Cannabis sativa species, especially cannabidiol (CBD) and Delta-9-tetrahydrocannabinol (Δ9-THC), have attracted medical attention in recent years. The actions of these two main cannabinoids modulate the cholinergic nervous system (CholNS) involving development, synaptic plasticity, and response to endogenous and environmental damage, as a characteristic of many neurodegenerative diseases. The dynamics of these diseases are mediated by specific neurotransmitters, such as the GABAergic nervous system (GNS) and the CholNS. The nematode Caenorhabditis elegans is an important experimental model, which has different neurotransmitter systems that coordinate its behavior and has a transgene strain that encodes the human β-amyloid 1–42 peptide in body wall muscle, one of the main proteins involved in Alzheimer´s disease. Therefore, the objective of this study was to evaluate the protective potential of terpenoids found in C. sativa in the GNS and CholNS of C. elegans. The effect of two C. sativa oils with variations in CBD and THC concentrations on acetylcholinesterase (AChE) activity, lipid peroxidation, and behavior of C. elegans was evaluated. C. sativa oils were efficient in increasing pharyngeal pumping rate and reducing defecation cycle, AChE activity, and ROS levels in N2 strains. In the muscle:Abeta1-42 strain, mainly when using CBD oil, worm movement, body bends, and pharyngeal pumping were increased, with a reduced AChE activity. Consequently, greater investments in scientific research are needed, in addition to breaking the taboo on the use of the C. sativa plant as an alternative for medicinal use, especially in neurodegenerative diseases, which have already shown positive initial results.Substances from the  Cannabis sativa plant has hundreds of chemical compounds produced by secondary metabolism, including cannabinoids, terpenes, and phenolic compounds, each with potential biological properties1. Among the cannabinoids, delta-9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) stand out as the two main ones2.The 3. These cannabinoids modulate the cholinergic nervous system (CholNS) involving development, synaptic plasticity, and response to endogenous and environmental damage, as a characteristic of many neurodegenerative diseases4. Through the interaction between cannabinoids and their receptors, an anti-inflammatory response is generated5, which is an important strategy for maintaining physiological homeostasis and acting on the central nervous system, including pain reduction6.THC is well known for its psychoactive properties, while CBD is a non-psychostimulant cannabinoid8. The GNS comprises the most abundant synapses in the central region of the central nervous system, which are mediated by the action of the neurotransmitter γ-aminobutyric acid (GABA), and plays a dominant role in inhibitory processes9. The GABAergic neurotransmission occurs in interneurons that modulate local neurotransmission including noradrenergic, dopaminergic, and serotonergic neurons10. Thus, it is believed that the reduction of GABAergic transmission over a long period is associated with the various deleterious impairments of different neurotransmitters observed in Alzheimer's disease (AD)11. The CholNS is responsible for cognition, memory, and senses, and its neurotransmitter is acetylcholine (ACh). ACh is an excitatory neurotransmitter formed from acetyl coenzyme-A and choline, which acts at neuromuscular junctions, memory, and areas of the brain related to learning12. Thus, the CholNS acts in the peripheral nervous system along with the neuromuscular connections, being, therefore, affected in neurodegenerative diseases such as Parkinson's and Alzheimer's diseases13.Some neurodegenerative diseases have an unknown etiology, but when it comes to diseases related to nerve synapses, the most studied systems include the GABAergic nervous system (GNS) and the CholNS15. In cholinergic signaling, the enzyme acetylcholinesterase (AChE) develops the nerve impulse through the hydrolysis of ACh into acetate and choline. Oh et al.16 showed that patients with AD enhanced when they were treated with AChE inhibitors. Similar investigations reported that acute CBD causes significant alterations in brain activity during relaxing conditions and performance of mental tasks in both healthy recruits and patients with a psychiatric illness17. The safety of CBD was also tested in C. elegans and no toxicity effects were observed18.Changes that occur in the CholNS are also responsible for behavioral disturbances in AD patientsC. elegans is consolidated in biochemical, toxicological, and genetic studies. The nematode C. elegans has different neurotransmitter systems that coordinate its behavior, including dopaminergic, cholinergic, serotonergic, glutamatergic, and GABAergic systems19. In addition, the C. elegans mutant strain muscle:Abeta1-42 has the addition of the gene that encodes the human β-amyloid protein 1–42 peptides in body wall muscle, which is transcriptionally activated by heat stress (25 °C)20. The β-amyloid protein is one of the main proteins involved in the onset and progression of AD21.To understand neurological disorders and their effects on physiological processes, model organisms have been used. Among these, the free-living nematode C. sativa-based CBD and THC oils, we hypothesized that these oils are effective in decreasing or delaying the symptoms caused by AD. This study aimed to evaluate the protective potential of different concentrations of CBD and THC cannabinoids obtained from C. sativa on behavioral parameters , cholinergic system (AChE activity), and reactive oxygen species (ROS) in C. elegans wild-type and two transgenic strains, a muscle:Abeta1-42 strain, that encodes the human β-amyloid 1–42 peptide in body wall muscle, and muscle:Abeta-control used as a control of muscle:Abeta1-42.Based on these studies and the gap in the potential