The initial test with the CSAC electrode, conducted in a 6 M KOH loaded symmetric supercapacitor, demonstrated an ultrahigh capacitance of 367 F g−1 at a current density of 0.2 A g−1 together with 92.09% retention after 10,000 cycles at 10 A g−1. More impressively, the zinc–ion hybrid supercapacitor using CSAC as a cathode achieves a high-rate capability (153 mAh g−1 at 0.2 A g−1 and 75 mAh g−1 at 10 A g−1), high energy density (134.9 Wh kg−1 at 175 W kg−1), as well as exceptional cycling stability . Such work thus illuminates a new pathway for converting biowaste-derived carbons into materials for ultrahigh-performance energy storge applications.In this research, we successfully produced hierarchical porous activated carbon from biowaste employing one-step KOH activation and applied as ultrahigh-performance supercapacitor electrode materials. The coconut shell-derived activated carbon (CSAC) features a hierarchical porous structure in a honeycomb-like morphology, leading to a high specific surface area (2228 m V) and the capacitance (C) of the supercapacitor can be elevated to increase the energy density (E) in accordance with the formula E = 1/2CV2 [In order to address the escalating demand for energy, continuous advancements have been achieved in the domain of energy conversion and storage devices ,2. Super= 1/2CV2 . General= 1/2CV2 ,8. As a = 1/2CV2 ,10.4 or Zn(CF3SO3)2) for a comparatively high working voltage (~1.8 V in the aqueous electrolyte) [−1) [−1 at 0.2 A g−1 in Zn(CF3SO3)2 electrolyte with 92.2% capacity retention after 10,000 cycles [−1 at 0.1 A g−1 in 2 M ZnSO4 electrolyte [Due to their high surface area and suitable porous structure, which are the primary factors affecting electrochemical energy storage in supercapacitors, hierarchical porous activated carbons (HPACs) have been acknowledged as active electrode materials in varying supercapacitors ,12. Electrolyte) ,27. Therte) [−1) ,29. Leve0 cycles . Chen etctrolyte .Herein, due to the enormous annual production of coconuts (~60 million tons worldwide), coconut shell was chosen in this study as the possible precursor for the large-scale manufacturing of HPAC and commercial application in energy storage devices. The coconut shell-derived carbon activated by KOH (CSAC) we have prepared features a honeycomb-like morphology, a large specific surface area and hierarchical porous structure. These attributes equip the CSAC with a high energy storage performance and enduring cyclability, performing well in both conventional symmetric and hybrid supercapacitors. Our findings underscore the potential of HPAC derived from the coconut shell as an exceptional electrode material for ultrahigh-performance supercapacitors.−1 makes it that the absorbed water in the coconut shell is difficult to be thoroughly released before 100 °C in time. A cliff-like mass decrease of 75.82% is ascribed to the elimination of pyrolysis products via the disintegration of bio-mass molecules at the green middle-temperature range of 150–600 °C. Further carbonization causes the TG curve to steadily diminish and reach a plateau at the last bule high-temperature area between 600 and 900 °C, leaving 15.75% of the original mass. −1 is ascribed to the impurities near carbon atoms, D band around 1355 cm−1 is attributed to the breathing mode of sp2-hybridezed structure units which is active in the presence of defects, Dʹ band positioned around 1490 cm−1 is caused by the defects from stacked graphene layers and G band, located at 1590 cm−1, is connected to the vibration of graphitic sp2-type carbon [DI/GI value (1.50) obtained from CSAC, as assessed by comparing the peak area ratio of D band and G band (DI/GI). In addition, the CSC and CSAC both have the obvious 2D bands around 2700 cm−1, indicating the existence of layer graphene. The amorphous-like structure of the activated carbon derived from coconut shell is shown by the results of the XRD pattern and Raman spectra discussed above.The thermogravimetric curve of the precursor in e carbon ,33. A hiThe SEM and TEM images in 2 physisorption isotherms of CSC and CSAC are presented in 2 g−1. According to IUPAC classification, the adsorption–desorption isotherm of CSC appears to be type-I curve, whereas the CSAC isotherm exhibits type-IV features with a typical H2 hysteresis loop in the relative pressure range of 0.4–1.0. In CSAC, such a hysteresis loop is assigned to a mesopore feature, which is further verified by the pore size distribution. Following KOH activation, there is a noticeable increase in pore volume within the range of 0.4 to 5 nm, as seen in 3 g−1 for CSC to 1.07 and 0.64 cm3 g−1 for CSAC. This enhancement can be primarily ascribed to the activation reaction of KOH with coconut shell at elevated temperatures. This process not only generates a greater number of micropores but also fosters the evolution of these micropores into mesopores. Consequently, such micro-, meso- and macropores characterized by N2 physisorption and SEM together formed the honeycomb-like hierarchical porous architecture, which facilitates swift ion transportation from the electrolyte to the adsorption site on the surface of internal micropores.The Ns and s. High-resolution C 1s spectra exhibit three resolved peaks located at 284.4, 285.7 and 288.5 eV, correspondingly associated with the C−C/C=C (C-1), C−O (C-2) and O−C=O (C-3) groups [s region, three fitted peaks are distinguished at 531.4 eV, 532.6 eV and 533.6 eV, which are credited to quinone oxygen (O-1), phenol groups (O-2) and carboxyl groups (O-3) [2 g−1). Such oxygen groups on the surface effectively hasten the electrochemical storage activities by enabling electron transfer and providing additional pseudocapacitance.The wide-scan XPS survey spectra, as depicted in ) groups . In termps (O-3) . Even th−1 display a quasi-rectangular shape within both the CSC and CSAC electrodes, implying ideal capacitive performance. In addition, CSAC displays a more extensive CV profile area than CSC, indicating a higher specific capacitance achieved. −1, illustrating excellent reproducible capacitive behavior. To delve further into the charge storage kinetics, CV curves of CSC and CSAC were explored at various scan rates, as in the following Equation (1):k1v refers to the current density related to a fast kinetic response, which are primarily surface-dominated, k2v1/2 equals the current density resulting from slow kinetic processes, which is mainly associated with the diffusion of ions. As plotted in −1, respectively. The GCD curves of CSC- and CSAC-based devices, performed at 0.5 A g−1  calculated from the intercept with a vertical coordinate gives 314 F g−1 for CSAC and 87 F g−1 for CSC, respectively. A detailed comparison of tC , EC and pC (pseudocapacitance) are tabulated in The electrochemical performances of pyrolyzed products are initially appraised in the symmetric supercapacitor in 6 M KOH electrolyte at 0–1.0 V. CV profiles a and at .5 A g−1 d, exhibi g−1  of 0.23 Ω as well as charge transfer resistance (ctR) of 0.41 Ω than the CSC-based device. According to the related Bode plots  at the high current density of 10 A g−1 after 10,000 cycles, highlighting superior reversibility. BETS, totalV, Vmicro, sR and ctR) for a clear comparison of the CSC and CSAC samples. The Nyquist diagrams in de plots b and Rande plots c, the re42− (3.79 Å), an aqueous ZHS device (Zn//ZnSO4(aq.)//CSAC) is further assembled to probe into its practical application. 2+ deposition/stripping onto the Zn anode as well as through the anion adsorption/desorption on the surface of the CSAC cathode. Therefore, all electrochemical results reported in 1vk refers to capacitive contribution and k2v1/2 denotes the diffusion contribution. As illustrated in −1. As the scan rate increased to 20 mV s−1, there was a gradual uptick in the capacitive contribution ratio to 82.89%, while the diffusion contribution ratio correspondingly declined to 17.11%. This change validates the capacitive-dominant nature from the CSAC cathode and expedited electrochemical kinetics at a high scan rate. GCD curves were applied to determine the specific capacity at varying current densities  and ctR (10.22 Ω) values. The exceptional electrochemical performance of the CSAC cathode-based ZHS device, in comparison to various ZHS devices as reported in The as-prepared CSAC exhibits a hierarchical porous structure, large specific surface area and high electrical conductivity, which all foster greater acceptability and a more concise transfer pathway for electrolyte ions and electrons. Encouraged by the above remarkable characteristics and the small hydrated radii of SOensities d. Based 0 W kg−1 ,38,39. I−1. The resulting mixture was washed with dilute HCl and deionized water until it reached a pH of 7, and then oven-dried overnight at 60 °C to yield the coconut shell-derived activated carbon (denoted as CSAC). For comparison, the coconut shell-derived carbon (abbreviated as CSC) was also prepared without adding KOH, following the same procedure. Coconut shells were sourced from a local fruit market near Jiujiang University. Initially, these shells were chopped into small fragments and thoroughly cleaned to remove the residual dust. The fragments were then oven-dried at 60 °C for 48 h, after which they were ground into powder. This obtained coconut shell powder was mixed with KOH in a weight ratio of 1:1, and then activated at 800 °C for 2 h under a nitrogen atmosphere, maintaining a heating rate of 2 °C min−1. The compositional phases and crystallographic structure of the carbon samples were examined by the XRD patterns generated by a Bruker Focus D8 Advance diffractometer  with Cu Kα radiation (λ = 1.54 Å). Raman spectra were procured with a Renishaw Inviaspectrometer , using an excitation wavelength of 532 nm, an excitation power of 1.5 mW and an acquisition time of 55 s. The surface structures and morphology characterizations of carbon samples were visualized by an S-4800 field-emission scanning electron microscope  and JEOL JEM-2100 transmission electron microscope , while the coconut shell was observed with an SZ810 optical microscope . The samples were degassed at 200 °C for 2 h, and then tested on a Micromeritics ASAP 2460 analyzer  at −196 °C to obtain the nitrogen physisorption isotherms. The specific surface areas were evaluated from the Brunauer–Emmett–Teller (BET) method, and the pore size distributions were calculated based on the nonlocal density functional theory (DFT) model. The surface chemical compositions were detected by an AXIS Ultra DLD X-ray photoelectron spectrometer  equipped with Al Kα radiation. All samples charging were calibrated using the C 1s peak (284.4 eV) as an internal standard.An STA800 thermogravimetric analyzer  was employed to perform the thermogravimetric analysis of coconut shell from room temperature to 900 °C under nitrogen with a heating rate of 5 °C min−2 active material. Two identical working electrodes were assembled in the 6 M KOH-loaded symmetric supercapacitor, where a piece of filter paper was applied as a separator. The electrochemical measurements, comprising cyclic voltammetry (CV), galvanostatic charge–discharge (GCD) and electrochemical impedance spectroscopies (EIS), were conducted on a coin-type device using a Chenhua electrochemical workstation . The specific capacitance  of the individual electrode in the symmetric supercapacitor was determined based on the CV and GCD curves, according to the following Equations (2) and (3):V (V) is the operation potential window, r (V s−1) is the scan rate, m (g) is the total mass of both electrodes and Δt (s) is the discharging time. i (A) in Equation (2) and I (A) in Equation (3) is the response current and the discharging current, respectively.All electrochemical performances of carbon samples were evaluated using CR2032 coin-type cells. For symmetric supercapacitors, a homogeneous slurry was created by combining the as-prepared carbon, acetylene black and polytetrafluoroethylene (PTFE) in ethanol and N-methyl-2-pyrrolidinone (NMP) solution at a ratio of 8:1:1. The working electrode was then created by pressing the dry slurry under 15 MPa on nickel foam. A single electrode had approximately 2 mg cm4 solution and a glass fiber membrane as the anode, the electrolyte and the separator, respectively. The CSAC cathode electrode included a CSAC powder (80 wt.%), conductive acetylene black (10 wt.%) and poly(vinylidene fluoride) . N-methyl-2-pyrrolidinone (NMP) solution was added to this mixture to form a uniform slurry. This was then coated on stainless steel foil and subsequently dried in a vacuum oven at 60 °C for 24 h. The CV and EIS measurements of ZHS were conducted on a Chenhua electrochemical workstation, while the GCD and long circles tests were evaluated by a CT2001 battery testing system .The ZHSs were also assembled in a CR2032 coin cell, employing zinc foil (12 mm in diameter), 1 M ZnSO−1 was achieved by a CSAC electrode in a 6 M KOH loaded-symmetric supercapacitor, maintaining 92.09% capacity retention even after 10,000 cycles at 10 A g−1. More notably, the ZHSs using CSAC as a cathode achieve a high-rate capability (153 mAh g−1 at 0.2 A g−1 and 75 mAh g−1 at 10 A g−1), high energy density (134.9 Wh kg−1 at 175 W kg−1), as well as outstanding cycling stability . Possessing excellent energy storage performance affirms that coconut shell-derived HPAC is a prospective candidate in ultrahigh-performance supercapacitors for industrial large-scale applications.In summary, we have successfully synthesized a hierarchical porous activated carbon from coconut shells via one-step KOH activation. The resultant CSAC materials feature exceptional properties including a honeycomb-like morphology, hierarchical porous structure, large specific surface area and commendable electrical conductivity. Thanks to these distinct advantages, a CSAC electrode manifests superior electrochemical performances in symmetric and hybrid supercapacitors. An ultrahigh specific capacitance of 367 F g


