption properties of nanocomposites.Many microwave absorbing materials have been reported in the last decade. At present, an increasing number of researchers is focusing on the electromagnetic properties of such materials at frequencies of 2 GHz to 18 GHzBy contrast, CoS nanocomposites and their wave absorption properties have not been researched. A previous study by the present authors revealed that polymer composites filled with inorganic semiconductors exhibit excellent microwave absorption. In this work, we used PVDF as polymeric matrix material for the composite due to its physical and dielectric properties, which are especially suitable for such composite.et al.27 analyzed the effects of carbon fiber (CF) with various content  on the mechanical properties of GF/PDMS composites. The experiment data suggested that the addition of CF significantly improved the mechanical properties of the GF/PDMS composites. The composite with 10 wt% CF (F/GF/PDMS composite) evidently increased in tensile strength (by 52%) and Young’s modulus (by 71%). Rajaei et al.28 studied how ammonium olyphosphate and talc affect the mechanical property of an epoxy/glass fabric composite. With the addition of flame retardants to the epoxy resin, the composite’s tensile and flexural moduli increased. Shin et al.29 investigated the mechanical properties of aluminium alloy 2024 (Al2024) matrix composites that were individually reinforced with either an MWCNT or few-layered graphene at 250 °C to 430 °C. Results showed that both resultant composites formed at 350 °C possessed high yield stress of approximately 110 MPa. Otto et al.30 analyzed the mechanical properties of several low-cost hybrid composites consisting of polyurethane and renewable natural fibers. A simplex-centroid mixture design model was used to evaluate the effects of the added fibres on composite properties, such as resilience, elastic modulus, and deformation, under permanent compression. The obtained hybrid composites demonstrated excellent performances of up to 32% resilience, 0.1 GPa elastic modulus, and 7.32% permanent deformation. Tarfaoui et al.31 investigated the relationship between the addition of carbon nanotube (CNT) additives and the elasticity of textile-based composites. The experimental results indicated that the mechanical properties of the composites increased with the CNT additive content and reached its peak value at 2%.Meanwhile, the mechanical performance of certain nano-porous materials has recently been studied by several researchers. Zhao In this research, cobalt sulfide/PVDF composites were prepared using a simple blending method and then studied with respect to their microstructure through scanning electron microscopy (SEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy. The improved wave-absorption ability of cobalt sulfide/PVDF composite was studied, and the mechanical properties were explored via tensile tests and finite element method (FEM) simulations.2 · 6H2O, analytical reagent) was obtained from Guangdong Guanghua Sci-Tech Co., Ltd. (China), and ethylenediamine  was supplied by Xilong Chemical Co., Ltd. (China). N, N-dimethyl formamide , sulfur powders , glycol , absolute ethanol , and deionized water  were acquired from Beijing Chemical Reagents Co. (China).Cobalt chloride hexahydrate , the grain morphology and size were analyzed through SEM and field emission SEM (FESEM) using a JEOL JSM-7500F microscope. Relative permittivity The molecular structure of cobalt sulfide and PVDF were established to obtain the mechanical properties of the reinforcement and matrix Fig. . The parFEM simulations were performed to further study the composite with respect to its mechanical properties. The FE models of the 5 wt%, 10 wt%, 20 wt%, and 30 wt% cobalt sulfide/PVDF composites are shown in Fig. The numerical model mainly includes the following steps: 1) Create a geometric model of the composite. A 100 × 50 × 5 μm rectangular shell was built to stand for the PVDF matrix in the software ANSYS FEM. (2) Define a random distribution of cobalt in the matrix. Several small internal rectangles with different sizes and positions represented the nano-porous cobalt sulfide. The nano-porous cobalt sulfide ranges in length from 4 μm to 10 μm and in width from 0.4 μm to 1 μm. The random distribution of cobalt sulfide is based on the modeling algorithm  cobalt sulphide, mechanical tests were carried out according to ASTM D882-12 standard. A 1000 N universal testing machine (Shimadzu AGS-X) was used at a loading speed of 50 mm/min. The thickness of the composite film, which was cut into several 9 mm × 3 mm rectangular pieces, was 55 μm. Two pieces of paper were pasted to both ends of each rectangular specimen for clamping of the tensile test  is a key factor that affects the prepared product structure. When Rs is 1:1, nanorods with uniform morphologies are obtained , which can be expressed as follows32:Zin stands for input impedance, μr stands for relative complex permeability , εr stands for complex relative permittivity, f stands for the microwave frequency, d stands for absorbent’s thickness, h stands for the Planck constant, and c stands for the propagation velocity of an electromagnetic wave in vacuum.The wave absorption properties were measured by reflection loss  and generally proportional to the load content  ε″/ε′ of the composites with different filler loadings was calculated; on this basis, the theoretical RLs of the PVDF and cobalt sulfide/PVDF composites with filler loadings of 5 wt%, 10 wt%, 20 wt%, and 30 wt% at 2.5 mm thickness could be obtained through Eqs ::ε′ and εε′ − ε″ curve will be a single semicircle. The semicircle is denoted as the Cole–Cole semicircle in general, and each semicircle and one Debye relaxation process share a one-to-one correspondence.Therefore, the ε′ − ε″ plots of the cobalt sulfide/PVDF with various content of cobalt sulfide, which are in semicircles, are presented in Supplementary Fig. The An increase in the composite’s conductivity can lead to significant dielectric loss according to free electron theory. Hence, the increased filler content of cobalt sulfide should increase the dielectric loss of the cobalt sulfide/PVDF composite because the superior conductivity of the cobalt sulfide can increase the conductivity of the composites.The equivalent stress (von Mises stress) results of the FE model are represented in Fig. The stress–strain curves of the elastic stage obtained by tensile tests and FEM simulations are shown in Fig. The elastic modulus of cobalt sulfide and PVDF are 300 GPa and 840 MPa, respectively. For cobalt sulfide/PVDF Composite Materials, under the condition of the same strain, the elastic modulus of cobalt sulfide is bigger, so the equivalent stress of cobalt sulfide is larger. With the addition of cobalt sulfide, the elastic modulus of cobalt sulfide/PVDF material increased significantly. The measured elastic modulus of 5 wt%, 10 wt%, 20 wt%, and 30 wt% is 524.73, 944.98, 1422.2, and 1746.6 MPa, respectively. The elastic