Calotropis gigantea leaves extract (CaG) was attained in silver-conjugated poliglusam nanomatrices (POL/Ag) to induce apoptosis in invasive ductal carcinoma (IDC) cells. The ethanolic CaG extract was prepared using percolation method and characterized by chemical tests for its active phytochemical compounds. The droplet-based microfluidic system was utilized as microscale encapsulation technique for CaG in nanomatrices at two different aqueous to oil flow rate ratios 1.0:1.5, and 1.0:3.0. Moreover, conventional batch system was utilized as macroscale encapsulation technique consisted of hot plate magnetic stirrer. The prepared nanomatrices were analysed for antioxidant activity using DPPH test and for cytotoxicity analysis using MCF-7 cells. The characteristic peaks of UV–Vis, FTIR and XRD spectrum confirmed the synthesis of CaG(POL/Ag) by both the encapsulation methods. However, microfluidic system was found to be more expedient because of attaining small and uniform sized silver nanoparticles (92 ± 19 nm) at high flow rate and achieving high encapsulation efficiency (80.25%) as compared to the conventional batch method (52.5%). CaG(POL/Ag) nanomatrices found to have significant antioxidant activity (p = 0.0014) against DPPH radical scavenging activity. The CaG(POL/Ag) of the smallest sized formulated by the microfluidic system has also shown the highest cytotoxicity (90%) as compared to batch method (70%) at 80 µg/mL. Our results indicate that the microscale technique using microfluidic system is a more efficient method to formulate size-controlled CaG(POL/Ag) nanomatrices and achieve high encapsulation of plant extract. Additionally, CaG(Pol/Ag) was found to be an efficient new combination for inducing potent (p < 0.0001) apoptosis in IDC cells. Therefore, CaG(Pol/Ag) can be further tested as an anti-cancer agent for in-vivo experiments.The encapsulation of plant extract in nanomatrices has limitations due to its adhesion to walls, size control, high cost and long durations that results in low yield. Macroscale and microscale level techniques for development of micro/nanoparticles may impact the encapsulation of plant extract. This study aimed to evaluate the relative efficiency of microscale and macroscale techniques for encapsulation of plant extract, which is not compared yet. Keeping this in view, encapsulation of  These particles can be formulated in distinctive ways to control particle size to not only act as anti-cancer agents but also serve as delivery vector for drug. As compared to conventional drugs, nanodrugs emerges as more significant improving stability, biocompatibility, biodegradability, crossing blood–brain barriers and especially providing a valuable targeted therapy2. In cancer treatment nano drug delivery is providing a more promising efficacy because they can be formulated to provide a shell (e.g. liposome), capsules (oil/water or polymeric membrane), embedding matrix like nanomatrices (polymeric) or pH-temperature sensitive drug-conjugation3. The conjugation of polymer embedding metallic nanoparticles is a hybrid combination and may provide an embedding and conjugation system to achieve high-definition encapsulation of any anti-cancer agent4.Nanoparticles have been studied extensively in cancer research because of their unique characteristics which facilitates scientists in diagnostic and treatment of cancer5. The recent research is focused to prevent and treat this cancer at early stages7. However, the synthetic chemo-drugs available in the market but comes with severe side effects. Medicinal plants are source of natural products with various biological activities like antiviral, hypoglycaemic, antibiotic, antioxidant, anticancer, antifungal, anti-hypertensive, and insecticide8. Calotropis gigantea (C. gigantea) is one of the abundantly occurring medicinal plant that usually found in northern areas of Pakistan10, and is comprised of many bioactive components including phenols, flavonoids, alkaloids etc.12. The rich source of phytochemicals makes it possible to use C. gigantea against several illness like inflammatory, microbial infection, wound healing,13 and apoptosis of cancer cells14. Furthermore, the extract of plant like CaG is a mixture of multiple bioactive components and their delivery at cancer site is very difficult in human complex body16. To overcome the problem of biodistribution and bioavailability of degradable plant extracts the advanced research has focused on preparation of nano or micromatrices17. These encapsulating matrices will not only safely deliver the plant extract but will also enhanced their therapeutic efficacy18. The extract of C. gigantea (CaG) has not been previously formulated with any nanoparticles for IDC cells and it is reported in this study.Invasive ductal carcinoma (IDC) is one of the most common globally occurring and lethal type of breast cancer18. Poliglusam is extensively used as microcarrier/micromatrices for safe delivery of anti-cancer and anti-microbial drugs as well as the plant-based drugs because of its natural biodegradable groups and performance delivery20. The poliglusam micromatrices can also be conjugated to the metals like silver nanoparticles (AgNPs) which will not only aid the encapsulation efficiency of poliglusam but also increase the cytotoxicity of nanoparticles21. These arrangements and modifications provide enhance surface interaction with cancer cells and allow extract to combat resistance in complex human body24.The encapsulation of plant extract in micro/nanoparticles such as polymers, cyclodextrins, solid dispersions and liposomes has emerged to be a most competent encapsulating agents26. The encapsulation methods can be divided into two techniques on basis of size of reactor for mixing reagents27. The microscale level consists of reactor that is 1 to 100 μm in diameter29. While reactor above 100 μm forms the macroscale level for encapsulation27. Microscale techniques may include the microemulsions30 formed by microfluidic system and phase inversion precipitation29. The macroscale techniques may include the spray drying, hot plate magnetic stirring, fluid bed coating, and ion gelation methods of encapsulation25. These methods and type of nanoparticles used in encapsulation depends upon the chemical nature of plant extract, i.e. oil based, volatile or high water soluble25. However, they have many different reported limitations such as extract adhere to the walls in spray drying, takes long duration for encapsulation, high cost due to use of high amount of nanoparticles and size control of encapsulation material25. These problems will not only cause denaturation of medicinal plant extract but will also results in low yield of encapsulation in nanomatrices. Microfluidic system is an efficient and precise technique for synthesis of size and shape control nanoparticles including nano/micromatrices. The PMMA microchip-based encapsulation of plant extract has been performed previously with up to 77.125% encapsulation efficiency (EE) of micromatrices31. The PMMA microchip was used for water in oil microemulsion in order to synthesize controlled sized nanoparticles and encapsulate the plant extract31. These microchips can also be modified for oil in water emulsion system depending upon the nature of plant extract.There are several methods, reported previously, for encapsulation of plant extract at micro and macroscale levelTo the best of our knowledge, the relative significance of microscale and macroscale techniques for encapsulation of plant extract has not been reported so far. Therefore, this research focuses on studying and comparing the two techniques at microscale and macroscale level for loading plant extract in nanomatrices. For this purpose, the CaG plant extract has been encapsulated in poliglusam-silver (Pol/Ag) nanomatrices at macroscale using conventional batch method and microscale using microfluidic system. We hypothesised that microfluidic system is a competent method for encapsulation of plant extract as compared to conventional wet chemical method . This new combinational nanomatrices were also examined for its antioxidant and anti-proliferative effect against IDC cells.3, Duksan), ethanol, acetic acid, canola oil (Sigma), sodium hydroxide (NaOH), 2,2-diphenyl-1-picrylhydrazyl , dimethyl sulfoxide (DMSO), chloroform, sulphuric acid, hydrogen chloride (HCl), Mayer’s reagent, ammonia solution and ferric chloride. All mentioned chemicals are of analytical grade.Poliglusam , silver nitrate  W.T. Aiton, http://legacy.tropicos.org/Name/2603210?projectid=32) were cut with clean scissors and brought for further identification by renowned plant taxonomist (Quaid-i-Azam University Islamabad). The research was in compliance with local policies and regulations of Federal region. The plant was washed with distilled water, dried under shade and ethanolic extract was prepared by following the reported percolation method31 at Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan. The percentage yield of extract was calculated using the following formula.The plant materials used in this study were collected from wild areas in Islamabad, Zero-point Latitude: 33° 41′ 37.00" N and longitude: 73° 03′ 54.00" E, after identification by the Dr. Mushtaq Ahmad, Director of Botanical Garden & Herbarium of Islamabad, Pakistan and Quaid-i-Azam University Islamabad, Pakistan. The plant specimen was authenticated by comparison with herbarium specimen of Islamabad Herbarium of Pakistan and submitted with voucher number ISL-769 in this herbarium for future reference. Following the rules of national action plan of the Pakistan, the leaves of plant  at 30 °C and weighed for percentage yield calculation31.Here, 33. For terpenoids detection, chloroform was added to plant extract in ratio of 2:5 and then 1 mL of sulphuric acid was added. For alkaloids, equal amount of HCL and extract was added. After mixing the three drops of Mayer’s reagent were added. The presence of flavonoids was indicated by the yellow colour when ammonia solution was added followed by few drops of sulphuric acid into the extract in equal ratio. Lastly, for phenols detection the ethanolic extract was diluted with distilled water and three drops of 10% ferric chloride was added33.The phytochemical analysis of ethanolic CaG extract was accessed by different chemical tests for terpenoids, flavonoids, phenols, and alkaloids by the reported testsThe Pol/Ag was prepared by the conventional batch method and microfluidic systems in order to compare the encapsulation of CaG using the macroscale and microscale techniques, respectively. The experimental setup is illustrated in Fig. 34 with few modifications as shown in Fig. 3 solution was added drop wise into poliglusam solution in ratio 2:1 (poliglusam : AgNO3) and left for 2 h of stirring. For encapsulation of CaG, 10 mg/mL of CaG was mixed into the poliglusam solution and AgNO3 was added drop by drop. After collection of nanomatrices the 10% of NaOH was added drop wise in volumetric ratio of 0.1:1; NaOH: AgNO3. These samples were washed three times with deionized water (d.H2O) by centrifugation at 10,000 rpm for 30 min each time and lyophilized for storage at room temperature and characterization.The experimental setup of conventional batch method consisted of hot plate magnetic stirrer to prepare the CaG loaded poliglusam-silver nanomatrices (CaG-(Pol/Ag)) at macroscale35 