Photobacterium. We postulate that propane biosynthesis is achieved through inorganic carbonate assimilation systems. The discovery of this strain may facilitate synthetic biology routes for industrial scale production of propane via microbial fermentation.Propane is a major component of liquefied petroleum gas, a major energy source for off-grid communities and industry. The replacement of fossil fuel-derived propane with more sustainably derived propane is of industrial interest. One potential production route is through microbial fermentation. Here we report, for the first time, the isolation of a marine bacterium from sediment capable of natural propane biosynthesis. Propane production, both in mixed microbial cultures generated from marine sediment and in bacterial monocultures was detected and quantified by gas chromatography–flame ionization detection. Using DNA sequencing of multiple reference genes, the bacterium was shown to belong to the genus  As LPG , or SOx . LPG pro, or SOx , 2019. B, or SOx . Current, or SOx . Switchissil LPG . The fir 4 years . Additio 4 years . The avaAn alternative route to bioLPG is through synthetic biology, but although researchers have genetically engineered bacteria capable of the production of propane, yield restrictions due to product or substrate toxicity, and maintaining cell integrity at high product concentrations remain a major challenge in industrial biotechnology . To datePhotobacterium that is carrying out the process. The natural propane biosynthesis identified here was achieved using simple feedstocks, which offers the prospect of the development of a scalable, economic process for propane production through synthetic biology. We also identified several genes that may contribute to potential biochemical pathways for propane production.After sampling differing anoxic environments where microbial biogenic propane production could predictably take place, we present, for the first time, clear evidence that naturally occurring microorganisms from marine sediment are capable of propane biosynthesis and identify a specific strain of Thermo Scientific) at pH 7.0. For marine samples, the slurry was generated under the same conditions; however, artificial seawater containing marine salts (Merck) at 30 gl−1, pH 7.4 was used in place of PBS. Initial liquid microbial cultures were prepared, in triplicate, in 20 ml GC headspace vials. Eight milliliter of either modified R2A or Zobell Marine Medium was inoculated with 2 ml of slurry generated from terrestrial or marine samples, respectively. For medium components see With the exception of sample FC.2, terrestrial soil samples were obtained using a soil auger. Sample FC.2 (anaerobic digest) was obtained directly from an agricultural anaerobic digester. Marine sediment sample FC.4 was obtained using a box corer. All samples were immediately placed in cool boxes for transport to the laboratory where they were routinely stored at 4°C. Inoculum slurry was generated within 24 h of obtaining each sample. To form an inoculum slurry approximately 20 g (wet weight) of each terrestrial sample was mixed with 25 ml sterile phosphate-buffered saline (PBS) (Merck).Mixed microbial cultures were maintained by subculturing 500 μl of the initial culture in 9.5 ml fresh media within GC headspace vials. Mixed microbial cultures were incubated without shaking at either 4°C for a minimum of 5 weeks or at 20°C for 7 days depending on experimental requirements. For propane analysis, negative controls were prepared as previously described with the addition of 5 mM sodium azide (Merck). After analysis for propane, a 10-fold dilution series of the mixed microbial culture was prepared in R2A or modified Zobell Marine media and 100 μl of 10−1, 10−2, and 10−3 dilutions were spread on the surface of solid media. After being allowed to dry, the agar plates were placed upside down in a sterile anaerobic chamber and a GasPak anaerobe sachet and CO2 indicator strip added before the chamber was sealed. Plates were incubated at 4°C for 10 weeks under anaerobic conditions. Individual bacterial species were isolated by repeated colony picking and sub-culture on solid media at 4°C for 10 weeks under anaerobic conditions. For propane analysis, all resultant bacterial strains were cultured in 10 ml liquid medium modified depending on individual experimental conditions for 7 weeks at 4°C. The positive control for propane analysis was a mixed microbial culture and the negative control, a mixed microbial culture treated with NaN3.Solid media was prepared as discussed in section “Sampling and generation of mixed microbial cultures”. with the addition of 1.5% (w/v) agar (Phenomenex) and a GasPro SI PLOT (30 m × 0.32 mm × 1.8 μm) (Agilent). GC conditions are provided in ® CANGasPropane 99.95% (Merck) and quantified using a six-point calibration curve. For the calibration curve, a tenfold dilution series to 10−3 was prepared in gas-tight headspace vials fitted with butyl rubber septa, using a gas-tight syringe. Concentrations for the 6-point calibration curve of 40, 30, 20, 15, 10, and 5 ppm were prepared from the 10−3 standard concentration in headspace vials containing 10 ml liquid growth medium. The limit of detection for propane, 3 times the standard deviation of multiple data points along the baseline divided by the slope was 0.816 ppm.Propane was analyzed by gas chromatography (GC) on a Varian 450 GC with flame ionization detection (FID). A CombiPAL autoinjector fitted with a gastight syringe was used to sample 250 μl headspace gas above the live bacterial cultures. To confirm the identity of propane, chromatography was carried out on two different column chemistries - a Zebron ZB-624 column (30 m × 0.32 mm × 1.8 μm) (Qiagen) as per the manufacturer’s instructions. Polymerase Chain Reaction (PCR) amplification and subsequent sequencing of the 16S rRNA gene were carried out via the methodology previously described by Bacterial isolates were initially identified by the sequencing of the gene encoding the 16S sub-unit of ribosomal RNA (16S rRNA). Chromosomal DNA was extracted from each strain of interest using a DNeasy UltraClean Microbial Kit  version 11.0.11 software was used to generate both multiple sequence alignments and phylogenetic trees  .Statistical analysis of propane production by both enrichment cultures and individual isolates was determined by one-way ANOVA followed by Dunnett’s in situ conditions were predicted as potential sources for microbial alkane production. Details of all samples are provided in Soil or sediment samples were obtained from terrestrial and marine locations where via GC–FID. Propane was identified and quantified by comparison with an authentic propane standard by GC on a Zebron ZB-624 GC column. Propane was detected in initial mixed microbial cultures derived from marine sediment samples (FC.4) at a significantly increased level in comparison to sterile media controls  were subjected to GC-FID headspace analysis. Propane was detected in both the sub-cultured and fresh marine sediment mixed microbial cultures at a level significantly higher than that detected in the NaN3 treated sample  were shown to produce propane at a level significantly higher than the NaNcontrols . Interesustonica . RepeatePhotobacterium .In early experiments, we observed that bacterial growth and/r propane production were not maintained over several rounds of sub-culture in fresh media. To determine whether the marine sediment itself, which was present in initial cultures, contained required nutrients for propane production, a sediment-free inoculum was prepared from initial cultures by gently resuspending microbial cells by gentle agitation. This was used to inoculate fresh media either devoid of any sediment or supplemented with pre-sterilized marine sediment (2% w/v). Significantly increased propane production was observed in cultures supplemented with pre-sterilized marine sediment compared to the cultures without sediment supplementation and to the NaN3 or 0.02 M CaSO4. We found that significant amounts of propane were produced in cultures supplemented with CaCO3 (38.53 ppm ± 5.61) compared to the NaN3 negative controls (2.03 ppm ± 0.34), with amounts statistically comparable to cultures supplemented with pre-sterilized marine sediment (48.05 ppm ± 7.38). In contrast, significantly less (3.97 ppm ± 0.007) propane was produced in cultures supplemented with CaSO4 compared to cultures supplemented with either pre-sterilized sediment or CaCO3. These results indicate that inorganic carbonate was required for propane production . The calculated mg propane per liter fermentation was 0.044 mg L−1, a 4,545 fold lower yield than that obtained through synthetic biology routes where maximum published propane yields are ca. 0.2 g L-1 fermentation   or CaCO3. Our functional analysis identified genes based on the specific biological process hypothesized to be putative orthologs for two carbonate transporters, bicA_1 and bicA_2 ; and a putative ortholog of carbonic anhydrase, cah (ON859039) , a Selenocysteine-specific elongation factor; selA (ON859040), an L-seryl-tRNA(Sec) selenium transferase; selD_1 and selD2 , Selenide water dikinases; and selU (ON859044), a tRNA 2-Selenouridine synthase  . An inteN859039) . EnzymesO enzyme . They arbacteria . The carescribed . The samsynthase . The enzsynthase . This evPhotobacterium sp. FC4.9. The study also identified several genes within the genome of this strain that are potentially involved in the propane biosynthesis pathway. As such this discovery opens the possibility of an alternative production pathway, via microbial fermentation, for the generation of propane for BioLPG. However, the quantity of propane produced was limited, and while there is scope for optimization and scale-up toward a fermentation route for bio-propane production, it is considered more likely that further elucidation of the mechanisms of propane biosynthesis by Photobacterium sp. FC4.9 could be utilized to generate recombinant bacteria with optimal production yields.To conclude, this study has identified, for the first time, a naturally occurring, propane-producing bacterium, The data presented in the study are deposited in the NCBI Nucleotide repository, accession numbers are provided in the main text of the article.FC: methodology, investigation, and writing – original draft. MT: formal analysis, writing – original draft, and writing – review and editing. NH: investigation. KS: funding acquisition, conceptualization, resources, and writing –review and editing. RM: conceptualization, supervision, and writing – review and editing. IB: project administration, funding acquisition, conceptualization, supervision, and writing – review and editing. All authors contributed to the article and approved the submitted version.This research was jointly funded through an industrial partnership with SHV Energy ; Invest Northern Ireland Proof of Concept grant 909 (Bio-propane); and a Department for the Economy (NI)/SHV Energy Co-operative Awards in Science and Technology (CAST) Ph.D. studentship.The authors declare that this study received funding from SHV Energy. