The excessive and inappropriate use of antibiotics is universal across all healthcare facilities. In Qatar there has been a substantial increase in antimicrobial consumption coupled with a significant rise in antimicrobial resistance (AMR). Antimicrobial stewardship programmes (ASPs) have become a standard intervention for effective optimization of antimicrobial prescribing.Pseudomonas aeruginosa was compared with antimicrobial consumption, calculated as DDD per 1000 patient-days (DDD/1000 PD). Susceptibility was determined using broth microdilution, as per CLSI guidelines. Antibiotic use was restricted through the ASP, as defined in the hospital’s antibiotic policy.A before–after study was conducted in Hamad General Hospital : 1 year before implementation of a comprehensive ASP compared with the following 2 years. The ASP included a hospital-wide pre-authorization requirement by infectious diseases physicians for all broad-spectrum antibiotics. Prevalence of MDR P. aeruginosa were collected prospectively over 3 years (2014–17). Susceptibility to certain antimicrobials improved after the ASP was implemented in August 2015. The prevalence of MDR P. aeruginosa showed a sustained decrease from 2014 (9%) to 2017 (5.46%) (P = 0.019). There was a significant 23.9% reduction in studied antimicrobial consumption following ASP implementation (P = 0.008). The yearly consumption of meropenem significantly decreased from 47.32 to 31.90 DDD/1000 PD (P = 0.012), piperacillin/tazobactam from 45.35 to 32.67 DDD/1000 PD (P < 0.001) and ciprofloxacin from 9.71 to 5.63 DDD/1000 PD (P = 0.015) (from 2014 to 2017).A total of 6501 clinical isolates of P. aeruginosa, pointing towards the efficacy of the ASP in reducing AMR.The successful implementation of the ASP led to a significant reduction in rates of MDR  Pseudomonas aeruginosa in particular is associated with wide varieties of HAIs,P. aeruginosa has emerged as a serious nosocomial problem with significant morbidity and mortality, with limited available treatment options, leading to prolonged hospital stays with cost implications.,,P. aeruginosa susceptibility, given the limited number of antibiotics with activity against this important pathogen.Healthcare-associated infections (HAIs) caused by Gram-negative bacteria (GNB) are of particular concern since more than 50% have been reported to be associated with MDR organisms. P. aeruginosa being the second most prevalent. From 2009 to 2014 there was a noticeable increase in resistance to both colistin and meropenem . In 2015, 8.1% of P. aeruginosa strains from the five major hospitals in Qatar were MDR.P. aeruginosa, the prevalence of MDR P. aeruginosa and antibiotic use in the hospital setting.According to internal monitoring records, Qatar has witnessed a decade of substantially rising antibiotic consumption, especially of carbapenems, coupled with a high prevalence of GNB isolated from patients at Hamad Medical Corporation (HMC) in Qatar, with The study obtained the necessary ethics approval from the Institutional Review Board Committee, HMC (protocol number IRGC-01-51-033), which complies with international ethical standards and regulations.P. aeruginosa over a 3 year period, between October 2014 and September 2017, compared with antibiotic consumption before and after programme implementation. The periods October 2014–September 2015 and October 2015–September 2017 were defined as pre-ASP and ASP analysis periods, respectively. Furthermore, we reviewed the trend of antimicrobial consumption across HGH wards since 2010.The present study was conducted in Hamad General Hospital (HGH), which has a capacity of 603 beds, dealing with acute care, including intensive care beds, and which serves most of the inhabitants of Doha, Qatar. We evaluated the effect of ASP implementation in August 2015 on resistance patterns of A formal ASP was introduced in August 2015 by a team consisting of an infectious diseases (ID) physician and a full-time pharmacist. The main strategy adopted by the ASP was a prospective-audit-and-feedback approach focused on IDSA recommendations for selected antimicrobials, based on their spectrum of activity and cost.The patients treated with restricted-use antibiotics were identified by the pharmacy department. A daily record was kept of the active prescriptions of restricted-use antibiotics and new ones were communicated by email to the ASP/ID group. The ASP team attempted to: (i) identify ineffective or excessive antimicrobial coverage; (ii) ensure that the orders adhered to policies and guidelines; (iii) discontinue unnecessary double coverage; (iv) identify patients whose treatment could be converted safely from parenteral to oral therapy; and (v) suggest ID consultations for difficult and complex cases. Antibiotics were considered to be inappropriately prescribed if one or more of the following criteria were met: (i) a narrower-spectrum antibiotic could be used, based on the culture results; (ii) there was no infection present ; (iii) hospital antibiotic guidelines were not followed without valid reasons; and (iv) dosage, duration of therapy and/or empirical treatment choice was suboptimal according to the available guidelines.Utilization data for antimicrobials were obtained for 2010 to 2017 from a previously created database. Medication orders were summed per medication per month in grams using the total number of doses and dose per administration. The grams were then converted into census-normalized DDD per 1000 patient-days (DDD/1000 PD) using the WHO anatomical therapeutic chemical classification system of 2012.We shared the antibiotic consumption data with prescribing units on a monthly basis.P. aeruginosa strains were isolated. The bacterial identiﬁcation and initial antimicrobial susceptibility testing was done using the BD Phoenix automated system, while Lioﬁlchem MIC Test Strips  and the standard reference strains Escherichia coli ATCC 25922, E. coli ATCC 35218 and P. aeruginosa ATCC 27853 were used for quality control, as per recommendations of the CLSI, and the results were interpreted using the CLSI reference breakpoints. Testing was performed for eight antimicrobial agents: gentamicin, tobramycin, amikacin, cefepime, ciprofloxacin, piperacillin/tazobactam, meropenem and colistin. MDR P. aeruginosa isolates were deﬁned as having in vitro non-susceptibility to at least one agent from ≥3 antimicrobial classes.P. aeruginosa species identification was confirmed by MALDI-TOF MS using a Bruker Daltonics MALDI Biotyper , according to the manufacturer’s instructions. A bacterial isolate of the same species and same antimicrobial susceptibility pattern in a patient isolated within 30 days, regardless of the site of isolation, was excluded. Isolates with major differences in susceptibility patterns were considered as new even if isolated within 30 days.During a 3 year period, from October 2014 and September 2017, a total of 525 MDR 2 test or Fisher’s exact test, as appropriate. A P value of 0.05 or less was considered statistically significant.All statistical analyses were done using Statistical Packages for Social Sciences v. 21.0 . For categorical variables, data were expressed as number and percentage and were analysed by χSince 2010, we were able to obtain DDDs per 1000 PD for the studied antimicrobial agents, as illustrated in P < 0.001), meropenem to 31.9 (2017) from 47.32 DDD (2014) (P = 0.012) and ciprofloxacin to 5.63 (2017) from 9.71 DDD (2014) (P = 0.015)  from 45.35 DDD (2014) (= 0.015) .P. aeruginosa  isolated from October 2014 to September 2017, from different clinical specimens taken from patients hospitalized in HGH   in 2015 (pre-ASP implementation) to 5.46% (122/2234) in 2017 (P = 0.019). This was similarly linked to an observed fall in resistance patterns of MDR P. aeruginosa to piperacillin/tazobactam (90.4% to 80.3%), meropenem (89.2% to 86.9%), ciprofloxacin (91% to 88.5%) and amikacin (58.4% to 47.5%) (The study included 6501 consecutive non-duplicate isolates of d in HGH . The ove405 270) . During o 47.5%) . It musto 47.5%) .P. aeruginosa and its susceptibility. Many published studies on antibiotic use and resistance have reported increased resistance with increased use of antibiotics.P. aeruginosa isolates with decreased meropenem, piperacillin/tazobactam and ciproﬂoxacin use. Slain and colleaguesSeveral strategies have been proposed to reduce and control antimicrobial use , but it has been suggested that antimicrobial restriction, either through formulary limitations or by the requirement for pre-authorization and justiﬁcation, is the most effective.P. aeruginosa isolates. In our study, 6501 P. aeruginosa clinical isolates were collected between October 2014 and September 2017. The ASP was strictly implemented in 2015 and the prevalence rate of MDR P. aeruginosa decreased from 9% to 5.46%, which was statistically significant. The study findings indicated that the trends in the resistance of P. aeruginosa to antimicrobial agents were influenced by the ASP implemented at HGH and that antibiotic usage was significantly related to the prevalence rate of MDR P. aeruginosa. Our data agreed with a study that looked into the relationship between carbapenem restriction in 22 university teaching hospitals and incidence rates of carbapenem-resistant P. aeruginosa.P. aeruginosa to imipenem and meropenem.We also investigated the prevalence and resistance to certain antibiotics in MDR P. aeruginosa. Although we studied the effect of the ASP on the susceptibility of MDR P. aeruginosa, showing favourable outcomes, we haven’t evaluated the effect on other GNB, particularly Enterobacteriaceae, which are associated with significant HAIs. Nevertheless, most of the broad-spectrum antibiotics used to treat P. aeruginosa are also used to treat HAI with Enterobacteriaceae. The expectation is that the resistance profile of other GNB will remain the same, as observed in other studies.Of note, there was a sustained consumption of anti-Gram-positive bacteria antibiotics for MRSA , which probably did not contribute towards the declining rates of MDR Amongst the notable observations is the fall of piperacillin/tazobactam consumption during 2015–16, which eventually returned to baseline, most likely reflecting the temporary international shortage associated with the stream of production.,It is also worth noting that one of the limitations of observational studies is the inability to establish causality. It has been previously established that propagation of AMR is not only linked to increased antibiotic consumption but also to poor infection control and prevention (ICP) measures.P. aeruginosa infections coupled with improved antibiotic susceptibility to standard treatment options. Epidemiological surveillance systems are necessary to monitor AMR organisms in conjunction with effective ASPs to deliver objectives. Such interventions are crucial for quality improvement and safeguarding patient outcomes.We demonstrated significant sustained reductions in both antimicrobial consumption, particularly broad-spectrum antibiotics, and the rates of MDR dlaa050_Supplementary_DataClick here for additional data file.