having T junction was used for the microemulsion formation. The straight channel carries the continuous phase (canola oil) and perpendicular channel carries the dispersed phase  shown in Fig. 3 and 10 mg/mL of CaG. First of all the dispersed phase is passed through the polytetrafluoroethylene (PTFE) microreactor immersed in water bath to heat the solution up to 90 °C31. The homogenous and stable microemulsions (water-in-oil) were formed by controlling the flow rates of both the phases. The total flow rate  ratio of aqueous: oil was set as 0.4: 0.6 (F1) and 1.8: 5.4 (F2) and relative flow rate (RFR) ratio of 1:1.5 (F1), and 1:3 (F2). The samples were collected and 10% of NaOH was added to them  for acceleration of reduction of AgNO3. The collected samples were washed and stored. In order to compare both methods with equal parameters, the stirring time set for batch system was 2 h which equals to the time taken by microfluidic system to collect equal volume of nanomaterial suspension.The microfluidic system was used for synthesis of CaG-(Pol/Ag) at microscale which is shown in Fig. 31. The %EE was calculated by following formula.Tp is total CaG used in encapsulation and Fp is free CaG detached from Pol/Ag.The percentage encapsulation efficiency (%EE) of Pol/Ag nanomatrices synthesized by microfluidic system and conventional batch method was calculated by measuring the free drug present in samples. For this, the CaG extract from Pol/Ag nanomaterial was separated using 10,000 rpm centrifugation for 30 min and the amount of free drug in supernatant was calculated using UV–Vis spectrophotometer at 340 nmThe physical characteristics, morphology and size of particles were characterized by scanning electron microscope (SEM), X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), Dynamic light scattering (DLS) and ultraviolet visible (UV–Vis) spectroscopy.32. 0.1 mM of DPPH solution was prepared in ethanol and immediately absorbance was taken at 517 nm for control reading using 96 well plate spectrophotometer (Thermo Fisher scientific). The different concentrations, 40, 60, 80, 100, 120 and 160 μg/mL of ascorbic acid, CaG, and CaG-(Pol/Ag) nanomatrices were prepared in DMSO, and DPPH solution was added in ratio of 1:3; DPPH: antioxidant. The mixtures were shaken vigorously and placed in dark for 30 min at room temperature. The absorbance of each sample was measured at 517 nm. Here, the ascorbic acid was used as standard.The TAC of CaG and CaG-loaded nanomaterial was evaluated by 1, 1-Diphenyl-2- picrylhydrazyl (DPPH) solution test by using the reported protocol31. The 96 well plate was prepared by seeding MCF-7 (1X107 cells/well) cells and cultured in RPMI media for 24 h at 37 °C. The different formulations of nanomaterial with different concentrations were added into the wells and further incubated for 24 h. Afterwards, the media was removed and MTT dye (5 mg/ mL) was added into each cell, incubated for 2 h and DMSO was added to solubilize the formazan crystals. The absorbance of each cell was taken at 570 nm using the micro-plate reader. the percentage (%) cell viability was calculated using the following formula.The CaG-(Pol/Ag) nanomatrices produced from batch and microfluidic methods were tested against the IDC cells (MCF-7) by MTT assayp < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. The nanomaterial and droplet size were measured by ImageJ software. The Mean and standard deviation (SD) were calculated by Graph Pad prism v.9The statistical data analysis was performed using GraphPad prism v.9. IC50 values were calculated and Dunnett’s multiple comparisons test was applied. Two-way ANOVA was also accessed. The threshold values were set as 0.05 and sstatistical significance was established when *12 In another research, the ethanolic extract of C. gigantea has 46.75 mg of flavonoid and 33.71 mg of phenolic content14. The previous reported studies from HPLC and GCMS data also confirms the presence of several pharmacological antioxidant compounds38 like terpenoids like Lupeol38, Urs-12-EN-28-Oic acid, oleanolic acid, 3-O-Acetyl-6-Methoxy-Cycloartenol., ketone like; 3.Alpha.-(Trimethylsiloxy) Cholest-5-Ene., sapogenin, natural steroids like; methylcholesta-4,6-Dien-3-One, terpenes like; caryophyllene, farnesene, and several flavonoids37 and cardenolides39.The CaG extract was prepared using the ethanol as solvent by percolation method. The 50 g of dried leaves of CaG has given 9.8 g of ethanolic extract and the yield of 19.6%. This extract was found to be positive for terpenoids, alkaloids, flavonoids and phenols as indicated by reddish-brown layer at interface, orange, yellow and green colour respectively. Those findings are in accordance with previous reported studies35. The UV–Vis characteristic peaks of Pol/Ag nanomatrices synthesized by conventional batch method (Ba-CaG(Pol/Ag)) and microfluidic system (Mi-CaG(Pol/Ag)) are shown in Fig. 40. The silver nanoparticles synthesized by microfluidic system has relatively more absorbance with narrow UV–Vis spectrum, which justify the narrow size distribution of nanoparticles synthesized by microfluidic system. The CaG has peak at 340 nm which is representing the effective encapsulation of CaG in nanomaterial. Furthermore, the peak around 280 nm shows the presence of poliglusam20. Altogether these peaks verify the formation of nanomaterial consisted of CaG loaded in poliglusam and silver nanoparticles31. The UV–vis spectrophotometry of CaG(Pol/Ag) prepared via the microfluidic system was performed after eight months to check the stability of the nanomatrices. A peak around 400 nm was observed which confirms the presence and stability of AgNPs as shown in Fig. The UV–Vis spectrophotometry of synthesized nanomaterial was performed to confirm the formation of CaG-(Pol/Ag) nanomatrices. The change in color from clear to reddish brown indicated the formation of Pol/Ag nanomatrices as shown in Fig. 41. Recently, Aftab et al., has achieved the 77.125 ± 6.9% EE of Calotropis procera in chitosan silver nanoparticles using the microfluidic system31, while we have not found any other similar study. However, other encapsulation techniques of micro and macro encapsulation has also been reported with many limitations25 as discussed in Sect. 1.The nanomatrices synthesized by microfluidic system were found to have % EE of 80.25%, while batch system has 52.5%. This shows that microfluidic system is relatively a more competent method to successfully encapsulate the high percentage of CaG extract in nanomaterial. This might be because of rapid mixing in microemulsions in the microfluidic chip42. The broadening and elevation of peak around 3280 cm-1 shown in Fig. -1 is indicating the role of hydroxyl and amino groups in stabilization of silver ions34. The CaG has displayed  the size of microemulsion as well the size of silver nanoparticles declined. This is because with the decrease in size of microemulsion the number of nuclei decreases that are formed within each microemulsion which result in decrease in average final size of the nanomaterialted Fig. A,B.42. At high flow rate, the Pol/Ag nanomatrices are more spherical and uniform. On the other hand, the Pol/Ag nanomatrices prepared via a batch method are more irregular in shape/surface. The irregular surface of poliglusam is also not displaying the silver nanoparticles clearly.The change in morphology of the nanomaterial captured by SEM was also observed. The SEM images has also shown the bright silver nanoparticles embedded on membrane-type- poliglusam collected from microfluidic systemThe SEM image was taken after eight months to check the stability of nonmaterices. As shown in Fig. 49. Yanar et al. has produced the size control liposomes48. While in this research we have formulated the size controlled poliglusam nanomatrices embedding silver nanoparticles conjugated with plant extract, which were not reported earlier.Overall, these finding shows that microfluidic system plays an important in homogenous mixing for synthesis of narrow size distribution and smooth surface of poliglusam microcarriers. These results also indicate that the size and morphology of nanomaterial can be tuned by microfluidic system.50. in this study the zeta potential of the nanomatrices was found to be 42.42 mV. Which suggests that they are highly stable and cationic. This strong cationic nature of the CaG(Pol/Ag) nanomatrices increase the endocytosis activity on the surface of negatively charged cancer cells due to electrostatic force  nanomatrices are evenly distributed with positive charge on their surface which make them an ideal nanoagent to induce cancer cell death.The hydrodynamic size and zeta potential of the microfluidic prepared CaG(Pol/Ag) nanomatrices was analysed using the DLS technique show in Fig. rce Fig. C. Theref12. The percentage inhibition of free radicals by CaG, CaG(POL/Ag) and standard is shown in Fig. The reported studies have found valuable phytochemical present in CaG including alkaloid, flavonoids, and phenols. Therefore, it can be used as a natural antioxidant50 values are shown in Fig. p < 0.0001) anti-cancer activity in increase concentration dependant manner. However, M-CaG(Pol/Ag) nanomatrices has shown more decrease in IDC cells viability as compared to Ba-CaG(Pol/Ag) nanomatrices. At 60 and 80 µg/mL of dose a difference of 30% and 20% of cell viability can be noticed in Fig. In this study, the IDC cells, MCF-7, were treated with the microchip-formulated (M-CaG(Pol/Ag)) nanomatrices of two different sizes, MF1-CaG(Pol/Ag) of 198 ± 36 nm and MF2-CaG(Pol/Ag) of 92 ± 19 nm, and batch method synthesized (Ba-CaG(Pol/Ag)) nanomatrices (213 ± 73 nm). Their cytotoxic effect and IC54. However, in previous studies scientist has also preferred the 50 nm size over 10 or 20 nm56, this may be because the smallest particles may escape the cancer cells easily even before performing their average activity. While, in our study, the size of particle is around 92 nm, which is quite optimal to enter and escape the cancer cells upon their average activity55.Furthermore, we have also found that smallest size particles 92 ± 19 nm (MF2-CaG(Pol/Ag) at 80 µg/mL has the minimum cell viability of 10% (% cytotoxicity = 90). While Ba-CaG(Pol/Ag) nanomatrices has lowest cell viability of 30% (% cytotoxicity = 70 ) at 80 µg/mL. These results indicate that with the change in size and morphology of nanoparticles, the cell viability of the cancer cells is also affecting. A small size and uniform morphology of Pol/Ag nanomaterial has more potent anticancer efficacy31. The two-way ANOVA test has confirmed those finding to be statistically significant, p < 0.0001.It can also be observed in this study that the antioxidant-CaG extract has shown 24% cell viability at 80 µg/mL, however conjugating it with tuned size and morphology Pol/Ag nanomaterial has increased the anti-tumour activityThe present study reports that encapsulation of plant extract at microscale using microfluidic system is more efficient to set out high yield encapsulation efficiency as compared to encapsulation at macroscale using conventional batch method. Also, the microfluidic system provides the flexibility to control the size, shape, and polydispersity of nanomaterial by changing the flow rates of the agents. This research reports the CaG(Pol/Ag) as a new nanotherapeutic agent which is competent to induce apoptosis in MCF-2 cells.