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, but were aware of the findings and encouraged us to submit for publication.KS was employed by the company SHV Energy.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lenvatinib is appropriate for reducing the production of nitric oxide (NO) and facilitating as block angiogenesis. However, to our knowledge, there are no data that support the correlation between NO and clinical response in patients who received lenvatinib therapy for HCC. Therefore, we investigated the correlation between the change rate of NO levels and clinical responses including adverse events (AEs) after lenvatinib therapy for unresectable hepatocellular carcinoma (HCC).3−) to nitrite (NO2−) with nitrate reductase, followed by quantitation of NO2− based on Griess reagent. To determine whether lenvatinib influences NO in unresectable HCC, we evaluated the influence of the change rate of NO from baseline after administration of lenvatinib on maximal therapeutic response and SAE.This study was conducted using previously collected data from another study. We enrolled 70 patients who received lenvatinib for advanced or unresectable HCC. NO was measured by converting nitrate (NOp = 0.632). However, the change rate of NO was significantly lower in patients with AEs than in those without AEs (p = 0.030). When a reduction in NO rate of < 0.8 was defined as a clinically significant reduction of NO (CSRN), the CSRN group had significantly worse progression-free survival (PFS) and overall survival (OS) than the non-CSRN group .After lenvatinib administration, a change rate in the NO from 0.27 to 4.16 was observed. There was no difference between the clinical response to lenvatinib and the change rate of NO (Decreased NO levels were associated with the occurrence of AEs and worse prognosis after lenvatinib administration. Change rate in serum NO can be used as predictive markers in patients receiving lenvatinib therapy for HCC.The online version contains supplementary material available at 10.1186/s12885-022-10002-x. Malignant tumors require the formation of mature blood vessels to promote their growth and contribute to pathological processes in the tumor environment , 2. NitrVEGF is secreted by tumor cells in response to hypoxia. Exposure of endothelial cells to VEGF leads to the phosphorylation and activation of NOS, resulting in the conversion of L-arginine and molecular oxygen into L-citrulline and NO . Thus, VOn this basis, simultaneously suppressing VEGF signals suppress tumor angiogenesis to the cancer cells, and VEGF inhibitors appropriately reduce the production of NO and facilitate antitumor drug delivery as block angiogenesis , 9. LenvAssuming more dramatic effects in unresectable HCC, the response to this drug is unpredictable. However, a previous study suggested that elevated NO levels in HCC patients were significantly reduced after radiofrequency ablation . LenvatiThis study was approved by the Institutional Review Boards and Ethics Committees of all hospitals involved (IRB number: 11000845). The study was registered in the Japan Registry of Clinical Trials (jRCT ID: 1030210283). These data were previously collected under another study (UMIN ID: 000036625). The data were collected after each patient wrote informed consent for the treatment.This study was performed using previously collected data under another study that was conducted across three medical institutions in Japan from 2017 to 2020 . The preHCC diagnosis was based on imaging modalities such as computed tomography and magnetic resonance imagery. Liver tumors with atypical imaging findings were histopathologically analyzed by biopsy. Lenvatinib was administered for unresectable or advanced HCC that was characterized by vascular invasion, metastatic disease, and/or progression after locoregional treatments.A starting drug dose was dependent on the patient’s weight: 12 mg and 8 mg/day for body weight ≥ 60 kg and <  60 kg, respectively. However, for patients with risk factors, such as low PS (performance status), Child-Pugh class B, and sarcopenia, an approved dose reduction from the initial dose depended on the attending physician’s discretion.3−) to nitrite (NO2−) with nitrate reductase, followed by quantitation of NO2−, using a Griess reagent. In this experiment, we used a colorimetric Nitric Oxide Assay kit . Absorbance was measured at 540 nm using a microplate reader  from the change levels and rate in NO after administration of lenvatinib. The maximal therapeutic response was evaluated by the modified Response Evaluation Criteria in Solid Tumors (mRECIST). SAEs were defined as events that result in death, are life threatening, require inpatient hospitalization or prolongation of existing hospitalization, or result in persistent or significant disability/incapacity according to ICH .p-values were two-sided, with differences < 0.05 considered to indicate statistical significance. Two categorical variables in the population were examined using the Chi-square test. The differences in OS and progression-free survival (PFS) were evaluated with the log-rank test and the Kaplan-Meier method in the two groups. PFS and OS were analyzed by the Cox proportional hazards regression model. The correlation between NO levels and the response to lenvatinib was analyzed by calculating the odds ratio and 95% confidence interval (CI) using univariate and multivariate logistic regression analyses. All analyses were performed using SPSS . Statistical analyses were reviewed by the Statista Corporation, Kyoto, Japan.All n = 38), nonalcoholic fatty liver disease (n = 16), alcohol (n = 10), and others (n = 6). There were 46 patients (65.7%) with liver cirrhosis. Lenvatinib was administered for vascular invasion (n = 24), metastatic disease (n = 22), and progression after locoregional treatment (n = 46). The types of treatment given were radiofrequency ablation (RFA) (n = 28), transcatheter arterial therapy (n = 50), and molecular targeted therapy (n = 6). There were 32 (45.7%) and 38 (54.3%) patients who were diagnosed as having Barcelona Clinic Liver Cancer (BCLC) stages B and C, respectively. There were 25, 35, and 10 patients who had daily initial doses of 12 mg, 8 mg, and 4 mg, respectively. The 10 patients, who received an initial dose of 4 mg, had liver cirrhosis with sarcopenia.Table n = 40), unmanageable AEs (n = 26), and withdrawal at the patients’ own discretion (n = 4). Table The numbers of complete response, partial response, stable disease, progressive disease (PD), and unevaluated response in maximal therapeutic response were 2 (2.9%), 24 (34.2%), 25 (35.7%), 15 (28.0%), and 4 (5.7%), respectively. The median PFS and OS in all patients were 163 and 403 days (95% CI 124–238 and 311–524 days), respectively. The median time to treatment failure values for all patients was 247 days (95% CI: 201–292). Reasons to discontinue treatment were PD   patients. After lenvatinib administration, change levels in the NO from 49.2 ± 39.8 to 45.1 ± 32.5 nmol/mL were observed (p = 0.864)    . Furthermore, the change rate of NO in patients with and without HBP were 1.1 ± 1.0 and 1.2 ± 0.9, respectively. No significant correlation was found between the presence of HBP and change rate of NO (p = 0.813).The change levels of NO in patients with and without HBP were − 4.1 ± 18.1 and − 3.9 ± 21.9, respectively. No significant correlation was found between the presence of HBP and change levels of NO (Receiver-operating characteristic (ROC) curve analysis was performed to assess the occurrence of SAE in patients with HCC. The respective cut-off points for SAE after lenvatinib treatment were estimated using ROC curves for the change rate of the NO  , respectively. Patients in the CSRN group experienced significantly worse PFS than those in the non-CSRN group  Fig. A.Fig. 4Pp = 0.005) , respectively. Patients in the CSRN group experienced significantly worse OS than those with non-CSRN . Table p = 0.033 and HR 2.107; 95% CI 1.130–3.599; p = 0.018, respectively).Table 3− and NO2−, can be easily detected by photometric means [To our knowledge, this is the first study to report the influence of NO levels after administration of lenvatinib regarding the clinical response and AEs for patients with unresectable HCC. A review clearly showed a significant reduction in NO levels after RFA for HCC . Therefoic means . Howeveric means , 21.On the other hand, the present study revealed that a significant reduction in the NO rate was associated with AEs after the lenvatinib therapy. Lenvatinib therapy improves HCC control by restoring the NO levels in tumor. However, excessively restored NO levels lead to the occurrence of AEs and shortened lengths of survival. Inhibition of angiogenic factor-mediated pathways, including VEGF, results in a subsequent reduction in NO production . NO is aWe also expected a correlation between the change in NO levels and HBP. Previous studies have shown that patients who experienced hypertension after lenvatinib administration had significantly better outcomes than those who did not develop AEs . A subseThus, the present study revealed the correlation between the change rate of NO and SAEs in patients with HCC who underwent the lenvatinib therapy. However, as evidenced previously, the distinct roles of NO in patients treated with VEGF inhibitors remain unclear . Previou3− and nitrite NO2−. Evaluation of NO by direct methods will be needed for future analysis. We only evaluated the correlation between the change rate of the NO levels and the clinical responses and AEs in the present study. Furthermore, this study did not exclude the effect on NO concentrations from any treatment given before the lenvatinib administration. There is insufficient evidence in the literature on the mechanism of how NO affects the therapeutic response and AEs. Fourth, patients with a total bilirubin of 2.0 mg/dL or higher were excluded because of the effect on absorbance measurements.There are a few limitations to this study. First, it was a retrospective study. Second, preserved blood serum samples could not be obtained on a scheduled day from all the patients receiving lenvatinib. Furthermore, we need to evaluate the most appropriate time after administration of lenvatinib in which AEs occur. Third, NO was indirectly assessed using nitrate NODecreased NO levels were associated with the occurrence of AEs and worse prognosis after the lenvatinib administration. Change rate in serum NO levels can be used as predictive markers in patients receiving lenvatinib therapy for HCC.Additional file 1. The measurement of NO Production of NO by NOS and its metabolism to nitrate and nitrite. Nitrate was converted to nitrite by nitrate reductase, and NO was measured using the Griess reagent. The microtiter plate after the reaction is shown. (PPTX 2781 kb)Additional file 2. Pretreatment NO levelsAdditional file 3. ROC. The occurrence of SAE using lenvatinib by ROC curves for patients with HCC. AUC, area under the curve. (PPTX 48 kb)