modulus calculated by the FE model is 895.62, 991.71, 1484.29, and 1784.09 MPa, respectively. These results show that the elastic modulus increases with the cobalt sulfide mass fraction. Therefore, cobalt sulfide is conducive to the improvement of the composites’ mechanical properties. The consistency between the FEM results and the experimental results validates the effectiveness of FEM.In this research, two-phase composites comprising cobalt sulfide and PVDF were synthesized and studied in terms of their microstructures and characterization, and the wave absorption and mechanical properties were explored. The results suggested that the cobalt sulfide/PVDF composites have strong microwave absorption intensity, reaching −43 dB at 6.6 GHz. This intensity can be tuned by regulating the thickness and the content levels of the cobalt sulfides. Moreover, the elastic modulus is increased with the cobalt sulfide mass fraction. With all the abovementioned improvements and advantages, the cobalt sulfide/PVDF composites are still as flexible as pure PVDF and also can be cut into desired morphologies. Therefore, cobalt sulfide is conducive to the improvement of the mechanical properties of composites. The cobalt sulfide/PVDF composites can be applied in commercial, military, and scientific electronic devices due to their outstanding microwave absorption performance and potential for industrial production.Supporting Information


Solenopsis invicta Buren, is an invasive ant species that commonly forages on trees. Our studies showed that S. invicta used multiple strategies to transport food items on vertical surfaces (tree trunks). Small food items (1 × 1 × 1 mm sausage) were carried and transported by individual ants, and larger food items were either collectively and directly transported or cut collaboratively first and small particles were then transported individually or collectively. Competition and deadlocks were frequently observed during individual and collective transport respectively. During cutting, groups of ants tightly fixed the food on the tree trunks by holding the edges of the food item, while other ants cut the food into smaller particles. All food items and particles were moved downward. We investigated the effects of food placement (placed on a platform or fixed on tree trunk), food shape (cuboid or flattened), particle sizes , and placement height  on the food transport on tree trunks. Our studies are the first to show how fire ants transport food on a vertical surface, and may provide insights into the development of novel fire ant baiting systems that can be placed on tree trunks.Many ants can cooperatively transport large food items (either coordinated or uncoordinated during transportation), which can be rarely observed in other animals besides humans. Although these behaviors have been extensively investigated on horizontal surfaces, few studies dealt with food transport on vertical surfaces. The red imported fire ant,  Among them, behaviors related to foraging processes are critical to the survival of ant colonies. Ants have evolved diverse foraging behaviors to acquire and utilize food in different habitats and environmental conditions. Some aspects of foraging behaviors, such as the cooperative food transport , are fairly common in ants but can be rarely observed in other animals besides humans8.Ants are eusocial insects that perform many collective behaviors for different tasks, such as nest construction, foraging, and defense7 reviewed cooperative transport by ants and defined two types of well-coordinated food transport: (1) encircling coordinated transport, and (2) forward-facing coordinated transport. The former type can be observed in ants such as Leptogenys diminuta Smith, Pheidologeton diversus (Jerdon), Novomessor cockerelli (Andre), and some Pheidole species11. During transportation, these ants gather around food items and hold the edge of the food with their mandibles and front legs. The latter type was only reported in the army ants that faced the same direction while transporting food items12. For both types, food can be transported fast with few or no deadlocks. Likewise, McCreery and Breed8 defined the efficient cooperative food transport in ants as “additional workers arrive at the food quickly, and transport progresses rapidly in a straight line toward the nest”.Previous studies showed that the food transport processes are highly efficient in some ant species. Czaczkes and Ratnieks15. The forming and maintaining of these “fast-track” paths largely depends on the short-lived but accurate trail pheromones dropped by individual ants on the substance. Importantly, the pheromone deposition by ants is flexible, and therefore the foraging paths can be rapidly adjusted in response to environmental changes17 and other situations (e.g. trail crowding18 and obstacle presence19). In addition, the cognition  of individuals may also play a role in the recruitment and food transportation on trails21.Some ants also use the two-way trails to connect nests and food sources. The trails provide navigation information and therefore may enhance the food transport efficiency22. McCreery and Breed8 pointed out that ant species with inefficient transport pattern lack the ability of modifying their behaviors based on other individuals that transported the same food item, and therefore cannot reach a consensus. One example is the red imported fire ants, Solenopsis invicta Buren, a significant ecological, agricultural, and medical pest with a worldwide distribution23. Wang et al.24 found that the cooperative food transport of S. invicta workers were typically uncoordinated because they “pull/push the food item in multiple directions, leading to a slow process with frequent deadlocks”.Many ants show the behavior of uncoordinated cooperative food transport, characterized by frequently occurring and long-lasting deadlockset al.25 and Wojtusiak et al.26). It is still unclear how food items were moved on a vertical surface. Yamamoto et al.25 studied the foraging strategies of 44 ant species that belong to 34 genera. They found that most ground-living ant species cooperatively transport large preys without the fragmentation process, whereas the tree-living ants commonly cut large preys and individually transported fragmented pieces to the nests. Interestingly, S. invicta not only forage on ground, but also captured preys (arthropods) in the tree canopies27. In a previous field study, we observed massive foraging activities (two-way ant trails) of S. invicta on tree trunks that transport preys downward to the nests (mounds) in the ground28. Although this process should have extra challenges such as gravity, we rarely observed the falling of preys during the transportation, indicating that the food transport by S. invicta workers is well organized on vertical surfaces with undetermined behavioral patterns.Most studies on ant foraging behaviors were performed on horizontal surfaces  What are the behavioral patterns and potential