The efficacy of therapeutic drug monitoring (TDM)-based antimicrobial dosing optimization strategies on pharmacokinetics/pharmacodynamics and specific drug properties for critically ill patients is unclear. Here, we conducted a systematic review and meta-analysis of randomized controlled trials to evaluate the effectiveness of TDM-based regimen in these patients.Articles from three databases were systematically retrieved to identify relevant randomized control studies. Version two of the Cochrane tool for assessing risk of bias in randomized trials was used to assess the risk of bias in studies included in the analysis, and quality assessment of evidence was graded using the Grading of Recommendations Assessment, Development, and Evaluation approach. Primary outcome was the 28-day mortality and secondary outcome were in-hospital mortality, clinical cure, length of stay in the intensive care unit (ICU) and target attainment at day 1 and 3.I2 = 0%). In-hospital mortality , clinical cure , length of stay in the ICU , and target attainment at day 1  and day 3  were not significantly different between the two groups, and all evidence for the secondary outcomes had a low or very low level of certainty because the included studies had serious risk of bias, variation of definition for outcomes, and small sample sizes.In total, 5 studies involving 1011 patients were included for meta-analysis of the primary outcome, of which no significant difference was observed between TDM-based regimen and control groups , registry number: CRD 42022371959. Registered 24 November 2022.The online version contains supplementary material available at 10.1186/s40560-023-00699-8. Antibiotic therapy is a cornerstone in the treatment of sepsis and severe infections, and appropriate antibiotic dosing design based on pharmacokinetics/pharmacodynamics (PK/PD) is necessary –4. The STherapeutic drug monitoring (TDM), which refers the management and adjustment of the patient's drug dosing based on the measured drug concentration in the blood, has been suggested as a method of drug exposure optimization , 16. PreRecent systematic review and meta-analysis evaluating TDM for beta-lactam antibiotics included five randomized control trials (RCTs), but two large RCTs on TDM have since been published, increasing the need for reevaluation . Hence, This systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement , and theOur research question was whether TDM should be used to adjust antimicrobial doses in patients with sepsis or treated in the ICU. Accordingly, RCTs comparing TDM to standard procedures in patients aged ≥ 18 years with either sepsis or who were critical ill were included. TDM studies that did not have a control were excluded. Case series, case reports, editorials, letters to editor, and conference abstracts were also excluded from this review.Two authors (N.T. and Y.O.) independently and systematically searched the MEDLINE (via PubMed), Cochrane CENTRAL databases, and Igaku-Chuo-Zasshi for peer-reviewed articles published as well as the ClinicalTrials.gov and World Health Organization International Clinical Trial Registry Platform for relevant completed trials between database inception and November 2022 (search date: 17 November 2022).Search formulas were created based on “Sepsis”, “Systemic Inflammatory Response Syndrome”, “Multiple Organ Failure”, “Critically ill”. “Therapeutic Drug Monitoring”, and “Randomized controlled trial” with Medical Subject Heading terms and text words along with a query starting with “NOT” to exclude ineligible studies. The terms were then arranged and entered in a form suitable for each database  independently screened the titles and abstracts of all articles to identify eligible studies by using a study by Ryyan  as the fTwo authors (N.T. and Y.O.) independently assessed the risk of bias for each included study using the Cochrane tool for assessing risk of bias in randomized trials, version two (RoB 2) . This toQuality assessment of evidence was graded using the GRADE system that provides a systematic approach for each outcome across studies . The GRAThe primary outcome of interest was 28-day mortality. Secondary outcomes were target attainment defined in each study at day 1 and 3, clinical cure , hospital mortality, and length of stay in the ICU. If TDM was simultaneously performed for multiple antibiotics, outcomes including target attainment were analyzed separately for each antibiotic. We also performed the analysis by the subgroup for the class of antibiotics when there was more than one study.2, and I2 statistic, in which I2 > 70% indicated severe heterogeneity. To calculate point estimates, when the mean and median were different, the mean was considered the same as the median, and the standard deviation (SD) was integrated by dividing the interquartile (IQR) by 1.35 (https://handbook-5-1.cochrane.org/chapter_7/7_7_3_5_mediansand_interquartile_ranges.htm).Extracted data were collected and statistically analyzed using RevMan ver 5.4.1 . Risk ratios (RRs) and corresponding 95% confidence intervals (CIs) were determined using a random-effects model with weights calculated using the Mantel–Haenszel method . For conP < 0.05 for hypothesis and < 0.1 for heterogeneity testing was considered statistically significant.A two-tailed n = 1011; TDM group, n = 510; standard dosing group, n = 501) . In the future, it is necessary to plan a search strategy that includes EMBASE in order to collect more appropriate literature.For critically ill patients, TDM-based antibiotic regimens were not associated with 28-day mortality, in-hospital mortality, clinical cure, target attainment at day 1 and day 3, and length of stay in the ICU when compared with standard protocols. Further RCTs in which outcomes are clearly defined, include disease severity, and focus on sepsis patients are warranted to verify the true efficacy of TDM.Additional file 1: Table S1. Literature search strategy for each database. Figure S1. Forest plot of TDM and control group in beta-lactam antibiotics. Figure S2. Funnel plot for the publication bias assessment.


The causal effect of obesity on diabetic retinopathy (DR) remains controversial. The aim of this study was to assess the causal association of generalized obesity evaluated by body mass index (BMI) and abdominal obesity evaluated by waist or hip circumference with DR, background DR, and proliferative DR using a two-sample Mendelian randomization (MR) analysis.−8) level were derived using GWAS summary statistics from the UK Biobank (UKB) with a sample size of 461 460 individuals for BMI, 462 166 individuals for waist circumference, and 462 117 individuals for hip circumference. We obtained genetic predictors of DR (14 584 cases and 202 082 controls), background DR (2026 cases and 204 208 controls), and proliferative DR (8681 cases and 204 208 controls) from FinnGen. Univariable and multivariable Mendelian randomization analyses were conducted. Inverse variance weighted (IVW) was the main method used to analyze causality, accompanied by several sensitivity MR analyses.Genetic variants associated with obesity at the genome-wide significance ; P=5.12×10-08], and hip circumference  were associated with increased risk of DR. BMI , waist circumference , and hip circumference  were correlated with the risk of background DR. MR analysis also supported a causal association between BMI , waist circumference , and hip circumference  and proliferative DR. The association of obesity with DR continued to be significant after adjustment for type 2 diabetes.Genetically predicted increased BMI [OR=1.239; 95% CI=;P=1.94×10This study using two-sample MR analysis indicated that generalized obesity and abdominal obesity might increase the risk of any DR. These results suggested that controlling obesity may be effective in DR development. Almost all type 1 diabetes patients and 60% of type 2 diabetes patients develop retinopathy within 20 years . Therefo2) is an emerging public health problem and a widely accepted risk factor for many diseases, such as type 2 diabetes, cardiovascular diseases (CVD), and cancer (Obesity (defined as a body mass index (BMI) ≥30 kg/md cancer –7. Variod cancer , but thed cancer , 10. In d cancer  and evend cancer , 13. Thed cancer . The terd cancer . Similard cancer  or to had cancer . In a red cancer . TherefoCompared to traditional retrospective studies, Mendelian randomization (MR) studies are less affected by confounding factors, and the causal sequence is more reasonable . This ap22.1via diabetes.We reported the MR study in adherence to the Strengthening the Reporting of Observational Studies in Epidemiology using Mendelian Randomization (STROBE-MR) . The MR p < 5 × 10−8); (2) SNVs were not associated with any potential confounders and were independent of each other to avoid biases caused by linkage disequilibrium ; and (3) SNVs are only linked to the outcome through exposure. An F statistic ; variance (se)) was calculated for each SNV  and DR, single-nucleotide variations (SNVs) were selected according to the following criteria Figure 1each SNV . Since a obesity . Informa2.2https://gwas.mrcieu.ac.uk/). Different stages of DR  were chosen as outcomes. The GWAS summary statistics of DR were extracted from the FinnGen (https://r5.finngen.fi/). Participants in the DR (GWAS ID: finn-b-DM_RETINOPATHY) analysis included 14,584 cases and 202,082 controls; participants in the background DR (GWAS ID: finn-b-DM_BCKGRND_RETINA) analysis included 2,026 cases and 204,208 controls; and participants in the proliferative DR (GWAS ID: finn-b-DM_RETINA_PROLIF) analysis included 8,681 cases and 204,208 controls. Cases of different stages of DR were identified based on the International Classification of Diseases-Revision 9/10 criteria from the hospital discharge registry (https://r5.risteys.finngen.fi/). We obtained genetic predictors of type 2 diabetes from Mahajan et al.  from the UK Biobank (UKB) as exposures. UKB was a UK-based cohort study that recruited about 500,000 participants aged 40–69 years between 2006 and 2010, from whom a series of medical and physical information was collected . BMI is 2.3p < 0.05 was considered to be statistically significant as evidence for a potential causal association.All statistical and MR analyses were performed using R software (version 4.1.1) using the R packages “TwoSampleMR” and “MR-PRESSO”. via Ruecker’s framework. p < 0.05 of Cochran’s Q and Rucker’s Q (Q–Q) indicates the MR−Egger analysis with the least heterogeneity (Q test and I2. p < 0.05 of Cochrane’s Q (I2 > 25% (The inverse variance-weighted (IVW) method was used as the primary method for calculating the causal effect. Given that the validity of the MR method is strictly dependent on the absence of pleiotropy, we used a series of MR analytical approaches to account for pleiotropy. First, we used MR–Egger  and weigogeneity , which iane’s Q  and I2 2 > 25%  were