of C. sativa oils on wild-type (N2) non-stressed C. elegans aiming to understand the effect of the oils applied alone in a healthy organism to observe the changes in the behavior  as well as on biochemical biomarkers such as acetylcholinesterase enzyme activity and ROS levels. In addition, evaluating in mutant worms the fluorescent antioxidant enzymes expression such as superoxide dismutase, catalase, glutathione-S-transferase, and heat shock protein even without stress.Here was evaluated the effect of the p = 0.008) in comparison to the control group  and olive oil  controls  and olive oil (p = 0.0001 for both) controls  strain after the 1.25% THC-treated group (p = 0.0001), olive oil (p = 0.0001), THC 2.5% (p = 0.0001), and THC 1.25% (p = 0.0001)  in comparison to the groups treated with water  and olive  controls, CBD 1.25%  and THC 1.25%   Fig. .Figure 3C. elegans Sod-3:GFP mutant strain was increased in the group treated with THC 2.5% in comparison to both water (p = 0.001) and olive oil (p = 0.04) controls, as well as CBD 5% (p = 0.04), and THC 1.25% (p = 0.008)  and olive oil (p = 0.0002) controls, CBD 5% (p = 0.0002), THC 2.5% (p = 0.004), CBD 1.25% (p = 0.0001)   and olive oil  controls, as well as CBD 5%  and THC 2.5%  treatments  under heat stress as an indicator of Alzheimer’s condition in humans. For this, the worms were heat stressed and treated with the C. sativa oils and had the behavior  and biochemical (acetylcholinesterase activity) biomarkers evaluated.Here was evaluated the effect of the p = 0.01) in comparison to CBD 1.25% in the muscle:Abeta1-42 transgenic strain  and olive oil (p = 0.02) controls, as well as the group treated with CBD 1.25% (p = 0.008) , CBD 1.25%, and THC 1.25% in comparison to water  and olive oil  control treatments , olive oil (p = 0.0001), CBD 1.25%  and THC 1.25%   strain muscle:Abeta1-42 strain without stress, increased in the group treated with CBD 5% in comparison to the water (p = 0.001) and olive oil (p = 0.0001) controls, and also in comparison with CBD 1.25% (p = 0.02) and THC 1.25% (p = 0.0001) treatments . Defecation cycle on muscle:Abeta-control strain was decreased in the groups treated with THC 2.5%, CBD 1.25%, and THC 1.25% in comparison to water (p = 0.0001) and olive oil (p = 0.0001) controls , olive oil (p = 0.0001), CBD 5% (p = 0.05), CBD 1.25% (p = 0.0001), and THC 1.25% (p = 0.0001)  Fig. C. The HSups Fig. D.muscle:Abeta1-42 strain in the group treated with CBD 1.25% in comparison to the groups treated with water (p = 0.01), olive oil (p = 0.001), CBD 5% (p = 0.007), THC 2.5% (p = 0.01), and THC 1.25% (p = 0.02) , olive oil (p = 0.03), CBD 5% (p = 0.0001), THC 2.5% (p = 0.0001), and THC 1.25% (p = 0.0001) treatments , olive oil (p = 0.002), and CBD 1.25% (p = 0.0001).Without stress, the pharyngeal pumping increased in the  p = 0.00, THC 2.5 p = 0.00, THC 2.5muscle:Abeta1-42 was increased in the group treated with CBD 5% in comparison to the controls water (p = 0.006), olive oil (p = 0.0001), THC 2.5% (p = 0.0003), CBD 1.25% (p = 0.0001), and THC 1.25% (p = 0.02) treatments , olive oil , CBD 5% , and THC 2.5%   and olive oil  controls  and olive oil (p = 0.007) controls, as well as in the CBD 5% (p = 0.008), and THC 1.25% (p = 0.05)  and olive oil (p = 0.01) controls  and olive oil  controls as well as with the CBD 1.25% group (p = 0.01)  and THC 1.25% , olive oil (p = 0.01), CBD 5% (p = 0.001), and THC 2.5% (p = 0.001)  Fig. C. On the25% Fig. D. In add01) Fig. D.Cannabis sativa-based CBD and THC oils have shown potential benefits for C. elegans nematodes. First, when evaluating the wild-type N2 strain, an increase in pharyngeal pumping was observed when CBD oil was added. In addition, a reduction in the duration of the defecation cycle was observed after exposure to the highest dose of CBD and the lowest dose of THC, as well as the activity of the enzyme AChE. Both the pharynx and the defecation cycle are could be modulated by AChE levels in the synaptic clefts.22. The ACh, in turn, is one of the main neurotransmitters that make up the nervous system, being responsible for behavior, memory, movement, and reasoning23. Inhibition of AChE enzyme activity is responsible for causing high excitability in cholinergic neurons, in addition to forcing ACh to remain longer in the synaptic cleft, inducing an excitatory effect in the system. The reflexes of this excitatory effect are mostly evidenced in high muscle contraction, such as the increase in the frequency of pharyngeal pumping25. In nematodes, the pharynx plays an important role, as it is a bilobed neuromuscular organ where the pharyngeal ring is located, which contains the highest concentration of neurons in the individual26. As the pharynx is mainly regulated by the action of the ACh neurotransmitter, the increase in pharyngeal rhythm is related to an improvement in neurodegenerative diseases27.AChE is a central nervous system enzyme that acts in the synaptic cleft and neuromuscular junctions and it is responsible for the hydrolysis of acetylcholine (ACh) into acetic acid and choline+) levels and the neurotransmitter γ-aminobutyric acid (GABA), which may indicate changes in the cholinergic and GABAergic systems due to a reduction in the time of the defecation cycle24. In addition, the accentuation of the reduction of the defecation interval is closely linked to the endocannabinoid system’s CB1 receptors, which are found throughout the central nervous system, peripheral, synaptic endings, intestine, and immune cells, being that in the intestine the endocannabinoid system plays an important role in its motility29. CB1 receptors act more actively in the presence of CBD, thus increasing the number of defective contractions and reducing the interval between them in C. elegans.The defecation cycle, resulting from muscle contractions, is also regulated by the ACh levels in the synaptic clefts. However, they are still