adaptations for vertical food transport? And (3) which factors affect the food transport on tree trunks?In the present study, we conducted a set of experiments under field condition to answer the following questions: (1) is vertical food transport by S. invicta workers when they moved food items on tree trunks. We did this by providing food items of various sizes  to ants on platforms set up on trees  individual transport – single ant carried and moved the food item; 2) collective transport – group of ants moved the food item without cutting it into smaller pieces; and (3) cutting – ants cut the food into small particles, which were then transported individually or collectively  were either placed on the platform or artificially fixed on trees , and the transportation (cutting) phases  were not significantly different between the two placements , durations of the searching  were cut during the transportation phase. The duration of searching phase was not significantly different between cuboid (4 × 4 × 4 mm) and flattened (8 × 8 × 1 mm) food  but more time during the transportation phase   Fig. .F = 3.78, df = 3, 52; P = 0.0158; Fig. F = 2.66, df = 3, 52; P = 0.0580; Fig. F = 3.42, df = 3, 52; P = 0.0237) and ants per gram of remaining food  were found in the tubes with particles ranging from 2–3 mm than in tubes containing the smallest particles  on tree trunks Fig.  and on gles Fig. .Figure 6F = 12.28, df = 2, 27; P = 0.0002; 2–3 mm: F = 13.33, df = 2, 33; P < 0.0001; Fig. F = 1.43, df = 2, 27; P = 0.2536; Fig. F = 3.83, df = 2, 33; P = 0.0319; Fig. This experiment tested the effect of placement height on food transportation and ant recruitment on tree trunks. Three tubes containing the similar amount of food were attached to tree trunks at the height of 20, 80, and 150 cm Fig. . For botS. invicta is one of the most extensively studied ant species, many aspects of its foraging behaviors have been studied just in recent years31. This study is the first to investigate how fire ants transport food items on the vertical surface. Results showed that multiple strategies  were used by S. invicta workers in response to different food sizes during vertical transportation, and food shape, particle size, and placement height had significant effects on the vertical food transport.Although et al.32 defined the self-assemblages of eusocial insects as the “physical structures comprised of individuals that have linked themselves to one another”. These structures played critical roles during foraging processes. For example, Peeters and De Greef 33 observed that some Leptogenys ant species linked their bodies in chains and walked backwards to drag large prey items such as millipedes. The self-assembling in S. invicta for foraging purpose was also reported in several recent studies. For example, Wang et al.30 reported that S. invicta workers could cover and feed upon the droplet of liquid food (sucrose water). In the present study, we found that S. invicta workers filled the gaps between food and tree trunks. We speculate that these ants may act like a smooth slope by linking the sticky tarsal pads to each other, as they did when forming other self-assembling structures such as rafts and sinking towers36. By so doing, the food item can be relatively easily pulled/dragged by other ants from the horizontal surface to the vertical surface. It is also possible that ants simply get under the food accidentally in an effort to access it.Anderson S. invicta was not significantly different on the horizontal surface24. In the present study, however, the efficiency of individual food transport was much higher than that of collective transport on tree trunks  when moving food items on horizontal and vertical surfaces, differences were detected between these two transportation conditions. For example, the efficiency of individual and collective food transport of 39. In these previous studies, ants stayed on top of food items and cut the food into small particles with their mandibles. In the present study, many ants stayed around the food items and held the edge of the food with their mandibles and front legs. Thus, the food was tightly fixed on the tree trunks  transporters caused lower food-moving speeds. However, contrasting results were reported in different ant species such as Paratrechina longicornis (Latreille), which showed higher speeds for larger group of transporters distributed around the food41. Interestingly, the opposite pattern was found for large-sized food, since ants spent significantly more time to cut the cuboid (4.0 × 4.0 × 4.0 mm) food compared to the flattened (8.0 × 8.0 × 1.0 mm) one  compared to the flattened one (3.0 × 3.0 × 1.0 mm) Fig. . In thisone Fig. . PerhapsS. invicta workers transported more food with the particle sizes ranging from 2–3 mm on the ground, which corroborates previous findings29. However, ants transported significantly more particles ranging from 1–2 mm on tree trunks , which removed more food mass regardless of particle sizes  in this area28. All studies were conducted during the daytime (9:30 am to 6:00 pm), with temperature ranging from 23–31 °C, and relative humidity 30–74%.The field studies were conducted from 25 October 2017 to 18 November 2017 in a green belt  along the street close to the campus of South China Agricultural University, Guangzhou, China. Our previous studies showed massive F. concinna trees with ant trails on trunks were used for this study (Supplementary Table Six 8:The food transport patterns (strategies) in response to different food sizes were identified and described for each phase. The transport path of the food (as well as the food particles cut by ants) was traced by recoding the location of individually transported food (see results) in every 2 s, and collectively transported food in every 30 s. For the food items cut by ants, the location of main part of the food was recorded in every 1 minute, and the location of cut particles was recorded in every 2 s  or 30 s (for collective transported particles). No location information was recorded during deadlocks and fixing (see results) when the food (or main part of the food) was unmoved or moved very slowly. During the transportation phase, the percentage of time when food was not moved  was calculated. For individual and collective transport, the transported efficiency was calculated using the formula provided by McCreery and Breedt-test , or one-way analysis of variance , followed by Tukey’s Honest Significant Differences (HSD) tests for multiple comparisons. Because the percentage of time accounting for the food that remaining unmoved were not normally distributed, these data were compared among different food sizes using the nonparametric Kruskal–Wallis test followed by Dunn’s post-hoc test for multiple comparisons43.Because some ants joined or left the food transport group over time, we counted the number of ant transporters in every 5 min across the transport process and then averaged. Transport efficiency as well as the time