con2 > 25%  and MR-P2 > 25% . In addi3F statistic of each SNV was greater than the empirical threshold of 10, and the minimum F statistics in subgroups are shown in –Our results indicated that 305, 252, and 275 SNVs in DR were associated with BMI and waist and hip circumference, respectively. A total of 306, 252, and 275 SNVs in background DR were associated with BMI, waist circumference, and hip circumference, while 305, 252, and 274 SNVs in proliferative DR were associated with BMI, waist circumference, and hip circumference, respectively Table 1.3.1p = 1.94 × 10−06] and waist circumference  by the IVW method were significantly associated with a higher risk of DR, consistent with results obtained by WM. Nonsignificant pleiotropy in BMI was detected by Cochrane’s Q test (p = 0.525), I2 = 0, MR–Egger intercept (p = 0.708), or MR-PRESSO global test (p = 0.535). Slight heterogeneity was present in waist circumference , but no significant outlier (p < 0.05) was identified by MR-PRESSO. Significant heterogeneity in hip circumference was detected by Cochrane’s Q test (p = 2.62 × 10−04), and a significant outlier (SNV:rs7903146) was detected by MR-PRESSO. Higher hip circumference was also suggestively associated with the risk of DR using the IVW method  after deleting the outlier. Moreover, our MVMR analysis suggested that the causal association between obesity and DR existed apart from diabetes. Using the MR–Steiger test, none of the variants were removed, and the results remained unchanged. Finally, the leave-one-out analysis found that no single SNV strongly drove the overall effect of obesity on DR.First, we explored the causal relationship between obesity and DR, as shown in 3.2p = 5.24 × 10−05], waist circumference , and hip circumference  were associated with a higher risk of background DR. The WM method showed similar results. Pleiotropy in BMI and waist circumference identified by Cochrane’s Q test, I2, MR–Egger intercept, and MR-PRESSO did not reach statistical significance . However, there was significant pleiotropy  in hip circumference, and MR-PRESSO detected one significant outlier (SNV:rs35506085). The IVW and WM methods still suggested a causal association between the hip circumference and background DR after deleting the outlier. The associations for obesity and background DR remained significant after adjustment for type 2 diabetes through MVMR. No one SNV was excluded by MR–Steiger. Additionally, the leave-one-out test showed that the MR results were not significantly affected by a single SNV.Next, we assessed the causal relationship between obesity and background DR, as shown in 3.3p = 1.46 × 10−08], waist circumference , and hip circumference  were suggestively associated with the increasing risk of proliferative DR using the IVW method. These results were supported by those of the WM method. No significant pleiotropy in BMI or waist circumference was detected by several sensitivity MR analyses. However, significant pleiotropy in hip circumference was found by Cochrane’s Q test (p = 3.00 × 10−05) or I2 = 27.5%. MR-PRESSO found two significant outliers . After deleting the two outliers, the causal relationship still persisted. Meanwhile, MVMR analysis indicated a causal relationship between obesity and proliferative DR aside from diabetes. Finally, MR–Steiger and the SNV leave-one-out method were used to further validate the data robustness, and no SNV was excluded.Finally, we further investigated the relationship between obesity and the risk of proliferative DR using MR analysis, as shown in 4The present study investigated the causal association between generalized obesity  and abdominal obesity  with different stages of DR using MR analysis. This study corroborated the conclusion that both generalized obesity and abdominal obesity are risk factors for DR (including background and proliferative DR). After adjusting for diabetes by MVMR, the causal relationship between obesity and DR still exists, suggesting that obesity may be an independent risk factor for DR.Obesity and diabetes are recognized as major public health problems worldwide. A series of scientific studies have indicated that obesity is involved not only in the pathogenesis of diabetes but also in the development of its complications . HoweverA recent meta-analysis pooled only prospective cohort studies, providing a high level of evidence that a higher BMI significantly increases the risk of DR incidence . Our MR BMI has limited value in accounting for fat distribution, as abdominal fat  is more strongly correlated with visceral fat than BMI . AbdominTo the best of our knowledge, this is the first study that has applied MR analysis to investigate the potential causal association between obesity and the risk of DR. The first advantage of the study is the MR design, which mitigates bias from reverse causation and confounding. The second advantage is that our MR study strictly utilized European subjects, thus minimizing bias due to population stratification. This study also has several limitations. The greatest concern in MR studies is horizontal pleiotropy, which occurs when genetic variants influence the outcome of more than one pathway. We designed a series of MR analytical approaches to minimize this bias. However, it is not possible to completely rule out residual pleiotropy. Moreover, MR analysis only made the assumption of a linear relationship  between 5In conclusion, these findings demonstrated a causal relationship between obesity and DR. Our MR analysis showed that obesity may be an independent risk factor for different stages of DR, which suggested that controlling obesity may be effective in DR development.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/CZ and XW performed the main data analysis and wrote the draft of the manuscript. XC supervised the whole research and is responsible for the integrity of the data analysis. All authors contributed to the article and approved the submitted version.


Migraine, a complex brain disorder, is regarded as a possible clinical manifestation of brain energy dysfunction. The trigeminovascular system is considered the basis for the pathogenesis of migraine, hence we depicted the proteomics profiling of key regions in this system, then focusing on protein alterations related to mitochondrial function. The aim of this study is to illustrate the role of mitochondria in migraine.A mouse model of chronic migraine (CM) was established by repeated nitroglycerin (NTG) stimulation and evaluated by von-Frey filaments, a hot plate and a light-dark box. Differentially expressed proteins (DEPs) in some subcortical brain regions of the trigeminovascular system were screened through liquid chromatography-tandem mass spectrometry (LC‒MS/MS) to analyse the specificity of key signaling pathways in different brain regions. And then mitochondrial function, structure and dynamics were determined by qPCR, ELISA, and transmission electron microscope (TEM). Finally, the effect of mitochondrial intervention-Urolithin A (UA) on CM was investigated.Repeated NTG injection triggered photophobia, periorbital and hind paw allodynia in mice. The proteomics profiling of CM model showed that 529, 109, 163, 152 and 419 DEPs were identified in the thalamus, hypothalamus, periaqueductal grey (PAG), trigeminal ganglion (TG) and trigeminocervical complex (TCC), respectively. The most significant changes in the brain region-specific pathways pointed to thalamic mitochondrial impairment. NTG induced mitochondrial structural disruption, dysfunction and homeostatic dysregulation, which could be partially attenuated by UA intervention.Our findings highlight the involvement of mitochondrial damage in the thalamus in central sensitization of CM, which provides evidence of possible metabolic mechanisms in migraine pathophysiology. Migraine is a common, recurrent, hereditary neurovascular headache disorder, which is the second leading cause of years lived with disability in the world according to the Global Burden of Diseases (GBD) . The curIn addition to the classical theory of the trigeminovascular system , impairmAs an increasingly powerful tool, liquid chromatography-tandem mass spectrometry (LC‒MS/MS) analysis has been applied to unmask the disease pathophysiology. This could make it possible to identify and quantify a few thousand proteins from a limited amount of sample in a single experiment and screen differentially expressed proteins (DEPs) in a complex mixture, thus providing new hints about disease pathogenesis . In prevCurrently, most studies emphasized the plasma or serum of humans, as well as the cortex of humans and rodents –17, whilhttp://www.randomizer.org/). The sample size was calculated by G*Power (ver. 3.1.9.7) based on the repeated measures design  and the results showed the total sample size was 18, that means 9 mice per group [All male C57BL/6J mice (21–32 g) were obtained from SiPeiFu Biotechnology Co., Ltd. . Mice were housed in a facility with a controlled temperature of 23 ± 2 ℃, humidity of 50 ± 10%, a 12 h light-dark cycle, and free access to food and water. Mice were left to acclimatize for 1 week prior to starting experiments. The animals were assigned to the experimental group using a randomized program  injection of nitroglycerin (NTG) 10 mg/kg) every second day for a total of 5 injections to closely mimic the recurrent attacks of CM in humans  was administered to the mice. As a natural gut microbiome-derived metabolite, UA displayed potent anti-inflammatory, anti-oxidative and anti-ageing properties –25. UA A. UA wasTo obtain accurate and objective results, the mice were habituated in the behavior room for several days prior to testing to reduce stress. The behavior room was quiet and hygienic, with moderate light, good ventilation and no smell. Behavior experiments were carried out at a fixed time every day (between 9:00 am and 3:00 pm). All the behavioral tests were performed in the same batch of mice. Eventually, six mice with significant changes in behavioral testing in each group were selected for further proteomics analysis. A fixed investigator was responsible for behavioral assessments and was blinded to the treatment during behavioral measurements.The mechanical withdrawal threshold was evaluated using von Frey filaments (range from 0.008 to 2 g). The periorbital and hind paw mechanical thresholds of the mice were assessed before and 2 h after i.p. injection of NTG or vehicle on each test day. Based on the traditional up-down method, a novel statistical algorithm that calculates the exact thresholds was used. Briefly, the middle weight 0.16 g was first applied. The stimulus would increase one weight if there is a negative response (O) and decrease one weight with a positive response (X). The interval between two stimuli was more than 30 s. Following the breaking point (OX/XO), four other stimuli were applied resulting in a certain pattern of