Aerogels have been attracting wide attentions in flexible/wearable electronics because of their light weight, excellent flexibility, and electrical conductivity. However, multifunctional aerogel‐based flexible/wearable electronics for human physiological/motion monitoring, and energy harvest/supply for mobile electronics, have been seldom reported yet. In this study, a kind of hybrid aerogel (GO/CNT HA) based on graphene oxide (GO) and carboxylated multiwalled carbon nanotubes (CMWCNTs) is prepared which can not only used as piezoresistive sensors for human motion and physiological signal detections, but also as high performance triboelectric nanogenerator (TENG) coupled with both solid–solid and gas–solid contact electrifications (CE). The repeatedly loading–unloading tests with 20 000 cycles exhibit its high and ultrastable piezoresistive sensor performances. Moreover, when the obtained aerogel is used as the electrode of a TENG, high electric output performance is produced due to the synergistic effect of solid–solid, and gas–solid interface CEs . This kind of aerogel promises good applications for human physiological/motion monitoring and energy harvest/supply in flexible/wearable electronics such as piezoresistive sensors and flexible TENG. A kind of conductive, ultralight, elastic, hybrid aerogel is prepared by simply physical crosslinking of graphene oxide nanosheets and carboxylated multiwalled carbon nanotubes (CMWCNTs). Piezoresistive sensors for multimode physiological/human motion signal detections with long‐term ultrastability are successfully achieved due to the 3D network structure. High‐performance triboelectric nanogenerator (TENG) is based on the coupling of solid–solid and gas–solid contact electrifications. In order to extend the practical applications of flexible/wearable electronics to adapt comprehensive monitoring requirements, it is indispensable to explore wearable electronics with multiple functions to measure and analyze comprehensive physical conditions. As the active materials for flexible/wearable electronics, excellent electrical conductivity, good flexibility, and reliable long‐term stability are always crucial issues to ensure an efficient and stable working process. Although excellent research works on flexible/wearable electronic devices have been reported recently, and most of them were achieved by directly doping an appropriate amount of conductive components  into commercial soft flexible substrates. However, the functional diversity of these flexible electronic devices is still not enough to adapt to the complex actual environment, for example, real‐time signals for monitoring different types and ranges of human motion . Besides, external power sources were always needed to maintain these electronics working, which require additional costs. To address these challenges, triboelectric nanogenerators (TENGs), based on the coupling effects of the triboelectric effect and electrostatic induction, have emerged as a compelling technology to realize the human physiological signal monitoring, human motion detection, and also power these electronics. The ideal electrode materials to meet these requirements should be lightweight, flexible, mechanical robust, and easy to regulate its electrical conductivity.In recent years, great efforts have been made to develop flexible/wearable electronics for physiological signal detections, and energy harvesting. Due to the merits of portability, high energy conversion/harvesting efficiency, flexibility, and stability, flexible/wearable electronics have been widely reported in a wide range of applications, such as piezoresistive sensors for health monitoring, and human motion detections in human–machine interaction, and triboelectric nanogenerators for harvesting tiny mechanical energy. However, most reported flexible electronics, based on a single working mechanism, are usually functionally monotonous. These monofunctional electronics are usually able to sense only one motion or physiological signal,1, 2 Conductive, compressible, and elastic aerogels have been considered as excellent candidates for fabricating flexible/wearable devices such as piezoresistive sensors, and energy harvesting devices. The flexible and compress 3D porous structure of aerogels can provide large numbers of adjustable contact areas which can modulate the electrical resistance under the external pressure. At the same time, compared with traditional TENGs, aerogel‐TENG generate charges from 2D to 3D.Aerogels, among the numerous reported materials for flexible/wearable electronics, is the lightest solid in the world. The unique 3D porous structures of aerogels provide high porosity, ultralow density, and high specific surface area.17 ConIn this work, we prepared a kind of hybrid aerogel (GO/CNT HA) composed of graphene oxide nanosheets (GO) and carboxylated multiwalled carbon nanotubes (CMWCNTs). It was demonstrated that when the obtained GO/CNT HA was used as a piezoresistive sensor, the real‐time signal detection of human physiological/motion was successfully achieved. More importantly, long‐term ultrastability of the piezoresistive sensor was demonstrated by 20 000 cycles’ repetitions of alternatively loading–unloading tests. Additionally, high electrical output performances of the flexible TENG were realized by the GO/CNT HA as the TENG's electrode (GO/CNT HA‐TENG). The enhancement of the GO/CNT HA‐TENG were proved to be due to the coupling of solid–solid CE between the two electrification layers, and gas–solid CE between the 3D framework of the inner GO/CNT HA and the filled air (3D electrification).22.1Figure 2 gas adsorption isotherms were performed. The average pore size was calculated to be 59.4 nm and the specific surface area to be 1.46 m2 g−1.As shown in 2.2Figure In order to explore the mechanical properties of GO/CNT HA, compress stress–strain experiments were carried out. To investigate the influences of the doped CMWCNTs on the mechanical properties of GO/CNT HA, aerogels with different amounts of CMWCNTs were prepared by the same preparing method. The corresponding compress stress‐strain curves of aerogels with different amounts of CMWCNTs were shown in I–V curves) under different compress strains were tested, respectively, and the results showed a good linear relationship, as shown in Figure I–V curves increased with the increase of the compress strain, the reason of which is due to the increase of the conductive contact points inside the GO/CNT HA. When external pressure is applied on the GO/CNT HA, excellent piezoresistive sensory properties were produced. Specifically, when the 3D macroscopical GO/CNT HA is compressed under external pressure, more GO nanosheets/CNT in the inner surfaces of the aerogel moved closer to each other, making the internal conductive contact points increase. The working mechanism of aerogel during compression is shown in Figure I–t curve) of GO/CNT HA, the current increases significantly with the increase of the corresponding compression strain . The contact regions of the inner surfaces of GO/CNT HA greatly increased, resulting in a greater increase in current and a steady current signal. Under different compress strains, the I–t curves of the compress–release were tested, as shown in Figure ε”, “R”, and “R0” respectively the compress strain, resistance during compression and resistance at initial stage of GO/CNT HA. As shown in Figure To evaluate the piezoresistive sensor performance of the GO/CNT HA, the current–voltage curves . The aerogel electrode was covered by a piece of commercial butyronitrile layer as one of the electrification layers, and PTFE film as the opposite electrification layer , a new kind of energy conversion and harvesting electronics invented by Wang in 2012.42 As 2.5−1. The contact area could be calculated to be S = 1.767cm2 . The GO/CNT HA‐TENG output performances under different compress strains (10–90%) are measured. The open‐circuit voltages (VOC) increased from 51.7 to 98.4 V  increased from 1.01 to 2.89 μA  increased from 16.6 to 31.9 nC , and the corresponding VOC was measured to be 62.5 V  was calculated to be 288 mW m−2 . Compared with the state‐of‐the‐art in the literature reports, the power density of GO/CNT HA‐TENG is relatively high  with 3D network structure was prepared by freeze‐drying method. The aerogel showed excellent mechanical and electrical properties . On this basis, multifunctional flexible/wearable electronics were constructed, including piezoresistive sensors for various human signal detections , and triboelectric nanogenerator for mechanical energy harvest. The excellent mechanical properties and long‐term stability of the aerogel was due to its unique physical structure  and chemical structure (hydrogen bonds formed between GO nanosheets and the CMWCNTs). Piezoresistive sensor with the advantage of detecting different types and ranges of human motion is due to the good elasticity, compressibility, and the 3D porous framework structure, which resulting the contact/separation between the conductive inner surfaces with the external pressure alternatively loading–unloading. The high enhanced electric output performances of the GO/CNT HA‐TENG were attribute to the coupling effect of solid–solid and gas–solid triboelectrification (3D electrification). The obtained GO/CNT composite aerogel in this work, to a certain extent, overcame the bottleneck of single function of flexible wearable electronic devices, and realized multi‐functional applications.4Water polyurethane  was purchased from Shanghai McLean Biochemical Co., Ltd. hydroxyethyl cellulose  was purchased from Aladdin. carboxylated multiwalled carbon nanotubes aqueous dispersion  was purchased from XFNANO. All reagents are analytical reagents. was added into 200 mL DI water, stir at room temperature for 30 min, and then ultrasonic treatment for 30 min until the aqueous suspension became dispersed homogeneously. (3) 4 mL WPU and 10 mL HEC was then added into 6 mL DI water (in 50 mL beaker) and keep stir. (4) Adding 10 mL 5 mg mL−1 GO and 10 mL CNT to the beaker. The mixed solution was stirred for 3 h, and then ultrasonic for 1 h (300 W), gel solution was obtained. (5) The gel was introduced into the template, frozen for 12 h (−20 C), and then freeze–dried for 24 h, and then the ultralight GO/CNT HA was obtained.(1) 3.0 g HEC was added into 100 mL DI water at room temperature and stir until it was completely dissolved. (2) 1.0 g GO nanosheets, prepared according to a modified Hummer method,51 wasI–t curve test. Linear motor (LinMot E1100) was used to provide driving force for TENG contact separation process. The electric output performance  of TENG was detected and recorded by Keithley 6514 electrometer.Microstructure of GO/CNT HA scanned by SU8020 (Hitachi) cold field scanning electron microscope. YueLian tension machine was used to compress stress–strain test for GO/CNT HA. Shanghai Chen Hua CHI660E electrochemical workstation was used for GO/CNT HA The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.Supplemental Video 3Click here for additional data file.