Strong evidence supports the benefits of exercise for healthy ageing, including reduced risk of neurodegenerative diseases. Recent studies suggested interorgan crosstalk as a key element of systemic adaptive response, however, the role of specific molecules in mediating exercise effects on the human brain are not fully understood. In the present study, we explored the exercise-related regulation of Growth Differentiation Factor 11 (GDF11) in cerebrospinal fluid (CSF) and blood.2max, cycle spiroergometry) and cognitive functions  were evaluated.The samples of serum, plasma and CSF were obtained before and 60min after acute exercise (90min run) from twenty healthy young individuals. Additional serum and plasma samples were collected immediately after run. GDF11 protein content (immunoblotting), body composition , physical fitness  as well as with the exercise-induced change in GDF11 levels in CSF . Indirect measure of blood-brain barrier permeability  tended to positively correlate with CSF/serum GDF11 ratio (p=0.060). CSF levels of GDF11 correlated positively with cognitive functions, including working memory, both before and after run (p<0.05).Running decreased GDF11 protein content in CSF (-20.6%. p=0.046), while GDF11 in plasma and serum were not regulated. Two GDF11-specific antibodies of different origin were used to corroborate this result. Individuals with higher physical fitness displayed greater exercise-induced decrease of GDF11 in CSF than those with lower physical fitness (p=0.025). VORunning-induced down-regulation of the GDF11 protein in the cerebrospinal fluid of healthy young individuals indicates the potential role of GDF11 in the exercise-induced cross-talk between periphery and the brain. ImportaExercise is a powerful physiological stimulus with a potential to prevent and even treat various age-related chronic diseases, enhance resilience against stress and reduce the burden of „diseasome of physical inactivity” . CirculaThe study protocol was described in detail elsewhere . BrieflyDay 1 (run) consisted of a 90 min monitored  run at the intensity of 75-80% maximal heart rate (HRmax), which was preceded by cognitive testing (computerized cognitive tests CogState & Memtrax). Blood was collected before, immediately after and 60 min after the 90 min lasting outdoor run. Cerebrospinal fluid (CSF) was collected 60min after the run by atraumatic lumbar puncture. During the run, each participant was provided with a drinking water (~3 dcl) to minimize effects of hemoconcentration on protein levels.Day 2 (baseline/rest) included collection of fasting blood, anthropometric and metabolic  measurements and an atraumatic lumbar puncture to collect CSF.2max by cycle spiroergometry) was performed on a separate day, one week after the Day 1.The assessment of cardiorespiratory fitness  and waist circumference measured at the mid-point between the lower border of the rib cage and the iliac crest. Habitual physical activity profile was evaluated by Baecke questionnaire , resting energy expenditure (REE) and metabolic substrate preference  by indirect calorimetry , blood pressure and heart rate by  and cardiorespiratory fitness by cycle spiroergometry  and cognitive functions by computerized cognitive tests (CogState and Memtrax). Cognitive testing was performed both before and after 90 min run. Body Mass Index (BMI) was calculated . To avoid blood contamination, the first 3 to 5 droplets of clear CSF have not been collected. The samples were immediately centrifuged at 400xg for 10 min at 4°C to remove the contaminating cells, aliquoted, placed on dry ice and stored at −80°C within 1h of collection. Paired CSF samples from both experimental days were obtained from 18 individuals, samples from additional 2 volunteers were taken only on Day 1 (after run). Post-puncture headache was reported in three out of thirty-eight individual punctures (7.9%), with a spontaneous resolution within 1–5 days.Cerebrospinal fluid (5–8 ml) was taken by an atraumatic lumbar puncture technique in the lateral decubitus position using , albumin  and insulin . HOMA-IR was calculated according to formula fasting insulin (mU/l) × fasting glucose (mmol/l)/22.5.Cerebrospinal fluid, plasma and/or serum  were mixed with SDS and DTT containing loading dye, incubated , separated on the 10%-SDS-PAGE and electroblotted to PVDF membrane . After blocking , membranes were incubated overnight with primary antibodies: (i) GDF11 N-terminus specific ,  was performed, with α error probability set to 0.05 and power (1-β error probability) to 0.95. Total sample size of 11 would be needed to determine relevant exercise-induced difference in this parameter. Pearson linear correlation analysis model was used. Values are presented as mean ± SEM and the statistical significance was set at p<0.05. Changes induced by 90 min run were calculated as a fold change (ratio between post-run and pre-run levels).Endurance exercise effect was assessed by either Student’s paired t-test (two-time points) or multiple comparison testing with ANOVA and Tukey post-hoc test (three time-points) using JMP . The characteristics of the study population is presented in 9/l, 0 min post exercise 267 ± 31 109/l, 60 min post-exercise 209 ± 58 109/l, p=0.036), which might reflect hemoconcentration present immediately after run. Interestingly, 90 min run increased lactate in CSF of all volunteers (baseline (resting)) 1.2 ± 0.1 mmol/l, 60 min post exercise 1.9 ± 0.4 mmol/l, p<0.001). Total serum creatinkinase increased immediately after run and compared to baseline remained elevated for another hour (baseline (resting)) 3.2 ± 2.4 umol/l, 0 min post exercise 4.5 ± 3.1 umol/l, 60 min post exercise 4.3 ± 2.9 umol/l, p=0.005).Serum lactate increased immediately after the run and near normalization was observed one hour post-run as compared to baseline (baseline (resting)) 1.1 ± 0.6 mmol/l, 0 min post exercise 3.3 ± 2.3 mmol/l, 60 min post exercise 1.6 ± 1.0 mmol/l; p=0.001). Similar pattern was observed for changes in serum albumin  45.1 ± 3.1 g/l, immediately post-exercise 49.1 ± 2.9 g/l, 60 min post-exercise 45.8 ± 2.7 g/l; p=0.001) and serum thrombocyte count (baseline (resting) 219 ± 43 10An acute bout of exercise led to the reduction of GDF11 levels in CSF, which was confirmed in paired CSF samples with two GDF11-specific antibodies for C and N terminus, respectively  or serum . Plasma and serum GDF11 levels did not differ (p=0.261), and not surprisingly there was a positive correlation between GDF11 in plasma and serum , but not between serum or plasma and CSF levels of GDF11 (data not shown).Neither serum nor plasma GDF11 levels changed immediately after or 60 min after the run (The running-induced changes of GDF11 protein in CSF tended to negatively correlate with changes in its plasma (but not serum) levels .Blood-brain barrier permeability, as indirectly assessed by the ratio of albumin in CSF and serum, was decreased after the 90 min run by 27.1%  as well as in plasma , but positively with GDF11/GDF8 in CSF .2max=47.2 mlO2/kgBW/min, cut off median of VO2max=42.2 mlO2/kgBW/min), individuals with lower physical fitness  displayed greater exercise-induced decrease in CSF levels of GDF11 . However, there was no fitness-related difference in GDF11 protein content in CSF neither at baseline (resting) state nor after run. The link between physical fitness and exercise-induced regulation of GDF11 in CSF was supported also by a positive correlation between running-induced change in GDF11 levels in CSF and VO2max   Figure 3Individuals with higher physical fitness decreased, whereas less fit volunteers rather increased GDF11 serum levels measured 60 min after 90 min run compared to baseline . This observation was further supported by a trend towards negative correlation between exercise-induced change in serum GDF11 and self-reported habitual sport activity . Moreover, baseline GDF11 serum levels positively correlated with physical fitness  Figure 3The pattern of associations between GDF11 levels, clinical & biochemical parameters and their running-induced changes is depicted in the heat map Figure 3Briefly, there were several associations between GDF11 levels in CSF and cognitive functions assessed by sensitive computerized tests. GDF11 levels in CSF were positively associated with psychomotor performance and attention, visual learning, as well as learning and working memory (p<0.05). Visual memory was also associated with CSF levels of GDF11/GDF8 . On the other hand, running-induced change in serum GDF11/GDF8 levels correlated positively with reaction time in two computerized tests , and negatively with learning and working memory .Running-induced change in serum GDF11 was positively associated with body fat (p=0.022) and negatively with muscle mass (p=0.011). Furthermore, it correlated positively with serum insulin immediately after the run (p<0.01) as well as with running-induced change in serum insulin (p=0.024). Running-induced change in GDF11 in CSF correlated negatively with change in serum insulin 60 min after run (p=0.015). Lower serum lactate before run (baseline) was associated with higher GDF11 levels in serum after an acute exercise bout (p<0.05), as well as with a greater running-induced decrease in GDF11 (p=0.01) and GDF11/GDF8 in CSF (p=0.05). Similarly, lower baseline serum creatinkinase was linked with a greater running-induced change in serum GDF11 (p=0.045). Adiponectin, adipokine with a neuroprotective potential which we showed to be regulated by acute intensive exercise , was posAn acute bout of intensive endurance exercise reduced GDF11 levels in CSF of young healthy physically active individuals. Levels of GDF11 in CSF and serum and/or their running-induced changes were associated with body composition, specific metabolic parameters, aerobic physical fitness and cognitive functions. Previous reports suggested neuroprotective effects of GDF11 in the brain  as well Exercise-mediated decrease of GDF11 in the brain, with no change in its blood levels, was previously reported in mice (3-month exercise intervention) . In humaInitial experiments with heterochronic parabiosis proposed GDF11 as an anti-ageing molecule with an ability to foster rejuvenation –6, howevBoth caloric restriction and physical activity are known to preserve or enhance cognitive functions and reduce risk of neurodegenerative disorders in humans , 28, howWe observed physiological changes in serum levels of glucose, insulin and lactate, expected in the population of healthy, young, trained individuals without known metabolic disorder in response to the exercise stimulus . Interestingly, lactate was elevated in CSF of all volunteers one hour after the run (~58%), in contrast to serum lactate which returned to baseline values after an original rapid increase. Thus, dynamic changes of lactate as a marker of exercise exhaustion in serum did not correspond to its changes in CSF. Similarly, there was a distinct exercise-induced dynamics of GDF11 in CSF compared to no change of GDF11 in serum/plasma, the pattern comparable to that of adiponectin, which we have reported previously . These oExercise has been shown as an effective strategy to counteract age-related cognitive decline, but molecular mechanisms underlying neuroprotective effects on human brain remain poorly understood . To elucThe levels of physical fitness was suggested as an important determinant of tissue-specific GDF11 expression, with higher expression found in skeletal muscle, but not in hippocampus, in sedentary old compared to young mice . In our In this work, we confirm in a bigger cohort our previous finding  that bloThe limitations of this report mostly originate from the nature of the study design and include smaller sample size as well as a relative heterogeneity of the study population. Taking into consideration anti-ageing properties of GDF11, it would be intriguing to apply this study design to individuals within different age groups as well as in those already diagnosed with an early phase neurodegenerative disease. The nature of the experimental design and potential health risks imposed by lumbar puncture hampered the possibility to explore dynamic changes of GDF11 levels in CSF (e.g. immediately after or 24 hours after the 90-min run). Further studies exploring the regulation of GDF11 by different exercise modalities and using animal models are warranted. Finally, mechanistic explanation of reported running-induced changes in CSF levels of GDF11 remains only speculative and further research is needed to fully elucidate the putative role of exercise in neuroprotective effects of GDF11 in human brain.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the University Hospital Bratislava. The patients/participants provided their written informed consent to participate in this study.MS performed the clinical study, analyzed and interpreted the data, wrote and revised the manuscript. KM performed immunoblotting, analyzed the data, contributed to the manuscript preparation and editing. MN, NA, performed immunoblotting. IS, ZK, PM, PV obtained CSF samples. JU co-designed and performed the study, contributed to data analysis, interpretation, writing and revision of the manuscript. BU developed the idea, designed, coordinated and performed the study, analyzed & interpreted data and wrote & revised the manuscript. All authors contributed to the article and approved the submitted version.