responses. The 50% threshold can be obtained by uploading the weight of the final filament and XO response pattern using the online tool .For the hind paw assessment, each mouse was placed separately in transparent plastic chambers (8 × 15 cm) which were positioned on a wire mesh grid. The mice were acclimated in the chamber for 30 min per day at least 3 days before testing. The von Frey filaments were perpendicularly applied to the plantar surface of the hind paw. Brisk paw withdrawal, flinching, lifting, or licking during stimulation or immediately after the removal of the filament were described as positive responses. For the periorbital assessment, each mouse was tested in a 4 oz paper cup that was placed in the plexiglass box. The mice were habituated in the cups for 30 min during each of the 3 days before measurements. The periorbital region was tested, including the caudal region of the eye to approximately the midline. A positive response included the mouse vigorously stroked its face with the ipsilateral fore paw, head shaking or retraction of head from the stimulus.The hind paw thermal thresholds were assessed before and 2 h after NTG or vehicle administration on each test day using a hot plate. After habituation, the mice were individually placed on the surface of a hot plate  with a preset constant temperature of 55 ℃. The response latency in seconds to the latency of hind paw withdrawal, licking, or jumping was recorded. To avoid tissue damage, the maximum response time was fixed at 20 Sect. . Each moA light-dark box was used to assess light-aversive behaviors of the mice 1 h after NTG or vehicle injection on the last test day. The light-dark box  consisted of two equal-size zones: a light chamber and a dark chamber (20 cm × 15 cm × 30 cm), which were separated by a dark insert (Med Associates) with an open channel (6 cm × 6 cm) to allow the mice to explore the two chambers freely. After injection of NTG or vehicle, mice were individually placed in the middle of the light chamber (inside = 1000 lx) and allowed to explore both chambers for 10 min. The trajectory of movement was video-recorded and analysed using Supermaze software. The total distance of motion, the transition between two chambers, and the time spent in each chamber were calculated.On test day 5, the mice were sacrificed 12 h after the last NTG or vehicle injection. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg), and the brain tissues  were harvested immediately according to Paxinos and Franklin’s Mouse Brain in Stereotaxic Coordinates (second edition) and stored at -80 ℃ -tandem mass spectrometry . Two micrograms peptides were separated with an analytical column  in 150 min. Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in 80% acetonitrile; gradient: 1 − 8% B in 4 min, 8 − 30% B in 136 min, 30 − 90% B in 6 min, 100% − 100% B in 4 min; flow rate: 400 nL/min. Mass spectrometer: spray voltage: 2.1 kV; capillary temperature: 300 ℃; S-lens: 50%; collision energy: 32% HCD; resolution setting: first level 60,000@m/z 200, two Level 30,000@m/z 200; Max IT: full MS 20 ms, full MS/MS 45 ms; parent ion scanning range: m/z 400–1040; product ion scanning range: start from m/z 200; number of windows: 80; Isolation window: 8 Th. One blank after each sample was run to prevent sample carryover.p-value < 0.05. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were conducted using the Database for Annotation, Visualization and Integrated Discovery  to identify enriched proteins. GO analysis was mainly divided into three categories: cellular component, biological process, and molecular function.Data were imported into DIANN V 1.8.0, and targeted extracted with a predicted mouse proteomics database  in library-free mode . Other pThe concentration of ATP was determined using an ATP assay kit  according to the manufacturer’s instructions. The activity of mitochondrial complex I was determined by a mitochondrial respiratory chain (MRC) complex I activity assay kit . Briefly, the respective reaction buffers were added to the mitochondria isolated from the mouse brain. The mixture was transferred to a prewarmed quartz cuvette and immediately put into a spectrophotometer. Then, the activity of mitochondrial complex I was determined by monitoring the decrease in nicotinamide adenine dinucleotide (NADH) at 340 nm for 2 min. The concentration of malondialdehyde (MDA) were measured using a lipid peroxidation MDA assay kit  according to the manufacturer’s instructions.Fundc1, forward primer: ACATTGTGATATCCAGCGG CTTCG and reverse primer: TGCCACAGTCTTCCTCTCATTGTTG; Fis1, forward primer: CCTGGTTCGAAGCAAATACAAT and reverse primer: CTTTTCATATTCC TTGAGCCGG; Mid49, forward primer: GTGACGGCTGACCATATCCAACTC and reverse primer: CTCACGACGAACCAGGAAGAAACC; OPA1, forward primer: CTTACATGCAGAATCCTAACGC and reverse primer: CCAAGTCTGTAACA ATACTGCG; PGC1α, forward primer: GGATATACTTTACGCAGGTCGA and reverse primer: CGTCTGAGTTGGTATCTAGGTC; TFAM, forward primer: GTG AGCAAGTATAAAGAGCAGC and reverse primer: CTGAACGAGGTCTTTTTG GTTT; CHOP, forward primer: CTCCAGATTCCAGTCAGAGTTC and reverse primer: ACTCTGTTTCCGTTTCCTAGTT; HSP10, forward primer: GGCCCGA GTTCAGAGTCC and reverse primer: TGTCAAAGAGCGGAAGAAACTT.Thalamus tissues were homogenized in TRIzol reagent (Invitrogen) and processed using Zymo Direct-zol RNA Microprep kit according to the manufacturer’s instructions . RNA froMice were anesthetized with sodium pentobarbital (50 mg/kg) and perfused with 0.1 M PBS solution (pH 7.4) followed by precooled 2.5% glutaraldehyde solution. The ventral posteromedial thalamic nucleus (VPM) was collected and cut into 1 × 1 × 1 mm tissue blocks. The tissue was fixed with 2.5% glutaraldehyde solution and then fixed in 10 g/L osmium tetroxide solution. The sample was dehydrated with a graded series of ethanol and embedded in Eponate 12 epoxy resin. Thin sections were counterstained with uranyl acetate and lead citrate. The micrographs were taken with TEM .p < 0.05 was considered to be statistically significant.Data are presented as the mean ± SEM. All data were statistically analysed and graphed by GraphPad Prism . The Shapiro‒Wilk test was applied to analyse the data for normality. An independent-sample t test was performed to assess differences between two groups. Comparison among three or more groups were investigated by one-way ANOVA followed by Tukey’s multiple comparison test to detect pairwise between group differences. Time courses of nociceptive behavioral data were analysed by two-way ANOVA with the Bonferroni post hoc test. With repeated administration of NTG, mice exhibited a decreased trend with time in the basal mechanical and thermal thresholds . Two hours after each single injection of NTG, an acute allodynia response was observed in mice. To evaluate the photophobic behavior of mice, a light-aversive test was applied. We found that mice in both groups spent significantly more time in the dark side of the box. Compared with those in the VEH group, mice in the NTG group showed a significant decrease in both the time spent in the light chamber and the number of transitions Fig. . Togethep-value < 0.05, 529, 109, 163, 152 and 419 DEPs between two groups were screened in five brain regions respectively. The number of DEPs in the thalamus was the highest. Compared with the VEH group, the NTG group had 268 up-regulated proteins and 261 down-regulated proteins   Fig. .Fig. 5Im2 in the NTG group was significantly increased compared with that in the VEH group (p < 0.01) . The number of abnormal mitochondria per 100 µm01) Fig. .Fig. 6CoFis1 and Mid49, two major mitochondrial fission markers were upregulated and OPA1, PGC1α and TFAM respectively related to fusion and biogenesis, reduced in CM  and mitochondrial UPR (CHOP and HSP10) were strikingly elevated in response to repeated NTG injection  is implicated in CM, molecular investigation was performed to evaluate mitophagy, fission, fusion, biogenesis and mitochondrial unfolded protein response (UPR). Our data illustrated both p < 0.05) and decreased damaged mitochondria , suggesting that the improvement of mitochondrial function has a certain therapeutic effect on allodynia in CM mice  and ellagic acid (EA), which is of great benefit to aging and chronic diseases . UA enhaOf course, our conclusion was supported by other groups’ studies. Based on a classic migraine rat model established by repeated dural infusions of inflammatory soup (IS), Xin Dong et al.  observed31P-MRS studies consistently highlighted the impairment of mitochondrial oxidative phosphorylation in the brains of migraine patients as evidenced by increased ADP levels and decreased phosphorylation potential (an index for readily available free energy) [ATP) channels may be a final common pathway in the genesis of a migraine attack. KATP channels are regulated by the intracellular ATP/ADP ratio, and the levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thereby supporting the hypothesis that brain mitochondrial energy metabolism is involved in the pathogenesis of migraine.Not only CM mouse models presented abnormal mitochondria took part in CM pathogenesis, but also human research provided the similar results. The latest research suggests that migraine is a brain dysfunction disorder, which occurs in genetically predisposed individuals with a mismatch between the brain’s energy reserve and workload , 47–49.  energy) –52. Usin energy)  found th energy)  believe 1 H-MRS study reported decreased concentrations of N-acetyl-aspartate (NAA) in the thalamus of patients with migraine without aura [A out aura . NAA is out aura , 56. Theout aura  integratout aura  with higout aura  to identThe present study provides a comprehensive assessment of protein signatures across five key subcortical brain regions in CM mice, indicating an important role of thalamic mitochondrial function in migraine chronification. We speculate that migraine chronification may be due to a decline in thalamic energy reserves caused by impaired mitochondrial metabolism, thus increasing the susceptibility to attacks.There are still some limitations in this study. Although the mouse model established by administration of NTG can partially mimic human migraine attacks, the current findings in mice cannot be extended to humans. Considering the effect of female hormones on pain processing in migraine, we performed the experiments using male mice. However, migraine is a female-prevalent disorder, so the results of this study did not take into account the role of female hormones. Despite the link between mitochondria and CM-related phenotypes, it has yet to be determined the molecular mechanism that the mitochondrial impairment could affect CM.Collectively, our study reveals that mitochondrial damage in the thalamus is involved in central sensitization of CM in mice. Further studies are needed to explore the specific molecular mechanism between mitochondrial metabolism and CM.