Unravelling the molecular mechanisms that account for functional pleiotropy is a major challenge for researchers in cytokine biology. Cytokine–receptor cross‐reactivity and shared signalling pathways are considered primary drivers of cytokine pleiotropy. However, reports epitomized by studies of Jak‐STAT cytokine signalling identify interesting biochemical and epigenetic determinants of transcription factor regulation that affect the delivery of signal‐dependent cytokine responses. Here, a regulatory interplay between STAT transcription factors and their convergence to specific genomic enhancers support the fine‐tuning of cytokine responses controlling host immunity, functional identity, and tissue homeostasis and repair. In this review, we provide an overview of the signalling networks that shape the way cells sense and interpret cytokine cues. With an emphasis on the biology of interleukin‐6, we highlight the importance of these mechanisms to both physiological processes and pathophysiological outcomes. The interleukin‐6 signalling cassette controls cytokine activities essential for homeostasis, immune defence and disease progression. In this review, we discuss the regulatory processes determining IL‐6 bioactivity and bioavailability. We consider the intracellular events that orchestrate and fine‐tune the delivery of Jak‐STAT signals and explore the transcriptional mechanisms that dictate the interpretation of IL‐6 signals in physiology and pathophysiology. DUSP, dual specificity protein phosphatasesELS, ectopic lymphoid‐like structuresGAS, gamma activated sequenceHDL, high‐density lipoproteinIL, interleukinISRE, interferon stimulated responsive elementJak, Janus activated kinasemiR, microRNAPIAS, protein inhibitor of activated STAT factorsPTP, protein tyrosine phosphataseSNP, single nucleotide polymorphisSOCS, suppressor of cytokine signallingSTAT, signal transducer and activator of transcriptionInterleukin (IL)‐6 controls multiple phenotypic traits that impact both immune effector functions and the regulation of homeostatic processes. Although traditionally viewed as a pro‐inflammatory cytokine involved in disease progression, the biology of IL‐6 is complex contributing to metabolism, development, tissue turnover and psychological well‐being , 6, 7, 8The functional pleiotropy of IL‐6 is typified by observations made in patients lacking a functional IL‐6 receptor cassette or downstream signalling intermediates, and children with anti‐IL‐6 autoantibodies , 14, 15.Significantly, studies in drosophila and mice re‐affirm the dynamic and pleiotropic properties of IL‐6 in physiology and pathophysiology . Here, tAlmost all stromal cells  and certain subsets of immune cells (e.g. macrophages and other monocytic cells) produce IL‐6 in response to Toll‐like receptor agonists, cytokines , lipid mediators (e.g. prostaglandins), adipokines, and as a consequence of cellular stress. Significantly, IL‐6 gene expression is subject to both homeostatic control and rapid induction following inflammatory challenge as a response to infection, trauma, autoimmunity or cancer. These involvements are equally reflected by the definition of IL‐6 as an adipokine, myokine, neurotrophic factor, lymphokine and monokine. How IL‐6 coordinates each of the activities is subjected to much research, and the complex nature of IL‐6 biology is highlighted by the various signalling mechanisms adopted by cells to sense and interpret changes in IL‐6 bioactivity (as discussed below).via separate interfaces . The. The4]. terfaces , 40, 41.terfaces , 32, 33.terfaces , 43, 44.terfaces . Beyond terfaces .This receptor system is elegantly poised to contain or limit the level of IL‐6 receptor signalling. Here, various mechanisms have evolved to restrain gp130‐mediated outcomes. For example, regulatory processes that affect receptor internalization and deactivation and signalling intermediates that inhibit or modify the signalling output of IL‐6. Many will be discussed in due course, but include protein tyrosine phosphatases (PTPs), microRNA (miR) that target mRNAs encoding components of the IL‐6 receptor cassette, and STAT‐inducible factors that act as negative feedback inhibitors of gp130 signalling  , 47, 48.gp130  are embryonically lethal with genetic mutation studies identifying essential roles for gp130 in development, haematopoiesis, tissue homeostasis, cell survival and growth, and immune regulation [Although gp130 was initially identified as the signalling subunit of the IL‐6 receptor complex, this glycoprotein displays a broad pattern of cellular expression and functions as the β‐cytokine receptor for IL‐11, IL‐27, oncostatin‐M, ciliary neurotrophic factor, leukaemia inhibitory factor, cardiotrophin‐1 and cardiotrophin‐like cytokine , 29. Micgulation , 50. IL‐gulation , 50, 51.gulation , 53. Thegulation , 29, 30.gulation , 55. Forgulation .−1) where it constitutes an IL‐6 buffering system that prevents penetrance of minor fluctuations in IL‐6 concentration being interpreted as a danger signal [In human serum, sgp130 is present at saturating levels , but these concentrations oscillate during the course of the day and are equally subject to seasonal variations between summer and winter months [−1 or μg·mL−1 quantities) in response to cytokines, Toll‐like receptor agonists, prostaglandins and other stress signals [IL6 expression [IL6R or IL6ST polymorphisms [IL6R polymorphism rs2228145 show enhanced sIL‐6R and IL‐6 levels, reduced C‐reactive protein and a lower risk of cardiovascular disease [The coordination of these modes of IL‐6 receptor signalling is influenced by changes in the bioavailability of IL‐6, sIL‐6R and sgp130. IL‐6 expression is tightly regulated at the transcriptional level to ensure physiological fluctuations in IL‐6 during homeostasis and rapid induction during infection, trauma or injury . In humar months , 63, 64. signals . Here, c signals , 67, 68.pression , 70. Carpression , 72. Simorphisms , 74. Ind disease , 76. Con disease , 68, 77.+ T‐helper cells, with sIL‐6R serving to retain circulating levels of IL‐6. Thus, there are shades of grey here, and the overall biological activity of IL‐6 is likely shaped differently in health and disease or different tissue compartments.A challenge for IL‐6 researchers is to understand the dynamics and kinetics of IL‐6 bioactivity. For example, what is the physiological role for classical IL‐6R signalling versus IL‐6 trans‐signalling and IL‐6 trans‐presentation? Whilst research of IL‐6 trans‐presentation is required, much work has centred on the distinction between classical IL‐6R signalling and IL‐6 trans‐signalling. Here, the application of sgp130 has provided considerable evidence that IL‐6 trans‐signalling broadens the types of cells that become IL‐6 responsive . The inflammatory regulation of sIL‐6R supporting a role for IL‐6 trans‐signalling in coordinating immune responses to disease. Classical IL‐6R signalling is, however, linked with the maintenance of immune or tissue homeostasis . However, these distinctions are not unequivocal. For instance, classical IL‐6R signalling supports the expansion of IL‐17‐secreting CD4In the subsequent sections, we will now turn our attention to the intracellular mechanisms that influence the transmission of IL‐6 receptor signals from the cell membrane to the nucleus and affect the biological properties of IL‐6.context is key to understanding IL‐6 signalling outcomes. This has been evidenced in models of bacterial peritonitis, a rare complication of peritoneal dialysis in patients with end‐stage renal failure [Staphylococcus epidermidis in mice revealed that IL‐6 acting on surrounding stromal tissue initiates the recruitment of leukocytes into the inflamed peritoneum by inducing the expression of chemokines, cell adhesion molecules, acute‐phase‐reactants and processes related to tissue barrier permeability [+ T cells within the local tissue [The signalling pathways associated with IL‐6 signalling are discussed at length in the literature , 31, 32. failure . Intrapeeability , 82, 83.eability . The releability , 87, 88.eability , 90. In l tissue . This il+ T cells [+ T cells reveal that STAT1 activity confers an interferon gene expression signature via an IRF1 feed‐forward loop that is not seen in the transcriptional profile of IL‐6 [+ T cells from Stat1−/− mice show that IL‐6 and IL‐27 induce very similar transcriptional programmes [A key question is how IL‐6 family cytokines, sharing the same signal transducing subunit namely gp130), can elicit unique STAT activation profiles and functional outputs. One explanation is likely to be found at the membrane interface of the cytokine‐receptor signalling complex. Here, our understanding rests on Cryo‐EM reconstruction of the extracellular region of the IL‐6‐IL‐6R‐gp130 hexamer . Less cl30, can e T cells , 95, 96. T cells . The Y61 of IL‐6 . Moreoveogrammes , 99. Thuogrammes , 101.Alterations in the cytokine control of STAT transcription factor phosphorylation have important consequences at the gene level . Studies+ T cells isolated from Stat1−/− mice show increased STAT3 phosphorylation when stimulated with IL‐6 or IL‐27, while the reverse is true Stat3−/− mice [vice versa [Stat3 deficient naïve CD4+ T cells shows that an absence of STAT3 results in a collapse in IL‐6 and IL‐27 gene regulation. In contrast, Stat1 deficiency reduces the diversity of cytokine‐induced transcripts [The significance of these processes is evidenced as follows. Activated naïve CD4−/− mice . As a coce versa . But whace versa . Transcrnscripts , 100. Thnscripts . TherefoSocs3−/− mice in vivo and patients lacking functional STAT1 and STAT3 transcription factors [SOCS3 transcript abundance in peripheral blood mononuclear cells from individuals with STAT1 gain‐of‐function and STAT3 loss‐of‐function mutations [Competition for phospho‐tyrosine residues is not the only contributing factor in determining the output of Jak‐STAT signals. This is because STAT transcription factors are not thought to be present at saturating levels in the cytoplasm . Instead factors , 110. SO factors . Moreoveutations .