Aspergillus fumigatus conidia is a necessary sequitur for infection. Germination of conidia starts with the breaking of dormancy, which is initiated by an increase of the cellular perimeter in a process termed isotropic growth. This swelling phase is followed by polarized growth, resulting in the formation of a germ tube. The multinucleate tubular cells exhibit tip growth from the hyphae, after which lateral branches emerge to form the mycelial network. The regulatory mechanisms governing conidial germination are not well defined. In this study, we identified a novel role for the transcription factor SltA in the orchestration of germination and hyphal development. Conidia lacking sltA fail to appropriately regulate isotropic growth and begin to swell earlier and subsequently switch to polarized growth faster. Additionally, hyphal development is distorted in a ∆sltA isolate as hyphae are hyper-branching and wider, and show branching at the apical tip. ∆sltA conidia are more tolerant to cell wall stressors on minimal medium compared to the wild-type (WT) strain. A transcriptome analysis of different stages of early growth was carried out to assess the regulatory role of SltA. Null mutants generated for three of the most dysregulated genes showed rapid germ tube emergence. Distinct from the phenotype observed for ∆sltA, conidia from these strains lacked defects in isotropic growth, but switched to polarized growth faster. Here, we characterize and describe several genes in the regulon of SltA, highlighting the complex nature of germination.Germination of inhaled Aspergillus fumigatus is the main human fungal pathogen causing aspergillosis. For this fungus, azoles are the most commonly used antifungal drugs for treatment of aspergillosis. However, the prevalence of azole resistance is alarmingly increasing and linked with elevated mortality. Germination of conidia is crucial within its asexual life cycle and plays a critical role during the infection in the human host. Precluding germination could be a promising strategy considering the role of germination in Aspergillus spp. pathogenicity. Here, we identify a novel role for SltA in appropriate maintenance of dormancy, germination, and hyphal development. Three genes in the regulon of SltA were also essential for appropriate germination of conidia. With an expanding knowledge of germination and its different morphotypes, more advances can be made toward potential anti-germination targets for therapy. Aspergillus species have a worldwide distribution and grow saprotrophically on a wide variety of dead organic matter but can also act as endophytes (A. sydowii) (A. flavus) (A. niger on onions and hyacinth bulbs)   and grai flavus) , and as h bulbs) . Aspergigillosis . The majumigatus .A. fumigatus produces multinucleate tubular cells termed hyphae. Hyphal tip extension occurs through the synthesis and addition of a new cell wall and membrane via vesicles fusing with the apical plasma membrane  SltA is functionally linked to the control of germination as it has a non-redundant role in controlling emergence from dormancy, germ tube formation and temporal and spacial control of hyphal branching. The ∆sltA strain was recently studied for virulence and showed a reduced capacity to adhere to, invade, and damage pulmonary epithelial cells  was amplified from pAN7.1 and around 1 kb of the 5′ and 3′ flanks from each target gene were amplified. The fusion PCR cassette was used to transform protoplasts as previously described (The MFIG001 strain (WT), a member of the CEA10 laboratory lineage deficient in nonhomologous end joining , was useescribed . The strsltA (AFUB_041100). Primers 041100recP1 and 041100recP2 were used to amplify the AFUB_041100 gene with 50 bp upstream flanking region and the terminator (500 bp), while primers 041100recP3 and 041100recP4 were used to amplify the downstream flanking region (500 bp) of the AFUB_041100 gene from MFIG001 genomic DNA. Primers ptrA_F_linker1 and ptrA_R_linker2 were used to amplify a 2 kb pyrithiamine resistance (ptrA) cassette from plasmid pSK485. The three products were fused together with the nested primers 041100recP5 and 041100recP6 and the common linker sequences on primers 041100recP2 and ptrA_F_linker1, and 041100recP3 and ptrA_R_linker2 using the fusion PCR protocol previously described . The knock-in cassette and RNP complexes were mixed with purified protoplasts, and transformation was carried out in the previously knocked out strain (∆sltA) as described by van Rhijn et al.  assembly method previously described by Al Abdallah et al. , 23. Brin et al. .Validation of the homologous recombination was performed by PCR using primers 041100P5 and 041100recP2, and 041100recP3 and 041100P4. The sequences of all primers and crRNAs used are given in N-morpholino)propanesulfonic acid) to a final concentration of 5 × 105 spores/mL in a 96-well plate, and incubated for 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 16 h at 37°C. Conidia were fixed in 4% formaldehyde, stained with Blankophor , and mounted on a microscope slide for viewing. Blankophor staining was imaged using a Zeiss Axio Scope A1 microscope with an objective EC “Plan-Neofluar” 40x/0.75 M27 lens using 330–385 nm excitation and 420 nm emission. Images were captured with a Zeiss Axiocam 208 color using Zen 3.3 blue edition software. Images were used to calculate the germination rate after 6 h and 8 h of development for the WT and ∆sltA strains. For this, three experiments using three replicates were performed, and a minimum of 100 conidia were scored. Conidia having an emerging germ tube (teardrop shape) were scored as germinated. P-values were calculated using the Mann-Whitney-Wilcoxon Test. Data were tested for equality of variance and normality using the Bartlett’s test and Shapiro-Wilkinson’s test, respectively  slants for 3 d at 37°C, followed by room temperature for 3–7 d. Conidia were harvested with a 0.05% (vol/vol) Tween 20 aqueous solution and filtered through a 40 µm nylon cell strainer . To analyze the germination of the mutant and WT strains, the appropriate amount of conidia was inoculated into liquid RPMI-1640 medium (Gibco Life Technologies) supplemented with 2% glucose, buffered with MOPS  conidia was selected under a binocular, excised with a surgical blade  as small agar (approx 3 × 3 mm) blocks, and transferred to a copper cup for snap-freezing in nitrogen slush. Agar blocks were glued to the copper surface with silicon crease . Samples were examined in a JSM-IT200LV scanning electron microscope  equipped with an Oxford CT1500 Cryostation for cryo-SEM. Electron micrographs were acquired from uncoated frozen samples, or after sputter-coating by means of a gold target for 2–3 times for 1 min. Micrographs of uncoated samples were taken at an acceleration voltage of 2 kV and at 5 kV in case of the coated samples.For the cryo-scanning electron microscopy (cryo-SEM), WT and ∆4 conidia of WT, ∆sltA, and sltArec were inoculated in a 24 well glass bottom plate (Greiner Bio-One) in RPMI-1640. Growth was assessed up to 16 h at 37°C by taking an image every 20 min on a Leica SP8 confocal microscope using a 10x/0.4 NA lens and a 514 nm argon laser. Images were compiled into videos using ImageJ.For the time-lapse live cell imaging, 5 × 103 conidia were inoculated per well in quadruplicate in RPMI-1640, and the 96-well plates were centrifuged for 10 min at 2,500 rpm  to collect them at the bottom of the wells. Objects were scanned every hour for the first 8 h and every 30 min during the next 8 h. Using the UniExplorer software, the area of the conidia was measured together with the circularity of the cells. To select only single conidia, the data were filtered by setting the area threshold between >10 μm2 and <25 μm2 and circularity >1.00. Additionally, non-growing cells were removed by setting the area threshold after 8 h of growth to >25 μm2. In total, between 52 and 107 cells were tracked for each analyzed strain over a growth period of 16 h. Area and circularity after each hour of growth was compared between the strains and P-values were calculated by Kruskal-Wallis’s test and Dunn’s correction  . For each strain, 5 × 10rrection -28. Darrection , 25. Gerrrection . To testsltA, and sltArec) after 12 h and 16 h of growth. For the analysis of the tip splitting phenotype, the distance from newly emerging branch to the apical tip was measured for 75 events during 16 h of growth for each strain. For analysis of the hyphal elongation rate, the length of 20 elongating hyphae was measured between 14 h and 16 h of growth to calculate the elongation speed. For analysis of the hyphal diameter, the width of 50 hyphae was measured at a random location but at least at 20 µm distance from the hyphal tip. P-values were calculated by Kruskal-Wallis’s test and Dunn’s test with Benjamini-Hochberg correction  containing per liter: 10 g glucose, 5.95 g NaNO3, 0.522 g KCl, 1.5 g KH2PO4, 50 mg MgSO4·7H2O, 1 mL trace elements. Trace elements contained (per 200 mL): 10 g EDTA, 4.4 g ZnSO4·7H2O, 1.01 g MnCl2·4H2O, 0.315 g CuSO4·5H2O, 0.22 g (NH4)6Mo7O24·4H2O, 1.0 g Fe(II)SO4·7H2O, and 2.2 g H3BO3 , Congo red (CR), and calcofluor white (CFW) were prepared in dimethyl sulfoxide (DMSO) to a concentration of 3,200 mg/L, 2,000 mg/L, and 2,000 mg/L, respectively. The 96-well plates were prepared with twofold dilutions in RPMI-1640 medium supplemented with 2% glucose and buffered with MOPS. The caspo, CR, and CFW concentrations ranged from 0.016 to 8 mg/L, 2 to 1,000 mg/L, and 2 to 1,000 mg/L, respectively. Conidia were harvested with a 0.05% (vol/vol) Tween 20 aqueous solution, and the suspension was adjusted to 80–82% transmission at 530 nm using a spectrophotometer  to create a suspension of 1–4.2 × 10sltA, and sltArec strain, radial growth was measured as described previously on MM plates supplemented with 0.5 mg/L caspo, 31 mg/L CR, or 31 mg/L CFW in triplicate. Additionally, serial 10-fold dilutions of conidia ranging from 1 × 104 to 1 × 102 cells in a volume of 5 µL were spotted onto MM supplemented with 0.5 mg/L CF, 31 mg/L CR, or 31 mg/L CFW in triplicate. Plates were incubated at 37°C for 48 h and imaged to determine the relative sizes of the colony.To study the effect of cell wall antifungals on the