duration for each phase were compared among different food sizes using the S. invicta workers need to transport and fix the large food items on tree trunks for cutting (see results), we conducted a study to investigate whether the food placement  affect the vertical food transport of ants. Eight F. concinna trees with ant trails on the trunks were used for this study . An observation area was created using the same method as described above. Two food-releasing platforms were fixed on the tree trunk, and each one was 1.5 cm apart from the center point .Because int Fig. . A food int Fig. . The pinint Fig. , and theF. concinna trees were selected to investigate the effect of food shape on transport behavior on tree trunks. The two food-releasing platforms were prepared as mentioned above. Two medium-sized food items with different shapes – cuboid (2.1 × 2.1 × 2.1 mm) or flattened (3.0 × 3.0 × 1.0 mm) – were randomly placed on either left or the right platform .Nine orm Fig. . We alsoorm Fig. . The triF. concinna trees were selected to investigate the effect of particle size on the food transport by S. invicta workers on tree trunks . Dog food  was ground with pestles and mortars, then sifted through 4-, 3-, 2-, 1-, and 0.45-mm sieves. As a result, the food particles with sizes (diameters) ranging from 0.45–1, 1–2, 2–3, or 3–4 mm were obtained. Approximately 500 mg food  at the height of ~130 cm . For each particle size, three tubes containing the similar amount of food , and we hypothesize that ants transport more food at the lower part of the tree. Twelve Supplementary materials


Francisella noatunensis subsp. orientalis (Fno) is the etiological agent of francisellosis in cultured warm water fish, such as tilapia. Antibiotics are administered to treat the disease but a better understanding of Fno infection biology will inform improved treatment and prevention measures. However, studies with native hosts are costly and considerable benefits would derive from access to a practical alternative host. Here, larvae of Galleria mellonella were assessed for suitability to study Fno virulence.Fno in a dose-dependent manner but the insects could be rescued from lethal doses of bacteria by antibiotic therapy. Infection progression was assessed by histopathology  and enumeration of bacteria recovered from the larval haemolymph on selective agar. Fno was phagocytosed and could survive intracellularly, which is consistent with observations in fish. Virulence of five Fno isolates showed strong agreement between G. mellonella and red Nile tilapia hosts.Larvae were killed by G. mellonella, can be applied to understand Fno infections, which will assist efforts to identify solutions to piscine francisellosis thus securing the livelihoods of tilapia farmers worldwide and ensuring the production of this important food source.This study shows that an alternative host,  Francisella noatunensis, with F. noatunensis subsp. orientalis (Fno) infecting warm water fish and F. noatunensis subsp. noatunensis (Fnn) affecting cold water species [Francisella spp. pathogens, F. noatunensis survives and replicates in host cells, particularly phagocytes such as monocytes, macrophages, neutrophils and phagocytic B-cells [Fno is a particular concern for tilapia producers as it can cause up to 95% mortality [Piscine francisellosis is a global disease caused by the bacterium  species . Once in B-cells –11. Fno ortality , 13 and ortality .Fno and a deeper fundamental understanding of virulence and pathogenicity may inform new and improved treatments, prevention measures and farm management practices. To this end, experimental studies have been performed in the native fish hosts and, though these trials have extended our knowledge of francisellosis, such an approach is costly, requires specialist infrastructure such as aquaria, raises ethical questions and can be constrained by legal statutes. Moreover, a lack of access to animals of the right age and size can also impact on these experiments. Hence, more practical alternative hosts that offer insights into the biology of F. noatunensis infections have been explored including zebrafish and their embryos [Relatively little is known of the infection biology of  embryos , 15; how embryos , 17.Dictyostelium discoideum [Tetrahymena thermophile [Caenorhabditis elegans [Artemia franciscana [Galleria mellonella [G. mellonella has considerable practical and biological benefits [G. mellonella and vertebrate hosts [Vibrio anguillarum isolates correlated strongly between the native Atlantic salmon  host and the G. mellonella alternative host [G. mellonella genome has been sequenced which allows for even better understanding of host-pathogen interactions at the molecular level and can further the interpretation of findings with greater biological relevance [in vivo when evading host defences and exploiting host tissues through conserved mechanisms of virulence, including cell adhesion and invasion, antioxidant protection measures, metal ion uptake, secretion systems, and toxin and enzyme production [G. mellonella has been used as an alternative host to understand infections by other Francisella spp., including the human pathogens Francisella hispaniensis [Francisella novicida [Francisella philomiragia [Francisella tularensis [As a result, non-vertebrate alternative hosts have been pursued as a way to study bacterial pathogens of fish, and this has led to investigations in the slime mould amoeba scoideum , 19, thermophile , the nem elegans , the crunciscana , and thellonella . Of thesbenefits , 25, white hosts , 27. Indive host . The insive host , 29. Fisive host –35. Moreelevance . Importaoduction –39. Of naniensis , Francisnovicida , Francisomiragia  and Franlarensis , 43.G. mellonella may prove suitable for studying the virulence and pathogenicity of Fno; however, first it is necessary to confirm that an infection occurs and virulence reflects that observed in the native host, including with respect to conserved mechanisms of virulence. Thus, the aim of the present study was to assess the suitability of G. mellonella as an alternative model for studying the virulence and pathogenicity of Fno.Therefore, G. mellonella larvae after injection with ca. 1 × 109 colony-forming units (CFU)/mL of Fno STIR-GUS-F2f7 , in each case there was a dose-dependent reduction in larval survival, with injection of greater CFU/mL causing greater reductions in larval survival , survival of larvae injected with sterile culture filtrate led to reductions in survival similar to injection with ca. 1 × 108 CFU/mL of live bacteria  supplemented to 10% bovine haemoglobin solution  to give CHAH medium and containing for this experiment 1 mg/L penicillin and 1 mg/L amphotericin B. While Fno CFU in the haemolymph reduced during the 264 h incubation for both isolates, it was the less virulent Fno Ehime-1 isolate that reduced in abundance more quickly than the Fno STIR-GUS-F2f7 isolate; indeed, Fno Ehime-1 was not detected at or after 192 h  were treated with three doses