+ T‐cell activation increases the expression of several protein phosphatases, including PTPN2, PTPN22 and DUSP2. Thus, the transcriptional output of a cytokine maybe altered by changes in the expression of these enzymes. Studies have shown that naïve CD4+ T‐cell activation impairs the IL‐6 control of STAT1 in activated and memory CD4+ T‐cell subsets [IL6 mRNA as part of a post‐transcriptional negative feedback mechanism. Significantly, mice lacking the Regnase‐1 encoding gene (Zc3h12a) die prematurely of systemic auto‐inflammatory complications [SOCS1, PTPN2, and Z3CH12A, synergistically potentiates the anti‐tumour efficacy of CD8+ T cells by promoting effector memory T‐cell functions [In addition to SOCS proteins, studies have shown that protein phosphatases also regulate Jak‐STAT cytokine receptor signalling. Examples include dual specificity protein phosphatases (DUSPs) and several PTPs , 115. Fo subsets , 120. Th subsets . Finallyications . The phyunctions .IL6 transcript. The most compelling are miR‐98 and miR‐223 [miR‐223−/− mice and contributes to exacerbated ethanol‐induced hepatic injury through an in increase in neutrophil infiltration and reactive oxygen species generation [miR‐223−/− mice where miR‐233 deficiency causes impaired muscle regeneration and increases in interstitial fibrosis due to the maintenance of a pro‐inflammatory macrophage population [Various miR interfere with the IL‐6 signalling cassette. These regulatory elements fine‐tune the expression and bioavailability of specific components within the receptor system and Jak‐STAT pathway Fig. . However miR‐223 , 126. In miR‐223 . IL‐6 isneration . A similpulation . Howeverpulation , which rmiR‐34a−/− mice show an increased incidence of colitis‐associated intestinal tumours that are typically larger in size and characterized by increases in IL‐6R expression and STAT3 activity [Several miR target components of the IL‐6 receptor complex or signalling intermediates within the downstream Jak‐STAT pathway. For example, miR‐34 and miR‐218, which are identified as tumour suppressors , 129. Cractivity . These factivity . Howeveractivity . Severalactivity , 134. Wiactivity .The negative regulators of the Jak‐STAT cascade are also subject to miR‐mediated repression. miR‐155 and miR‐19 have been shown to target SOCS1 and SOCS3, respectively , 136. In+ T cells (Th1 cells) and IL‐4‐secreting CD4+ T cells (Th2 cells) where the localization of STAT4 and STAT6 to P300 enhancers promote the expression of the lineage defining transcription factors TBET and GATA3, respectively [STAT transcription factors rarely bind at gene promoter regions. More frequently, STAT binding occurs at intron‐localized motifs, sometimes intergenic regions, and often in close alignment with distal enhancer elements , 140. Onectively . Thus, tectively , 150.In vitro, STAT1 binding to P300 triggers a trans‐auto‐acetylation reaction, which catalyses the recruitment and activation of neighbouring P300 molecules. Here, the phosphorylation‐induced dimerization of the STAT1 transcription factor complex is important for bridging P300 molecules and drawing them into proximity with one another [Various biochemical studies have explored the interaction of STAT transcription factors with P300 , 153. In another . The cry another , 152. Th another .in silico motif enrichment analyses and biochemical approaches support the idea that STAT transcription factors engage with highly dynamic enhancer interactomes in a context‐dependent manner [Advances in next‐generation sequencing technologies  are now affording exciting opportunities to understand how STAT transcription factors function in cooperation or competition at promoter sites (e.g. through cross‐regulation), and form meaningful partnerships with other transcription factors at enhancers. Here, t manner , 154. Thin vitro have shown that the latent enhancer repertoire is ligand‐specific, delivering overlapping and distinct chromatin remodelling in response to different TLR ligands  and cytokines  [ChIP‐seq and ATAC‐seq have evidenced the dynamic and complex nature of STAT transcription factor interactions with the genome (e.g. P300 sites as discussed) , 150. Thβ, IFNγ) . Latent β, IFNγ) , 158. Onβ, IFNγ) , 149. Laβ, IFNγ) . Consequβ, IFNγ) . To undeAlu elements functioning as either tissue‐specialized (poised/primed) or latent enhancers [Alu elements belong to a family of endogenous retroelements, which comprise up to 10% of the human genome and are often transcriptionally repressed through methylation [Alu enhancer function across tissues. This analysis identified involvements in processes integral to tissue specialization. These included neuronal involvement in the brain, immunological processes in the spleen, and metabolism in the Liver [Alu elements with P300 occupancy. Thus, reflecting a proximity to genes controlling tissue identity [Alu enhancers were shown to promote cellular proliferation by regulating chromatin looping through interactions between RNA Polymerase III, general transcription factor III (TFIIIC), CCCTC‐binding factor (CTCF) and the cohesion complex [Alu enhancers appear to govern the overall chromatin architecture influencing gene expression over long distances through the organization of three‐dimensional units of gene regulation termed topologically associated domains [Alu elements and various interferonopathy‐like conditions [Alu enhancer may coordinate de novo genomic architectures that facilitate adaptation to immune challenge. Further work is, however, required to characterize the definition of latent enhancers that are unveiled following exposure to immune or environmental challenge.An emerging literature points to a subset of nhancers , 163. Alhylation , 164. Onhe Liver . This fuidentity . These ridentity , 143. El domains , 163. Al domains , 165. Henditions , 168. Wein silico motif enrichment analyses are supportive of the notion that STAT transcription factors form unique complexes in a highly context‐dependent manner [Alu enhancers are notable for their particularly high density of transcription factor binding motifs with at least one study proposing that evolutionary divergence has created Alu subsets that harbour distinct sets of concensus motifs [Alu enhancers [In summary, t manner . Thus, wt manner . For exas motifs . Analysis motifs . Other snhancers .Genetic ablation studies in mice showcase roles for IL‐6 in both normal physiology and pathophysiology. These are often recapitulated in humans where biological drugs and small molecule inhibitors acting directly on the IL‐6 cytokine receptor or downstream signalling intermediates identify the importance of IL‐6 signalling through STAT1 and STAT3 , 173.The Jak‐STAT pathway has evolved to sense and interpret cytokine cues essential for cellular proliferation and functional identity . These ivice versa) [The transcriptional output delivered by the Jak‐STAT pathway is highly dynamic and incorporates partnerships with other transcription factors . Workinge versa) . Cross‐re versa) , 91. WhiIl6−/− mice with arthritis [In mouse models of experimental autoimmune or inflammatory disease, IL‐6 deficiency often limits the histological signs of pathology. For example, hallmarks of synovial hyperplasia, synovial infiltration (or associated exudate), and joint erosion are all less pronounced in rthritis , 179. Thrthritis , 182. Derthritis , 184. Inrthritis , 186. Thrthritis , 189.+ T cells  [gp130Y757F:Y757F mice with antigen‐induced arthritis provide some evidence of STAT transcription factor cross‐regulation [gp130Y757F:Y757F mice with antigen‐induced arthritis displayed exacerbated joint pathology with synovitis revealing evidence of lymphoid aggregates [+ T cells from inguinal draining lymph nodes of gp130Y757F:Y757F mice with arthritis showed that genetic ablation of Stat1 in these animals enhanced the expression of several STAT3 regulated cytokines involved in T cell‐driven synovitis [gp130Y759F:Y759F) [gp130Y757F:Y757F and gp130Y759F:Y759F mice [The Jak‐STAT pathway senses and interprets cytokine cues targeted by drugs  commonly prescribed for the treatment of rheumatoid arthritis , 171. Asnctions) , 200. Stgulation , 201. Thgulation , 201. Hegregates , 199. MoF:Y759F) , 203. ThF:Y759F) . In this59F mice , 206.Il6−/− and Il27ra−/− mice with antigen‐induced arthritis, studies show that these mice develop joint disease resembling the heterogeneous features of synovitis commonly seen in humans with rheumatoid arthritis [Il6−/− mice develop a low‐inflammatory pathology (lacking an immune cell infiltrate) resembling fibroblast‐rich synovitis [Il27ra−/− mice where the presence of organized ectopic lymphoid‐like structures (ELS) resembled lymphoid‐rich synovitis [Extending the investigation of STAT1 and STAT3 cytokine signalling to wild‐type, rthritis , 208. Foynovitis , 208. Thynovitis , 210. Reynovitis , 98, 211ynovitis , 211. Hevia the Jak‐STAT pathway is controlled at multiple levels [+ T cells from Lck‐Cre:Ptpn2fl/fl (T‐cell restricted deletion) and Ptpn22−/− mice, PTPN2 and PTPN22 have been reported to inhibit STAT1 tyrosine‐phosphorylation (pY‐STAT1) in IL‐6‐treated effector memory CD4+ T cells [+ T cells, this form of STAT1 regulation altered the types of genes under STAT3 control in memory CD4+ T cells [+/−Ptpn2 mice develop synovitis showing enriched expression of synovial Th17 cells and ELS in the SKG model of autoimmune arthritis, consistent with the development of synovitis in Il27ra−/− mice with AIA [+ T cells showed that PTPN2 inhibition of pY‐STAT1 enhances STAT3 gene regulation through promoters displaying P300 super‐enhancer architectures [PTPN2 in human synovial biopsies showed the highest expression in lymphoid‐rich synovitis [As discussed, cytokine signalling e levels , 171. The levels , 106. Ote levels . For exae levels , 214. The levels . Focussi T cells , 216. Wh T cells . In muri T cells , 222. Fowith AIA , 219. Motectures , 139. Thtectures . Here, aynovitis . Thus, Pynovitis . This maynovitis , 228. CoA key question is whether IL‐6 utilizes different enhancer subtypes to affect its homeostatic and disease‐causing properties. Latent enhancers, as an adaptive response to environmental stimuli, might be expected to drive processes relevant to supporting tissue adaptation. Meanwhile, enhancers linked to IL‐6 homeostatic properties are likely to be to be constitutively active as homeostasis is ongoing under tonic signalling. Indeed, genes contributing to set biological processes often share common mechanisms of transcriptional control . FunctioAlu enhancers is significant due to the historic association of retroelements with SNPs linked to most types of human pathology. For example, systemic lupus erythematosus and Sjörgren's syndrome, and human genetic disorders such as Aicardi‐Goutieres syndrome and familial chilblain lupus [Alu‐RNA linked autoimmunity, or interference with cellular processes including ribosome function. Retroelements are not static, and have the capacity, once expressed, to interfere with cellular processes and in rare cases, reintegrate into the genome [Alu insertions often contribute to hereditary disease, blood disorders and neurological conditions. In inflammation, Alu RNA expression is interferon inducible and has the capacity to activate intracellular Pattern Recognition Receptors including MDA5 and RIG‐1 to further propagate the immune response [Alu‐RNA expression may contribute to neuroinflammation and neurodegeneration in Alzheimer's disease, and the functional properties of peripheral blood cells isolated from patients with Systemic Lupus Erythematous (SLE) [The connection between STAT transcription factors and in lupus , 167. Thin lupus . Thus, pe genome . Here, sresponse , 232. Itus (SLE) . FurtherAdvances in cytokine biology increasingly identify novel mechanisms of cytokine involvement in physiology and pathophysiology. Studies in animal models and cell systems have now been aided by the widespread use of cytokine targeting therapies in patients with complex immune‐mediated conditions, infectious diseases and cancer. This is epitomized by studies of the Jak‐STAT pathway, which frequently combine methods common to the evaluation of cellular immunology, histology, functional genomics and the holistic analysis of the proteome and transcriptome. These approaches provide a high‐level understanding of how cells and tissues sense and interpret cytokine cues during health and disease. Various challenges, however, still remain. Focussing on the biology of IL‐6, there is a pressing need to understand how this major inflammatory cytokine contributes to normal physiology, competent anti‐microbial host defence, and the transition to pathophysiology and systemic chronic inflammation. Here, the estate agents  adage ‘Location, Location, Location’ takes on an important meaning and offers opportunities to understand how the systemic and local inflammatory context is shaped to promote disease susceptibility, heterogeneity in patient pathology, multimorbidity and response to drug therapy.