growth of the WT, ∆sltA strains were grown on SDA slants for 4 d at 37°C. For RNA extractions, 1 × 105 spores/mL of both strains were grown in 50 mL of RMPI-1640 medium supplemented with 2% glucose and buffered with MOPS for 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 16 h at 37°C with constant shaking at 150 rpm in triplicate. Liquid cultures were centrifuged for 5 min at 2,000 rpm , RPMI-1640 medium was removed, and pellets were transferred to 1.5 mL tubes containing glass beads, immediately frozen with liquid nitrogen and kept at –70°C until use. Frozen samples were homogenized with a MagNa lyser (Roche) by three times shaking for 30 s at 7,000 speed. One milliliter of TRIzol reagent (Ambion Life Technologies) was added, immediately vortexed for 1 min and incubated at room temperature for 5 min. Two hundred microliters of chloroform was added, vigorously shaken for 15 s, incubated at room temperature for 3 min, and centrifuged at 12,000× G for 15 min at 4 °C (Hettich® MIKRO 220R). Clear supernatant was transferred to a new 1.5 mL centrifuge tube, 200 µL of chloroform was added, vigorously shaken for 15 s, incubated at room temperature for 3 min and centrifuged at 12,000× G for 15 min at 4°C. Clear supernatant was transferred to a new 1.5 mL centrifuge tube, 500 µL of isopropanol was added, and samples were mixed by inversion and incubated for 2 h at –20°C. Next, samples were centrifuged at 12,000× G for 10 min at 4°C, supernatant was removed and pellets were washed with 70% ethanol; 0 h and 2 h pallets were resuspended in 30 µL Diethyl pyrocarbonate (DEPC)-treated H2O, and 4–16 h pallets were resuspended in 50 µL DEPC-treated H2O. The concentration of RNA was measured with a Qubit 4 fluorometer  using RNA High Sensitivity Assay kit, and the quality was assayed with a TapeStation 4150 system (Agilent) using a High Sensitivity Screentape.WT and ∆The mRNA library was constructed using Illumina Stranded mRNA prep protocol according to manufacturer’s instruction (Illumina Inc.). Briefly, RNA samples were purified, fragmented, and used to synthesize cDNA. An adenine (A) nucleotide was added to the 3ʹ ends of the blunted fragments. Next, preindex anchors were ligated to the double-stranded cDNA fragments. Finally, the adapter-ligated fragments were purified using magnetic beads and amplified to add indexes and primer sequences for cluster generation. Sequencing was performed on a NovaSeq6000 with a 2 × 150 bp S1 flow cell (Illumina Inc.).A. fumigatus A1163 from Ensembl release 52) using STAR version 2.7.3a (sltA and WT) and growth phase , isotropic growth (2 h and 4 h samples), polarized growth (6 h and 8 h samples), and early hyphal growth (EHG) (12 h and 16 h samples) to compare transcript levels of ∆sltA to WT in each growth phase. The normalization and dispersion estimations were performed with DESeq2 using the default settings. Differentially expressed genes (DEGs) were identified by filtering the results for each growth phase using the following thresholds: log2 fold change (Log2FC) ≥1 or ≤–1; adjusted P-value ≤0.05 (Benjamini-Hochberg)  .A. fumigatus A1163 annotation using the FungiFun2 2.2.8 Beta web-based tool  enrichment analyses were carried out with the sed tool . DEGs shjustment ), exceptjustment . Manual A. fumigatus at the isotropic growth phase were compared with those from dormant spores or when the polarized growth phase was compared with the isotropic growth phase . Our exploratory microscopic data suggest that SltA is involved in regulating genes involved in germination and hyphal growth. Consequently, the ∆sltA strain was selected for more in-depth analysis of germination and EHG considering its abnormal morphological phenotype.We have previously shown that 13 TFs have enhanced expression  when the transcriptomes of th phase  39). To. ToA. futh phase  by brighth phase  and a masltA, additional germination and growth experiments were performed. Conidia were tracked over a period of 16 h, cell area and loss of circularity (a proxy for germination) were measured, and the data of the first 8 h were used to analyze germination kinetics (see Supplementary files 1 at https://figshare.com/s/a7900b7d20abc2b421a9). ∆sltA conidia started to break dormancy before WT conidia and those from a reconstituted ∆sltA isolate (sltArec) as observed by a significant difference in conidia size after 3 h  , and 1% (95% CI 0.16–5.10) in WT  and sltArec  conidia, respectively. After 6 h, the percentage-germinated ∆sltA conidia increased to 83% (95% CI 70.26–90.62) compared with 33% (95% CI 23.70–44.58) and 30% (95% CI 22.05–39.15) in WT  and sltArec  conidia, respectively. To assess if the conidia of each strain swelled similarly in terms of area increase, we calculated the percentage of swelling per conidium before the germ tube emerged. All strains showed a similar area increase before switching to polarized growth  . ∆sltA cmergence , which wmergence . Fifteend growth . The rapsltA strain (see Supplementary files 2 at https://figshare.com/s/59bae2cf7e50b357cc5e). In ∆sltA, hyphal tips displayed a hyper-branching phenotype with blunted tips , which suggests a role for SltA in maintaining hyphal polarity and normal hyphal elongation. Hyphae of the ∆sltA strain were wider than their WT and sltArec hyphae counterparts  and during isotropic growth , polarized growth , and EHG . Principal component (PC) analysis performed on the transcriptomic data revealed that the largest variance was caused by time with a large change from dormant state to isotropically swelling, and from polarized growth to EHG . The larsltA was compared with the transcriptome of the WT strain during each growth phase , and DEGs were identified  and dormant conidia [gliM (AFUB_075730) and gliG (AFUB_075740)]. In dormant conidia, levels of gliM, gliG, gliA, gliF, and gliT transcripts were vastly higher (10.84- to 98.43-fold) in the ∆sltA strain compared with the WT strain  in the ∆sltA strain compared with the WT strain (The classification ontology showed secondary metabolism was enriched in dormant conidia (99 genes), swelling conidia (42 genes), and during polarized growth (37 genes) in ∆rs (BGC)  (Table Srs (BGC) . ComponeT strain . In isotT strain . HoweversltA conidia start to swell earlier than the WT strain, the expression levels of the genes involved in the signaling pathways associated with the onset of germination were assessed. The alpha subunit gpaB (AFUB_012410) of the heterotrimeric G protein was upregulated in the ∆sltA strain together with the catalytic subunit pkaC (AFUB_027890) of the cAMP-PKA signaling pathway . The average of all Log2FC values per gene were calculated and sorted from smallest to largest; genes were only included when significance thresholds were met for all analyzed growth phases. The top 12 genes with the lowest average expression values are shown in To identify SltA target genes that might be responsible for the early germination and distorted EHG phenotype observed in ∆https://figshare.com/s/a7900b7d20abc2b421a9). We compared the ∆AFUB_035430, ∆AFUB_071880, ∆AFUB_085360, ∆AFUB_084520, and ∆AFUB_099730 strains with the WT strain for area increase and drop of circularity during 8 h of growth , 10% in ∆AFUB_084520 , and 20% in ∆AFUB_099730  compared with 4% in the WT strain (95% CI 1.28–10.45). After 6 h, percentage of germinated conidia in strains ∆AFUB_071880, ∆AFUB_085360, ∆AFUB_084520, and ∆AFUB_099730 increased to 71% , 57% , 76% , and 75% , respectively, compared with 33.75% (95% CI 24.35–44.64) in the WT strain  showed an early onset of isotropic growth and slower growth of fatter germ tubes and early hyphae. Further hyper-branching, meandering growth, and tip splitting were observed. We explored, in detail, the germination and EHG of ∆sltA and the transcriptomic situation during these phases. Previous studies have demonstrated that the C2H2 zinc finger TF SltA functions as a regulator for cation homeostasis  and EHG. One of the null strains   . In anotT strain . However-glucans ; therefosltA strain. The place of vesicle fusion and, therefore, the direction of growth are orchestrated by the position of the Spk, cell end markers, and other proteins involved in the process of exocytosis  showed rapid germ tube emergence compared with the WT strain. The conidia, however, did not start to swell at an earlier time point, but increased less in terms of conidial area before the germ tube emerged. Interestingly, the genes AFUB_084520 and AFUB_099730 are unique to the genus Aspergillus and not present in all species. Both genes are coding for transmembrane proteins, AFUB_084520 encodes a nucleotide sugar transporter, and AFUB_099730 is a very small (78 amino acids) protein containing transmembrane domains. Further research is needed to unravel how these transmembrane proteins play a role in rapid emergence of the germ tube. Taken together, we have identified two different mechanisms for rapid germination in A. fumigatus conidia: conidia started to swell earlier and subsequently switched to polarized growth faster, and conidia swelled at a similar rate as seen in the WT strain, but switched to polarized growth faster.To assess which genes may be involved in the rapid germination and unstable hyphal elongation phenotype, we selected highly downregulated genes and observed the first 16 h of development of five knockout strains. No difference in hyphal development was observed, but three of the selected strains  and EHG. Phenotypic analysis showed rapid swelling of the conidia and rapid germ tube emergence. After germ tube emergence, the elongating hyphae are wider and curlier compared to the WT strain. Additionally, we observed a hyper-branching and splitting of the apical tip phenotype. A transcriptomic analysis of dormant conidia, germinating conidia, and EHG did not show a dysregulation of polar hyphal growth-related genes. The elaborate phenotypic analysis suggests that the Spk in ∆sltA may be unstable in terms of positioning, speed, and behavior of the Spk.In summary, we have screened 13 TF null mutants for their importance during isotropic swelling, polarized growth, and EHG, and were able to identify the C