strongly influenced by two other factors, namely calcium , inducing irreversible damage2O2, demonstrate a considerable impact of high THC cannabis extracts to fight ROS formation31. However, CBD did not show as high an antioxidant action as THC under these test conditions31.Moreover, in vitro studies exploring the impacts of cannabis extracts with various THC and CBD percentages on oxidative stress in differentiated neuronal SH-SY5Y cells using Hmuscle:Abeta1-42 strain that encodes the human β-amyloid 1–42 peptide in muscles, it was possible to observe that the highest CBD concentration increased worm movement and pharyngeal pumping, and reduced AChE activity in relation to the strain muscle:Abeta-control, used as a control. This indicates that for the muscle:Abeta1-42 strain, where β-amyloid expression occurs, the release and/or action of ACh in the synaptic cleft needs to be more accentuated, so that correct signaling occurs, which demonstrates that Cannabis-based oils, especially CBD, could act as an aid in the process of improving cholinergic signaling in individuals with β-amyloid expression. Patients affected by Alzheimer's have aggregations of different isoforms of β-amyloid protein in the synaptic clefts, which under normal conditions would be precipitated and eliminated periodically21. These aggregations, in addition to hindering communication between neurons causing neurotransmitters to lose efficiency, are also responsible for the death of cholinergic neurons and thus leading to the progression of neurodegenerative diseases. In addition, in aging tests, a gradual decrease in the pharyngeal pumping is reported, which may be accelerated by neurodegenerative diseases32. Furthermore, the increase in the time between defecation cycles, related to the increase in AChE activity with the inhibition of the excitability of the cholinergic system, allows the reduction of the defecation cycle20.In the transgenic C. elegans mutant strains for neurodegenerative diseases, as well as in patients affected by neurodegenerative diseases, there is a harmful decrease in the levels of neurotransmitters and also in the activity of enzymes that act in the synapse process33. To reverse this effect, defining compounds capable of activating and stabilizing these enzymes at normal levels is an important process to reduce the progression of neurodegenerative diseases. The modulation of enzymes such as AChE can positively influence neurotransmission in neurodegenerative patients since in many diseases such as Alzheimer's and Parkinson's, intense and dense protein neurofibrillary tangles are formed in the synaptic clefts. Therefore, the activation of nervous system enzymes intensifies the processes, equalizing the synapses34.Naturally, in Cannabis is known to increases appetite, particularly in high-fat foods35. This occurs as the endocannabinoid system regulates energy balance and food intake at various functional levels, both in the brain and in the gastrointestinal tract36. Thus, the increase in the pharyngeal rate in the mutant strains can also be related to the food-inducing effect of these organisms. This is particularly important in patients suffering from neurodegenerative diseases with loss of appetite, where the use of oils can also favor an improvement in food intake.In addition, the endocannabinoid system receptors, mainly CB1, are important biomarkers of body weight gain. Studies such as the one presented are important to clarify the true mechanisms by which fragmented or small improvements are observed in chronic diseases linked to the central nervous system. However, even more importantly, it is necessary to show through science, the need not discriminate between these therapies. Considering that, in many cases, it is the most effective and perhaps the only alternative available.Cannabis Research and Patients), a non-profit initiative that aims to support research and also the individual cultivation of Cannabis for medicinal purposes. As the oils obtained were donated and the extraction protocols were not provided by APEPI, they were used for analysis on nematodes in the same way that they are marketed for medicinal purposes for patients with various diseases, including AD. The vehicle used was ultrapure olive oil, also received by the APEPI – RJ, and used as a control treatment.The oils were donated by APEPI—RJ  of crude CBD and THC oils were added to 10 mL of methanol:n-hexane solvent , homogenized in a vortex mixer for 1 min, and kept in an ultrasound bath for 30 min.The samples were then refrigerated at − 20 °C for 30 min and centrifuged at 4000 rpm for 20 min. The supernatant was filtered through polytetrafluoroethylene membranes  and stored in vials, in a refrigerator, for further analysis. For the analyses, a gas chromatograph coupled to the detector by mass spectrometry  was used with the following chromatographic conditions: column, DB5-HT (30 m × 250 µm × 0.1 µm); oven, 150 °C (1 min); 15 °C/min; 270 °C (6 min); 20 °C/min; 300 °C (2 min); running time: 18.5 min; flow: 0.6 mL/min; 1 µL injection, split: 50:1; injector and transfer line temperatures, 250 °C; mass spectrometer programmed for acquisition in Full Scan mode 50–500 m/z, electron ionization source (EI) 70 eV. The obtained mass spectra were compared to those from the Nist library.Crude CBD oil contains 8.98% CBD and 1.22% THC, while crude THC oil contains 50.94% THC and 6.08% CBD.Caenorhabditis Genetics Center (CGC), University of Minnesota, Minneapolis, USA. The nematode strains were maintained in a nematode growth medium (NGM) with Escherichia coli OP50 at 20 °C37. The study was carried out in the Biochemistry Laboratory of the Federal Institute of Rio Grande do Sul, campus Sertão, Sertão, Brazil. All used strains are detailed in Table All the strains used in the study were obtained from the muscle:Abeta1-42 is widely used in DA. It encodes the human β-amyloid 1–42 