Sepsis causes multiple organ injuries, among which the heart is one most severely damaged organ. Melatonin (MEL) alleviates septic myocardial injury, although a systematic and comprehensive approach is still lacking to understand the precise protective machinery of MEL. This study aimed to examine the underlying mechanisms of MEL on improvement of septic myocardial injury at a systematic level. This study integrated three analytic modalities including database investigations, RNA‐seq analysis, and weighted gene co‐expression network analysis (WCGNA), in order to acquire a set of genes associated with the pathogenesis of sepsis. The Drugbank database was employed to predict genes that may serve as pharmacological targets for MEL‐elicited benefits, if any. A pharmacological protein–protein interaction network was subsequently constructed, and 66 hub genes were captured which were enriched in a variety of immune response pathways. Notably, PIK3CG, one of the hub genes, displayed high topological characteristic values, strongly suggesting its promise as a novel target for MEL‐evoked treatment of septic myocardial injury. Importantly, molecular docking simulation experiments as well as in vitro and in vivo studies supported an essential role for PIK3CG in MEL‐elicited effect on septic myocardial injury. This study systematically clarified the mechanisms of MEL intervention in septic myocardial injury involved multiple targets and multiple pathways. Moreover, PIK3CG‐governed signaling cascade plays an important role in the etiology of sepsis and septic myocardial injury. Findings from our study provide valuable information on novel intervention targets for the management of septic myocardial injury. More importantly, this study has indicated the utility of combining a series of techniques for disease target discovery and exploration of possible drug targets, which should shed some light on elucidation of experimental and clinical drug action mechanisms systematically. Sepsis is believed the main culprit for high morbidity and mortality in the intensive care unit.Melatonin (MEL) is a neuroendocrine hormone synthesized by the pineal gland, with an inherent role in circadian rhythm control.Although several studies have shown a protective role for MEL in sepsis and septic myocardial injury, the current understanding with regards to pharmacological actions behind MEL is mainly derived from its intervention with common molecules or signaling pathways. A more thorough, systematic and comprehensive approach is urgently needed to unveil the full spectrum behind MEL‐elicited protective mechanisms. Network pharmacology provides a practical strategy for elucidation of potential mechanisms of multicomponent and multitarget drugs by analyzing multiple networks of complex interactions, commonly performed to discover the key targets for disease treatment.22.1Saa3,Csf3,Fpr2To obtain genes associated with the pathogenesis of sepsis, we performed transcriptome analysis on cecal ligation and puncture (CLP) and sham groups. A total of 24 RNA‐seq libraries were constructed and analyzed, among which 12 biological replicates were included in each group. After removing low‐quality reads, the remaining reads were aligned to mouse genome reference  was chosen as the soft‐thresholding parameter to ensure a scale‐free network , stroke volume (SV) , and cardiac output (CO) , and a positive correlation with sepsis score   Figure . Subsequ) Figure  (details) Figure  and show2.3A total of 109 known sepsis‐related genes were collected from multiple databases . Therefore, this level of MEL was used for the remaining studies. Subsequently, sepsis score and anal temperature were monitored 8 h post‐CLP. In this study, the MSS system was introduced to monitor experimental mice based on their physical appearance, level of consciousness, activity, response to stimuli, eyes, respiration rate and quality (from 0 to 4 points for each criteria).p < 0.05).To validate the hub genes detected, both in vivo and in vitro experiments were performed. CLP is one of the most widely used model for experimental sepsis. The establishment of CLP model mainly consists of two steps: cecal ligation and puncture Figure . Mice we2.5p < 0.05). In addition, levels of blood biochemical parameters (lactic dehydrogenase (LDH), creatine kinase (CK), blood urea nitrogen (BUN), and aspartate aminotransferase (AST)) were significantly increased, and level of albumin (ALB) was decreased after CLP injury, the effects of which were reverted by MEL treatment . Myocardial function was assessed using echocardiography. As shown in Figure p < 0.05), the effects of which were mitigated by MEL . Besides, H&E staining showed obvious structural abnormalities and myocardial fibrosis in CLP‐injured myocardium, while MEL treatment remarkably restored these CLP‐induced pathological changes , lymphocytes (LYM), monocytes (MON), granulocytes (GRA), and platelets (PLT), decreased the level of red blood cells (RBC) . However, MEL treatment reversed these culprit effects . Moreover, the IHC staining also confirmed that MEL decreased PIK3CG expression . Moreover, the qRT‐PCR assay demonstrated that LPS treatment significantly increased Akt, IL‐6, TNF‐α, Myc, and Pdk1 mRNA expressions in LPS‐injured HL‐1 cells, the effect of which was abrogated by MEL . Immunofluorescence and western blot results also indicated that MEL countered LPS‐induced hyperactivated PIK3CG signaling cascade . Moreover, the levels of p‐AKT/AKT, MYC, PDK1, NLRP3, and IL‐6 rose significantly after LPS injury, the effects of which were reversed by MEL . Subsequently, PIK3CG was silenced by small interfering RNAs (siRNAs). The qRT‐PCR assay demonstrated that PIK3CG siRNA significantly decreased PIK3CG, NLRP3, Caspase‐1, and IL‐6 mRNA expressions but increased Bax mRNA expressions in HL‐1 cells .HL‐1 cells were pretreated with various concentrations of MEL  prior to exposure to LPS for 3 h. The results showed that MEL exerted the most significant protective effect against LPS‐injured cells, as evidenced by increased cell viability and a decreased intracellular ROS production  and catalytic subunit (p110). PI3K is reported to be involved in cell proliferation, apoptosis, migration, and inflammation among other pathophysiological processes, affecting the onset and development of a variety of cardiovascular diseases.Saa3 and Fpr2) as potential targets of septic myocardial injury in previous studies.As mentioned above, a systematic and comprehensive approach is urgently needed to explore the mechanisms of MEL in the protection against septic myocardial injury. Network pharmacology provides a practical strategy for elucidating the potential mechanisms of multicomponent and multitarget agents by analyzing various networks of complex interactions, which is widely used to discover key pathways and target molecules for disease treatment.4This study clarified the mechanisms of MEL intervention in septic myocardial injury involved multiple targets by integrating multiple approaches. Additionally, PIK3CG‐governed signaling cascade plays an important role in the etiology of sepsis and septic myocardial injury. However, the regulation pattern and upstream/downstream pathways of PIK3CG need to be further investigated. This finding provides valuable information on novel intervention targets for the management of septic myocardial injury.55.1N), and low‐quality reads containing more than 20% of bases with qualities of <20. Hisat2 software was employed to align clean reads to mouse genome (version: mm10 NCBI). The SVA/Combat softwarep < 0.05. The heat map and volcano map were generated based on the top 200 DEGs (top 100 upregulated genes and top 100 downregulated genes) in the CLP group using the ggplot2 package in R language.In this study, a total of 24 male BALB/c mice were divided into sham and CLP groups. Myocardium (100 mg) was collected at post‐8 h, and TRIzol reagent  was employed to extract total RNA from mouse tissues. Quality and quantity of RNA extracts were checked using a Bioanalyzer 2200 . The cDNA libraries were sequenced using a HiSeqXTen (Illumina) platform and finished the sequencing in three batches. Raw reads were mined for clean reads by removing the adaptor sequences, reads with >5% ambiguous bases |β.i and gene j. The power of β (weighted coefficient) was set to be 8 (R2 = 0.85) to establish a signed scale‐free network. Next, a topological overlap matrix (TOM) was derived using the adjacency matrix data and the dynamic