Lampropedia strains predicted a repertoire of heavy metal resistance genes. However, we are still lack of experimental evidence regarding to heavy metal resistance of Lampropedia, and their potential in mixed heavy metal removal remain elusive.Heavy metal contamination has been a severe worldwide environmental issue. For industrial pollutions, heavy metals rarely exist as singular entities. Hence, researches have increasingly focused on the detrimental effect of mixed heavy metal pollution. Genome analysis of Lampropedia aestuarii strain GYF-1 was isolated from soil samples near steel factory. Heavy metal tolerance assay indicated L. aestuarii GYF-1 possessed minimal inhibition values of 2 mM, 10 mM, 6 mM, 4 mM, 6 mM, 0.8 mM, and 4 mM for CdCl2, K2CrO4, CuCl2, NiCl2, Pb(CH3COO)2, ZnSO4, and FeCl2, respectively. The biosorption assay demonstrated its potential in soil remediation from mixed heavy metal pollution. Next the draft genome of L. aestuarii GYF-1 was obtained and annotated, which revealed strain GYF-1 are abundant in heavy metal resistance genes. Further evaluations on differential gene expressions suggested adaptive mechanisms including increased lipopolysaccharides level and enhanced biofilm formation.In this study, a L. aestuarii GYF-1 exhibited mixed heavy metal resistance, which proven its capability of being a potential candidate strain for industrial biosorption application. Further genome analysis and differential gene expression assay suggest enhanced LPS and biofilm formation contributed to the adaptation of mixed heavy metals.In this study, we demonstrated a newly isolated The online version contains supplementary material available at 10.1186/s12866-023-03093-4. Heavy metal contamination has been a severe worldwide environmental issue due to their toxicity, accumulative, and nonbiodegradable properties. The primary cause of heavy metal pollution has emerged from industrial activities, such as mining, electroplating, paints and pigments, batteries, tanning and textile, steel industries. Use of pesticides, insecticides, fertilizers in agricultural fields have been secondary source of heavy metal contamination . RecentlBacillus sp., Pseudomonas sp., Alcaligenes sp., Rhizopus sp., Sphingomonas sp., Azospira sp. and Cupriavidus sp., etc [Microorganisms have developed various mechanisms for adaptation of mixed heavy metal stress, resulting in an eco-friendly and cost-effective strategy called biosorption. The detoxification of mixed heavy metals includes biosorption via production of extracellular polymeric substances (EPS), efflux of toxic metals by active transporters, intracellular sequestration, surface precipitation, metal reduction. To date, many bacterial groups are considered as potential bioagents for mixed heavy metal removal, such as sp., etc , 6. TherLampropedia spp. is a Gram-negative, Neisser-positive, non-spore forming coccus. L. hyalina was firstly isolated from polluted water sample by Schroeter in 1886 [L. hyalina strains, ATCC 11,041, ATCC 43,383, DSM 15,336 were identified from dairy farm yard [L. hyalina DSM 15,536 was identified as phosphorus removal bacteria because its capability of synthesizing polyphosphate and polyhydroxyalkanoates accumulating bacteria [Lampropedia hyalina might also be resistant to heavy metal stress via polyphosphates-mediated detoxification. Later, researches isolated three other species of Lampropedia, including Lampropedia aestuarii YIM MLB12 from a sediment sample of the Maliao River estuary [Lampropedia puyangensis 2-bin from cankered bark tissue of Populus × euramericana [Lampropedia cohaerens CT6 from arsenic rich microbial mats of a Himalayan hot water spring [L. cohaerens CT6 also indicated a repertoire of heavy metal resistance genes against arsenic, copper, cobalt, zinc, magnesium, and cadmium [Lampropedia, and their potential in mixed heavy metal removal remain elusive. in 1886 . Since tarm yard , and actarm yard , respectbacteria . This sur spring . To note cadmium . HoweverLampropedia aestuarii GYF-1 isolated from soil samples near steel industry. Results demonstrated resistances of L. aestuarii GYF-1 to Cd2+, Cr6+, Cu2+, Ni2+, Pb2+, Zn2+ and Fe2+ as well as its capability of removing mixed heavy metal contamination. Further integrated genomic analysis and relative gene expression results elucidate adaptive mechanisms for reducing mixed heavy metal stress.The present study aimed to evaluate the heavy metal resistance and application value of 6+ and Fe2+ are usually more toxic than Cr3+ and Fe3+. Thus, a stimulated heavy metal medium (SHMM) containing seven heavy metals 2, 80 µM ZnSO4, 100 µM FeSO4) was formulated that mimics the heavy metals that present in the soil digested solution  near steel industry in Liuzhou Guangxi Province, where is biggest steel industrial district of south China. The concentrations of heavy metal were evaluated by AAS . The overnight cultured seeds were inoculated to LB medium supplemented with individual heavy metal at OD600 of 0.05, and incubated at 30oC for 48 h. The MIC values suggested that 2 mM of CdCl2, 8 mM of K2CrO4, 6 mM of CuCl2, 4 mM of NiCl2, 6 mM of Pb(CH3COO)2, 0.8 mM of ZnSO4, or 4 mM FeCl2, respectively, is able to inhibit the growth of strain GYF-1 2, 0.6 mM of ZnSO4, or 2 mM FeCl2, respectively, was selected  are missing, which probably because a few sequence gaps are missing in the L. aestuarii GYF-1 genome.Next, the adaptive mechanisms used by bacteria . Annotatis Table . HoweverL. aestuarii GYF-1 also use this strategy to possess mixed heavy metal adaptation, we first compared LPS level in between control and mixed heavy metal condition , kdsB (GYF_02650), kdsC (GYF_02059) kdsD (GYF_02058) for 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis are upregulated under mixed heavy metal stress   Fig. d, suggesL. aestuarii GYF-1 increased level of lipopolysaccharides and to adaptively respond to mixed heavy metal stress.On the other hand, the up-regulation of the KDO and lipid A biosynthesis was consistent with the higher relatively expressions of components in Type II and Type III secretion pathway, which are probably required for exportation of LPS synthesis-related enzymes Fig. . Taken tL. aestuarii GYF-1 formed clear, thick, and tight biofilm under mixed heavy metal stress compared to the vague, smooth, and loosen shape under normal LB growth  and pgaB (PGA N-deacetylase) were at least 2-fold higher in mixed heavy metal stress that in normal growth; the expressions of two PGA synthase, pgaC and pgaD, were slightly increased but to a lesser extent. In addition, the up-regulated secretion pathway might also contribute to biofilm formation  encodes multicopper polyphenol oxidoreductase laccase was found in Scaffold 1. The copZ is a known encodes cytoplasmic copper chaperone, which was predicted in Scaffold 13 (GYF_00913). A MerR-family transcription factor cueR was predicted in Scaffold 8 (GYF_03485). In addition, GYF_01534 was predicted as a potential copper(I)-binding protein although its function was not annotated in Lampropedia spp. Analysis of nickel transporter predicted many genes encode nikABCDE transporter in GYF-1 genome. This could explain previous finding that strain GYF-1 have high nickel tolerance but with no adsorption in nickel stress, because the main mechanism of nickel resistance used by GYF-1 is via efflux mediated by transporters. A whole nikABCDE gene cluster (GYF_01655–01659) was annotated in Scaffold 2, and partial nik gene clusters were predicted in Scaffold 3, 8, 11, 16, 20, suggest the nik transporter complex in GYF-1 might be different with those classic systems reported in model bacteria. Besides those single heavy metal specific transporter, many multiple heavy metal transporters were also annotated. The czcABC gene cluster (GYF_02595–02597) was annotated in Scaffold 4, which encode heavy metal efflux pump of cobalt, zinc, and cadmium. Two P1B-type ATPase genes zntA (GYF_03486 and GYF_03587) which confer resistance specifically to Pb2+, Zn2+, and Cd2+ were found in Scaffold 8. The ABC-type Mn2+/Zn2+ transport system znu was predicted in Scaffold 5, with two znuB (GYF_02916 and GYF_02917) and one znuC (GYF_02918), suggest the difference of znuABC complex in L. aestuarii GYF-1 from other studied species. The presence of multiple efflux transporters could also explain L aestuarii GYF-1 exhibited high tolerance to individual heavy metal stress as indicated by MIC values  and ppk2 (GYF_02770), are involved in polyphosphate metabolic process. The expression of ppk1 and ppk2 was assessed to evaluate if inorganic polyP contributes to intracellular heavy metal sequestration. The expression of ppk1 was 2.18-fold higher in 1 × SHMM than in control medium, and no significant up-regulation was found with regards to ppk2 expression , as polyanions, are involved in detoxification of heavy metals . Annotation Fig. . This inBiosorption is defined as adsorption of substances by using passive physiochemical pathways, such as electrostatic forces and ion/proton displacement, while bioaccumulation is active metabolic event in which heavy metals are taken up into the cell . In thisL. aestuarii GYF-1 showed biosorption efficiency of greater than 90% in removing Cd2+, Cr6+, and Pb2+, however, relatively low efficiency in adsorbing Cu2+ and Zn2+ and no affinity to Ni2+, individually. The biosorption efficiency is impacted by various factors, such as pH, temperature, initial concentration of the heavy metals, cell density, treatment time, etc [L. aestuarii GYF-1 could be used as biosorbent for bioremediation of soil samples in a lab-scale. Several bioprocess factors also should be taken into considerations when scale-up to industrial level, for example type of bioreactor, pH, and temperature control, mixing and agitation, feeding strategy (in batches or in continuous mode) [For biosorption application, we applied two-round adsorption strategy into removal of mixed heavy metals pollution. ime, etc . Thus, ous mode) , 28.L. aestuarii strain from soil samples near steel factory. Heavy metal tolerance assay indicated L. aestuarii GYF-1 possessed MIC value of 2 mM, 10 mM, 6 mM, 4 mM, 6 mM, 0.8 mM, and 4 mM for CdCl2, K2CrO4, CuCl2, NiCl2, Pb(CH3COO)2, ZnSO4, and FeCl2, respectively. The biosorption assay demonstrated its capacity in bioremediation of soil polluted by mixed heavy metals. Genome analysis revealed abundance of heavy metal