peptide in body wall muscle38. The strain muscle:Abeta1-42 had the induced expression of the protein β-amyloid through heat stress (25 °C), in larval stage L1. Strain muscle:Abeta-control is a negative control of strain muscle:Abeta1-42.The transgenic strain C. elegans were also used to evaluate oxidative stress with the green fluorescent protein (GFP) assays: the strain CF1553, a mutant for superoxide dismutase (sod-3 gene), GA800, a mutant for catalase , CL2166, a mutant for glutathione-S-transferase (gst-4 gene), and CL2070, a mutant used to determine the heat shock protein (hsp-16.2).Different strains of muscle:Abeta1-42 (GMC101), muscle:Abeta-control (CL2122), Sod-3:GFP (CF1553), Gst-4:GFP (CL2166), Hsp-16.2:GFP (CL2070), and Cat-1,2,3:GFP (GA800) were maintained at 20 °C. The nematodes were exposed to six different treatment groups: water control, olive oil control (used to prepare the CBD and THC oils), CBD 5%, THC 2.5%, CBD 1.25%, and THC 1.25%. They are exposed in the L4 stage.For the non-stressed assays, the strains wild-type N2, C. elegans nematodes were transplanted and sown in NGM, and kept in growth chambers at 20 °C, until they reached the L4 larval stage and after that they were synchronized (pregnant or with eggs)42. Nematodes feed with E. coli. At the L4 larval stage, the worms were washed 3 times, and centrifuged at 3000 rpm, for 2 min. Then, the nematodes were exposed to a synchronization in a 5 mL solution containing hypochlorite (1.5 ml) and sodium hydroxide (0.250 ml). The reagents break the cuticle and release their eggs. The eggs were placed on plates with the M9 buffer solution36, and the eggs were maintained at 20 °C, for approximately 24 h, until they reached the L1 larval stage. After that, they were transferred to NGM and fed with E. coli. On the next day, when they reach the L4 stage, they are able to be exposed to the treatments.The E. coli and acclimated for another 30 min for further behavioral evaluation.The nematodes were exposed to the treatments for 1 h. After that period, the nematodes were washed 3 times in M9 buffer solution, transferred to Petry dishes containing muscle:Abeta1-42 and muscle:Abeta-control were raised to a temperature of 25 °C, after their synchronization. In this condition, the transgenic strain muscle:Abeta1-42 encodes the human β-amyloid 1–42 peptide in body wall muscle. The exposition of the treatments was carried out with the same methods used for non-stressed nematodes.For the assays with heat-stress (HS), the transgenic strains Body bend rate, defecation cycles, and pharyngeal pumping rate were evaluated. All evaluations were performed in triplicate.43.To evaluate the locomotor parameters of body bends, twenty-four nematodes were exposed to each treatment in the larval stage L4, where they were evaluated individually 2 times for 30 s, with a total time of 60 s for each nematode. These individuals were collected and placed in a Petry dish (5 cm diameter) containing agar for 1 min. Each of the nematodes was kept in a horizontal position (X axis) and the number of body curves generated in 30 s was counted. Body curvature was defined as a change in the direction of locomotion of the nematode's anterior part, i.e., the pharynx, along the Y axis, and expressed per minute43.The duration of the defecation cycle was performed using a microscope (100 ×) . Twenty-four individuals, synchronously grown young adults, were examined per strain, per treatment. The time between two evacuative intervals was measured. Each nematode was observed for three consecutive defecation cycles and the time between the two consecutive cycles was calculated44. They are fed by sucking bacteria and grinding them in their terminal bulbs. As a complete cycle of synchronous contraction and relaxation of the terminal bulb is called a “pump”, the pharyngeal pumping rate (pumps per minute) was measured by visually observing the nematodes under a microscope (100×) .To measure the pharyngeal pumping, twenty-four nematodes were observed three times for 10 s each, closing a total of 30 s (10 s-10 s-10 s)45. After treatment exposure, 10.000 nematodes were washed three times with M9 buffer solution and transferred to microcentrifuge tubes. Samples were frozen and thawed 3 times in liquid nitrogen, followed by 5 rounds of sonication, each of 15 s in the ice, at 30% amplitude, with 10-s intervals between each. Then, the samples were centrifuged for 30 min at 15.000 rpm and the supernatants were collected. An aliquot (160 μL) of the supernatant was mixed with a solution containing DTNB (0.25 mM) and acetylthiocholine iodide , and incubated at 30 °C for 5 min. The absorbance was measured in a spectrophotometer  at 405 nm, at 30 s-intervals, for 4 min. Kinetic measurements were recorded and converted to the total cholinesterase activity using the extinction coefficient (11.5 M-1 cm−1) of the 5-thio-2-nitro-benzoic acid. AChE activity was expressed as μmol ASChI min−1 mg−1 protein46.The AChE activity was determined in L4 stage nematodes, using a colorimetric assayC. elegans mutant strain for each analysis. SOD activity was determined using the strain CF1552 [muls84 (pAD76) sod-3p::GFP + rol-6(su1006)], CAT with GA800 strain [wuls151 ctl-1 + ctl-2 + ctl-3 + myo-2::GFP], GST with CL2166 strain [dvIs19 (pAF15) gst-4p::GFP::nls], and HSP with CL2070 strain (dvIs70 [hsp-16.2p::GFP + rol-6(su1006)]). These strains were exposed to the treatments as mentioned before. After that, the individuals were washed and transferred to 96-well microplates with M9 buffer solution, using excitation at 485 nm and emission at 530 nm to measure total fluorescence. Fluorescence-based assays were conducted on a microplate reader , according to Tamagno et al.41 and Tamabara et al.47.Determination of the green fluorescent expression of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), and heat shock protein (HSP) was performed using a