tree cut algorithm was performed to identify gene modules (which corresponded to colored branches of dendrogram). To decipher connections between gene modules and biometric, plasma and echocardiographic parameters of septic mice, Pearson's correlation coefficients between mouse traits and the module eigengene  were calculated.Quality‐controlled count matrix was applied to identify significant gene modules and determining the core genes related to biometric, plasma and echocardiographic parameters of septic mice using the WGCNA packagep = 0.05. Within a given module, gene significance (GS) described the relationship between gene expression profile and each trait. Module membership (MM) was defined as the correlation between the gene expression profile and module eigengene. The core genes were picked out according to the strict cut‐off criteria |MM| > 0.8, |GS| > 0.8. For details in each procedure, please refer to the previous work of Langfelder and Horvath.In order to screen the core gene candidates, modules associated with the traits of interest, in our case “SV,” “CO,” “group,” and “sepsis score” were further considered for the intranodal analysis. The significance threshold of module selection was set as 5.4https://pubchem.ncbi.nlm.nih.gov/). Based on structural information, MEL putative targets were surveyed based on DrugBank 5.0 with similarity >0.6.https://www.tcmsp-e.com/) and TCMIPhttp://www.tcmip.cn/TCMIP/index.php/Home/), which integrates information from the DisGeNET database,Structural information of MEL was acquired from the PubChem database . The structural information of MEL was obtained from the PubChem database, and then the sdf format was converted to the mol2 format using the OpenBabel v3.1.1 software. The proteins were preprocessed with AutoDock v4.2.6 software before docking, including removing water molecules, adding hydrogens and calculating the Gasteiger charge. The ligand was also preprocessed via AutoDock, including adding all hydrogens and detecting torsional bonds. The active pocket sites for small molecule ligand binding were obtained from the POCASA website . The pockets were sorted by volume depth, and the top five were picked for docking.The structures of proteins  were obtained from the RSCB PDB database  aged 8–10 weeks were obtained from the animal center of Air Force Military Medical University of PLA . All animal experiment protocols were performed in accordance with the guidelines of Animal Care and Use Committees at Northwest University  and carried out according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . All mice had free access to food and water and were bred at 26°C in a 12 h light/12 h dark cycle.5.8The CLP model was used to induce sepsis as previously described5.8.1MEL was dissolved in DMSO and given to mice every 2 days for 6 days before CLP injury. Mice were randomly assigned to the following groups: (a) mice injected with 1 ml/kg DMSO and subjected to sham surgery; (b) mice injected with 1 ml/kg DMSO and then subjected to CLP surgery; and (c) mice treated with MEL and then subjected to CLP surgery. In the third group, different levels of MEL  were administrated to mice to monitor survival rate for 72 h, among which 30 mg/kg was selected for further analysis, including sepsis score, anal temperature, blood routine parameters, blood biochemical parameters, cardiac function, myocardial structure, and molecular signaling Figure .5.9At 8 h post‐CLP, at least 10 μl of blood was collected from the left eyeball into a heparin‐coated tube. The levels of WBC, LYM, MON, GRA, RBC, and PLT were detected by an automatic blood analyzer . After that, 150 μl of serum was isolated from the rest of the whole blood by being centrifuged at 3000 rpm for 10 min. The levels of LDH, CK, AST, ALB, and BUN were detected by an automatic blood biochemical analyzer .5.10Transthoracic echocardiography was performed using an animal‐specific instrument  at 8 h post‐CLP in mice. Mice were anesthetized with 3% isoflurane and 1 L/min 100% oxygen in an induction chamber for 1–2 min. Next, mice were, respectively, laid supine on a warm platform and kept anesthetized by 2% isoflurane until they lost body‐righting reflex. Then, series of M‐mode images at the level of papillary muscles were obtained. SV, CO, LVPWs, LVPWd, LVEDV, LVESV, and HR were measured using Vevo LAB 3.0.0. All measurements were based on three consecutive cardiac cycles.5.11The myocardium was fixed in 4% paraformaldehyde and sectioned at a thickness of 4–5 μm. Morphological changes in myocardium were observed by hematoxylin–eosin (H&E) staining. The degree of myocardial fibrosis was examined by Masson staining . For immunostaining, paraffin‐embedded slices were stained with the respective primary antibody against NOX2, IL‐6, TNF‐α , PIK3CG , then incubated with a secondary biotinylated anti‐rabbit IgG, stained with 3,3′‐diaminobenzidine (DAB), and imaged using a microscope . Finally, immunoreactive areas were quantified using the ImagePro Plus 4.5 software .5.12Total RNA was extracted from tissues using the TRIzolTM total RNA extraction kit , and reverse transcription was performed using the Prime Script RT Master Mix . Then TNF‐α, IL‐1β, IL‐6, NLRP3, PIK3CG, Akt, Myc, Pdk1, and Caspase‐1 mRNA level were detected using quantitative real‐time reverse transcriptase PCR analyses with SYBR Premix Ex Taq . The primers that were used are shown in Table 5.13HL‐1 cells were seeded and cultured as previously described.5.14HL‐1 cells were seeded and cultured as previously described. The cell viability and intracellular ROS generation were examined according to our previous study.5.15Isolated cells were homogenized in RIPA buffer containing protease and phosphatase inhibitors . Thirty‐fifty μg of total protein extract was loaded to SDS‐PAGE (8% or 10%) and transferred onto PVDF membranes , which were next blocked by 5% skim milk and incubated with primary antibodies at 4°C for 24 h. In this study, used primary antibodies were as follows: PIK3CG , AKT, p‐AKT, MYC, NLRP3  PDK1 , IL‐6 , and β‐actin . Then, membranes were washed three times with TBST and incubated with secondary antibodies  at room temperature for 1 h. Subsequently, used the fluorescence signal to visualize the membrane and used the MiNiChemi610 imaging system for detection . Finally, the signal was quantified by Image J 5.0 software .5.16The sequences of siRNA targeting murine PIK3CG  were synthesized by Sangon Biotechnology . The siRNA was used to knockdown PIK3CG in HL‐1 cells. The specific step can be referred to our previous studies.5.17t test was used for comparison between two groups, and one‐way ANOVA was used for multiple group comparison. A p < 0.05 indicated significant differences.Data were analyzed using the GraphPad Prism 9.0.0 software . All values are presented as the mean ± standard deviation (SD). For analysis of experimental data, Qiong Liu: Data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); validation ; writing – original draft (lead); writing – review and editing . Yushu Dong: Data curation (lead); investigation (lead); methodology (lead); writing – original draft . Germaine Escames: Data curation ; investigation (lead); methodology ; writing – original draft . Xue Wu: Investigation ; methodology ; writing – original draft . Jun Ren: Investigation ; methodology ; writing – review and editing . Wenwen Yang: Investigation ; methodology . Shaofei Zhang: Investigation ; methodology . Yanli Zhu: Investigation ; methodology . Ye Tian: Investigation ; methodology . Yang Yang: Conceptualization (lead); funding acquisition (lead); project administration (lead); resources (lead); supervision (lead); validation (lead); writing – review and editing (lead). Darío Acuña‐Castroviejo: Conceptualization (lead); investigation ; methodology ; project administration ; validation (lead); writing – original draft ; writing – review and editing .This work was supported by the National Natural Science Foundation of China (81871607 and 82070422), Innovation Capability Strong Foundation Plan of Xi'an City , Key Research and Development Program of Shaanxi (2020ZDLSF04‐03), Major Research Projects of Xi'an Science and Technology Plan (201805104YX12SF38 (2)), and the Instituto de Salud Carlos III , Spain, grants PI19‐01372 and CB16/10/00239.The authors declare no conflict of interest.https://publons.com/publon/10.1002/btm2.10384.The peer review history for this article is available at Appendix S1 Supporting InformationClick here for additional data file.