The carcinogenic effect of arsenic (As) has been documented in lung, bladder and skin cancers but remains unclear for digestive cancers, although metabolic pathways of As and recent data suggest that it may be an important determinant in these malignancies as well.This study aimed to systematically review the available literature investigating the potential association between As and digestive cancers.An extensive search was conducted in Medline Ovid SP, Cochrane, PubMed, Embase.com, Cochrane Library Wiley, Web of Science and Google Scholar. Studies providing original data in humans, with As measurement and analysis of association with digestive cancers including esogastric cancers (esophagus and stomach), hepato-pancreatico-biliary (HPB) cancers  and colorectal cancers were eligible.A total of 35 studies were identified, 17 ecological, 13 case–control and 5 cohort studies. Associations between As and digestive cancers were reported for both risks of incidence and cancer-related mortality. Overall, 43% (3/7) and 48% (10/21) studies highlighted an association between As and the incidence or the mortality of digestive cancers, respectively.A substantial proportion of studies exploring the potential link between As and digestive cancers suggested an association, particularly in HPB malignancies. These findings emphasize the need to further investigate this topic with dedicated and high-quality studies, as it may have an important impact, including for prevention strategies.The online version contains supplementary material available at 10.1186/s12940-023-00988-7. Digestive malignancies represent a major health problem. In 2018, 4.8 million of new cases and 3.4 million of deaths were reported, worldwide  [Arsenic (As) is a heavy metal and component of the earth crust that may contaminate water, air, soil and food. Exposure to arsenic in humans occurs mainly through contaminated subsoil water, industrial exposure, food and tobacco. Metabolized by the liver, its metabolites are excreted through bile -for the most toxic ones- and urine . The maglargest mass poisoning of a population in history” [The geographical distribution of As is heterogeneous, with areas like Chile, India or Bangladesh showing particularly high concentrations associated with devastating repercussions. As an illustration, the WHO estimated that the As crisis in Bangladesh was the “history” . The carhistory” .The role of As in digestive malignancies is not clear. Some recent studies suggest a carcinogenic effect of As in biliary cancers -10; howeThe exact carcinogenic mechanisms of arsenic in humans is not fully elucidated but several elements have been postulated, including cell damage induced by the generation of reactive oxygen species and nitrogen species, genotoxic damage induced by As and signaling pathways activation/inhibition related to gene expression variations , 13.This study aimed to systematically review the available data on the association between As and digestive cancers.This systematic review was conducted according to the recommendations of the Cochrane Handbook for Systematic Reviews and Interventions  and accoAn extensive review of the literature was conducted to identify studies investigating the association between As and digestive cancers. The following databases were queried until February 28, 2022: Medline Ovid SP, Cochrane, PubMed, Embase.com, Cochrane Library Wiley, and Web of Science (Core Collection). A supplementary search has been conducted in Google Scholar, Embase, Ovid SP, Wiley and Web of Science. All searches were conducted without language or date restrictions. Algorithms of search with specific syntaxes for each database are detailed in Additional File Two investigators (S.K. and L.M.) independently applied the selection criteria provided in Table n = 6), stomach (n = 9), biliary (n = 7), liver (n = 17), pancreas (n = 5) and colorectal (n = 10). Of note, 8 studies investigated different types of digestive cancers in the same report. Relevant findings generated by the selected studies will be detailed for each organ.A comprehensive search of the literature identified 2622 studies after removal of duplicates Fig. . A thoroSix studies were selected  . A varieBased on previous findings identifying nickel (Ni) and As as risk factors for oral cancers, Lee et al. launched a subsequent study on Ni and As in EC, showing an association between EC prevalence and Ni but not As . BlackfoIn a case–control study conducted in Pakistan, investigators used hair samples as a proxy of overall exposure to heavy metals including As in a cohort of patients with various types of cancers. EC patients showed higher levels of As, as opposed to healthy controls .p = 0.328) [A recent cohort study conducted in Iran measured As in EC and non-cancer tissue samples showing comparable median concentrations of 0.6 and 0.8 µg/kg, respectively (= 0.328) .Nine reports investigated the consequences of As on gastric cancer (GC), including 6 ecological and 3 case–control studies. López-Abente et al. found no association between As and GC , conversp < 0.01). Furthermore, the quasi-Poisson regressions for the effects of As and Ni in soils showed significantly increased RR in men  and in women .Likewise, a Japanese study enrolling a large cohort of patients identified no association between air concentration of As and GC-related SMR . Chen etKohzadi et al. performed inductively coupled plasma mass spectrometry (ICP-MS) to measure concentrations of several heavy metals in tissue . TherewiIn 2013, an ecological study conducted in Ireland aimed to investigate the relationship between trace elements in soil and cancer incidence, including GC . CorrelaLike most South American countries, Argentina also encompasses regions with high concentrations of As in drinking water. Its association with SMR related to GC was evaluated in Córdoba, showing insignificant results  .In Taiwan, mortality related to GC appeared higher in BFD areas, both in men  and women  as compared to control regions .Nozadi et al. also utilized ICP-MS to measure As in tissues samples of GC patients compared to controls but did not find any difference .A total of 25 articles investigating hepato-pancreatico-biliary (HPB) cancers were selected  was the most frequently investigated digestive malignancy, with 17 studies identified: 9 ecological, 5 case–control and 3 cohort studies.Five studies were conducted in Taiwan, including 3 analyses of BFD regions. Tsai et al. also analyzed cancer mortality related to hepatocellular carcinoma (HCC) in BFD areas and found that LC mortality was higher in men  and women  compared to control regions .Chen et al. reported similar findings  whereas Lin et al. analyzed As concentrations and LC-related mortality and showed that As concentrations in drinking water > 0.64 mg/L were associated with higher LC-related mortality. Conversely, no association? was detected for low As concentrations (< 0.64 mg/L) . A studyIn 1955, the western part of Japan suffered from a mass As poisoning due to contaminated milk powder. A study hypothesized that this event may have been associated with long-term injuries in the exposed population, including digestive cancers (liver and pancreas) . The autAlso tackling the question of early-life exposure, Smith et al. leveraged the unique epidemiological scenario described in the city of Antofagasta in Chile where the population was exposed to very high concentrations of As in drinking water (870 µg/L) in 1958, with an abrupt stop in 1970 . This nap = 0.24).In 2008, Baastrup et al. published a study with prospectively collected data . They usvs. 0.019 ± 0.008 mg/L, p < 0.001), in a case–control study conducted in Egypt [As concentrations in blood of 314 patients with HCC were found to be significantly higher as compared to control subjects .Hsu et al. evaluated the interaction between arsenic exposure and HBV or CV infection in chronic liver disease . Among sA total of 7 articles were identified, with 6 studies on gallbladder (GBC) and one on bile duct cancer (BDC). It included 3 ecological, 2 case–control and 2 cohort studies.Tsai et al. analyzed cancer mortality related to GBC but found no effect of As .p = 0.03). These findings were confirmed by national-scale analyses in the US, Taiwan and India. The same group performed a study in BDC, showing similar results [In 2020, Ganesan et al. specifically explored the effect of As-contaminated water on GBC incidence . First,  results .p = 0.001) and those with high concentrations of As in soil  showed higher risk of GBC.A study conducted in India aimed to assess the geographical pattern of GBC according to the proximity to River Ganga, a widely known source of heavy metal intoxication . Odds rap < 0.001) and T3 . Intrigued by these results, a European group of researchers utilized Mendelian randomization analysis to decipher the association of As [AS3MT) variants permitted to distinguish the impact of each As species. While the data confirmed a protective effect of iAs and MMA, it highlighted a deleterious effect of DMA on the risk of GBC.A recent study performed metallomic analyses in serum with a panel of 18 metals in a large cohort of patients including 259 with GBC, 701 with gallstones and 851 controls . Surprison of As . In contr = 0.005, p < 0.05), in a case–control study [Finally, Kumar et al. detected a significant correlation between age and As levels in blood samples from 175 patients with GBC (ol study .Five articles explored the association between As exposure and pancreatic cancer (PC), including 2 ecological and 3 case–control studies.An ecological study conducted in Taiwan showed comparable mortality related to PC between BFD endemic areas and control regions, in men  and women  .The Japanese case–control study investigating the As-mass poisoning in milk powder also analyzed PC-related mortality . Resultsp = 0.009). In a follow-up study, the authors aimed at deciphering a potential link between these trace elements and oncogenic driver mutations in KRAS [A Spanish consortium took advantage of the multicentric cohort PANKRAS II  to asses in KRAS . Sample Additional file p = 0.1) [The previously cited Danish study also investigated the effect of low As levels in drinking water on colon cancer incidence, showing no significant association  . Despitep < 0.01) but a protective effect in men  [The incidence of colon cancer was monitored in Córdoba (Argentina), a region known for high concentrations of As in water. A gender-specific effect of As was detected, showing a detrimental impact in women  .The Taiwanese ecological study by Tsai et al., mentioned above for esogastric and HPB cancers, also analyzed the effect of As in BFD endemic areas for colorectal cancer (CRC)-related mortality . A signiIn the early 1960s, Taiwan implemented a tap water supply system in BFD-endemic areas to prevent As-induced deleterious effects. Yang et al. aimed at analyzed the effect of this measure on CRC-related SMR between 1971 and 2006 . During Cancer incidence rates in Appalachian Kentucky are particularly concerning. A study compared Appalachian Kentucky counties—known for their exposure to coal contaminants including As—with control urban county . A similvs. 1.08 µg/kg, respectively (p = 0.007) [Measurement of As concentrations in toenail samples from 239 patients showed higher As levels as well as higher incidence and mortality rates related to CRC in the study group. Analyses revealed lower As concentrations in cancer as compared to non-cancer samples, with median values of 0.27 = 0.007) .The present systematic review identified studies exploring the potential association between As and digestive cancers with different perspectives and endpoints, focusing either on incidence or on mortality. Forest plots aimed to illustrate these associations. For esogastric cancers Fig. , availabThis study systematically reviewed the available data on the potential association between As and digestive cancers.As is a documented carcinogen for bladder, lung and skin cancers . These oThis systematic review identified 35 studies. First, this is a very small number, considering that it explored all digestive cancers and that these are responsible for almost 5 million new cases each year . In addiSome limitations need to be discussed. The main one is intrinsic to the topic rather than to the methodology of the present study: selected studies are few and of overall modest quality. In addition, they showed a high degree of heterogeneity for various aspects: study design, sample size, endpoints and technique of measurement of As concentrations. In addition, most studies failed to provide precise data regarding the types of cancers . Finally, a meta-analysis was not feasible due to the different endpoints reported in the selected studies. Tackling the question of As in digestive cancers by a systematic review revealed evidence emphasizing the importance of further studies in this area. There is an urgent need to develop new strategies of prevention, which is likely the best—if not the only—way of controlling mortality related to these aggressive cancers . Mitigating As effects and controlling the levels of As on a large-scale is a challenging task, particularly in developing countries. As exposure includes multiple paths; if one only considers drinking water, WHO has set a precise threshold (i.e. 10 µg/L) and made clear recommendations . The issThe present study also emphasizes specific points that need to be considered in future studies, like the importance to distinguish the varying effects according to the different species of As.This systematic review identified 35 studies that investigated the potential assocation of As on digestive cancers. Results suggest that As may be a determinant in digestive cancers, in particular in hepatobiliary cancers. It reinforces the rationale and underscores the need to conduct future studies focusing on this question to provide data of high quality on an individual basis. Documenting a carcinogenic effect of As in digestive cancers may have a substantial and potentially beneficial impact, especially in terms of prevention.Additional file 1. Search algorithms.Additional file 2. Quality assessment of the selected studies.Additional file 3. Selected studies investigating the effect of arsenic (As) in esogastric cancers.Additional file 4. Selected studies investigating the effect of arsenic (As) in hepato-pancreatico-biliary cancers [ cancers –54.Additional file 5. Selected studies investigating the effect of arsenic (As) in colorectal cancers [ cancers .