resistance genes in the genome of L. aestuarii GYF-1. Further evaluation on differential gene expressions under stress condition suggest enhanced LPS and biofilm formation contributed to the adaptation of mixed heavy metals. This study demonstrated L. aestuarii GYF-1 can be selected as a potential candidate strain for biosorption application.In this study, we isolated a mixed heavy metal resistant For soil sample collection, 10–20 g soils a depth of 10 cm below the surface, and a total of 10 samples were collected using an ordinary shovel. The sampling site was 3-km away from a steel factory in Liuzhou, Guangxi Province, South China. Each sampling site was spaced at least 500 m apart.To determine the heavy metal concentration, the collected sludge samples were treated according to GB 15,618 − 2018 . Briefly2, 25 µM K2CrO4, 20 µM CuSO4, 15 µM NiCl2, 15 µM Pb(CH3COO)2, 80 µM ZnSO4, and 100 µM FeSO4.A stimulated heavy metal medium (SHMM) containing seven heavy metals was formulated that mimics the heavy metals that present in the soil digested solution. Thus, 1 × SHMM contains basal LB medium and supplemented with 0.2 µM CdCl2O, vortexed at room temperature (RT) for 10 min. One microliter of suspension was used for 10-fold serial dilution, and spread 1 ml of each diluted gradients on 0.1 × SHMM agar plate. The plates were aerobically incubated at either 30oC or 37oC for 48 h, and then individual colony was purified on 0.1 × SHMM agar plate.For isolation of heavy metal resistant bacteria, 0.5 g of sieved soil sample was mixed with 25 ml sterile ddHoC for indicated time. The samples were collected, cell pellets were removed by centrifugation at 7,000 rpm for 5 min. The supernatant was digested using HNO3 to oxidize to a single high-valence state or converted into inorganic compounds, and digest was diluted to an appropriate concentration. The metal concentrations were determined by using Agilent AA 240FS (Agilent). The parameters including air flow, acetylene flow, characteristic wavelength, slit width, lamp current were according to the manufacturer’s instructions. The removal of heavy metal was equal to the initial concentration of heavy metal in the medium subtracted by the residual heavy metal concentration in the supernatant.The biosorption of heavy metals was determined by using atomic absorption spectroscopy. The overnight cultured bacteria were inoculated in 100 mL of LB broth supplemented with individual heavy metal or with 1 × SHMM at 30The isolate was cultured in Luria-Bertani (LB) medium to observe the colony morphology, and cell shape was microscopically checked. Colony PCR was directly used for amplification of 16S rDNA by universal primers (8F: 5’-AGA GTT TGA TCC TGG CTC AG-3’ and 1492R: 5’-CGG TTA CCT TGT TAC GAC TT-3’). The PCR fragments were electrophorized, sent for Sanger sequencing , and blasted on NCBI website. For phylogenetic analysis, the 16 S rDNA sequences of close bacterial strains were clustered by ClustalW , and theThe genomic DNA of GYF-1 was prepared by using Quick-DNA Kits (ZymoResearch). The genomic DNA was quantified by Qubit dsDNA HS Assay Kit (ThermoFisher). The 500 ng of genomic DNA was used for DNA library preparation by using NEB Next Ultra DNA Library Prep Kit for Illumina. The quality of the DNA library with length of ~ 500 bp was electrophoresed and quantified by Thermo Qubit 4.0 (ThermoFisher). Sequencing was performed on Illumina HiSeq 4000 platform with 150 bp paired-end mode.in silico DNA-DNA hybridization (DDH) using Genome-to-Genome Distance Calculator (GGDC) [The acquired sequence reads were subjected to quality filtering and de novo assembly using SPAdes 3.5.0 . The gapr (GGDC)  was perfFor genome annotation, the coding genes and non-coding RNAs were predicted by Prokka 1.10 , interspL. aestuarii GYF-1 were tested for heavy metals, including CdCl2, CrCl3, K2CrO4, CuSO4, NiCl2, Pb(CH3COO)2, and ZnSO4. The LB medium supplemented with 0.1 M of individual heavy metal (pH was adjusted either by NaOH or HCl to 6.8) was diluted to indicated concentration. The overnight culture broth was inoculated to final OD600 of 0.05. The strain was considered tolerant to a particular heavy metal concentration if OD600 higher than 0.2 at 30 °C for 48 h incubation. The MIC was defined as the lowest concentration of a heavy metal inhibiting bacterial growth for 2 days.The minimum inhibitory concentration (MIC) values of L. aestuarii GYF-1, LB medium was supplemented with 0.8 mM CdCl2, 2 mM CrCl3, 2mM K2CrO4, 2mM CuSO4, 2mM NiCl2, 6 mM Pb(CH3COO)2, or 0.6 mM ZnSO4, respectively. The pH of the LB-heavy metal medium was adjusted to 6.8 either by NaOH or HCl. For biosorption assay, 100 ml of medium was inoculated with overnight culture to OD600 of 0.05, and incubated at 30oC with shaking. Every 12 h, 5 ml culture was sampled for measurement of OD600 and residual heavy metal concentration by AAS. The removal rate of individual heavy metal in the sample was calculated by Eq. 1.For evaluating the biosorption capacity of init is the initial concentration of heavy metals in the medium; Csample is the concentration of heavy metal in collected sample.where CL. aestuarii GYF-1 in mixed heavy metal stress was firstly assessed in 1 × SHMM that mimics the heavy metals that present in the soil digested solution. The overnight culture broths were inoculated at OD600 of 0.05, and cultured at 30oC for 72 h. To prepare the GYF-1 concentrate used for bioremediation, the culture broth was first prepared in LB supplemented with 1 × SHMM for 24 h at 30oC, the cell suspension was washed twice by PBS, concentrated by centrifuge, and kept at 4oC for further use.The survival of Removal of mixed heavy metals were using a sequential removal strategy. The soil samples were air-dried and sieved. Then, 2 g of dry soil were digested with an acid mixture (hydrofluoric acid-nitric acid-hydrochloric acid). The digested solution was cooled, filtered, adjusted pH to 6.8 by NaOH and diluted to 50 mL, and then 50 mL 2 × LB medium was added for preparation of soil extract. Five milliliter of soil extracts were sampled to determine the initial heavy metal concentrations.600 of GYF-1 concentrate was inoculated to 100 mL of soil extract, and shaking at 30oC for 24 h. The culture was centrifuged, 5 ml of supernatant was sampled, and the rest was inoculated again with GYF-1 concentrate in the same growth condition as second-round adsorption. The removal of heavy metals in collected samples were determined by AAS and calculated by Eq. 1.In first-round remediation, 1 OD2 and 0.5 mM MgCl2. Pellets were then resuspended in PBS, and sonicated for 10 min on ice. To eliminate the contamination of proteins and nucleic acids, 10 µg/mL of proteinase K (Roche) was added to the cell mixtures and tubes were kept at 65oC for 1 h, the mixtures were subsequently treated with 20 µg/mL of DNase I (ThermoFisher), 40 µg/mL of RNase  at 37 oC overnight. Next, an equal volume of 90% hot phenol (65oC) was added to the mixtures, vortexed vigorously, and incubated at 65oC for 15 min. The mixtures were cooled down on ice, and centrifuged at 12,000 rpm for 10 min at 4oC, supernatants were transfer to new tubes, and phenol phases were re-extracted with 100 µL of water twice. The upper aqueous phase from each step were combined. The crude extracts were further precipitated by two volume of 0.375 M MgCl2 in 95% ethanol at -20oC. After the samples had cooled to 0oC, they were centrifuged at 12,000 rpm for 15 min at 4oC. The pellets were re-suspended in 1 × sampling buffer, boiled at 70oC for 5 min, electrophoresed by using NuPAGE 12% Bis-Tris Gel system (ThermoFisher), and stained by either silver staining by Mass Silver Stain kit  or coomassie blue R-250  according to the manufacturer’s instructions.Extraction of LPS was using hot phenol-water method as described previously with some modifications . Briefly600 of 0.05. The cultures were incubated for 24 h at 30oC at 200 rpm, and harvest by centrifugation at 4,000 rpm at 4oC for 10 min, and washed once by cold 1 × TE . Extraction of total RNA was performed by using Bacteria Total RNA Isolation Kit  following the manufacturer’s instruction. The resuspended RNA was treated with DNase I (ThermoFisher), and purified with the RNA Clean & Concentrator kit (ZymoResearch). The resulting RNA concentration was measured with a Biospectrometer (Eppendorf).Overnight culture of strain GYF-1 was inoculated to LB with and without supplemented with 1 × SHMM at final OD2O was used as no-RT negative control to evaluate genomic DNA contamination. Primers used for qPCR were designed using Primer designing tool and list of primers was available (Table T method.cDNA synthesis was performed using the PrimeScript RT Reagent Kit (Takara) according to the manufacturer’s protocol with 1 µg of total RNA as template, and aliquot without adding PrimeScript RTase with DEPC-treated ddHle Table . The qPCBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5