specific 2DCF-DA, 0.5 mM), for 1 h. The nematodes were washed again, containing the fluorescence dye, and then exposed to hydrogen peroxide (0.4 mM). The ROS levels were analyzed inside the nematodes by measuring fluorescence using excitation at 485 nm and emission at 535 nm, for 1 h in the presence of hydrogen peroxide. The results were expressed as function-time curves per mg of protein.The ROS levels were evaluated in 1500 nematodes of the wild-type N2 strain. Nematodes in L4 stage were exposed to the six treatments, as previously described. After that period, they were washed out of treatment, and exposed to 2.7- dichlorofluorescein-diacetate  and homogeneity (Bartlett test). Parametric data were analyzed using a one-way analysis of variance (ANOVA), followed by Tukey's post hoc, and non-parametric data were compared using Dunn’s test. All analyses were evaluated at a 5% significant level. Statistical analysis was performed using Graph Pad Prism version software, version 8.0.1 .


Painless gastrointestinal endoscopy is widely used for the diagnosis and treatment of digestive diseases. At present, propofol is commonly used to perform painless gastrointestinal endoscopy, but the high dose of propofol often leads to a higher incidence of cardiovascular and respiratory complications. Studies have shown that the application of propofol combined with ketamine in painless gastrointestinal endoscopy is beneficial to reduce the dosage of propofol and the incidence of related complications. Esketamine is dextrorotatory structure of ketamine with a twice as great anesthetic effect as normal ketamine but fewer side effects. We hypothesized that esketamine may reduce the consumption of propofol and to investigate the safety of coadministration during gastrointestinal endoscopy.A total of 260 patients undergoing painless gastrointestinal endoscopy (gastroscope and colonoscopy) were randomly divided into P group , PK1 group (propofol + esketamine 0.05 mg/kg), PK2 group (propofol + esketamine 0.1 mg/kg), and PK3 group (propofol + esketamine 0.2 mg/kg). Anesthesia was achieved by 1.5 mg/kg propofol with different doses of esketamine. Propofol consumption per minute was recorded. Hemodynamic index, pulse oxygen saturation, operative time, induction time, awakening status, orientation recovery time, adverse events, and Mini-Mental State Examination (MMSE) were also recorded during gastrointestinal endoscopy.P = 0.021) and 18.76% (P = 0.000), respectively. In all four groups, systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), but not pulse oxygen saturation (SpO2) significantly decreased (P = 0.000) immediately after administration of induction, but there were no significant differences between the groups. The induction time of groups P, PK1, PK2, and PK3 was 68.52 ± 18.394, 64.83 ± 13.543, 62.23 ± 15.197, and 61.35 ± 14.470 s, respectively (P = 0.041). Adverse events and psychotomimetic effects were observed but without significant differences between the groups.Propofol consumption per minute was 11.78, 10.56, 10.14, and 9.57 (mg/min) in groups P, PK1, PK2, and PK3, respectively; compared with group P, groups PK2 and PK3 showed a decrease of 13.92% . Gastrointestinal (GI) endoscopy is the gold standard for the diagnosis of digestive tract diseases and is also widely used for the treatment of digestive tract diseases . Almost When facing all these challenges, anesthesiologists must make an anesthesia plan before the GI procedure, ensuring that patients are stable and comfortable during the procedure, wake up quickly, and leave the hospital safely as soon as possible . In clinEsketamine, an antagonist of the N-methyl-D-aspartic acid receptor, has analgesic and anesthetic effects and causes less cardiorespiratory and respiratory depression. It also leads to fewer psychotomimetic effects and less secretion , 13. TheChina Ethics Committee of Registering Clinical Trials approved the protocol (Ethics Number: CHIECRCT20210050), and the study was registered in the Chinese Clinical Trial Registry . All participants signed the informed consent. The full trial protocol can be accessed at the following website: chictr.org.cn.A prospective, double-blind, randomized controlled study was conducted at the First Affiliated Hospital of Jinan University.Between November 9, 2020, and September 1, 2021, patients scheduled for elective painless GI endoscopy were eligible for participation in this trial if they: (i) were 18–60 years old; (ii) had American Society of Anesthesiologists Physical Status (ASA PS) I to II; (iii) had a full understanding of the purpose and significance of this trial; and (iv) were able to sign an informed consent form.Exclusion criteria were as follows: (i) a history of unregulated diabetes, hypertension, and hypotension; (ii) myocardial infarction within 6 months or unstable angina pectoris; (iii) III degree atrioventricular block; (iv) severe snoring; (v) sleep apnea syndrome; (vi) decompensated liver function or renal function; (vii) dialysis treatment; (viii) psychosocial disease or cognitive dysfunction; (ix) a history of psychotropic drugs and narcotic drug abuse; (x) allergy to or contraindication for the drugs used in this study; (xi) participation in clinical trials of other drugs within 3 months; (xii) other circumstances in which the investigator determined that a patient was not suitable for participation in the clinical trial.According to our pre-experiment, the mean value and standard deviation of propofol consumption per minute in each group were obtained, and the inspection efficiency was set at 0.90, while the inspection level was set at 0.05. A required sample size of 62 subjects per group was calculated using PASS 11 software . Considering a loss rate of 5%, a total of 260 subjects (65 subjects per group) were required.The random sequence generator of STATA MP 14 software generated 260 random numbers and divided