Cognitive–behavioural therapy (CBT) and counselling for depression (CfD) are recommended first-line treatments for depression. While they approach change differently, there is little understanding of the impact those approaches have on change during treatment.This study aimed to identify whether CBT and CfD target different symptoms and explore the implications of modelling choices when quantifying change during treatment.Symptom-specific effects of treatment were identified using moderated network modelling. This was a retrospective cohort study of 12 756 individuals who received CBT or CfD for depression in primary/community care psychological therapy services in England. Change was modelled several ways within the whole sample and a propensity score matched sample (n=3446).CBT for depression directly affected excessive worry, trouble relaxing and apprehensive expectation and had a stronger influence on changes between suicidal ideation and concentration. CfD had a stronger direct influence on thoughts of being a failure and on the associated change between being an easily annoyed and apprehensive of expectation. There were inconsistencies when modelling change using the first and second appointments as the baseline. Residual score models produced more conservative findings than models using difference scores.CfD and CBT for depression have differential effects on symptoms demonstrating specific mechanisms of change.CBT was uniquely associated with changes in symptoms associated with anxiety and may be better suited to those with anxiety symptoms comorbid to their depression. When assessing change, the baseline should be the first therapy session, not the pretreatment assessment. Residual change scores should be preferred over difference score methods. Cognitive–behavioural therapy (CBT) and counselling for depression are recommended first-line treatments for depression and are considered equally effective on average. However, little is known about how change comes about.This study investigates symptom-specific effects and identifies specific symptoms and symptom interactions associated with each intervention. In addition, it highlights methodological considerations when modelling change.CBT was uniquely associated with changes in symptoms associated with anxiety so may be better suited to those with anxiety symptoms comorbid to their depression.There is a strong preference among patients for psychological therapies over antidepressant medications.The symptom experiences of people with depression are heterogeneousCfD aims to engender change by exploring the emotional meaning associated with experiences and developing alternative ways of understanding these experiences to inform a new self-concept.10.1136/bmjment-2022-300621.supp1Supplementary dataThe aims of this study were to (1) identify the direct influence of CBT compared with CfD on symptom change using network intervention analysisRoutine clinical data were gathered from eight Improving Access to Psychological Therapies (IAPT) services. All were part of the North Central and East London IAPT Service Improvement and Research Network.Patients are assessed by a clinician to determine their needs and consider the most suitable intervention(s). Patients receive a diagnosis based on International Classification of Diseases, 10th Revision; this represents the focus of treatment agreed on a patient and a clinician. Patients are offered treatment(s) recommended by the National Institute for Health and Care Excellence (NICE) in guidance specific to the patient’s diagnosis.CfD and CBT were delivered by clinicians with doctoral qualifications in clinical or counselling psychology or with postgraduate diplomas in CBT. Sessions lasted 50–60 min and typically 8–16 sessions were offered. Prior to treatment, patients completed an initial assessment (session 1), and those offered CfD or CBT were placed on a waiting list to start treatment. As such, session 2 represents the first treatment session, typically occurring 4–12 weeks after the assessment session.For details of the theory underlying these therapies and the competence frameworks, see IAPT services are mandated to collect sessional outcome data with all patients as well as numerous sociodemographic and treatment-related variables,Changes scores were estimated for all 16 symptoms of the PHQ-9 and GAD-7. We estimated the residual and difference scores with both session 1 and session 2 as baselines, to account for regression to the mean. Scores were calculated as follows: Difference Score (DS)=postscore−prescore; Final Score (FS)=postscore; Proportional Change (PC)=100*DS/prescore; Residual Score (RP)=postscore−predict value (relationship of prescore–postscore); Residual Change Score (RC)=DS−predict value (relationship of prescore–DS).Given the potential for topological overlap, we investigated multicollinearity cross-sectionally using the goldbricker function in the networktools package.Moderated Network Modelset al, 2002) total score. Propensity score matching was performed using MatchIt package.Estimation of the residual models was conducted using the whole sample and a propensity score–matched sample. Propensity score matching was used to control for confounding as the intervention type was not randomly assigned. Matching variables included session 1 item scores (PHQ-9 and GAD-7), gender , employment status (employed/unemployed), taking psychotropic medication (yes/no), age (continuous), ethnicity  and baseline functional impairment as measured using the Work and Social Adjustment Scale  with false discovery rate (FDR) co rrected p values within both the whole and propensity score–matched samples.https://osf.io/ak4ev/.The study has followed the STROBE  reporting guidelines  differed between the groups see . There wThe propensity score model is plotted in Results using the difference score with session 1 as the baseline were different than other change models , with the direct associations negatively correlated will all other estimates, including modelling the difference score with session 2 as the baseline, r=−0.60. Direct associations found with residual score models using the session 1 baseline were different from those found using session 2 data. The associations found when using session 2 as the baseline were consistent whether using the final score or residual score outcome (r>0.98).The whole sample residual models using the session 2 baseline were similar (r>0.99), and similar to the propensity score–matched models (r>0.98). Fewer direct associations were identified in the propensity score–matched sample using the residual change score outcomes. In these models, using the session 2 baseline, there was consistency across four items identified as having direct associations, three positively associated with CBT and one positively associated with CfD. Across the propensity score–matched models, there was a larger change of scores on thoughts of being a failure with CfD (RCX2: 0.03) and a larger effect on excessive worry (RCX2: 0.02), troubling relaxing (RCX2: 0.02) and apprehensive expectation (RCX2: 0.02) with CBT.When looking at the influence of treatment on symptom-to-symptom interactions , there wWithin the whole sample, there was evidence of stronger related change between anhedonia and appetite during CBT than CfD . Further, the CBT group showed an associated change between suicidal ideation and restlessness (0.03), suicidal ideation and being easily annoyed/irritated (0.04) and between depressed mood and psychomotor disturbance (0.01); these were absent for the CfD group.Between the propensity score–matched models, only two effects were identified in both models: the CfD group showed a stronger related change between feeling annoyed and apprehensive expectation . There was also a difference between groups on the associated change between suicidal ideation and concentration , with the CBT group displaying stronger associated change than the CfD group. Given the difference between interventions on the number of sessions attended, we controlled for the number of sessions within the RCX2 model. This did not alter any of the direct or indirect effects see . Within p²=0.002, estimated marginal means±SE (CBT: 8.28 (0.05), CfD: 8.98 (0.13)) than when controlling for session 1 scores: F=17.94, p<0.001, ωp²=0.002 (CBT: 8.29 (0.06), CfD 8.9 (0.14)). There was no evidence of a difference between groups for the final PHQ-9 total score when controlling for session 1 PHQ-9 scores: F=1.3, p=0.254 (CBT: 9.58 (0.06), CfD: 9.77 (0.16) or session 2 scores: F=4.385, p=0.036 (CBT: 9.56 (0.06), CfD: 9.90 (0.15)). Within the propensity score–matched sample, there was a greater degree of change in both anxiety and depression during CBT than CfD when controlling for the session 2 score, PHQ total score: F=6.836, p<0.009 (CBT: 8.89 (0.14), CfD: 9.40 (0.14), and GAD-7 total score: F=18.35, p<0.001, ωp²=0.005 (CBT: 7.72 (0.13), CfD:8.47 (0.12)).Within the whole sample, there was a greater degree of change in anxiety but not depression during CBT than CfD. This difference was larger for the final GAD-7 score when controlling for session 2 scores: F=24.255, p<0.001, ωSymptom change is plotted in This study investigated differences in symptom-specific effects of CBT and CfD, and the impact of modelling symptom changes in a variety of commonly used ways for adults with depression treated in primary/community care psychological therapy services. We found that CBT for depression may work by directly affecting excessive worry, trouble relaxing and apprehensive expectation, while CfD may work by affecting thoughts of being a failure. These effects were specific to the type of treatment, that is, they were not shared effects  or indirect effects of changes in other symptoms influenced by the treatments. There were also treatment-specific effects on symptom-to-symptom interactions. CfD had a stronger influence on the associated change between feeling annoyed and apprehensive expectation than CBT. The associated change between suicidal ideation and concentration was greater for CBT than CfD.We found variability in the results obtained from different ways of measuring change. There was little consistency in the results between using session 1 and session 2 as a baseline. This is important because many observational studies and clinicians use pre–post change in a symptom measure score as their primary outcome. Further, within treatment settings, there can be a period (weeks to months) between initial assessment (session 1) and commencing treatment (session 2). Hence, session 2 appears to be a more appropriate baseline for measuring treatment-related symptom change. Differences between the whole and propensity score–matched samples would suggest that there is an influence of covariates, but it is less evident when estimating direct associations, although propensity score matching cannot fully redress selection biases or confounding given the potential influence of unmeasured variables.The results provide evidence to elucidate how these therapies may work. For example, compared with CBT, CfD was directly associated with a change in the thoughts of being a failure. CfD also demonstrated a greater associated change between feeling annoyed and apprehensive expectation (feeling afraid that something bad will happen) than CBT. This fits with the theoretical underpinnings of CfD targeting the development of self-concept and conditions of worth and their link to emotional processes.CBT for depression was uniquely associated with changes in symptoms associated with anxiety. Some of the observed symptom effects could be considered mechanistic  others are more descriptive.There was a stronger associated change between suicidal ideation and concentration for CBT than CfD. Within this sample, we cannot identify temporal precedence. However, in a dynamic network model of change during psychotherapy, temporal influence was stronger for concentration on suicidal ideation than the other way around.We attempted to balance groups on observed covariates, but they may have differed on important, unmeasured confounders such as those related to aspects of severity,It is important to understand how interventions work so that more effective and efficient treatments can be developed, and so that interventions can be more acceptable to patients. This study suggests that as CBT was uniquely associated with changes in symptoms associated with anxiety it may be better suited to those with anxiety symptoms comorbid to their depression.The study also highlights methodological considerations. When assessing change, the baseline should be the first therapy session (or second session) not the pretreatment assessment. This will address potential sources of bias such as regression to the mean. When calculating change, residual change scores should be preferred over difference score methods.