Elizabethkingia anophelis has emerged as a critical human pathogen, and a number of isolated reports have described the successful treatment of Elizabethkingia infections with vancomycin, a drug that is typically used to target Gram-positive bacteria. This study employed in vitro broth microdilution checkerboard and time-kill assays, as well as in vivo zebrafish animal models to evaluate the individual and combination antimicrobial effects of vancomycin and rifampin against E. anophelis. The minimum inhibitory concentration ranges of vancomycin and rifampin against 167 isolates of E. anophelis were 16–256 mg/L and 0.06–128 mg/L, respectively. The checkerboard assay results revealed a synergistic effect between vancomycin and rifampin in 16.8% (28/167) of the isolates. Time-kill assays were implemented for 66 isolates, and the two-drug combination had a synergistic interaction in 57 (86.4%) isolates. In vivo zebrafish studies revealed that treatment with vancomycin monotherapy, rifampin monotherapy, or vancomycin–rifampin combination therapy yielded a higher survival rate than the control group treatment with 0.9% saline. The results of this study support the use of vancomycin to treat E. anophelis infections. Elizabethkingia are characterized as Gram-negative, aerobic, pale yellow-pigmented, nonmotile, incapable of fermenting glucose, non-sporulating, positive for the oxidase test, showing weak indole production, and are negative for nitrate reduction  amino} ethanesulfonic acid). Subsequently, 50 µL of this broth suspension was placed in a 96-well plate and incubated at a temperature between 34 and 36 °C in a non-CO2 incubator. After 18 to 24 h of incubation, the results were examined, and the MIC was determined as the lowest concentration of antibiotics that prevented visible bacterial growth.The MICs of vancomycin and rifampin  against Enterococcus faecalis because the Clinical and Laboratory Standards Institute do not provide interpretive criteria for these antibiotics against “other non-Enterobacteriaceae” [The breakpoints for susceptibility testing were determined based on the interpretive standards for riaceae” . A MIC oE. anophelis isolates, as described in a relevant study [5 colony-forming unit (cfu)/mL. MICs were then determined. The fractional inhibitory concentrations (FICs) were calculated as follows: FIC of vancomycin (FICVA) = MIC of vancomycin in combination therapy/MIC of vancomycin monotherapy; FIC of rifampin (FICRIF) = MIC of rifampin in combination therapy/MIC of rifampin monotherapy; and FIC index = FICVA + FICRIF. The combination was defined as synergistic when the FIC index was ≤0.5, indifferent when the FIC index was >0.5 to ≤4, and antagonistic when the FIC index was >4 [To analyze the synergistic effect of vancomycin and rifampin combination therapy, this study implemented a two-agent broth microdilution checkerboard assay for all 167 nt study . Bacterix was >4 .8 cfu/mL. Subsequently, 25 µL of the adjusted bacterial suspension was introduced into a flask containing 25 mL of cation-adjusted Mueller–Hinton broth, resulting in a bacterial concentration of approximately 5 × 105 cfu/mL. Solutions of vancomycin and rifampin, each at a concentration equivalent to 1 × MIC, were added to these bacterial suspensions. After an incubation time of 0, 2, 4, 6, 8, 10, and 24 h, 50-µL aliquots of the bacterial suspensions were added to 450 µL of phosphate-buffered saline. The colony number was counted on Mueller–Hinton agar plates inoculated with 100-µL aliquots of 10-fold serially-diluted bacterial suspension after 20–22 h of incubation at 37 °C. Antibiotics were classified as bactericidal if the number of colonies decreased ≥ 3 log10 within 24 h compared with the initial inoculum [10 decrease in colony count compared with the most active single agent. Antagonism was defined as a ≥2 log10 increase in colony count compared with the most single active agent. Indifference was defined as the change in colony count between synergy and antagonism [In this study, a total of 66 isolates from different strains were randomly chosen for the time-kill assay because of their time-consuming, labor-intensive, and costly nature. The bacterial strains were cultured overnight at a temperature of 35 °C in cation-adjusted Mueller–Hinton broth. The bacterial suspension was then adjusted to a 1.7 McFarland standard, which corresponds to approximately 5 × 10tagonism .E. anophelis strains with high MICs of vancomycin and rifampin  were arbitrarily chosen from the collection of the 167 isolates for the evaluation of antimicrobial effects using the zebrafish animal model. A 10-µL bacterial solution (7.5 × 109 cfu/mL) was injected into the peritoneal cavity of zebrafish using a 35G beveled steel needle. Intraperitoneal antibiotic injections were administered 2 h after bacterial injection. The following dosages of vancomycin and rifampin were administered: vancomycin, 15 mg/kg every 12 h for 72 h after a loading dose of 25 mg/kg; and rifampin, 5 mg/kg every 12 h for 72 h. Each study group comprised five zebrafish, and the experiment was repeated twice. Kaplan–Meier survival analysis, and the log-rank test were implemented using SPSS version 19.0 . Statistical significance was indicated at a two-tailed p value of <0.05. The animal study followed the guidelines for the care and use of laboratory animals, and was approved by the Institutional Animal Care and Use Committee of E-Da Hospital (IACUC-EDAH-110028). For this study, we utilized approximately 7-month-old wild-type zebrafish (AB strain) that were acquired from a commercial distributor in Taiwan . To acclimate the zebrafish to the laboratory environment, all experiments were conducted at least one week after their arrival at our laboratory. The zebrafish were accommodated in multiple 1.5-L tanks and subjected to a 14-h light/10-h dark cycle at a temperature of 28 °C. They were provided with a diet containing crude fiber (maximum 2.5%), crude moisture (maximum 6.32%), crude fat (minimum 6.8%), and crude protein (minimum 46.8%), along with essential vitamins such as vitamin A, vitamin B1-3, vitamin B6, vitamin D3, vitamin E, vitamin K3, and calcium pantothenate. These zebrafish were fed twice daily. Two E. anophelis has emerged as a critical pathogen that causes life-threatening infections in patients. Therapeutic options for this multidrug-resistant microorganism are limited. This study revealed the excellent antimicrobial efficacy of vancomycin against E. anophelis, either as a monotherapy or in combination with rifampin. Further clinical trials are warranted to evaluate the efficacy and outcomes of vancomycin monotherapy and vancomycin–rifampin combination therapy for E. anophelis infections in humans.


NDM) or class D (e.g. OXA-48-type) carbapenemases, because those latter enzymes readily hydrolyse this antibiotic, often leading to high MICs of temocillin.5 A recent study evaluating the use of temocillin against ESBL- and AmpC-producing Enterobacterales confirmed the excellent activity of temocillin, and the few high MICs observed were linked mostly to the carriage of multiple β-lactamases by corresponding isolates rather than any single β-lactamase type.6 In recent years, a number of countries in Europe, including Belgium, France, Luxembourg and the UK, have revived the use of this antibiotic, predominantly for the treatment of urinary tract infections but also for bloodstream and lower respiratory tract infections.8 Subsequently, the surveillance of resistance to temocillin is essential and can be easily determined using either routine broth microdilution or disc diffusion testing; however, such tests require 18–24 h to achieve a result. In this study, we designed and evaluated a rapid (∼4 h) test that can determine resistance or susceptibility to temocillin in Enterobacterales.Temocillin is a semi-synthetic 6-α-methoxy derivative of ticarcillin, first developed in 1981.E. coli, 33 Klebsiella pneumoniae, 6 Enterobacter cloacae, 4 Klebsiella aerogenes and 2 Klebsiella oxytoca isolates (Table JAC Online). Isolates were submitted to the Swiss National Reference Center for Emerging Antibiotic Resistance (NARA) from hospitals and clinics throughout Switzerland, and speciated by either MALDI-TOF MS  or API-20E tests . Susceptibility testing was performed by broth microdilution according to CLSI guidelines,9 and resistance was determined according to EUCAST breakpoints (R > 16 mg/L),10 because there are no available guidelines for temocillin resistance according to CLSI. This identified that 38 tested isolates were temocillin susceptible, with MICs ranging from 2 to 16 mg/L, and the remaining 62 were resistant, with MICs ranging from 32 to >256 mg/L. The β-lactamase content of the tested isolates was determined by multiplex PCRs targeting CTX-M, CMY, DHA, SHV, TEM and OXA-1-like encoding genes,11 and the carbapenemase gene content had previously been determined by PCR, and subsequent Sanger sequencing of amplicons by NARA.12A total of 100 clinical Enterobacterales isolates were tested, comprising 55 https://www.bio-rad.com), as follows: a 0.5 MacFarland suspension was prepared then diluted 1/10 in fresh MH broth; 50 µL of this suspension was then used to inoculate the plates , resulting in a final temocillin concentration of 16 mg/L, before incubation at 37°C for 4 h. Negative control wells, both with and without temocillin, were inoculated with 50 µL 0.85% NaCl, diluted 1/10 in fresh MH broth, instead of the bacterial suspension. After 3 h and 4 h, plates were read and a colour change from red to yellow was interpreted as positive (resistant), confirming acid production via the metabolism of glucose in the test solution, whereas red was interpreted as negative (susceptible)  broth (CA-MHB) powder (Bio-Rad), 0.0125 g of phenol red (Sigma Aldrich) and 225 mL of distilled water. The pH was adjusted to 7.3 by adding drops of 1 mol/L hydrochloric acid prior to autoclaving. After cooling to room temperature, 25 mL of a 10% anhydrous D(+)-glucose  (sterilized by filtration) was added, resulting in the final concentrations of 2.5% CA-MHB, 0.005% phenol red indicator and 1% glucose. The temocillin test solution was prepared with 21.3 mg/L of temocillin and 150 µL was added into one well of a 96-well polystyrene plate; 150 µL of test solution without temocillin was added into a second well. Inoculums were prepared from overnight cultures, grown on URISelect 4 agar  harbouring blaTEM as a sole acquired ß-lactamase gene, exhibiting an MIC of temocillin at 32 mg/L, therefore corresponding to a 100% specificity and 98.4% sensitivity. Most of the isolates exhibiting high-level temocillin resistance (MIC ≥64 mg/L) resulted in a positive test result after only 3 h; however, those with resistant MICs closer to the breakpoint (32 mg/L) required 4 h. The test was shown to be effective regardless of the Enterobacterales species, β-lactamase content and overall mechanism of resistance. This rapid test could be easily implemented in a clinical laboratory and can be set up alongside routine antimicrobial susceptibility testing methodologies (AST), but providing a result 14–20 h earlier than traditional AST and potentially sparing the use of other β-lactams such as the carbapenems. These results show that as the use of temocillin to treat Gram-negative infections becomes more commonplace, such a test can prove useful in determining targeted rather than simply empirical therapy in a relatively short time frame.The test presented a single false-negative result, this being an dkad243_Supplementary_DataClick here for additional data file.