Targeted photodynamic therapy (TPDT) is considered superior to conventional photodynamic therapy due to the enhanced uptake of photosensitizers by tumor cells. In this paper, an amphiphilic and asymmetric cyclo-Arg-Gly-Asp-d-Tyr-Lys(cRGDyK)-conjugated silicon phthalocyanine (RSP) was synthesized by covalently attaching the tripeptide Arg-Gly-Asp (RGD) to silicone phthalocyanine in the axial direction for TPDT of triple-negative breast cancer (TNBC). RSP was characterized by spectroscopy as a monomer in physiological buffer. Meanwhile, the modification of RSP with RGD led to a high accumulation of the photosensitizer in TNBC cells overexpressing ανβ3 integrin receptors which can bind RGD, greatly reducing the risk of phototoxicity. In vitro photodynamic experiments showed that the IC50 of RSP was 295.96 nM in the 4T1 cell line, which caused significant apoptosis of the tumor cells. The tumor inhibition rate of RSP on the orthotopic murine TNBC achieved 74%, while the untargeted photosensitizer exhibited no obvious tumor inhibition. Overall, such novel targeted silicon phthalocyanine has good potential for clinical translation due to its simple synthesis route, strong targeting, and high therapeutic efficacy for TPDT treatment of TNBC. Targeted therapy of the TNBC remains a major challenge due to the lack of the effective targets and drug resistance. Based on the intrinsic characteristics of TNBC, preclinical trials of various targeted therapy have been performed for TNBC.In this study, we designed and synthesized a novel amphiphilic and asymmetric cyclo-Arg-Gly-Asp-d-Tyr-Lys(cRGDyK)-conjugated silicon phthalocyanine (RSP) Scheme . The cRGThe basic principle for the design of targeted photosensitizers is to ensure the in vivo tumor targeting and cellular affinity while effectively reducing photosensitizer aggregation. We axially modified one end of silica phthalocyanine with cRGDyK to obtain amphiphilic, asymmetric targeting photosensitizers. In order to examine the rationality and feasibility of the molecular design as much as possible, we simulated the lowest energy conformation of RSP Fig. . As a co32 Then, the silicon dichloride phthalocyanine was axially substituted with p-hydroxyphenyl propionic acid under alkaline conditions to obtain a silicone phthalocyanine with two free carboxyl groups . Finally, the RGD targeting group was conjugated to the phthalocyanine. In order to avoid self-cyclization of the cRGDyK molecule and the formation of modified molecules at both ends, activation of one of the carboxyl groups of SC was first performed with the activating ester. After the activation of the carboxyl group, a simple amide condensation reaction at room temperature by strictly controlling the ratio of cRGDyK and SC (1:3) was carried out to initially obtain cRGDyK-substituted silicon phthalocyanines . The structures of SC and RSP were verified by NMR and MS . Therefore, we dissolved RSP in physiological saline containing 1% CEL, DMEM medium containing 0.05% CEL, and DMF (as a control) to measure its absorption and fluorescence spectrum. From the absorption spectra Fig. , it coulIn order to better simulate the situation of photosensitive molecules in practical applications, the singlet oxygen yield of RSP in physiological saline containing 1% CEL was measured Fig. . The sloTo assess cellular uptake of RSP, we incubated RSP and SC (untargeted control) with triple-negative breast (4T1) cancer cells , which was consistent with the singlet oxygen yield. Then, apoptotic, necrotic and live 4T1 cell populations were quantified by flow cytometric after TPDT , SC, RSP, SC + light (SCL) and RSP + light (RSPL). As shown in Fig. PDT Fig. . The apoThe ideal targeted photosensitizer should be able to quickly accumulate at the tumor site and metabolize from normal organs after injection. The investigation of the pharmacokinetics of RSP showed that plasma half-time was 45.22 min and elimination half-time was 463.02 min Fig. . After 2The orthotopic and subcutaneous mouse model of triple-negative breast cancer were established and tumor imaging assay was conducted to investigated the in vivo tumor targeting of RSP. In the subcutaneous tumor model, the fluorescence signals for RSP and SC gradually increased with time after tail vein injections Fig. . For RSPThe encouraging fluorescence imaging results thrilled us to further investigate the in vivo photodynamic efficacy of RSP in 4T1-tumor bearing mice. As shown in Fig. By H&E staining and TUNEL staining analysis of the tumor site, the strong green fluorescent signal of tumor cell sections in the RSPL group indicates a high rate of apoptosis of cancer cells, which accelerates the inhibition of tumor growth. The SCL (SC + Light) group showed only a slight green fluorescence demonstrating that the RSP targeting in vivo was much better than SC. After quantitative analysis of the fluorescence signals of the tissue, it was found that the apoptosis in the RSPL group was twice that of SCL group Fig. . These r34 ADC can precisely deliver drugs to tumor cells through highly specific antibody-receptor-specific recognition.35 While enhancing the uptake by tumor cells, it reduces the nonspecific uptake of photosensitizers by normal cells. In addition, smaller-sized nano-antibodies have been shown to improve the targeting ability of drugs. However, the large size of antibodies limits their penetration into tumors, and the high cost of production limits their use in a few clinical sites. In contrast, modification of drugs using small molecule peptides with high tumor specificity can provide comparable targeting ability to antibody drugs while reducing the production cost. We synthesized amphiphilic and asymmetric targeted silicone phthalocyanines using targeted peptides. Its smaller molecular size has better penetration ability. The cost of targeted modifications has been reduced remarkably, making it more suitable for large-scale synthesis. Therefore, RSP has better prospects for clinical translation than ADC.As photodynamic therapy progresses toward clinical translation, more precise photosensitizer enrichment becomes mandatory. Targeted modification of PS to increase the peak concentration of photosensitizer at the tumor site is the current focus of research. The predominant approach for targeted modification of small molecule drugs is antibody-drug conjugate (ADC).38 can effectively improve the sensitivity of contrast agents for tumor imaging, which confirms the broad tumor-targeting effect of cRGDyK. In addition, RGD has been widely used to improve the targeting of small molecule photosensitizers or nano-photosensitizers. Previous studies have reported overexpression of integrins in some tumors, including triple-negative breast cancer, which have led to the development molecular probes for the treatment of the RGD base.42 Although these studies modified phthalocyanine with cRGDyK peptide, they still failed to achieve successful clinical translation. The most important reasons for this are poor water solubility and complex synthesis routes. Poor water solubility leads to aggregation in aqueous solutions of phthalocyanines with reduced single-linear oxygen yields. To increase water solubility, drug excipients that promote solubility or inhibit aggregation of photosensitizers are added, which adds a lot of unnecessary burden to metabolism. Considering these problems, we modified the phthalocyanines axially rather than on the rings. The formation of axial chemical bonds hinders the interaction between the phthalocyanine rings, which effectively avoids the aggregation of photosensitizers. Moreover, RGD was introduced not only as a targeting group but also as a hydrophilic group. The increase of water solubility further reduces the aggregation of phthalocyanine. cRGDyK molecule enhances both the targeting ability and hydrophilicity of RSP, which effectively solves the problem of targeting phthalocyanine aggregation.The cRGDyK peptide is the most widely studied class of tumor-specific peptides. There have been many preclinical experiments showing that the use of cRGDyK coupled with imaging agents 43 The fluorescence spectroscopy experiments showed that RSP buffer under physiological conditions had higher fluorescence intensity than in DMF. Meanwhile, the Q-band absorption peak of RSP in physiological saline containing 1% CEL was sharp. These results indicated that RSP is in the form of monomer in physiological buffers. The axial modification of phthalocyanine effectively avoided the formation of H-aggregates and J-aggregates. Further experiments also demonstrated that RSP exhibited good singlet oxygen yield and photostability in physiological buffers. The modification of RGD resulted in a high accumulation of photosensitizer in triple-negative breast cancer cells. In vitro photodynamic experiments showed that the IC50 of RSP in 4T1 cell line was 295.96 nM, which could cause significant apoptosis of tumor cells. And RSP showed high tumor suppression rate for triple-negative breast cancer tumors in orthotopic mice in vivo. In addition, RSP has a circulating half-life of approximately 45 min and is metabolized by the liver and kidney. In conclusion, RSP showed superior tumor targeting than non-targeted silicone phthalocyanine and could inhibit tumor growth more effectively.In our further study, we found that after axial modification of RGD, phthalocyanine not only increased targeting but also significantly reduced aggregation in aqueous solution. In previous studies, the fluorescence intensity of silicone phthalocyanine in aqueous solution was significantly lower than that in organic solvents.RSP showed great promise for improving the photodynamic therapeutic efficacy of triple-negative breast cancer versus the untargeted photosensitizer. Both in vivo and in vitro, the targeted photosensitizer RSP exhibited better comparative targeting and tumor suppression in triple-negative breast cancer, as compared to the non-targeted silicone phthalocyanine. Although