Trust is a key determinant of health, but has been undermined by the COVID-19 pandemic and the associated infodemic. Using data from eight countries, we aimed to epidemiologically describe levels of trust in health, governments, news media organisations, and experts, and measure the impact of political orientation and COVID-19 information sources on participant’s levels of trust.We simultaneously conducted a stratified randomised online cross-sectional study across eight countries on adults aged ≥18 years between 6 and 18 November 2020. We employed crude and adjusted weighted regression analyses.We included 9027 adults with a mean age of 47 years (range = 18-99), of whom 4667 (51.7%) were female. Trust in health experts ranked highest across all countries (mean (x̄) = 7.83; 95% confidence interval (CI) = 7.79-7.88), while trust in politicians ranked lowest . In adjusted analyses, political orientation and utilised information sources were significantly associated with trust. Individuals using higher levels of health information sources trusted health authorities more than those using lower levels . Similarly, individuals using higher levels of government information sources  and those using higher levels of new media information sources  had highest trust in governments/politicians and news media, respectively. However, there was little difference in trust in health, government, or news media between individuals using higher or lower levels of social media information sources.Trust is a key determinant of health, but has been politically fragile during this infodemic. High compliance with public health measures is key to combatting infectious diseases. In terms of people’s trust, our findings suggest that politicians and governments worldwide should coordinate their response with health experts and authorities to maximise the success of public health measures. With a conservatively estimated 6.95 million attributable deaths reported to the World Health Organization (WHO) , the corPeople’s trust is a key component for population compliance. It directly impacts people’s risk perception, social cohesion, and behavioural changes and has been recognised as foundational in determining a country’s mortality and morbidity trajectory -7. CountTrust can take several forms, ranging from an individual’s trust in national governments and officials to one’s interpersonal trust in their surrounding community. A 177-country study investigating pandemic preparedness found significantly lower standardised infection rates with higher government and interpersonal trust . While aPolitical orientation and its relationship to trust have been found to significantly impact COVID-19 prevention behaviours -13. The The WHO identified the “infodemic”, referring to the rapid spread of both accurate and inaccurate information in the age of the internet and social media, as a key challenge in responding to COVID-19 and other disease outbreaks . MisinfoUsing an eight-country cross-sectional study design, which recruited representative samples of adults, we aimed to investigate people’s level of trust and how it was affected by their political orientation and information sources. Our primary goals were to epidemiologically describe levels of trust in health, government, and news media organisations and experts and measure the impact of participant’s political orientation and COVID-19 information sources on their levels of trust.We conducted a stratified randomised online cross-sectional study in seven countries , England, Switzerland, Belgium, Philippines, New Zealand) and one territory (Hong Kong) simultaneously between 6 and 18 November 2020. As Hong Kong is a special administrative region of China and enjoys governing and economic autonomy, we referred to it as a country rather than a territory. The timing of this study coincided with the beginning of the second COVID-19 wave [We included adults aged ≥18 years who resided of participating countries at the time of the survey.Informed by the Organisation for Economic Co-operation and Development (OECD) guidelines , we desiIn scientists, doctors, and health experts;In national health organisations;In global health organisations;In government;In politicians;In news organisations.Response options for each used a 10-point scale, ranging from 1 (very low) to 10 (very high) levels of trust.We asked participants about their political orientation via the question “When it comes to politics, people talk about ‘left’ and ‘right’. Where would you place yourself on a scale, where 0 stands for far left and 6 for far right?”. Due to the political sensitivities in Hong Kong preceding instrument development , we did Regarding information sources, the participants were asked “To what extent do you use the following sources to inform yourself about the coronavirus ?”. We devised 11 possible responses:The federal government;Provincial government;Politicians;World Health Organization (WHO);Health professionals in the media;Public health authorities;Television;Radio;Newspapers (including online);Social networks ;Other internet sources.Each question had additional response options which ranged from 1 to 4 . We collapsed questions into four domains: health (questions 4-6), government (questions 1-3), news media (questions 7-9), and social media (questions 10-11). We calculated summed response scores for each domain and standardised them by dividing by the number of contributing variables. We dichotomised these re-scaled scores into higher (score ≤2.5) and lower (score >2.5) categories, with this 2.5 threshold representing the mid-point on the four-point response option scale, falling between “often” and “sometimes”.A detailed account of the sociodemographic variables is presented elsewhere . In brieA detailed description of the procedure is available elsewhere ,22. CounWe set a minimum sample size of 1000 adults for each participating country, except for Canada (the research program host), which we set at 2000. This was to obtain a largely balanced sample sizes for each country, thereby maximising statistical power for intercountry comparisons, to gain reasonable intra-country power (detecting differences in proportions of ≥10% or a relative risk of ≥1.2 exceeding 80% for a two-tailed α = 0.05), and to maximise country inclusion and diversity within a constrained budget and timeframe.We used the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) guidelines in reporting this study . We assiR). Next, we conducted crude and adjusted complete case regression analyses relating participants’ political orientation and information source level to their trust in health authorities, government/politicians. Rather than employing bivariable analyses to screen sociodemographic and potential confounding factors, as suggested by Sun et al. [2 statistic to determine the significance of variables within the regression models.We initially described and compared participant numbers and sociodemographic characteristics by countries using Pearson design-based F-test. We assessed correlation between primary variables using weighted Pearson pairwise correlation coefficients  = 17.0 years), with the oldest participant being 99 years; 4667 (51.7%) were female, 4318 (47.8%) male, 28 (0.3%) neither female nor male, and 14 (0.2%) preferred not to answer the question. Regarding household composition, 2680 (29.7%) participants lived with their spouse/partner and 2069 (22.9%) resided with their spouse/partner and child(ren). The majority (n = 6450 (73.3%)) were non-essential workers, although 800 (9.1%) participants reported being essential workers in health (P = 0.79) between countries, we observed a statistically significant difference in age (P < 0.001), usual household composition (P < 0.001), and essential worker (P < 0.001) distributions. Reflecting cultural differences, participants from the Philippines had a younger average age and were more likely to live with extended family, and Hong Kong participants were more likely to be non-health essential workers , and trust in global health organisations ranked third  (P < 0.001). For instance, although ranked lowest in both countries, trust in politicians for New Zealand participants was higher than in the USA  except for between government (x̄ = 6.28) and news organisations .We observed significant differences in the mean level of trust between countries for each of the six separate variables  declaring as far left, 553 (6.9%) as far right, and 3427 (42.7%) as moderate  to R = 0.39 .For the combined information sources domain scores, complete non-missing responses ranged from 92.0% (n = 8290) for the derived social media variable to 96.0% (n = 8688) for health. Overall, participants sourced information to a higher degree  from health (n = 4664 (53.7%)), government (n = 3448 (40.0%)), news media (n = 3791 (43.9%)), and social media (n = 2694 (32.5%)) sources  had correlations ranging between 0.71-0.81   apart from social media information source level related to trust in news media (P = 0.01) and social media information source level related to trust in health authorities (P = 0.30)  and the highest in New Zealand’s x̄ = 6.18)  to10 (very high)). At the beginning of the pandemic, USA and New Zealand had very different approaches to health messaging from their respective leaders, with New Zealand’s prime minister at that time globally heralded for her leadership in the nation’s COVID-19 response . Messagi8 . HoweveTrust is a key determinant of health; it is central to the successful implementation and uptake of public health measures. Although it shifts easily and significantly impacts society, it has received relatively little public health research attention. This study empirically highlights the universally high levels of public trust placed in health experts and authorities across eight countries. We recommend that governments and policymaker coordinate their response with health experts and authorities, thereby maximising the likely population health impact. COVID-19 is not the first, and certainly will not be the last, global health crisis that we have faced. However, it is the first to exist on this scale during the “infodemic” in the new digital landscape of the internet and media. We need to learn the lessons from COVID-19 to inform successful response for future health crises and challenges.