of tetracycline  and in each case antibiotic treatment led to significant (p < 0.05) increases in larval survival, indicating that infections leading to mortalities could be prevented through antimicrobial therapy  when performed with polyclonal anti-Fnn NCIMB 14265 antibodies that cross-react with Fno .In a final experiment, the virulence of each of four Fno and relatively little is known of the infection biology of this bacterium, with efforts towards improved understanding hampered by difficulties associated with performing fish trials, including cost, legislative burden and ethical acceptability. Non-vertebrate alternative hosts offer solutions to many of these problems and can deliver valuable insights in host-pathogen interactions given the similarities in host innate immunity due to the universal ancestry of all organisms. G. mellonella is an alternative host used widely for understanding virulence and pathogenicity of bacterial pathogens, including those causing disease in fish [G. mellonella would be suitable for studying Fno infections.Francisellosis is an emerging bacterial disease in tilapia farming caused by  in fish , due to  in fish , 44. HenFno into the G. mellonella larva appeared to cause infection, as evidenced by the far greater mortalities caused by living bacteria compared to heat-killed counterparts; the dose-dependent increase in mortalities caused by greater doses of Fno; and significant improvement in larval survival after treatment with an antibiotic to which Fno was susceptible. Antibiotic therapy kills or inhibits the replication of the bacterium and allows the host immune system to counter successfully this microbial invasion. Importantly, there was good correlation in the relative virulence of four Fno isolates between G. mellonella and O. niloticus hosts, although Fno STIR-GUS-F2f7 did differ in virulence between the two hosts. This finding suggests similar virulence factors are involved in insect and fish infections, though this needs to be confirmed by further approaches such as testing of knockout strains, and these investigations might uncover the reason for the discrepancy between hosts in the virulence of Fno STIR-GUS-F2f7. Still, Fno STIR-GUS-F2f7 was detected intracellularly in G. mellonella, as observed with other Francisella spp. in this insect model [G. mellonella to be a suitable alternative host for studying Fno infections. The progression of Fno infection in G. mellonella is similar in nature to previous findings where these larvae have been inoculated with other pathogens, with evidence of non-host recognition, an inflammatory response, the formation of melanised nodules and tissue necrosis [Fno were recognised by the insect and mounted an immune response, as evidenced by the darkening of the larval colour shortly after injection due to activation of prophenoloxidase pathway leading to melanisation [Direct injection of ct model , and thict model , 11, 45,necrosis –49. Heatnisation . MortaliFno appeared to cause greatest mortality in G. mellonella at 28 °C, which is close to the optimum for replication of this bacterium in vitro [Fno typically causes infections in tilapia when the water temperature reduces and this becomes more prevalent below 25 °C, probably due to increased host stress [G. mellonella typically lives around 28 °C in its natural environment and thus is probably in better physiological condition [G. mellonella model provides an opportunity to investigate the temperature-regulation of virulence factor expression in Fno more ethically, as it can be incubated at a range of temperatures [in vitro , 51. In t stress , which mondition . Nevertheratures , 25 and eratures .Fno infection, though oxidative stress response proteins , type-4 pili, iron sequestration mechanisms and a type-VI secretion system have been detected in the Fno genome, all of which are key virulence factors for other pathogenic Francisella spp. [Fno infection and certainly G. mellonella lends itself to high-throughput screens, which are often necessary for these types of studies. Moreover, the detection of lethal activity in sterile culture filtrates in this present study suggests the presence of extracellular virulence factors, such as toxins or degradative enzymes, and fish-pathogenic Francisella spp. do produce outer-membrane vesicles embedded with virulence factors [G. mellonella system would be useful for determining the relative virulence of isolates, as such information is useful for identifying particularly problematic  strains.Relatively little is known of the key virulence factors involved in lla spp. , 55–57.  factors , 58. BeyFno did not appear to replicate, which is in contrast to a previous study on V. anguillarum where more virulent isolates replicated inside the haemolymph to a far greater extent than less virulent isolates [V. anguillarum was confirmed to be mostly in the haemolymph compared to the rest of the body, it was not possible to obtain Fno CFU counts for whole-larva homogenate, due to the difficulty of selecting for this fastidious bacterium against the abundant bacteria found on the larva surface and in the gastrointestinal tract (data not shown). Nevertheless, the histopathological analyses appeared to support replication of Fno inside the G. mellonella as the bacteria appeared to become more abundant during the progression of the infection, though the Fno were increasingly detected inside haemocytes, in tissues including the fat body, or in aggregates in the haemolymph, all of which might explain the lower than expected Fno CFU counts in the haemolymph when plated on agar. Indeed, the protocol in this present study was refined to lyse the host cells in the haemolymph because this increased Fno CFU abundance (data not shown), likely by releasing the bacterium from the confines of host cells. Hence, agar counts are probably not a true representation of Fno replication in the larvae and quantification of Fno abundance during infection from pathology preparations, or by molecular methods such as quantitative PCR, likely offer more accurate estimates.Interestingly, when haemolymph was collected from inoculated larvae and plated on to agar, isolates . Howeverisolates , where VG. mellonella to be a useful model for studying infections caused by Fno and thus can be applied to increase our understanding of the virulence and pathogenicity of this pathogen. Such an approach will support efforts towards solutions that prevent and reduce outbreaks of francisellosis in tilapia and improve production in this important industry.In conclusion, this present study demonstrates 2PO4.2H2O , 2.56 g Na2HPO4.2H2O  and 8.77 g NaCl. Culture media, PBS and water were sterilised by autoclaving at 121 °C for at least 15 min. Antibiotic solutions were sterilised by passing through a sterile polyethersulfone 0.22-μm filter .Unless stated, all reagents were sourced from Sigma Aldrich Ltd. , while solvents were purchased from Thermo Fisher Scientific . One-litre of PBS  consisted 0.876 g NaHFno were collected