FST) among localities of bee‐ than vertebrate‐pollinated species with increasing geographical distance, topographic barriers and historical climatic instability. High admixture among montane populations further suggested relative climatic stability of Neotropical montane forests during the Pleistocene. Overall, our results indicate that pollinators may differentially impact the potential for allopatric speciation, thereby critically influencing diversification histories at macroevolutionary scales.Animal pollinators mediate gene flow among plant populations, but in contrast to well‐studied topographic and (Pleistocene) environmental isolating barriers, their impact on population genetic differentiation remains largely unexplored. Comparing how these multifarious factors drive microevolutionary histories is, however, crucial for better resolving macroevolutionary patterns of plant diversification. Here we combined genomic analyses with landscape genetics and niche modelling across six related Neotropical plant species  differing in pollination strategy to test the hypothesis that highly mobile (vertebrate) pollinators more effectively link isolated localities than less mobile (bee) pollinators. We found consistently higher genetic differentiation ( Two of the six species are pollinated by bees, three are pollinated by mixed assemblages of vertebrates  and one is pollinated by nonterritorial passerine birds Figure , 2021. W22.1Adelobotrys adscendens (Sw.) Triana, cloud forest bee‐pollinated Meriania maxima Markgr., passerine‐pollinated Axinaea costaricensis Cogn., and three Meriania species pollinated by different combinations of vertebrates—M. phlomoides (Triana) Almeda and M. tomentosa (Cogn.) Wurdack, pollinated by hummingbirds and bats; M. sanguinea Wurdack, pollinated by hummingbirds, bats and rodents, Dellinger et al., M. sanguinea, 2: M. tomentosa and M. phlomoides [part of the same subclade of Merianieae], 3. Ax. costaricensis; Dellinger et al., Merianieae (Melastomataceae) are a Neotropical plant tribe of ~300 species, which has radiated recently in the tropical Andes . Bee pollination is ancestral and common both among lowland rainforest and cloud forest Merianieae, while shifts to vertebrate pollination  are restricted to cloud forest species  individuals per locality across a distance of 1 km  or Ecuador , we estimated genome sizes to select appropriate restriction enzymes for RADseq (restriction site‐associated DNA sequencing). We prepared fresh leaf material for three individuals per species in Otto's buffer  as an internal standard to calculate 1C values for each run (three runs per sample) and calculated the mean 1C per species over all runs and individuals. Average genome sizes  for the three species were estimated as follows: Ad. adscendens 0.344 pg, SD 0.003; Ax. costaricensis 0.587 pg, SD 0.01; M. phlomoides 0.639 pg, SD 0.006. According to the Kew C‐value database, these genome sizes are the second, third and fourth estimates for the family  for single‐digest RADseq  to the restricted samples at 16°C overnight. The P1 adapters included both index and inline barcodes, which were different from each other by at least three nucleotide positions. After P1 ligation, we sheared the DNA by sonication in a Bioruptor Pico (Diagenode) using two cycles  to obtain DNA fragments of 400 bp on average. After P2 adapter ligation, PCR (polymerase chain reaction) amplification and clean‐up steps , we performed a final size selection for the range 220–850 bp using a 1.5% precasted dye‐free cassette and a Pippin Prep (Sage Science). All libraries were sequenced on an Illumina HiSeq 2500 machine at Vienna BioCenter Core Facilities (VBCF) (http://www.viennabiocenter.org/vbcf/next‐generation‐sequencing/) as 100‐bp single‐end reads.In accordance with the relatively small genome sizes, we selected the restriction enzyme 2.4bamindexdecoder version 1.03 , and quality‐filtered and further demultiplexed the reads to individual accessions (inline barcodes) using process_radtags.pl from stacks version 1.47  of 4 and 5, subsequently increased M  starting from 1, and n  as n = M or n = M + 1  using export_sql.pl from stacks. We then mapped the raw reads of each accession to this pseudoreference separately using the mem algorithm of bwa version 0.7.12‐5  with  et al.,  for paraAd. adscendens, the lowland bee‐pollinated species, is part of a clade that diverged from core Merianieae (to which the other five species belong) ~25 million years ago . Whereas the common pseudoreference we used for mapping should be appropriate for the five core Merianieae species, ascertainment bias may affect the inference for Ad. adscendens based on this reference. To address this issue, we tested whether the mapping rates for the six study species to the pseudoreference differ significantly from each other using ANOVA and Tukey tests as post‐hoc tests. Indeed, the mapping rate of Ad. adscendens was significantly lower than of all other species . Using this approach, we increased the mapping rate for Ad. adscendens from 31.7% to 35.1%  using the genotype‐free method implemented in angsd  on genotype likelihoods as the degree of deviation from Hardy–Weinberg equilibrium  mating patterns by testing for isolation‐by‐distance (IBD) between individuals using Mantel's tests on pairwise genetic distances , starting from genotype‐frequency‐based covariance matrices . We then tested for significant differences in genetic diversity between populations of the same species  as a post‐hoc test to assess which populations were significantly different. To test whether certain populations were more disparate  than others, we used betadisper and TukeyHSD as post‐hoc test  or folded SFS (Ad. adscendens) using realSFS in angsd.To assess population genetic differentiation, we calculated ngsadmix, Skotte et al., K = 1 to K = n + 1 ancestral populations, n being the total number of sampled localities in each species. We compared the rate of change in the log‐likelihood of different successive K‐values  to select the K‐value best describing clustering in the data and used bar plots for visualization.We further calculated the admixture coefficient for each individual by estimating the likelihood of genetic clustering in the data , current climatic suitability , climatic suitability since the Last Glacial Maximum (LGM)  and the environmental niche (IBE) on population genomic differentiation (FST). For each species’ entire distribution range, we downloaded occurrence data from GBIF  and calculated topographic complexity using the Terrain Ruggedness Index . Finally, we calculated all pairwise least‐cost distances (paths with least‐accumulative cost over cost surface) between the sampling localities (costDistance function).We used landscape genetic approaches to estimate the degree of connectivity among localities, assessing the relative impact of isolating barriers : Annual Mean Temperature (bio1), Mean Diurnal Temperature Range (bio2), Annual Precipitation (bio12), and Precipitation Seasonality . We created 10,000 pseudo‐absence points (the default number of background points used by maxent) from raster cells lacking GBIF occurrences . We set 80% of the GBIF occurrences of each species as the training data set and 20% to validate the models, using 500 iterations. We fit models 10 times and validated model fit using AUC  as for the current climate, but derived from three different general circulation models for the LGM and mid‐Holocene  and averaged suitability of each raster cell. A raster cell providing 100% suitable conditions at all three times hence received a value of 1, while a raster cell providing unsuitable conditions at all three times received a value of 0. Again, we subtracted the suitability raster by 1 to arrive at a resistance cost surface and calculated all pairwise least‐cost distances between localities of each species  per population and calculating the Euclidean distances in environmental space between sampled localities using the Terrain, IBRHabitat and IBI represent refined geographical matrices and may thus be strongly correlated with the Euclidean geographical IBD matrices. We hence first ran separate Mantel tests  on these four matrices  to assess collinearity ). Then, for each species, we selected the distance measure with the highest R² and used multiple matrix regressions with randomizations , unique contributions to the explained deviance.Given recent criticism on the use of multiple matrix regressions  and 77,720 polymorphic loci (core Merianieae). The average mapping rates ranged between 35.1% and 54.3%, with a coverage of 5.7–16.8×  across all sites ranged between 0.0006 and 0.0073 and was variable among species  were generally low for all species , followed by M. sanguinea  while all other species had lower F values  and M. tomentosa (4–5/10 comparisons) and no differences in M. sanguinea and Ax. costaricensis  confirmed patterns found in admixture and co‐ancestry analyses and indicated moderate to high population structuring, ranging from 0.0178  to 0.661  and Ad. adscendens highest genetic differentiation (FST = 0.422). Comparing the two adjacent localities in each species, we found lowest FST in bee‐pollinated M. maxima (0.018) and highest in passerine‐pollinated Ax. costaricensis . In vertebrate‐pollinated M. phlomoides, M. sanguinea, M. tomentosa and Ax. costaricensis, FST also correlated most strongly with geographical distance (IBD). In bee‐pollinated Ad. adscendens, correlation was strongest (but not significant) with geographical barriers (IBRTerrain) and historical habitat suitability , while in M. maxima, FST was associated most strongly with current climatic suitability . In all species except Ax. costaricensis, we also detected a significant correlation with Pleistocene climatic instability  and IBD  and IBD  are generally considered as relatively mobile  across localities of bee‐ than vertebrate‐pollinated species Table . These rAdelobotrys adscendens has a wide distribution in lowland rainforests in tropical Latin America, while the other five species inhabit montane cloud forests with relatively continuous  or patchy  distributions . In Ad. adscendens, the marked separation into two clusters . Whether these observed phenotypic differences are the result of random genetic drift, or a response to different selection pressures, remains to be investigated.Ectothermic insect pollinators may be affected more strongly by harsh abiotic climatic conditions  than vertebrate pollinators . Accordingly, we found some isolating effects related to current climatic conditions Table  only in  Figures  and 3a cs Figure . In M. mHabitat) or environment , a pattern documented for other montane Neotropical plant lineages  coast of Central America  or underwent down‐ and upslope migrations (“moist‐forest” hypothesis), remains a major conundrum (Carnaval et al., 2009, Valencia et al., 2010, Ramírez‐Barahona & Eguiarte, e Figure . These re Figure , and eves Figure c. Furthe Figures . Indeed, Figures . Finallya Figure . Such bra Figure  whether M. sanguinea, and hummingbird and bat pollinators in northern Ecuador. Obtaining such natural history information, in addition to population genomic data, will be key to adding a more realistic understanding of the processes governing speciation. This will ultimately help in addressing broad‐scale questions such as how the tropics worldwide have become exceptionally biodiverse, but also in tracing similarities and differences in drivers of diversification across different tropical habitats and continents, namely Andean uplift in the Neotropics (Lagomarsino et al., Together, our results are highly valuable in adding to the limited data available on the diverse processes shaping population genetic differentiation of tropical plants, including differences in pollination strategy. While we highlight that a wider sampling across bee‐pollinated Merianieae is required to firmly establish the role of pollinators in promoting population differentiation, our result on stronger isolation among bee‐ than vertebrate‐pollinated populations suggests a critical role of pollinator mobility in shaping population‐level processes. Extrapolating to a macroevolutionary perspective, pollinator shifts are often invoked as “key innovations” spurring diversification (van der Niet et al., ASD, JS and OP conceived and designed the study, ASD and DFF performed fieldwork, ASD, JB and EMT conducted laboratory work, ASD and OP analysed the genomic data, ASD performed niche modelling and wrote the first draft; all authors contributed to revising the manuscript.Supplementary MaterialClick here for additional data file.