them into four groups. The patients were assigned into four groups: group P (propofol); group PK1 (propofol + eskatemine 0.05 mg/kg); group PK2 (propofol + eskatemine 0.10 mg/kg); and group PK3 (propofol + eskatemine 0.20 mg/kg). For each patient enrolled, a researcher drew an envelope to determine the patient’s grouping, but the patient was not informed of the grouping. Except for propofol, clinical researchers were given syringes with clear solutions in the same bottles with codes according to the randomization order. The researchers who performed the randomization and blinding procedure did not participate in the follow-up study. Other investigators were not informed of the grouping of the study and the experimental drugs. To ensure allocation concealment, randomization results were sealed until the end of the study.After routine GI endoscopy preparation and fasting for 6 h before the procedure, the patients arrived at the endoscopy center and were asked about basic information. If the patients met the inclusion criteria, they signed an informed consent form. Grouping was determined according to the randomization. Before sedation, the study drugs were diluted with normal saline in a 10 mL syringe by a blindless nurse, with final concentrations of 0.5 mg/mL, 1 mg/mL, and 2 mg/mL for esketamine.2) at T0, followed by Mini-Mental State Examination (MMSE) [The procedure of sedation is shown in Fig. n (MMSE) . Data co2 at T1 were recorded 5 min before anesthesia induction. The patient was sedated by the same blindless anesthesiologist using the prepared test drug and propofol. The same dose of normal saline (0.1 mL/kg), eskatemine  for the P group, PK1 group, PK2 group, and PK3 group, respectively, were injected slowly (more than 6 s) followed by 1.5 mg/kg propofol for anesthesia induction. BP, HR, and SPO2 at T2 were recorded immediately after administration of induction. When the patient’s eyelash reflex disappeared and there was no significant movement after the gastroscope introduction, the same doctor at the endoscopy center began to perform the endoscopy. BP, HR, and SPO2 at T3 were recorded immediately after gastroscope insertion. A rescue dose of propofol (0.5 mg/kg) was used if body movement, cough, or swallowing occurred during the procedure. BP, HR, and SPO2 were recorded immediately before (T4) and after (T5) colonoscopy. After the GI endoscopy examination, the patient was roused every 2 min. If the patient was able to open their eyes and nod their head, the time of awakening was recorded. Accordingly, 10 min after the patient awakened, MMSE was evaluated by the same anesthesiologist.Oxygen supplementation of 3 L/min was delivered via a nasopharynx tube throughout the study. BP, HR, and SPO2 < 95% was corrected by chin lifting, while SpO2 < 90% was corrected by face mask ventilation. Phenylephrine 0.1 mg was injected when BP was below 30% of the baseline level, and atropine 0.25 mg was injected when HR was below 50/min. Other respiratory and cardiovascular adverse events were recorded and managed in accordance with the clinical operation standard. Before leaving the operating room, BP, HR, and SPO2 (T6) as well as the ambulation time were recorded.During the operation process, SpO0–T6), induction time (from the injection of the study medication to the disappearance of eyelash reflex), procedure time , delayed awakening (awakening time more than 6 min is defined as delayed awakening), orientation recovery time , MMSE results, and adverse events. Adverse events included propofol injection pain, hypoxemia (SPO2 < 95%), hypotension , hypertension , arrhythmia, nausea and vomiting, dizziness, cough, body movement, apnea, excessive oral secretion, and psychotomimetic effects.The primary outcome of this study was propofol consumption per minute (mg/min). The total consumption of propofol and the duration of the procedure were recorded and then propofol consumption per minute was calculated. Secondary outcomes included hemodynamic index  was used for statistical analysis. Continuous variables were shown as mean ± SD, and categorical variables were expressed as N (%) of patients. All data were checked for normal distribution using the Kolmogorov–Smirnov test and histograms. For continuous variables, ANOVA followed by Student–Newman–Keuls test was performed. For discrete variables, Kruskal–Wallis Figure P = 0.021) and 18.76% (P = 0.000), respectively. There were no significant differences in propofol consumption per minute among the three esketamine groups.Propofol consumption per minute was 11.78, 10.56, 10.14, and 9.57 (mg/min) in groups P, PK1, PK2, and PK3, respectively  at T2, the time immediately after administration of induction. SBP, DBP, HR, and SpO2 were significantly higher at T6 (P > 0.05), the time before patients left the operating room, compared with the baseline value , DBP (P = 0.253), HR (P = 0.838), and SpO2 (P = 0.087) did not differ among the groups . The induction time of group PK3 was 7.17 s shorter than that of group P. There were 8, 9, 8, and 5 patients who experienced an awakening delay in groups P, PK1, PK2, and PK3, respectively (P = 0.716). There were no significant differences in procedure time, orientation recovery time, and awakening status between the groups  and body movement (P = 0.004). One patient in group PK1 developed an unintentional tremor, which gradually disappeared after half an hour of rest. One patient in group P and one in group PK3 had unintentional lacrimation without depression or sadness. One patient in group PK3 was agitated, reporting a very pleasant mood and involuntarily wanting to laugh. One patient in group PK3 had nightmares and felt tormented. No arrhythmia, nausea and vomiting, apnea , hypertension (one patient in group PK1), and psychotomimetic effects Tables  and 5. CP = 0.210) in any of the groups. There were also no significant differences among the four groups . Finally, multicenter studies with larger sample sizes are needed to confirm our findings.In this study, we observed that in painless gastrointestinal endoscopy, propofol combined with 0.2 mg/kg esketamine had a shorter induction time, lower incidence of cough and body movement, and less propofol consumption per minute than propofol alone; there was no effect on recovery time, hemodynamic stability, postoperative cognitive function, and the incidence of adverse events and psychotomimetic effects.