from separate outbreaks of francisellosis: Austria , and STIR-GUS-F2f7  supplemented with 0.1% glucose and 2% IsoVitaleX (Becton Dickinson BBL). Glycerol stocks (20%) were prepared for long-term storage at − 70 °C. The bacterial isolates were confirmed as Fno according to the methods described by Frerichs and Millar [Five isolates of cichlids ), Ehime- in 2001 ), Franc- in 2012 ), PQ1104d Millar , includiG. mellonella larvae were purchased from UK Waxworms Ltd. . Moribund, discoloured and dead larvae were removed and only those with uniform cream colouration and of 250–350 mg were used for experiments. Routinely, larvae were kept in the dark in Petri dishes at 4 °C and used within one week of receipt.Final instar stage Fno were inoculated into 15 mL supplemented MHB and cultured for 20 h at 150 rpm to mid-logarithmic phase of growth. Bacterial cells were harvested by centrifugation  and then washed twice by resuspension in 10 mL PBS, before finally re-suspending in 15 mL PBS. Cell density was determined by measuring absorbance at 600 nm (A600) using a spectrophotometer  and then adjusted by dilution with PBS to the desired CFU/mL according to a standard curve (data not shown). Typically, serial 10-fold dilutions of bacterial suspensions in PBS were plated on CHAH (6× 20 μL of each dilution) to determine accurate CFU/mL after incubation , or for fish trials by the drop plate method as described by Chen et al. [A few colonies of n et al. .G. mellonella larvae experiments were performed in a bacteriological laboratory according to the methods described by McMillan et al. [n = 24, with mean percentage group survival calculated prior to preparing Kaplan-Meier plots. Two control groups were included in each experiment: one group of G. mellonella larvae received injections of ‘PBS only’ to assess the impact of physical trauma, while a second ‘unmanipulated’ group received no injections and was used to assess background larval mortality.n et al. . BrieflyG. mellonella larvae were injected with a ca. 1 × 109 CFU/mL suspension of Fno STIR-GUS-F2f7 and incubated at 15, 22, 25, 28 or 37 °C for 264 h to determine the effect of temperature on larval survival after injection with live Fno. This experiment was performed only once.Groups of G. mellonella larvae were injected separately with ca. 1 × 108, 5 × 108, 1 × 109 or 5 × 109 CFU/mL suspensions of each of the Fno isolates. In addition, the supernatant from the first centrifugation step to harvest the Fno cells  was removed aseptically with sterile scissors and harvesting the haemolymph according to McMillan et al. [Fno and against other bacteria. Still, primary identification tests  and end of the experiment.To assess the abundance of n et al. . The haen et al. ). Ten-fosts see ‘’ were peG. mellonella larvae from lethal doses of each of the Fno isolates (ca. 1 × 109 CFU/mL), sterile-filtered tetracycline in PBS (ca. 10 mg/g body weight) was administered by injection at 2, 24, and 48 h post-infection. Each Fno isolate had been shown to be highly susceptible to the action of tetracycline by disk diffusion (data not shown). In addition to the PBS injected and unmanipulated controls, two extra control groups were prepared: one group of G. mellonella larvae was injected with PBS instead of bacteria and then with tetracycline (to assess the toxicity of the antibiotic), and another group was injected with bacteria and then with PBS instead of tetracycline (to confirm the virulence of the Fno). The multiple injections were administered to different prolegs, as described in Desbois and Coote [To assess whether antibiotic therapy would rescue nd Coote .Fno STIR-GUS-F2f7 in the G. mellonella larvae tissues, 20 G. mellonella larvae were injected with 1 × 109 CFU/mL and incubated as above. Three larva were sampled at 48, 72 and 96 h for histopathological analyses by haematoxylin and eosin (H & E) staining, Gram Twort staining and IHC. Each larva was anesthetized on ice for at least 30 min, injected with ca. 100 μL of 10% (v/v) neutral-buffered formalin and then kept in this solution for 24 h at 4 °C to fix the internal organs and block melanisation [To observe the progression of infection and localise nisation ; unmanipTissue sections were prepared from whole larvae that were dissected transverse to the median plane of the body into six equal sections using a scalpel , and then each section was wrapped in biopsy tissue paper prior to placing into standard tissue cassettes for processing overnight on a processor  and subsequent embedding in paraffin wax . The procedure was performed carefully to avoid squeezing the larval tissues. Each wax block was trimmed with a microtome  to expose the tissue and soaked in water for 30 min prior to cutting. Four micrometer-thick sections were mounted onto glass slides  and dried in an oven overnight at 60 °C. Then the sections were deparaffinised in xylene for 3 min then 2 min (twice), rehydrated in absolute ethanol (2 min) and methylated spirit (1 min), before rinsing in tap water (1 min).Sections were stained with Mayer’s haematoxylin ‘Z’ stain  for 5 min and then rinsed in tap water. Next, the sections were dipped three times in 1% acid alcohol , rinsed in tap water, counterstained with eosin solution  before rinsing again in tap water. The slides were dehydrated in absolute ethanol for 2 min then 1 min (twice), before being cleared with xylene (5 min) and mounted with Pertex medium . Once dry, the slides were examined using an upright light microscope  and images were collected with a digital camera  and cellSens 1.17 software (Olympus).Sections were stained with 2% Lillie’s crystal violet solution  for 1 min and then rinsed in running tap water. The slides were treated with 0.4% Lugol’s iodine solution  for 1 min, before being rinsed in tap water, and flooded with acetone (Thermo Fisher Scientific) for 2–5 s. The slides were rinsed again in running water and counterstained with Twort’s stain in a closed Wheaton Coplin staining jar  for 5 min. Five hundred millilitres of Twort’s stain consisted 100 mg of neutral red and 900 mg of 0.2% fast green (Thermo Fisher Scientific) in 95% ethanol, with a working solution prepared by diluting this stock solution in distilled water (1:3). After staining, the slide was rinsed in tap water. Finally, each section was rapidly dehydrated by dipping twice in absolute ethanol for 5 s each time, and then cleared, mounted and examined as described in Section 5.10.2.Fno in larvae tissues using polyclonal anti-Fnn NCIMB 14265 antibodies that cross-react with Fno. First, sections were pre-treated with 3% (v/v) hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity and then washed thrice in PBS. All incubations were performed in a humidified chamber at room temperature (ca. 22 °C). Non-specific binding of the secondary antibody was blocked by incubation with normal goat serum  diluted 1:10 in PBS for 15 min. The serum was discarded, slides tapped dry and then rabbit antisera containing the primary antibodies (diluted 1:300 in PBS) was