The aim of this study was to estimate the long-term health and economic consequences of improved risk factor control in German adults with type 2 diabetes.1c by 5.5 mmol/mol (0.5%), of systolic BP (SBP) by 10 mmHg, or of LDL-cholesterol by 0.26 mmol/l in all patients, and (2) achievement of guideline care recommendations for HbA1c (≤53 mmol/mol [7%]), SBP (≤140 mmHg) or LDL-cholesterol (≤2.6 mmol/l) in patients who do not meet the recommendations. We calculated nationwide estimates using age- and sex-specific quality-adjusted life year (QALY) and cost estimates, type 2 diabetes prevalence and population size.We used the UK Prospective Diabetes Study Outcomes Model 2 to project the patient-level health outcomes and healthcare costs of people with type 2 diabetes in Germany over 5, 10 and 30 years. We parameterised the model using the best available data on population characteristics, healthcare costs and health-related quality of life from German studies. The modelled scenarios were: (1) a permanent reduction of HbA1c by 5.5 mmol/mol (0.5%), SBP by 10 mmHg or LDL-cholesterol by 0.26 mmol/l led to per-person savings in healthcare expenditures of €121, €238 and €34, and 0.01, 0.02 and 0.015 QALYs gained, respectively. Achieving guideline care recommendations for HbA1c, SBP or LDL-cholesterol could reduce healthcare expenditure by €451, €507 and €327 and gained 0.03, 0.05 and 0.06 additional QALYs in individuals who did not meet the recommendations. Nationally, achieving guideline care recommendations for HbA1c, SBP and LDL-cholesterol could reduce healthcare costs by over €1.9 billion.Over 10 years, a permanent reduction of HbA1c, SBP and LDL-cholesterol control among diabetes patients in Germany can lead to substantial health benefits and reduce healthcare expenditures.Sustained improvements in HbAThe online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-023-05950-3. Achieving guideline care recommendations for HbA1c, SBP and LDL-cholesterol over 10 years resulted in QALYs gained of 0.03 , 0.05  and 0.06 , respectively, and decreased healthcare costs by €451 , €507  and €327 , respectively, per person with ABC levels above the recommended threshold. Generally, QALY gains and healthcare savings were substantially smaller for the 5-year time horizon and substantially larger for the 30-year time horizon, with the exception that healthcare cost savings were smaller and negative for LDL-cholesterol control over the 30-year time horizon, i.e. the costs increased.Sustained population-wide decreases of HbA1c by 5.5 mmol/mol (0.5%), SBP by 10 mmHg and LDL-cholesterol level by 0.26 mmol/l , 0.07  and 0.25  over 5, 10 and 30 years, respectively, and healthcare cost savings of €400 , €581  and €451  per person for people with at least one value above the ABC thresholds.Simultaneously decreasing HbAESM Table 1c by 5.5 mmol/mol (0.5%), of SBP by 10 mmHg and of LDL-cholesterol by  0.26 mmol/l would result in 54,381, 98,753 and 78,535 QALYs gained, respectively, and €543 million, €1047 million and €60 million cost savings, respectively, for people with type 2 diabetes in Germany; achieving recommended levels of HbA1c, SBP and LDL-cholesterol for every patient with type 2 diabetes in Germany would lead to 43,358, 45,418 and 185,381 QALYs gained, respectively, and reduce healthcare expenditures by €681 million, €457 million and €921 million, respectively.The nationwide cost savings and QALY gains resulting from improved ABC control from a national healthcare system perspective are shown in Table Shifting the ABC risk factor distribution simultaneously for all type 2 diabetes patients in Germany would gain 210,333 QALYs and reduce healthcare costs by over €1.8 billion within the national healthcare system over 10 years. Achieving recommended levels of ABC control for everyone with levels above the threshold would lead to a decrease of over €1.9 billion in healthcare expenditure, and an increase of 268,783 QALYs.1c, SBP or LDL-cholesterol should not exceed €549, €760 or €958, respectively  than those for achieving recommended levels of SBP (€457 million). This highlights the importance of complementing clinical disease management with population-based intervention strategies .Achieving recommended levels for ABC control would reduce the healthcare expenditures of adults with diabetes by around €289 million annually (€1443 million saved over 5 years). In relative terms, this would reduce annual healthcare expenditures for patients with diabetes in Germany by approximately 1% .1c led to an increase of 0.35 life years over lifetime  to 7% [53.0 mmol/mol]) resulted in 0.08 QALYs gained over a time horizon of 10 years, and a mean decrease in SBP of 10 mmHg resulted in 0.1 QALYs gained [1c sustained over 10 years resulted in 0.02 QALYs gained, and a sustained decrease of SBP by 10 mmHg (from 154 to 144 mmHg) resulted in 0.02 QALYs gained  would lead to gains of 0.81 QALYs and healthcare expenditure savings of US$10,800 per person over their lifetime from a US private payer perspective [Other previous modelling studies also reported higher QALY gains and cost savings from treatment scenarios that improve ABC control than those found in our study. A study by Palmer et al using the CORE Diabetes Model suggested that a decrease in HbAspective . These dspective  and othespective . In addispective .1c control be cost-effective at a willingness-to-pay threshold of €50,000 per QALY gained over a 30-year time horizon range between €190 and €549. In context, the per-person annual costs for metformin, sulfonylurea, sodium–glucose cotransporter 2 (SGLT-2) inhibitors, dipeptidyl peptidase 4 (DPP-4) inhibitors and glucagon-like peptide-1 (GLP-1) antagonists in Germany are approximately €33–99, €31–131, €377–884, €350–511 and €861–1463, respectively [As the aim of the analyses was to highlight the long-term health benefits and cost saving potential of improved risk factor control, we did not consider any resources and costs needed to achieve these improvements. However, all real-world risk factor improvement strategies of course come with costs to achieve these improvements. We show that the maximum per-person annual costs for interventions that improve HbAectively . It is iectively .We used one of the most established type 2 diabetes simulation models . We para1c, SBP and LDL-cholesterol may have further decreased since 2014. If this is the case, we may have slightly overestimated the health benefits, as the given changes in risk factors in people with lower baseline disease risk would mostly translate into lower absolute health benefits. Sixth, a population-wide shift in HbA1c, SBP or LDL-cholesterol may lead to adverse complications such as hypoglycaemia for individuals at the lower end of the HbA1c distribution, but we did not model these potential adverse outcomes. Due to the low proportion of people in these very low ranges, we assume that this would affect our results only marginally. Finally, cost estimates for complications of diabetes and estimates on diabetes prevalence were taken from samples of Germans insured in the statutory health insurance scheme in which approximately 89% of Germans are included. Compared with the statutory health insurance scheme, the per-person cost estimates for treating complications are likely to be higher and the prevalence of type 2 diabetes is likely to be lower in individuals with private health insurance, and thus the estimates for the national healthcare system perspective may be biased in either direction. We again assume that that this would affect our results only marginally.First, the results of this modelling study are potentially limited by the underlying model assumptions. We demonstrated in a previous study that the risk equations used in the UKPDS-OM2 predict cardiovascular events well but over-estimate mortality. However, the performance of the model for the prediction of other outcomes remains unknown . Second,1c level, SBP level and LDL-cholesterol level should not cost more than €549, €760 and €958 a year, respectively.Both population-level shifts in risk factor distribution and the achievement of guideline care recommendations for patients exceeding recommended risk factor levels can result in substantial health benefits and cost savings. The expected long-term health and economic benefits described in this study may help German decision makers to assess the clinical effects of interventions and their costs from an efficiency perspective. Using an internationally accepted willingness-to-pay threshold, treatment aiming for better adherence to the guideline-recommended HbASupplementary file1 (PDF 591 KB)Below is the link to the electronic supplementary material.


However, it is unclear for how long continuous warming due to global warming affects the microbial composition and GHG release from soils along Arctic glacial meltwater rivers.Global warming is caused by greenhouse gases (GHGs). It has been found that the release of methane (CHin situ temperature), 10°C, and 20°C. The incubation was carried out under anoxic conditions and lasted for few days. Bacterial composition and CH4 production potential were determined based on high-throughput sequencing and physiochemical property measurements.In this study, the soil upstream of the glacial meltwater river (GR) and the estuary (GR-0) in Svalbard, with strong soil heterogeneity, was subjected to short-term field incubation at 2°C (4 concentration at the GR site was higher than that at the GR-0 site, while the mcrA gene was lower at the GR site than that at the GR-0 site.Our results showed no significant differences in bacterial 16S rRNA gene copy number, bacterial composition, and methanogenic potential, as measured by mcrA gene copy number and CH4 concentration, during a 7- and 13-day warming field incubation with increasing temperatures, respectively. The CH4 concentration less than the spatial scale, highlighting the importance of warming time in influencing CH4 release from soil. In summary, our research implied that microbial composition and CH4 emissions in soil warming do not increase in the first several days, but site specificity is more important. However, emissions will gradually increase first and then decrease as warming time increases over the long term. These results are important for understanding and exploring the GHG emission fluxes of high-latitude ecosystems under global warming.Based on the warming field incubation, our results indicate that short-term warming, which is measured in days, affects soil microbial composition and CH Correspondingly, the soil of the Svalbard Glacier basin has great heterogeneity  for measuring environmental parameters, and approximately 32 g of the soil was added into a 50 mL centrifuge tube with 20 mL RNA later  for determining the composition of the microorganism community at 20°C. However, 3 mL of 2 mol/L NaOH was added to a 20-mL serum bottle along with 5 g soil for determining CH4 concentration and stored at 4°C  upstream of the glacial meltwater river, while the other was taken from the soil  at the estuary of the glacier meltwater river  at 2°C. d at 4°C .® SPIN Kit for Soil . Before following the manufacturer's instructions, all the samples were washed with 1 × PBS twice and centrifuged at 12,000 × g for 5 min. The DNA concentration was then measured using a NanoDrop 2000 spectrometer . The V4 region of the bacterial 16S rRNA gene was amplified using the primers 533F (5′-TGCCAGCAGCCGCGGTAA-3′) and Bact 806R (5′-GGACTACHVGGGTWTCTAAT-3′) . The sequencing of purified DNA on the Illumina MiSeq platform was performed by the Personalbio Biotechnology company in Shanghai, China.The genomic DNA of six initial samples and 81 incubation samples stored in RNAlater at −20°C was extracted from approximately 0.5 g fresh, homogenized soil using the FastDNAThe analysis used the pipeline QIIME2 (version 2022.2)  using bac341f (5′-CCTACGGGWGGCWGCA-3′) and prokaryotic 519r (5′-TTACCGCGGCKGCTG-3′)  gene in these river sediments were estimated by qPCR. The DNA fragments encoding the mcrA gene were amplified using PCR with the universal primers mlas-mod-F (5′-GGYGGTGTMGGDTTCACMCARTA-3′) and mcrA-rev-R (5′-CGTTCATBGCGTAGTTVGGRTAGT-3′) , 2 μL of template DNA, and 1 μL of each forward and reverse primer. The standard curve consisted of a diluted known amount of purified PCR product obtained from plasmid DNA using the bacterial 16S rRNA gene-specific primers bac341f/519r between 103 and 109 copies/μL. The amplification efficiency was between 90–110%, and the R2 of the standard curve was above 0.90. The thermal cycle program was for 2 min at 50°C and 3 min at 98°C, followed by 40 cycles for 15 s at 98°C, 30 s at 55°C, and 30 s at 72°C. The standard curve consisted of a known amount of diluted purified PCR product obtained from plasmid DNA using the mcrA gene-specific primers mlas-mod-F/mcrA-rev-R between 102 and 107 copies/μL. The thermal cycling conditions were followed by heating at 50°C for 2 min, 98°C for 3 min, followed by 40 cycles of heating at 98°C for 30 s, 58°C for 40 s, and 72°C for 30 s. Three replicates were performed for each sample, and the statistical analysis was performed using Student's t-test.This template used in qPCR for 16S rRNA and III, Elementar, Germany). The procedure for measuring the total nitrogen content was similar but lacked the reaction with acid. Based on repeated determinations, the detection limits for carbon and nitrogen were 8 μg, with a precision better than 6%. The soil samples used in the above parameters were stored at −20°C. The CH4 and CO concentrations in the 20 mL serum bottle headspace were measured on a gas chromatograph with a flame ionization detector   was measured using an element analyzer  belonging to different phyla have been found in 87 incubation samples at two different sites, totaling up to 1,011,481. The taxa at the GR site were dominated by six bacterial phyla: Actinobacteria (22–61%), Proteobacteria (11–50%), Firmicutes (8–18%), Bacteroidota (3–17%), Desulfobacterota (5–10%), and Acidobacteriota (2–6%) followed by Gemmatimonadota and Chloroflexi . Actinobt groups . Meanwhin groups .mcrA, which encodes methylcoenzyme M reductase and is a key enzyme in methanogenesis  was 1.3 μmol/L. Under 10°C, the average net increase of CH4 for 2 days was 3.9 μmol/L. At 20°C, the average net increase of CH4 was 2.4 μmol/L for 2 days  in incubated samples. However, it only exists at the GR-0 site. At all three different incubation temperatures, the concentration of CO was higher in the early stage of incubation  and reached its maximum on the third day , CH4 , and TN  were the major factors affecting soil microbial composition  and time (7 and 13 days) during incubation  in our short-term incubation did not increase with the anoxic warming experiment  can be found in the article/JL: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Z-YZ: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing. ZY: Data curation, Formal analysis, Investigation, Methodology, Resources, Writing – review & editing. WL: Methodology, Software, Visualization, Writing – review & editing. YL: Software, Writing – review & editing. YZ: Conceptualization, Funding acquisition, Validation, Writing – review & editing.