monoclonal antibodies remain the mainstay of targeted therapies, their large size limits their penetration into tumors, and high production costs limit their usage in a few clinical centers. However small molecule agents like RSP, which can be synthesized inexpensively on a large scale, represents a promising next-generation PDT modality. Our work addresses current research needs and most definitely study will help expand the currently limited range of PDT cancer therapies available. Based on RSP, silicone phthalocyanine can not only be conjugate to cRGDyK, but also can be replaced with different peptides according to different tumor types to achieve photodynamic therapy for different kinds of tumors. Further it can be connected to different imaging contrast agents, such as MRI contrast agent, SPECT contrast agent, to achieve impact-guided photodynamic therapy, making it a multifunctional molecule for theranostics.In summary, we demonstrated that covalent linkage of RGD in RSP can effectively enhance the uptake of integrin-expressing tumor cells, hence improving the efficiency of PDT. In physiological buffers, this photosensitizer RSP has excellent fluorescence properties and singlet oxygen production, and exhibits high stability under light. Both in vivo and in vitro, the targeted photosensitizer RSP exhibited better comparative targeting and tumor suppression in triple-negative breast cancer, as compared to the nontargeted silicone phthalocyanine. Consequently, the targeted silicon phthalocyanine synthesized in this paper has a simple synthesis route, strong targeting, high photosensitivity, and has a strong prospect for transformation and application. There are ongoing efforts to evaluate the efficacy of our small molecule probes for clinical imaging and therapy. We expect that the successful development of these agents will expand the currently limited range of cancer therapies available.44 All reagents, including 3-(4-Hydroxyphenyl)propionic acid, sodium hydride, EDC·HCl, NHS, cyclo(Arg-Gly-Asp-d-Tyr-Lys) (cRGDyK), diethyl ether and dichloromethane are commercially available and were used without further purification. Nuclear magnetic resonance (NMR) spectra were recorded on a Brüker Avance 500 spectrometer at 25 °C. Chemical shifts were reported in parts per million (ppm) downfield from the Me4Si resonance which was used as the internal standard when recording 1H NMR spectra. Mass spectra (MS) were obtained on Solarix XR. UV–vis absorption spectra were obtained on Thermo Fisher UV–vis spectrophotometer. Fluorescence spectra of the different samples were obtained on Thermo Fisher fluorescence spectrophotometer. HPLC assays were performed on a C18 column . solvent A: 100% H2O containing 0.1% TFA; solvent B: 100% DMF containing 0.1% TFA. The column temperature was 30 °C and the flow rate was 1.0 ml/min. The elution gradient was: 10-95% B for 20 min, then 95% B for 20 min.Silicon phthalocyanine dichloride was synthesized according to the reported method.50H35N8O6Si [M + H] + , 871.2443; found 871.2426.Silicon phthalocyanine dichloride (100 mg), 3-(4-Hydroxyphenyl)propionic acid (108 mg), and sodium hydride (16.4 mg) were added to a 100 mL round bottom flask, dissolved in 15 mL anhydrous toluene, and heated to reflux for 24 h. Then the mixture was cooled to room temperature. The solvent was spin-dried, and the obtained solid was washed with water and filtered. The obtained residue was fully dissolved in DMF and filtered. The filtrate was spin-dried and dried to obtain 2[4-(3-carboxypropyl)phenoxy]silicon phthalocyanine (SC). HRMS (ESI): m/z calcd for C1H NMR  δ 9.67 , 9.15 ,8.86 , 8.54 , 8.29 , 7.93 , 7.76 ,7.20-6.99 , 6.64 , 6.42 , 5.97 , 5.41 ,5.14 , 4.50 , 4.08 – 3.93 , 3.21-2.90 , 2.76-2.58 , 2.34-2.09, 1.84 , 1.47 , 1.04.) HRMS (ESI): m/z calcd for C77H74N17O13Si [M + H] + , 1472.5443; found 1472.5406. HPLC (680 nm): >96%.SC (20 mg), EDC·HCl (4.4 mg) and NHS (2.6 mg) obtained in the previous step were dissolved in 2 mL of anhydrous DMF, stirred at room temperature for 4 h, and then cRGDyK (4.27 mg) and the solution were added to the mixture and stirred overnight at room temperature. The mixture was poured into excess diethyl ether and filtered. After the filter residue was collected and fully washed by water after being fully washed with dichloromethane. The resulting precipitate was vacuum dried to obtain cRGDyK modified targeted silicon phthalocyanine (RSP) .  as a singlet oxygen sensor. DPBF is bleached by singlet oxygen to its corresponding endoperoxide. The reaction was monitored spectrophotometrically by recording the decrease in optical density at 414 nm. The bleaching rate of DPBF can also be used as a method to compare the yield of singlet oxygen. In the actual measurement of singlet oxygen yield, a certain amount of DPBF ethanol solution and test solution are mixed together in a dark room. A rotor is added to the cuvette, and the laser is irradiated for different times while stirring. The absorption spectra of the mixed solution was tested after different time of irradiation with an ultraviolet spectrophotometer, then the change of the absorption value of DPBF at 414 nm was obtained.Luciferase labeled murine triple-negative breast cancer (4T1-Luc2 ATCC® CRL-2539-LUC2) cells were routinely cultured in DMEM medium (GIBCO) containing 10% fetal bovine serum (FBS). All experiments were performed at room temperature unless otherwise stated. CEL was obtained from Sigma. CCK8 cell activity assay kits were from Beyoncé. DCFHDA reactive oxygen species assay kits were purchased from KGI. Lysosomal probe, DAPI, and apoptosis assay kits were purchased from Solebro.4T1 cells were seeded and cultured overnight at a density of 10,000 cells/well. On the second day, the medium was changed to DMEM medium containing 1 μM RSP or SC (containing 0.05% CEL) or DMEM medium. Three cells were set up in duplicate wells at each observation time point. At different time points after incubation, the cells were taken out of the incubator, the medium containing the photosensitizer was discarded, and the cells were incubated with a fresh medium. The fluorescence inside the cells was observed under a fluorescence microscope and photographed at least three fields at each time point. Image J was used for quantitative analysis.Subcellular colocalization pictures were taken under a laser confocal microscope . 4T1 cells were cultured in 35 mm cell culture dishes at a density of 20,000 cells/dish, and cultured overnight. After 24 h, the medium was replaced with DMEM medium (containing 0.05% CEL) containing 1 μM RSP or SC. After 4 h of incubation, the plates were washed with PBS and then stained with DMEM medium containing Lysotracker for 30 min and washed again with PBS. Then fixed and counterstained with DAPI. The light was always avoided during the entire operation. The dynamic change of fluorescence on any straight line passing through the whole cell was analyzed by Image J. The result was plot to qualitatively analyze the co-localization situation of lysosome and phthalocyanine. In order to more completely reflect the co-localization of lysosomes and phthalocyanines, the Pearson co-localization coefficient was analyzed on the fluorescence fields of cells obtained from three photographs, and the difference in lysosomal localization of RSP and SC in the cells was quantitatively analyzed.DCFHDA, a small molecule probe that can freely cross the cell membrane, was used as the probe to detect intracellular singlet oxygen. The generation of singlet oxygen can be judged by whether or not it produces green fluorescence. 4T1 cells were seeded in a 96-well plate at a density of 10,000 cells/well. After 24 h, the medium was replaced with DMEM medium containing 1 μM RSP or SC (containing 0.05% CEL). The cells were incubated for 4 h. Then the cells were taken out of the incubator, add DMEM medium containing 10 μM DCFHDA, and continue incubating for 30 min. After the incubation, the original medium was discarded, replaced with a fresh DMEM medium, and irradiated with a xenon lamp. After irradiation for different periods of time, it was detected with Enzyme Labels to determine the production of reactive oxygen species in the cells. Then observe the green fluorescence generated in the cells under a fluorescence microscope.4T1 cells were seeded in a 96-well plate at a density of 10,000 cells/well, and each well was cultured overnight with 200 μL of DMEM medium containing 10% FBS. On the second day, the original medium was discarded and replaced with DMEM medium containing different concentrations of SC or RSP (containing 0.05% CEL). After 4 h of incubation, the cells were incubated with fresh medium and irradiated with a xenon lamp. Then 10 μL of CCK8 was added to each well, and the incubation continued for 1 h. After the incubation, the absorbance value at 450 nm was measured with enzyme labels . At the same time, a blank control was set to determine cell viability. The dark toxicity test method was similar, except that no light was applied to the cells.5 cells/well overnight. On the second day, the medium was changed to DMEM medium containing 500 nM RSP/SC and 1% CEL, and the cells were incubated for 4 h. Then the medium was replaced with fresh DMEM. The cells were applied light/ no light, and then digested.The apoptosis rate and necrosis rate of the cells were detected on the flow cytometer according to the operating procedure of the apoptosis detection kit. 4T1 cells were seeded in a six-well plate at a density of 5 * 107 4T1-luci tumor cells under the fourth pair of mammary glands in mice. Treatment was initiated approximately 6 days after inoculation when the tumor volume grew to approximately 100 mm3.Balb/c female mice aged 6 to 8 weeks were purchased from Beijing Weitong Lihua Laboratory Animal Technology. All animal experiments were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University. In situ breast cancer tumor models were established by injecting 2 * 10In order to