added to the slides and incubated for 1 h (PBS was added in place of antisera in a negative control). The slides were washed with PBS and then goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase conjugate  was added for 30 min. Slides were washed in PBS and incubated with the Immpact DAB peroxidase substrate  for 10 min, before the reaction was stopped by immersion in tap water. The slides were counterstained with Mayer’s haematoxylin for 4 min, rinsed in tap water, dehydrated in a graded series of ethanol (70% followed by 100% for 5 min each) and cleared in xylene (5 mins twice) prior to adding a coverslip with Pertex mounting media. Slides were examined by light microscopy.IHC was performed to localise O. niloticus) of 10 ± 0.5 g and 7.0 ± 0.19 cm were purchased from a private farm in Prachinburi, Thailand and transported to the research aquarium of Fish Vet Group Asia Ltd. (FVGAL), Chonburi, Thailand. Upon arrival, the fish were transferred to 100-L circular tanks in a recirculation system for acclimation. Water conditions were maintained as follows: 28 ± 1 °C; 6.5–7 mg/L dissolved oxygen; pH 7–7.5; ≤0.1 mg/L free ammonia; ≤0.25 mg/L nitrite; and ≤ 0.2 mg/L nitrate. Fish were acclimated for 2 weeks and fed at 3% body weight per day with a commercial tilapia feed . The Fno-free status of the fish was determined prior to challenge using samples of spleen and head kidney from four fish by bacteriological analyses and a Francisella genus-specific PCR performed as described previously [Red Nile tilapia (eviously .Fno isolate was passaged two times in three fish (each fish was ca. 20 g) by intraperitoneal (i.p.) injection of 1010 CFU/fish in PBS after being anaesthetised , and then incubated for 4 d at 23 ± 2 °C. As Fno Ehime-1 did not cause any mortalities in either passage it was not included in the subsequent challenge trial (data not shown). Ten fish were allocated randomly into each of 40 3-L tanks containing 2.5 L of dechlorinated water and fish were not fed for 48 h prior to the Fno challenge. Tanks were divided into four main groups (one for each Fno isolate) and five sub-groups of duplicate tanks. Then the fish in each sub-group (n = 20) were i.p. injected separately with 100 μL of ca. 1 × 104, 1 × 105, 1 × 106, 1 × 107 or 1 × 108 CFU/mL in PBS of each of the Fno isolates, with the bacterial inoculums prepared according to Section 5.4. A further two tanks with 10 fish in each contained the controls that received an injection of PBS only. Fish were maintained for 20 d at 23 ± 2 °C, fed ad libitum and examined four times per day for mortalities. To confirm recovery of Fno from dead and moribund fish, these animals were removed and tissues collected (including the head kidney and spleen) for: i) direct PCR with Francisella genus-specific primers  isp-value of < 0.05 was considered to indicate a significant difference. Relative virulence of the Fno isolates in O. niloticus and G. mellonella models of infection were calculated using area under the curve of cumulative survival of each group of injected larvae and fish  were calculated and standard deviations determined.Where required, survival differences between groups were compared with the logrank test in Prism  and a n et al. ). For CF


Over the last decade, damage control surgery (DCS) has been emerging as a feasible alternative for the management of patients with abdominal infection and sepsis. So far, there is no consensus about the role of DCS for acute perforated diverticulitis. In this study, we present the outcome of a multi-institutional series of patients presenting with Hinchey's grade III and IV diverticulitis managed by DCS.All the participating centers were tertiary referral hospitals. A total of 34 patients with perforated diverticulitis treated with DCS during the period 2011–2017 were included in the study. During the first laparotomy, a limited resection of the diseased segment was performed followed by lavage and use of negative pressure wound therapy (NPWT). After 24/48 h of resuscitation, patients returned to the operating room for a second look. Mortality, morbidity, and restoration of bowel continuity were the primary outcomes of the study.2 (SD ± 3.33). Thirteen cases (38%) were Wasvary’s modified Hinchey's stage III, and 21 cases (62%) Hinchey's stage IV. Mean Mannheim Peritonitis Index (MPI) was 25.12 (SD ± 6.28). In 22 patients (65%), ASA score was ≥ grade III. Twenty-four patients (71%) had restoration of bowel continuity, while 10 (29%) patients had an end colostomy (Hartmann’s procedure). Three of these patients received a temporary loop ileostomy. One patient had an anastomotic leak. Mortality rate was 12%. Mean length of hospital stay was 21.9 days. At multivariate analysis, male gender (p = 0.010) and MPI (p = 0.034) correlated with a high percentage of Hartmann’s procedures.There were 15 males (44%) and 19 females (56%) with a mean age of 66.9 years (SD ± 12.7). Mean BMI was 28.42 kg/mDCS is a feasible procedure for patients with generalized peritonitis secondary to perforated diverticulitis, and it appears to be related to a higher rate of bowel reconstruction. Due to the open abdomen, stay in ICU with prolonged mechanical ventilation is required, but these aggressive measures may be needed by most patients undergoing surgery for perforated diverticulitis, whatever the procedure is done. Despite the ongoing progress for resuscitation of septic patients, Hartmann’s procedure (HP) is still considered the safest treatment for generalized peritonitis secondary to complicated acute diverticulitis (Hinchey’s classification stages III and IV) –3. HowevThe charts of a series of patients who underwent DCS for purulent or fecal diverticular peritonitis in four Italian institutions during the period 2011–2017 were evaluated. Participating centers were tertiary care referral hospitals . Diverticulitis was staged according to Wasvary’s modified Hinchey classification . All patAt laparotomy, once the affected bowel segment was resected and the abdominal cavity copiously irrigated with saline solution, the open abdomen (OA) technique was set up by using a negative pressure wound therapy . The decision to restore continuity or create a terminal colostomy was postponed to a second-look operation, 24–48 h later. Occasionally, at the surgeon’s discretion, the OA was maintained following the second-look procedure if the surgical scenario was believed still contaminated and not safe for bowel reconstruction. In such instances, a fascial mesh was placed in order to avoid fascial retraction of the abdominal wall. A defunctioning ileostomy was performed based on the surgeon’s choice when the bowel continuity was re-established. Alternatively, an end colostomy according to Hartmann’s procedure was the choice for those patients in which an intestinal anastomosis was judged unsafe.