Escherichia coli (E. coli).This study was conducted to investigate the effects of each phytogenic feed additive  on the growth performance, nutrient digestibility, intestinal morphology, and immune response in weaned pigs infected with E. coli challenge, 2) PC; basal diet with E. coli challenge, 3) T1; PC + 0.04% PFA1, 4) T2; PC + 0.01% PFA2, 5) T3; PC + 0.10% PFA3, 6) T4; PC + 0.04% PFA4, 7) T5; PC + 0.10% PFA5. The experiments lasted in 21 d, including 7 d before and 14 d after the first E. coli challenge. In the E. coli challenge treatments, all pigs were orally inoculated by dividing a total of 10 mL of E. coli F18 for 3 consecutive days. The PFA-added groups significantly increased (P < 0.05) average daily gain and feed efficiency and decreased (P < 0.05) the fecal score at d 0 to 14 post-inoculation (PI). Tumor necrosis factor α was significantly lower (P < 0.05) in the PFA-added groups except for T1 in d 14 PI compared to the PC treatment. The T3 had a higher (P < 0.05) immunoglobulin G and immunoglobulin A concentration compared to the PC treatment at d 7 PI. Also, T3 showed significantly higher (P < 0.05) villus height:crypt depth and claudin 1 expression in ileal mucosa, and significantly down-regulated (P < 0.05) the expression of calprotectin compared to the PC treatment.A total of 63 4-week-old weaned pigs were placed in individual metabolic cages and assigned to seven treatment groups. The seven treatments were as follows: 1) NC; basal diet without E. coli alleviated the negative effects of E. coli and improved growth performance. Among them, the mixed additive of bitter citrus extract, thymol, and carvacrol showed the most effective results, improving immune response, intestinal morphology, and expression of tight junctions.Supplementation of PFA in weaned pigs challenged with  Escherichia coli (E. coli) is known to be a major cause of PWD  with initial body weight (BW) of 8.03 ± 0.43 kg were used in this study. All pigs were assigned to a completely randomized seven treatment groups based on the initial BW. There was one pig in a cage and nine replicate cages per treatment. Pigs were individually placed in 45 cm × 55 cm × 45 cm stainless steel metabolism cages in an environmentally controlled room. Pigs were housed in individual pens for 21 d, including 7 d before and 14 d after the first E. coli challenge treatments, all pigs were orally inoculated by dividing a total of 10 mL of E. coli F18 for 3 consecutive days from d 0 post-inoculation (PI) after 7 d of adaptation.In the E. coli challenge.Pigs were weighed individually at the beginning (d −7), d 0 before inoculation, and d 7, 14 PI. Feed intake (FI) was recorded daily the diet supply amount and remaining amount. Average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (G:F) were calculated for each interval from d − 7 to 0, d 0 to 7 PI, d 7 to 14 PI and d 0 to 14 PI. The fecal scores were individually recorded at 08:00 and 17:00 by the same person during the entire experimental period. The fecal score was assigned as follows: 0, Normal feces; 1, Soft feces; 2, Mild diarrhea; 3, Severe diarrhea. The fecal score of each pig was calculated as an average within the period before and after the 2O3, 2 g/kg) was added to the diets as an indigestible marker to measure digestibility [Chromium oxide , and on d 7 and 14 PI. At the time of collection, blood samples were collected into vacuum tubes containing K3EDTA for complete blood count analysis, and nonheparinized tubes for serum analysis, respectively. After collection, serum samples were centrifuged at 3000×g for 20 min at 4 °C. Thereafter, the blood sample tubes were stored in − 20 °C refrigerator until analysis. The white blood cell (WBC), basophil, neutrophil, and lymphocyte levels in the whole blood were measured using an automatic blood analyzer . Immunoglobulin G (IgG) and immunoglobulin A (IgA) levels were gauged using an automatic biochemistry blood analyzer . Interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) concentrations were determined using commercially available ELISA kits  and the absorbance was measured at 450 nm.Blood samples were collected from the jugular vein of all pigs before the At the end of the experiment (d 14), pigs were anesthetized with carbon dioxide gas after blood sampling and euthanized by exsanguination. After euthanization, intestinal tissues of about 10 cm from the ileum  were collected and fixed in 10% neutral buffered formalin  for intestinal morphology and expression analysis of intestinal tight junction proteins. After cutting the intestine sample, it was dehydrated and dealcoholized. The samples were then installed on slides, treated with paraffin, and stained with hematoxylin and eosin. The slides were examined using an Olympus IX51 inverted phase-contrast microscope. Intestinal morphological measurements included the villus height (VH), crypt depth (CD), and villus height:crypt depth ratio (VH:CD).The expression of calprotectin and claudin 1 (CLDN1) was studied by using immunohistochemistry (IHC). The ileal tissue fixed in 10% NBF solution was cut, embedded in a paraffin wax block and sectioned to a thickness of 5 μm. After deparaffinizing the paraffin sections and rehydrating the tissues, they were placed in running tap water for 10 min. This section was reacted in 0.03% hydrogen peroxide solution for 15 min, and then dipped in distilled water (DW) for 10 min. Then, the