investigate the blood clearance rate of RSP, healthy male Balb/c mice were selected as experimental subjects, and blood was collected from the orbit to detect the drug concentration in the body at different time points. Fluorescence labeled Organism Bioimaging Instrument  was used to measure the relative concentration of phthalocyanine in mouse plasma. Mice were injected with RSP through the tail vein at a dose of 2 mg/kg. At different time intervals, blood samples were collected from the orbits of mice with capillary tubes coated with heparin, placed on ice, and centrifugated to separate blood cells . The resulting plasma samples were analyzed for phthalocyanine concentration.For in vivo fluorescence imaging, RSP or SC dissolved in physiological saline (containing 1% CEL) was injected intravenously at a dose of 2 mg/kg into tumor-bearing mice (both in situ and subcutaneous models). Then IVIS Spectrum In Vivo Imaging System (PerkinElmer) was utilized to collect the fluorescence signals of RSP (660 nm/710 nm) in subcutaneous models at different times after the injection. The small animal fluorescence imaging system was used for in situ tumor-bearing mice imaging, taking pictures, and recording. The excitation wavelength was 660 nm, and the fluorescence signal was collected with a 700 nm filter. Then, the FOBI software was used to process the data to analyze the enrichment of phthalocyanines in the tumors of the mice after different injection times. The mice after 24 h fluorescence imaging were sacrificed, and the hearts, livers, spleens, lungs, kidneys, and tumors were collected for analysis of fluorescence semiquantitative biodistribution.3. The test groups were as follows: Group 1: PBS; Group 2: Light; Group 3: SC; Group 4: RSP; Group 5: SC + Light, Group 6: RSP + Light. First, 200 μl of RSP (2 mg/kg), SC (2 mg/kg), and PBS were injected intravenously. 4 h after the injection, the tumor area was irradiated with 671 nm laser (0.1 W/cm2) for 6 min with a total laser intensity of 36 J/cm2 and laser treatment was performed on groups 2, 5, and 6. After PDT treatment, the tumor size was measured every day using electronic vernier calipers until 12 days. The maximum width (X) and length (Y) of the tumor were measured, and the following formula was used to calculate the tumor volume: V = (X2Y)/2. From the first day of treatment, the weight change of each group of mice was recorded until the end of the treatment. Mice in each group were selected randomly, and excised tumors for pathological analysis. In order to investigate the biological safety of this PDT method, after the treatment, the main organs  of each group of differently treated mice were dissected, and H&E section staining was performed to evaluate their Pathological changes.PDT treatment was performed on the 7th day when the 4T1 tumor model was established in mice, and the average initial tumor volume was 100 mmSupplementary information


Tracheobronchial adenoid cystic carcinoma (TACC) is a rare tumour. About one-third of patients miss their chance of surgery or complete resection as it is mostly detected in the advanced stage; hence, photon radiotherapy (RT) is used. However, the outcomes of photon RT remain unsatisfactory. Carbon ion radiotherapy (CIRT) is thought to improve the therapeutic gain ratio; however, the outcomes of CIRT in TACC are unclear. Therefore, we aimed to assess the effects and toxicities of CIRT in patients with TACC.The inclusion criteria were as follows: 1) age 18–80 years; 2) Eastern Cooperative Oncology Group Performance Status 0–2; 3) histologically confirmed TACC; 4) stage III–IV disease; 5) visible primary tumour; and 6) no previous RT history. The planned prescription doses of CIRT were 66–72.6 GyE/22–23 fractions. The rates of overall survival (OS), local control (LC), and progression-free survival (PFS) were calculated using the Kaplan-Meier method. Treatment-induced toxicities and tumour response were scored according to the Common Terminology Criteria for Adverse Events and Response Evaluation Criteria in Solid Tumors, respectively.Eighteen patients with a median age of 48 (range 30–73) years were enrolled. The median follow-up time was 20.7 (range 5.8–44.1) months. The overall response rate was 88.2%. Five patients developed lung metastasis after 12.2–41.0 months and one of them experienced local recurrence at 31.9 months after CIRT. The rates of 2-year OS, LC, and PFS were 100, 100, and 61.4%, respectively. Except for one patient who experienced grade 4 tracheal stenosis, which was relieved after stent implantation, no other ≥3 grade toxicities were observed.CIRT might be safe and effective in the management of TACC based on a short observation period. Further studies with more cases and longer observation are warranted. Salivary gland tumours are rare diseases that could affect head, neck, breast, pelvis, gynaecological tract, and trachea –6. TwentX-ray radiation therapy (RT), as an adjuvant or definitive treatment method, has been used for TACC. In the early years, the clinical outcomes of definitive RT for TACC were not satisfactory as it is a slowly-growing malignancy and resistant to RT , 15–17. Charged particle beams, including proton and carbon ion beams, have physical advantages, such as specific dose distribution of the Bragg peak and narrow penumbra. Therefore, they could provide better sparing of normal tissue –28. In aOur centre, Shanghai Proton and Heavy Ion Center (SPHIC) was officially opened in 2015. Since then, we have treated 23 patients having TACC with CIRT. Here, we retrospectively summarize the preliminary results.Patients who met the following criteria were enrolled onto in this study: 1) 18–80 years old; 2) Eastern Cooperative Oncology Group Performance Status 0–2; 3) histologically confirmed TACC; 4) stage III–IV disease according to the modified Bhattacharyya’s protocol ; 5) visiPretreatment evaluation in all patients included complete disease history and physical examination, complete blood count, serum electrolytes, renal and liver function tests, electrocardiogram, pulmonary function tests, and a mandatory fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) scan for clinical staging, same as pretreatment evaluation for lung cancer patients we reported before . BronchoThe procedures of CIRT, including preparing, planning and delivery, was performed similar to previously described . The patThe scanning scope of the simulation computer tomography (CT) starts from the angle of the mandible to the adrenal glands to include tumour lesions, entire lungs, whole neck, and all the organs/tissues through which the beams were likely to pass. All patients were evaluated for tumour motion using 4-dimensional (4-D) simulation CT. If the tumour motion in any direction was less than 5 mm, the patient was treated under FB; if the motion exceeded 5 mm, a breath control technique, either active breathing control  or respiratory gating , was required to mitigate the residual motion (RM) to < 5 mm during treatment. For patients using gating, 10 phases of the whole respiratory cycle were reconstructed on 4-D CT. The gating window  was selected to restrict the RM to < 5 mm, same as breath control methods for lung cancer patients we reported before .The target delineation was also similar as our protocol for lung cancer patients . Gross tThe target coverage requirements were as follows: (i) at least 99% of the GTV was covered by 99% of the prescription dose, (ii) 99% of the CTV by 95% of the prescription dose, and (iii) 90% of the PTV by 90% of the prescription dose. The dose constraints for the main organs at risk (OARs) included: (1) lung: mean dose (Dmean) of bilateral lungs < 15 GyE, the percentage volume of the lung receiving 20 GyE or more in total lung (V20) < 20%, V5 < 50%; maximum dose (Dmax) of the main bronchial tree < 105% of the prescription dose; (2) heart: V30 < 30%, V40 < 25%; (3) oesophagus: Dmean < 34 GyE, Dmax < 105% of the prescription dose; (4) spinal cord: Dmax < 45 GyE; (5) thyroid: Dmean < 45 GyE; and (6) stomach: Dmax < 45 GyE.The Siemens Syngo Planning System® was used for planning in all patients. Beam energy 85–430 MeV (CIRT) plans were designed using 2–4 beams with the PBS technique. Two orthogonal X-ray images were taken to verify the patient’s position according to bone structures before each daily irradiation. A set-up error ≤ 3 mm before the treatment was allowed. Carbon ion beams were delivered under the same breath control mode used for simulation CT. All the patients underwent review CT before the first treatment and every week during treatment, and plan recalculation on the latest CT was conducted for every patient to check the dose distribution. Replanning was demanded when recalculation revealed poor coverage of targets or overdosing to the organs at risk. These procedures were same as treatment protocols for lung cancer patients we treated before .This part was similar to previously described . All patThe objective response rate (ORR) was defined as the proportion of patients who reached complete response (CR) or partial response (PR) and kept for more than 3 months in all patients. The rates of overall survival (OS), local control (LC), and progression free survival (PFS) were calculated using the Kaplan-Meier method. The times to events were calculated from the start of CIRT until the first documented treatment failure. OS was defined till the date of death or the last follow-up. LC was defined till the date of local failure or the last follow-up. PFS was defined till the date of disease progression at any site or death, or the last follow up. All analyses were performed using SPSS® statistics version 26 .From March 2016 to December 2019, 23 consecutive patients with TACC received CIRT in the SPHIC. Among them, three patients previously received RT; two treated by radical surgery with positive surgical margins but had no visible residual tumour (R1 resection). Hence, 18 patients who met the inclusion criteria were enrolled