à la demand and/or on a telephone interview.Data analysis included age, gender, Body Mass Index (BMI), American Society of Anesthesiologists (ASA) score, Wasvary’s modified Hinchey's classification stage III and IV rate, severity of peritonitis according to Mannheim Peritonitis Index , type ofU test was carried out to analyze quantitative variables. Fisher’s exact test was performed to study categorical variables. Univariate analysis of age, male gender, BMI, ASA score > 2, MPI, and Bjӧrck grade at the II look were performed in order the evaluate a statistical difference in patients presenting with end colostomy versus those with an intestinal anastomosis. Any variable with p < 0.2 at univariate analysis was entered in a multivariate backward stepwise logistic regression model with Hartmann’s procedure as a dependent variable. The statistical analysis was performed using the IBM SPSS software package, version 21.0 for Mac OsX.Quantitative data are expressed as mean or median ± standard deviation (SD). The qualitative data were elaborated as frequency and percentage. The Mann-Whitney 2 (SD ± 3.33). ASA score was > 2 in 22 patients (65%). Based on the severity of the disease, 13 cases (38%) were classified as Wasvary’s modified Hinchey's stage III, and 21 cases (62%) as Hinchey's IV. The mean MPI was 25.12 (SD ± 6.28). A NPWT technique for open abdomen was performed in all patients. In seven cases (21%), there was the need for a third look for the persistence of peritoneal contamination: four of these had an intestinal anastomosis eventually. The modified Björck classification at II look were 1A = 27, 1B = 3, 1C = 2, 2A = 0, 2B = 1, 2C = 1, 3A = 0, 3B = 0, and 4 = 0.During the period of study, 34 patients underwent DCS for septic shock secondary to purulent or fecal diverticular peritonitis. Mean age was 66.9 years SD ± 12.7) (Table 2.7 (TablIn 24 cases (71%), the intestinal continuity was restored, and in 3 of these (12%), a protective loop ileostomy was performed. Ten patients had an end colostomy as a result of the DCS. The overall morbidity rate was 41% (14/34). According to the Clavien and Dindo classification, there were 0 grade I (0%), 6 grade II (18%), 6 grade III (18%), and 2 grade IV (6%). In three cases (9%), a re-operation was needed: in one case for an anastomotic leak and two cases for dehiscence of the laparotomy wound. In the patient with the bowel leak, the anastomosis was dismantled, and an end colostomy was warranted.p = 0.067; odds ratio 3.067 [95% CI 0.555–16.935]). All deaths were related to the multiorgan failure. Mean follow-up was 47 months (SD ± 35.67); all patients with loop ileostomy underwent closure of ileostomy. None of the patients presenting with an end colostomy had a Hartmann’s reversal. So far, the incisional hernia developed in 8 out 30 patients (27%) seen on our patient visit. No recurrence of diverticulitis episodes occurred.The mean ICU length was 13.98 days (SD ± 13.47), and the mean postoperative stay was 21.9 days (SD ± 16.24). Four deaths (12%) were recorded: three patients following a Hartmann’s procedure and one after bowel anastomosis (p = 0.010) and MPI (p = 0.034) correlate with a high percentage of Hartmann’s procedures  and PA (4%) and that morbidity for both resection and stoma reversal operations was comparable (39% HP vs. 44% PA). Furthermore, they found that at 18 months, 96% of PA patients with loop ileostomy and 65% of HP patients had a stoma reversal. Thus, they concluded that PA with diverting ileostomy is preferable respect to HP in patients with diverticular peritonitis . CPrimary anastomosis has been widely accepted as an alternative to HP with comparable mortality and a low risk of anastomotic leakage –23. The The recent World Society of Emergency Surgery guidelines about the management of intra-abdominal sepsis state that there is insufficient evidence to advocate for DCS as a general strategy in patients with secondary peritonitis (Recommendation 1 C). On the other hand, even though with a low grade of recommendation (Recommendation 2 C), the same guidelines suggest that DCS may be an option in selected significantly physiologically deranged patients with ongoing sepsis .. They analyzed 15 patients who underwent DCS with lavage, limited resection of the diseased colonic segment, VAC, and second-look operation; 9 of them had intestinal continuity restored during a second-look operation, with 1 patient developing anastomotic leakage. More recently, Sohn et al. reported on 58 patients undergone DCS for Hinchey III (81%) and Hinchey IV (19%) acute diverticulitis; they recorded a secondary bowel restoration rate of 83% with a 24% of loop ileostomy rate. Ten percent of their patients experienced an anastomotic leak that eventually required the creation of a stoma. At the end of the hospital stay, they scored a 50% rate of patients without a stoma [Only a few case series focusing on DCS for perforated diverticulitis are so far reported. Perathoner et al. were the first to describe the combination of emergency laparotomy with the application of abdominal vacuum-assisted closure (V.A.C.®) to treat patients with generalized peritonitis in 2010 . They an a stoma . Kafka-R a stoma .A careful selection of patients that may have benefited from DCS is mandatory in order to avoid overtreatment. It is evident that OA with multiple steps surgery is a risky and costly procedure, and for these reasons, it should not be carried out to merely restore bowel continuity. The primary target of the procedure should be to save lives while to avoid a stoma comes as a reasonable consequence of the first. In our study, 68% of all patients were discharged with a colonic anastomosis. The mortality rate was 12%, slightly higher than data reported in the literature (9–9.8%) , 26. In Moreover, a dynamic fascial closure and early removal of the NPWT system allowed for primary closure of the abdominal wall in all patients, although 27% of patients developed an incisional hernia in the follow-up. Anastomotic leak occurred in one patient (3%), and an end colostomy managed it at re-operation. At the multivariate analysis, male gender and MPI turned out to be the predictive factors for an end colostomy creation at the II look.We are fully aware of several limitations affecting the present study. Firstly, it has a relatively low number of enrolled patients, and it cannot be excluded a bias in patients’ selection. Moreover, the study is retrospective, and it encompasses a quite large period. Finally, reliable criteria backing the decision to restore bowel continuity or to bail out with an end colostomy at the time of second look are lacking.Damage control strategy represents a feasible strategy for patients with generalized peritonitis secondary to perforated diverticulitis. This surgical approach seems to offer a high rate of bowel reconstruction with less stoma, without increasing mortality and morbidity. It requires a stay in ICU with prolonged mechanical ventilation, a hefty price that however these patients should have paid to septic shock independently from the type of surgery. Further randomized, controlled, and comparative studies are needed to achieve significant conclusions.