in this analysis. Five patients were treatment-naive, two underwent R2 resection, one underwent exploratory surgery, three had recurrent tumour 1.2–30 years after surgery, and seven underwent endoscopic debulking surgery  before CIRT. One patient had one lung metastatic lesion and one had multiple lung metastases before CIRT. The lesions in six patients involved the carina or bilateral main bronchus. Thirteen patients had lesions that were longer than 5 cm. Patient characteristics are detailed in Table Among 18 patients, the methods used for breathing control were ABC in 1 patient, respiratory gating in 9 patients, and FB in 8 patients.Sixteen patients received a total dose of 66–72.6 GyE/22–23 fractions Table . The twoAll patients tolerated CIRT well. No patient experienced grade 3 or higher acute toxicities. Grade 2 acute toxicities included oesophagitis (two cases), pneumonitis (one), tracheal stenosis (one), hoarseness (one), and haematological toxicities (one). Regarding late toxicities, airway stenosis was observed in three patients. Among them, one patient who underwent argon helium laser ablation plus cryosurgical ablation before CIRT experienced grade 4 tracheal stenosis 4.5 months from the first fraction of CIRT, and the stenosis was relieved after stent insertion. The second patient received similar endoscopic debulking treatment before RT and developed grade 2 tracheal stenosis 1.2 months from the first fraction of CIRT, and symptoms improved after symptomatic treatment. The third patient, whose lesion was located close to the glottis, experienced grade 2 laryngostenosis 8 months after the first fraction of CIRT, and the symptoms were relieved after symptomatic therapy. The median maximal doses to the trachea/larynx of patients with or without airway stenosis were 72.91 GyE (range 68.69 ~ 73.19) versus 72.66 GyE (range: 69.73 ~ 87.41). Other grade 2 late toxicities included radiation-induced lung injury (RILI) (three cases), hypothyroidism (one), and tracheitis (one)  months. Tumour response at 3 months after CIRT was evaluated in 17 patients, except for 1 patient, whose tumour was not shown clearly on CT due to the atelectasis. ORR was 88.2% with CR in 7, PR in 8, and stable disease (SD) in 2 patients. Five distant metastases occurred in five patients during follow-up. Among them, one patient with stage IV (T4N0M0) disease developed multiple lung metastases after 14.8 months and experienced local recurrence 31.9 months from the first fraction of CIRT with a dose of 69 GyE/23 fractions. One or more newly emerged metastatic lung lesions were observed in the other four patients 12.2–41.0 months from the first fraction of CIRT. For salvage treatments, one patient received radioactive seed implantation for local recurrence, and one SBRT for lung metastases. The median PFS time in all patients was 41.0  months. The rates of 2-year OS, LC, and PFS were 100, 100, and 61.4%, respectively, for the whole cohort. A typical case is shown in Fig. TACC is a very rare tumour, and the clinical experience is limited. Although surgery plays the most critical role, radical resection is often difficult. RT has not been recognized for its role in the management of TACC. Published data on radical or definitive RT for TACC patients have been limited to small sample sizes. Compared to surgery with or without adjuvant RT, definitive RT usually resulted in inferior clinical outcomes in several retrospective analyses  in TACC, a few studies have been published. Bittner et al.  reportedp = 0.528). Whether the stenosis was caused by the intra-tracheal laser ablation, the nature of an intraluminal malignancy, or individual radiation sensitivity of trachea, etc. remains unclear and need to be further investigated. No other grade ≥ 3 adverse effects were observed. The 2-year LC and OS rates for the whole cohort reached 100% with a median follow-up time of 20.7 months. Only one patient developed local recurrence at 31.9 months from the first fraction of CIRT and, including this patient, five had distant metastatic (DM) lesions to the lungs. Our results using CIRT are promising compared to those using photon or neutron beams.TACC is thought to be a slowly growing malignancy and with radio-resistance. CIRT has been demonstrated to have biological advantages on radio-resistant malignancies in in-vitro and in-vivo experiments, as well as in clinical practice . Its capThere are currently no up to date guidelines on the CIRT technique that we could follow. TACC tends to have mucosal or sub-mucosal spread along the tracheal wall; the subclinical lesion is at least 1 cm beyond the visible or palpable tumours . To coveIn this study, the most prominent late toxicity was airway stenosis, which can be life threatening. Airway stenosis was observed in other published reports, using either surgery or radiation therapy, and the incidence varied from 5 to 23% , 22, 25.Lung metastasis was an important outcome in the patients in this study. Five developed new metastases to the lungs after CIRT; the 2-year DM rate was 38.6%. This is consistent with previous reports that the 5-year lung metastasis rate could be as high as 77.8% (20–77.8%) , 13, 18.There were some limitations to this study. First, the number of patients was small. Second, the compensated dose for interruptions of CIRT was decided by the physician’s discretion as there was no established compensation method and dose constraints to OARs for CIRT to follow. Third, the follow-up time was not long enough, especially for this slowly growing tumour, and thus, local control and survival rates may have not reached their final values. Prospective studies with large sample sizes are warranted to better define the role of CIRT in patients with TACC. Two prospective phase II clinical trials involving patients with different surgical statuses are ongoing in our centre.To the best of our knowledge, this is the first report on TACC treated only by CIRT for all the patients. This study showed that CIRT was feasible; toxicities were mild, and overall survival and local control at 2-years were satisfactory. However, more patients and long-term follow-up are needed to confirm the findings shown in this study.


Vibrio parahaemolyticus is an ongoing source of shellfish-borne food illness in the Northeast United States, and there is keen interest in understanding the environmental conditions that coincide with V. parahaemolyticus disease risk, in order to aid harvest management and prevent further illness. Zooplankton and chitinous phytoplankton are associated with V. parahaemolyticus dynamics elsewhere; however, this relationship is undetermined for the Great Bay estuary (GBE), an important emerging shellfish growing region in the Northeast United States. A comprehensive evaluation of the microbial ecology of V. parahaemolyticus associated with plankton was conducted in the GBE using 3 years of data regarding plankton community, nutrient concentration, water quality, and V. parahaemolyticus concentration in plankton. The concentrations of V. parahaemolyticus associated with plankton were highly seasonal, and the highest concentrations of V. parahaemolyticus cultured from zooplankton occurred approximately 1 month before the highest concentrations of V. parahaemolyticus from phytoplankton. The two V. parahaemolyticus peaks corresponded with different water quality variables and a few highly seasonal plankton taxa. Importantly, V. parahaemolyticus concentrations and plankton community dynamics were poorly associated with nutrient concentrations and chlorophyll a, commonly applied proxy variables for assessing ecological health risks and human health risks from harmful plankton and V. parahaemolyticus elsewhere. Together, these statistical associations (or lack thereof) provide valuable insights to characterize the plankton-V. parahaemolyticus dynamic and inform approaches for understanding the potential contribution of plankton to human health risks from V. parahaemolyticus for the Northeast United States.Microbial ecology studies have proven to be important resources for improving infectious disease response and outbreak prevention. IMPORTANCE The Vibrio-plankton interaction is a focal relationship in Vibrio disease research; however, little is known about this dynamic in the Northeast United States, where V. parahaemolyticus is an established public health issue. We integrated phototactic plankton separation with seasonality analysis to determine the dynamics of the plankton community, water quality, and V. parahaemolyticus concentrations. Distinct bimodal peaks in the seasonal timing of V. parahaemolyticus abundance from phyto- versus zooplankton and differing associations with water quality variables and plankton taxa indicate that monitoring and forecasting approaches should consider the source of exposure when designing predictive methods for V. parahaemolyticus. Helicotheca tamensis has not been previously reported in the GBE. Its detection during this study provides evidence of the changes occurring in the ecology of regional estuaries and potential mechanisms for changes in V. parahaemolyticus populations. The Vibrio monitoring approaches can be translated to aid other areas facing similar public health challenges. Vibrio parahaemolyticus has been the focus of numerous intensive studies due to its role as a human and animal pathogen  that vary in strength  in the Northeast United States  to identify water quality variables that covaried with plankton and associated V. parahaemolyticus in order to improve V. parahaemolyticus modeling and risk forecasting in this region. This study is the first comprehensive, multiyear assessment of plankton and concentrations of V. parahaemolyticus from plankton in the GBE and the Northeast United States.Little is currently known about plankton community dynamics in the GBE, and this type of study has not been conducted previously. Therefore, the goals of this work were (i) to identify the taxa in the plankton community, (ii) to determine whether individual taxa or overall plankton concentrations covaried with V. parahaemolyticus concentrations, plankton community analysis, water quality, and nutrients were conducted. V. parahaemolyticus concentrations and plankton total abundance varied widely overall  concentrations were consistent with data in previous reports  and May (2015). Sampling ended for the same reason in November (2015) or December (2014 and 2016). Thirty-one total sampling events with complete data for  overall ; however reports , 22.V. parahaemolyticus (detection limit is a most probable number [MPN] of >0.03/g) was detected in only 54.8% of phytoplankton samples (17/31) and 45.1% of zooplankton samples (14/31). Phytoplankton concentrations (cells/liter)  of phyto- and zooplankton taxa sampled during this 3-year period had been previously identified in the GBE  . Diatoms the GBE  23, 24), 24ChaetChaetoceros spp. and Helicotheca tamensis were present at high concentrations and dominated the plankton community during summer months (Shannon diversity index [H] < 2). Samples from the spring and fall months were generally more diverse (Shannon H > 2), with the exception of early spring samples, which were predominantly Skeletonema spp. For example, in 2016, Skeletonema sp. concentrations exceeded 7,700 cells/liter in three samples in the spring months, and these samples were comparatively less diverse than other samples from spring months. Nauplii and Navicula spp. were generally present at low levels throughout the study period, except in July 2015, when both were detected at >1,000 cells/liter.Each plankton sample contained an average of 14 unique phyto- and zooplankton taxa  . ChaetocSkeletonema spp., Chaetoceros spp. and Helicotheca tamensis, and Rhizosolenia spp. were differentiated by indicator species analysis (ISA) into distinct spring, summer, and fall assemblages, respectively  analysis but did not alter the season-specific outcomes.The plankton community was strongly seasonal, and ectively . These tHelicotheca tamensis (0.63 variance explained), followed by Rhizosolenia spp. (0.55 variance explained), Chaetoceros spp. (0.39 variance explained), and Skeletonema spp. (0.35 variance explained), was the most strongly seasonal and best fit with harmonic regression (model 2). The estimated peak timing of the summer-specific Helicotheca tamensis and Chaetoceros spp. occurred in August around day 223 ± 11 and day 217 ± 18, respectively. The seasonality of V. parahaemolyticus concentrations from phytoplankton were also well fit by the harmonic regression in model 2, and peak timing was estimated to occur in August at day 224 ± 16.The season-specific patterns of the plankton community were also identified by seasonality models 1 and 2  in indivV. parahaemolyticus were well fit by model 1 and 2, and peak timing was similar . Taxa that were infrequently detected and at low abundance were not well fit by the seasonal variables in model 1 or 2 (see Table S2).Nauplii and zooplankton-associated 4 were best fit by harmonic regression in model 2 (>0.65 deviance explained) and peak timing occurred after day 213. NO3 + NO2 was well fit by models 1 and 2, with peak timing around day 200 ± 14. Chlorophyll a, pheophytin, turbidity, and the other measured nutrients had little or no seasonality based on poor fits to both models .The variables water temperature, salinity, dissolved oxygen (DO), and POV. parahaemolyticus concentrations in phytoplankton, Helicotheca tamensis, Chaetoceros spp., PO4, salinity, and water temperature is present in the upper left corner of V. parahaemolyticus concentrations in zooplankton, photoperiod, nauplii, and copepods are also present in the upper middle section of 3 + NO2, Rhizosolenia spp., and Stephanopyxis spp. produced a separate significant cluster but were negatively correlated with variables that clustered with V. parahaemolyticus concentrations in phyto- or zooplankton. No significant correlations were identified between chlorophyll a, pheophytin, total suspended solids (TSS), particulate nitrogen (PN), and particulate carbon (PC) and the majority of individual plankton taxa or V. parahaemolyticus concentrations.A cluster of significant positive correlations between V. parahaemolyticus concentrations from plankton were bimodal  were highest prior to the warmest water temperatures, and the highest concentrations of V. parahaemolyticus from phytoplankton (peak timing at day 224 ± 16) occurred approximately one week after the peak timing of water temperature. bimodal . V. para3 + NO2, DO, and photoperiod were most strongly related to nauplii and V. parahaemolyticus concentrations from zooplankton in both correlative relationship and seasonal timing. Salinity and PO4 were most strongly correlated with V. parahaemolyticus concentrations from phytoplankton and individual phytoplankton taxa. These environmental variables covaried positively with water temperature, unlike DO and NO3 + NO2, though their maxima occurred approximately a month after the maxima of water temperature and were similar in timing to the individual phytoplankton taxa and V. parahaemolyticus from phytoplankton.NOV. parahaemolyticus concentrations cultured from plankton in the GBE are highly seasonal, are bimodal, and are most closely associated with individual plankton taxa rather than total plankton abundance. V. parahaemolyticus concentrations from phyto- and zooplankton are differentially associated with water temperature, salinity, pH, DO, NO3 + NO2, and PO4, which suggests niche-specific preferences. Importantly, common water quality variables and indicators of plankton abundance or V. parahaemolyticus associated with plankton, such as TDN and chlorophyll a, were not statistically or temporally associated with the dynamics of either group, which provides important insight into why these variables did not contribute well to previous V. parahaemolyticus modeling efforts  did not correspond with peak plankton concentrations , regardless of the taxa. Similarly, V. parahaemolyticus from zooplankton was intermittent and at low abundance, and often V. parahaemolyticus was not cultured from zooplankton samples, even though zooplankton taxa were present in the water column.6 cells/liter (Chaetoceros spp. (n = 5), Navicula spp. (n = 1), and Skeletonema spp. (n = 1). Skeletonema spp. were detected in the cooler spring months in the GBE, and elevated concentrations of Skeletonema spp. were detected when sampling could begin safely in early March or April. The single midsummer “bloom” of Navicula spp. in 2015 was unexpected, as Navicula spp. were generally detected year-round at low levels. Chaetoceros spp. dominated most warm-water samples, and their abundance was statistically associated with Helicotheca tamensis.The majority of plankton taxa observed throughout this study were diatoms, which is consistent with previous reports on the GBE ecosystem , 24. Onlls/liter , 26. ThrH. tamensis was absent in prior analyses of the plankton community of the GBE  and were most correlated with and temporally similar to patterns in water temperature, salinity, PO4, and H. tamensis. V. parahaemolyticus concentrations from zooplankton, however, were highest earlier in the year around mid-June to early July (day 188 ± 34), preceding the warmest water conditions and at approximately the time when nauplii were most concentrated in the water column. Nauplii and V. parahaemolyticus from zooplankton were also positively correlated and temporally associated with photoperiod and inversely with DO, pH, and NO3 + NO2, i.e., a different set of water quality parameters than those associated with phytoplankton. Work from other regions has also observed that V. parahaemolyticus from different matrices can have distinctly associated water quality conditions . Chlorophyll a was not correlated with either plankton dynamics or taxon blooms, nor was it associated temporally or statistically with V. parahaemolyticus in zoo- or phytoplankton. There is work that has shown that V. parahaemolyticus can also support a free-living lifestyle in the water column by subsisting off nutrient-rich floccules or polysaccharide exudate  bed and is a long-term monitoring location , which has an important oyster  ammonium (NH4), orthophosphate (PO4), dissolved organic nitrogen (DON), total suspended solids (TSS), particulate carbon (PC), particulate nitrogen (PN), chlorophyll a (CHL) and pheophytin (PHEO) measurements. Data for these parameters were also obtained from the SWMP database (https://cdmo.baruch.sc.edu/dges/). Separate water temperature, salinity, pH, and dissolved oxygen (DO) measurements using YSI 6600 and EXO multiprobe sondes , were collected concurrently with sampling at low tide from the NI study site.Continuous  water temperature, salinity, dissolved oxygen (DO), pH, and turbidity data were obtained from the Stormwater Management Program (SWMP) from 2014 to 2016 for times simultaneous with and preceding sampling events at a SWMP datasonde site (station GRBGB) in close proximity  to the NI study site. Grab water samples were collected on each sample date and processed for water quality analyses, including nonpurgeable organic carbon (NPOC), total dissolved nitrogen (TDN), nitrate and nitrite  was used during 10 weighted tows to collect ∼140 liters of water. In year 2, a Niskin sampler with a student net was used to collect 160 liters. In year 3, a 30-liter Schindler-Patalas trap  with a student net was used to collect 180 liters. All sample counts were transformed to cells per liter to standardize comparisons between years. One sample was phototactically separated using published methods and the plankton separation equipment described previously . The sepThe second plankton sample was analyzed with a phase-contrast microscope at ×400 magnification  to count 10 nonconsecutive columns (100 quadrants of the Sedgwick rafter grid). Plankton enumeration and concentration determinations were consistent with standard methods for plankton analysis  using a V. parahaemolyticus concentrations according to previously published methods with a 3-tube MPN alkaline peptone water enrichment and Vibrio CHROMagar , relative abundance (RA), species richness, evenness, and Shannon H diversity were calculated for the study. The taxa observed in collected samples were classified as abundant , common (≤4% and ≥0.1%), or rare (<0.1%) in the GBE plankton community. Rare taxa (abundance < 0.1%) were not included in univariate, multivariate, or seasonality analysis.0 is the intercept, t is the daily time series, and β1 indicates a general trend in the variable of interest. Model 1 contains the photoperiod variable βpphotoperiod, and model 2 uses harmonic regression terms for the calendar day in the study where βs and βc are the coefficients of the harmonic terms and ω is the term representing the annual cycle . The peak timing of the periodic oscillations identified by model 2 was determined by calculating the phase shift:Seasonal community assemblage was assessed between calendar seasons  and years  by permutational multivariate analysis of variance (PERMANOVA), multiresponse permutation procedure (MRPP), and indicator species analysis (ISA). The seasonality and general trend for all variables were also explored with models 1 and 2:k was 0. When k was equal to 2π, and when k was equal to π. The phase shift (ψ) was then multiplied by 365.25 to account for leap years. Confidence intervals (CI) of the peak timing were calculated by determining the estimated variance of the phase shift (ψ), where When r2) value of models 1 and 2 (CI values for the peak timing estimates were determined as  1 and 2 , 52.V. parahaemolyticus dynamics in plankton.Spearman rank correlation analyses were performed for all variables to assess the relationship of environmental variables to


Acute kidney injury (AKI) is one of the most common and serious complications in patients with type B aortic dissection (TBAD). This study aimed at investigating the incidence and risk factors of in-hospital AKI in TBAD patients involving the renal artery who underwent thoracic endovascular aortic repair (TEVAR) only.A total of 256 patients who were diagnosed as TBAD combined with renal artery involvement were included in this retrospective study. All patients were divided into the AKI group and the non-AKI group according to the KDIGO criteria. The risk factors for AKI were identified using a multivariate logistic regression model.p = .038) were prone to AKI, and lower estimated glomerular filtration rate (eGFR) , higher diastolic blood pressure (DBP) , and fasting blood glucose (FBG) ≥7 mmol/L on admission  were independent risk factors for AKI.A total of 256 patients were included in this study, and the incidence of AKI was 18% (46/256). Patients in the AKI group were more likely to have a higher proportion of the youth, a higher level of body mass index, and a shorter time from onset to admission. Multivariate logistic regression analysis revealed that the youth (age ≤40 years) (OR: 2.853, 95%CI: 1.061–7.668, Higher incidence of AKI had been perceived in this study, most of them were young and middle-aged patients. Renopreventive measures should be considered in those high-risk patients with younger age, lower eGFR, higher DBP, and higher FBG on admission. All patients underwent TEVAR only and none of them had previously received renal replacement therapy. The patients’ medical records were collected and analyzed, including medical history, physical examination, laboratory measurements, and imaging examinations. Institutional review board approval was granted for the use of an aggregate data set for research and quality improvement by the Ethics Committee of Beijing Anzhen Hospital, Capital Medical University, and the principles outlined in the Declaration of Helsinki were followed. All participating patients signed informed consent.2.2.Patients presenting with renal insufficiency underwent preoperative hydration to make sure the urine output was over 100 mL/h. We gave the patients isotonic saline (1 mL/kg/h) from 12 h pre-procedure to 24 h post-procedure.2.3.Percutaneous TEVAR was conducted following standard procedures which began with the puncture of the common femoral arteries (CFAs) under fluoroscopy. 5 F or 6 F Perclose ProGlide devices were deployed in the CFA, and a platinum pigtail catheter was sent along the femoral artery to perform angiography of the abdominal aorta, descending thoracic aorta, and ascending aorta. After confirming the formation of Stanford TBAD and the location of the dissection lesion, the coated stent was transported along the hardened guidewire and released after precise positioning of the lesion. Aortography was performed again to confirm that the lesion had been successfully isolated.All these patients use 100–120 mL contrast, and fluid resuscitation and vasoactive agents were give according to hemodynamics. Vasopressors mainly included epinephrine and norepinephrine, and antihypertensive drugs included nicardipine, isosorbide mononitrate, nitroglycerin, and esmolol.2.4.AKI was defined as an increase in serum creatinine (SCr) by ≥0.3 mg/dL (26.5 μmol/L) within 2 days or an increase in SCr to ≥1.5 times baseline within 7 days based on the Kidney Disease: Improving Global Outcomes (KDIGO) criteria . BaselinAll patients included in this study had undergone computed tomography angiography (CTA), and they were diagnosed as TBAD with renal artery involvement  according to Stanford classification . Based o2.5.t-test or Mann–Whitney U-test when the distribution and variance met the appropriate conditions. Categorical variables were expressed as frequency (percentage) and analyzed using the chi-squared test or Fisher’s exact test when needed. Variables with less than 20% data missing and found to be statistically significant (p < .05) between two groups were included in univariate analyses (unadjusted), then the variables that had statistical differences (p < .05) were included in the multivariate logistic regression analysis. Odds ratios (OR), with 95% confidence intervals (CI) and p-value, were reported. Statistical significance was defined as a p < .05.Statistical analysis was carried out using the SPSS software version 21.0. The continuous variables were presented as the mean ± standard deviation (SD) or median (interquartile range), and inter-group comparisons were performed using independent 3.3.1.2 vs. 90.9 ± 19.9 mL/min/1.73 m2, p = .022), while diastolic blood pressure (DBP) and fasting blood glucose (FBG) on admission in AKI group were higher . However, no statistically significant differences were observed in sex, history of hypertension, systolic blood pressure (SBP) on admission, and classification of renal arteries between the two groups  of them developed AKI. In those 46 patients who develop AKI, 12 (26.1%) of them had AKI before TEVAR, and 34 (73.9%) of them developed AKI after the procedure, including 39 (15.2%) in stage I, 4 (1.6%) in stage II, and 3 (1.2%) in stage III. The mean age of the patients was 52.8 ± 9.8 years, 217 (84.9%) were male, 228 (89%) were young and middle-aged people (age ≤65 years), 201 (78.8%) had a history of hypertension, and the median time elapsed from symptom onset to hospital admission was 7 days (1–19). Compared to the non-AKI group, patients in the AKI group were more likely to have a higher proportion of the youth, have a higher level of body mass index (BMI), shorter time from onset to admission. Moreover, the eGFR on admission of patients in the AKI group was significantly lower than that of the non-AKI group  was carried out for all variables which had statistical differences except for BMI (absence of effective case number >20%), including the youth, eGFR, DBP, and FBG. Multivariate logistic regression analysis revealed that the youth (age ≤40 years) were prone to AKI, other risk factors for AKI in TBAD patients involving the renal artery including lower eGFR, higher DBP and FBG ≥ 7 mmol/L on admission .4.AKI is a common and serious complication of TBAD, and it is associated with prolonged hospital stay duration and reduced survival . AKI mayWe studied the risk factors of AKI in TBAD patients with renal artery involvement. Multivariate analysis revealed that the youth, lower eGFR, higher DBP, and higher FBG on admission were independent risk factors for AKI. Several clinical risk factors for AKI were defined in previous studies of TBAD, including SCr on admission, SBP on admission, CKD, coronary heart disease, congestive heart failure, and history of hypertension ,17,24,25In recent years, several studies explored the association between the type of renal arteries involvement based on CTA  in TBAD patients receiving TEVAR or drug treatment ,16,17 T. TakahasThere were a few limitations to our study. First, this was a retrospective single-center study, therefore, selection bias and confounding bias were unavoidable. Besides, the diagnosis of AKI was based on SCr value only, for the urine output data was missing in those hospitalized patients with urgent and serious conditions, which may underestimate the rate of AKI. Meanwhile, the present study could provide reliable evidence for randomized controlled trials controlled trial which a have more scientific clinical design.5.In summary, the high incidence of in-hospital AKI had been perceived in TBAD patients with renal artery involvement, most of them were young and middle-aged people, and the youth were prone to AKI. Our study provided evidence that eGFR, DBP, and FBG on admission were significant independent risk factors of AKI. In TBAD patients, especially young patients or those who have lower eGFR, higher DBP, and higher FBG on admission need more attention. Therefore, the preliminary assessment of AKI and active management for the high-risk groups are essential to improve the prognosis of TBAD by preventing AKI. However, once the AKI has occurred, prompt treatment is critical for improving survival.


Postoperative gastrointestinal function influences postoperative recovery and length of hospital stay for patients undergoing colorectal surgery. Goal-directed fluid therapy (GDFT) restricts fluid administration to an amount required to prevent dehydration. Although the fluid management of GDFT could decrease the incidence of postoperative complications in patients who undergo high-risk surgery, certain patients may not respond to GDFT. Thus, to achieve optimal treatment, identification of patients suitable for GDFT is necessary. Metabolomic profiling of 48 patients undergoing surgery for colorectal cancer was performed. Patients were divided into delayed- and enhanced-recovered groups based on gastrointestinal function within 72 hours, and the results of omics analysis showed differential serum metabolites between the two groups of patients in the post anesthesia care unit 24 hours after surgery. A support vector machine model was applied to evaluate the curative effects of GDFT in different patients. Four metabolites, oleamide, ubiquinone-1, acetylcholine, and oleic acid, were found to be highly associated with postoperative gastrointestinal function and could be used as potential biomarkers. Moreover, four pathways were found to be highly related to postoperative gastrointestinal recovery. Among them, the vitamin B6 metabolism pathway may be a common pathway for improving postoperative recovery in various diseases. Our findings proposed a novel method to predict postoperative recovery of gastrointestinal function based on metabolomic profiling and suggested the potential mechanisms contributing to gastrointestinal function after surgical resection of colorectal cancer under the fluid management of GDFT. Postoperative gastrointestinal (GI) recovery significantly influences perioperative complications and prognosis in patients undergoing colorectal surgery, particularly for elderly patients who are at risk of increased hospital stay and organ dysfunction owing to delayed GI recovery . The amoBy adjusting arterial pressure and blood flow parameters, including cardiac output, cardiac index, stroke volume index, stroke volume (SV) variation (SVV), and oxygen delivery, GDFT can modify perioperative fluid administration to achieve suitable end-organ perfusion . SVV is The ERAS program has facilitated progress in promoting the recovery of intestinal function and has been proposed as a standard strategy for enhancement of postoperative recovery in northern Europe and other countries , 12. HowMetabolomics is a novel approach with many applications in biological research. With the development of advanced analytical techniques and bioinformatics, metabolomics has been extensively applied as a holistic diagnostic tool in clinical and biomedical studies. In contrast to proteomics, genomics, and other methods that reflect indirect changes in the system, metabolomics reveals the direct response of the physiological environment . In the Recently, researchers have demonstrated that some patients exhibit poor postoperative recovery condition under the intra-operative fluid management of GDFT . TherefoThe demographic data for all patients were shown in 2 of 0.255 and R2X of 0.553. For ESI- ion mode  and at 24 hours after surgery for all 48 patients. Delayed PACU samples were separated as group A, enhanced PACU samples were separated as group B, delayed 24 hours samples were separated as group C, and enhanced 24 hours samples were separated as group D. Representative ESI+ and ESI- ion chromatograms for each group are presented in ion mode , there wof 0.584 . From th2 of 0.343 and R2Y of 0.97 in ESI+ ion mode  scores greater than 1.0 in multivariate statistical analysis and p values of less than 0.05 in univariate analysis were considered as the most significant metabolites. Twenty metabolites for ESI+ ion mode and eight metabolites for ESI- ion mode were selected as the most significant differentiating metabolites  results of enhanced and delayed postoperative recovery samples in the PACU revealed differences in their distributions, with a Qion mode  and a Q2ion mode . Althougabolites .2 of 0.691 and R2Y of 0.985 for ESI+ ion mode . Importantly, not all abdominal surgeries involve the gastrointestinal tract, and different types of surgeries may result in different procedures for recovery of intestinal function. Colorectal surgery is known to alter the integrity of the intestinal tract, and the recovery of intestinal function may mainly depend on mechanisms mediated by the intestinal tract itself. The PLS-DA model used in this study to identify significant differential metabolites showed low prediction accuracy; thus, some metabolites with the same variation tendencies in groups A–B and C–D were not recognized as significantly different. However, the model showed good performance without excessive lifting, indicating that although we could ignore some significant differential metabolites, the selected metabolites were reliable.In summary, we proposed a novel method to predict postoperative recovery of GI function using metabolomics. From only 10 metabolites isolated from blood samples collected 24 hours after surgery blood samples or 22 metabolites from blood samples collected in the PACU, we may predict the patient’s possible long-term postoperative recovery condition of GI function. Four potential biomarkers were identified. Further studies are needed to evaluate whether these biomarkers may have potential therapeutic applications in promoting postoperative GI function.2 or > 25.0 kg/m2; presence of cardiovascular diseases, such as coronary artery or valvular diseases, New York Heart Association Functional Classification class II or higher heart failure, and arrhythmia; presence of metabolic diseases, such as severe diabetes, ketoacidosis, hyperosmolar syndrome, or long-term hypoglycemia; presence of systemic immune disease; presence of liver and kidney dysfunction; presence of chronic obstructive pulmonary disease; and refusal to participate in the study or presence of mental illness and inability to communicate correctly. In addition, because of the pneumoperitoneum in laparoscopic surgery may affect the change of chest pressure, and the accuracy of SVV monitoring may be influenced, thereby the laparoscopic colorectal surgeries were also excluded.This was a single-center, prospective, observational cohort study. Two parallel groups (enhanced and delayed recovery) were enrolled according to the postoperative time of flatus. Patients who had undergone elective surgery for colorectal cancer resection were enrolled from December 2018 to October 2019 at Shanghai Changhai Hospital, Second Military Medical University. Written informed consent was obtained from all participants on the day before surgery. Patients who met any of the following criteria were excluded: ASA IV or V; age < 18 years or > 70 years; fasting time < 8 hours or > 20 hours; pre-operative ileus; body mass index (BMI) < 18 kg/m2 between 35 and 45 mmHg to improve the operating conditions, and the positive end-expiratory pressure was set at 6–8 mL H2O when necessary. For intra-operative analgesia, each patient received bilateral transverse abdominal fascia nerve blocks containing 20–30 mL ropivacaine (0.375%) after induction.General anesthesia was induced by midazolam (0.04 mg/kg), propofol (1–2.5 mg/kg), fentanyl (2.5–6 μg/kg), cis-atracurium (0.2–0.3 mg/kg), and dexamethasone (10 mg). Anesthesia was maintained by inhalation of sevoflurane (0.8–1.3 MAC) with intermittent supplementation using other auxiliary drugs, such as remifentanyl (0.1–0.2 μg/kg/min), dexmedetomidine (0.2–0.5 μg/kg/h), cis-atracurium, and fentanyl. To accelerate postoperative extubation, inhalation was discontinued, and total intravenous anesthesia by propofol (100–200 μg/kg/min) was applied before abdominal closure. Patients were ventilated in a volume-controlled mode with a tidal volume of 8 mL/kg (air: 40–100% oxygen). The respiratory rate was set to maintain the PaCOOn arrival at the operating room, patients received intravenous ringer lactate (5 mL/kg), and hemodynamic parameters were recorded as the baseline. After induction of anesthesia, a 20-G radial arterial line was inserted and connected to an electrocardiogram monitor and fourth-generation Vigileo/Flotrac system  to determine changes in arterial blood pressure waveforms, SVVs, and cardiac indexes. A double-lumen 5.5-Fr catheter  was inserted into the right internal jugular vein for colloid supplementation. A continuous infusion of ringer lactate was given through peripheral veins during the operation at a rate of 3 mL/kg/h. Colloid  management was conducted under the guidance of the Vigileo/Flotrac system. If the SVV changed by more than 10% 2–3 consecutive times, the colloid was injected via the central vein at 4 mL/kg. After 5 min, SVV changes were observed again. Vasoactive agents were given according to changes in intra-operative hemodynamics based on the recommendations of the anesthesiologist. Transfusions were applied according to recent guidelines .The study was designed as a two-arm, parallel, prospective, observational cohort. The primary endpoint of the study was the timepoint of flatus and differences in blood metabolomics between the two groups. Hemodynamic parameters at different timepoints, surgical resection methods, total intra-operative time, and perioperative fluid volume were recorded. Additionally, the number of hemodynamic fluctuations, such as hypotension or hypertension, was collected. The timepoints of flatus and feeding were recorded, and the time of flatus was used as an indicator of postoperative recovery. The occurrence of flatus 72 hours after surgery was identified as delayed postoperative recovery. The length of hospital stay and duration of postoperative complications were calculated.Blood samples were collected from all patients at PACU admission and 24 hours after surgery. Blood was collected into heparinized tubes, and plasma was immediately separated by centrifugation at 3000 rpm for 10 min and stored at –80° C until metabolomics analysis.LC/MS analysis was carried on an Ultimate 3000LC Q Exactive platform . The parameters for electron spray ionization (ESI)+ and ESI- ion modes were as follows: heater temperature, 300° C; sheath gas flow rate, 45 arb; auxiliary gas flow rate, 15 arb; sweep gas flow rate, 1 arb; spray voltage, 3.0 kV; capillary temperature, 350° C; S-Lens RF level, 30%. The data were analyzed using feature extraction and preprocessed with Compound Discoverer software (Thermo). In total, 2015 features in ESI+ ion mode and 1601 features in ESI- ion mode were evaluated, and editing of data was performed with the Multivariate Analysis (MVA) feature of SIMCA-P software .A supervised learning model was built by a support vector machine (SVM) to classify the delayed and enhanced postoperative recovery groups. Finally, we used radial as SVM kernel function under type C classification and selected 22 features (A–B) and 10 features (C–D) to construct the two SVM models separately. Under these parameters, the predictive accuracies reached 92.8% and 88.8%, and the area under the curve (AUC) values were 0.983 and 0.941 for groups A–B and C–D, respectively, indicating that the model was effective.P values less than 0.05 were considered statistically significant.Statistical analysis was performed using GraphPad Prism version 8.0 . Baseline demographics and postoperative complications were evaluated using Pearson’s chi-square tests for categorical variables, with Fisher’s exact test for small counts. Analysis of variance with Tukey’s correction was used for continuous data. Results with http://www.chictr.org.cn/index.aspx; identifier: ChiCTR1800015899). Written informed consent was obtained from all patients or legal guardians.The study was approved by the appropriate Institutional Review Board (IRB) and Changhai Hospital Ethics Committee  and was registered at the Chinese Clinical Trial Registry (Patients gave consent for their personal or clinical details to be published in this study. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Figure 1Supplementary Tables