antigen was retrieved and dipped in DW for 10 min, followed by blocking in 4% bovine serum albumin solution for 30 min. The tissue section was then incubated with primary antibodies, anti-calprotectin  and anti-claudin1 . Then, it was incubated with the secondary antibodies, envision anti-mouse  and envision anti-rabbit (Dako). The sections were washed with tris-buffered saline-tween and incubated with 3,3′-diaminobenzidine (DAB) to visualize immune complexes. The processed slide sections were slide-scanned through Axio Scan Z1 . The completed file was analyzed using Zen Image Analysis, an Axio Scan Z1 program (Zen 3.4 blue edition). Analysis was analyzed by the contrast of DAB of IHC and positive reaction color to total tissue area, and the results were expressed as a total area, a positive area, and the percentage of positive area.P < 0.05 was indicated to be statistically significant, and a level of 0.05 ≤ P < 0.10 was considered to have such a tendency.All data were analyzed via the general linear model procedures of SAS , using each pen as the experimental unit. Differences between treatment means were determined using Tukey’s multiple range test. A probability level of P < 0.05) BW than the other treatment groups. Compared with the NC treatment, the PC treatment reduced (P < 0.05) ADG and G:F at d 0 to 7 PI. Also, at d 0 to 7 PI, the PFA-added treatment groups showed higher (P < 0.05) ADG and G:F compared to the PC treatment. At d 7 to 14 PI, the T3 had a significantly lower (P < 0.05) ADFI than other treatments, but showed a higher tendency (P = 0.073) in G:F. Compared with the PC treatment, the PFA-added treatment groups significantly increased ADG and G:F (P < 0.05) at d 0 to 14 PI, but there was no difference in ADFI. There was no difference between treatment groups in the initial BW of pigs Table . At d 7 E. coli inoculation, there was no difference in the fecal score at d −  7 to 0 (Table P < 0.05) in the PC treatment than in other treatment groups. Also, in d 0 to 14 PI, the PC treatment showed significantly higher (P < 0.05) fecal scores than other treatment groups, and the PFA-added treatment groups showed similar or lower (P < 0.05) fecal scores to the NC treatment.Before  0 Table . The fecP < 0.05) in the PFA-added treatment groups at d 14 PI compared to the PC treatment. No difference was observed in CP digestibility at d 14 PI among treatment groups.At d 7 PI, there were no significant differences in DM and CP digestibility between treatment groups Table . The DM E. coli inoculation (Table P < 0.05) in T2 and NC treatment at d 7 PI compared to other treatment groups. Also, T2 had a significantly lower percentage (P < 0.05) of neutrophils and a significantly higher percentage (P < 0.05) of lymphocytes at d 7 PI. The neutrophils and lymphocytes of the PFA-added treatment groups except for T1 were recovered to the NC treatment level at d 14 PI, and among them, T2 showed significantly similar values (P < 0.05) to the NC treatment.WBC, basophil, neutrophil, and lymphocyte levels did not differ significantly between treatment groups on d 0 before on Table . The numE. coli inoculation (Table P < 0.05) compared to PC treatment. TNF-α was significantly lower (P < 0.05) in the PFA-added treatment groups than in the PC treatment at d 7 PI, among which T3 and T5 were the lowest (P < 0.05). At d 7 PI, T3 showed the lowest IL-6 (P < 0.05) compared to other treatments. At d 14 PI, the IgG concentration showed a tendency for T3 to be higher (P = 0.077) than the PC treatment. TNF-α was significantly lower (P < 0.05) in the PFA-added treatment groups except for T1 in d 14 PI compared to the PC treatment.In serum IgG, IgA, TNF-α, and IL-6, there was no significant difference between treatment groups at d 0 before P < 0.05) than in PC treatment (Table P = 0.067) than those of the PC treatment. VH:CD showed significantly higher values (P < 0.05) in T3 than PC treatment.VH in T3 and NC treatment was significantly higher  the expression of calprotectin in the ileum mucosa compared to other treatments (Table P < 0.05) in the PFA-added treatment groups than in the PC treatment, and the T3 showed the highest (P < 0.05) among the PFA-added treatment groups.T3 and NC treatment significantly down-regulated  from E. coli, the addition of essential oils showed improved WBCs and neutrophil results compared to pigs fed the basal diet after the LPS challenge due to the anti-inflammatory response to essential oils [An increase in the total number of WBCs indicates the presence of systemic inflammation . Lymphocial oils . E. coli challenge significantly decreased serum IgG and IgA concentrations at d 7 PI compared to the NC treatment, and showed a decreased tendency at d 14 PI. Furthermore, the feeding of a mixture of bitter citrus extract, thymol and carvacrol to pigs infected with E. coli showed significantly higher IgG and IgA concentrations at both d 7 PI and d 14 PI compared to PC treatment. Ahmed et al. [E. coli challenge condition increased the IgG levels of weaned pigs, consistent with the results of the present study. Thymol contained in essential oils can increase goblet cells in the animal ileum and inhibit the growth of pathogenic bacteria [E. coli challenge significantly increased TNF-α at both d 7 PI and d 14 PI. Also, the E. coli challenge increased IL-6 at d 7 PI and showed a high trend at d 14 PI. However, both TNF-α and IL-6 were significantly lower in the T3 group fed a mixture of bitter citrus ext ract, thymol and carvacrol than in the PC treatment, suggesting that this PFA positively modulated the immune and inflammatory responses. TNF-α levels in the blood have been linked to gut inflammatory disease, and a previous study found that a combination of carvacrol and thymol lowered TNF-α mRNA expression in the intestine of weaned pigs, leading to improved gut health and growth performance [E. coli in weaned pigs and have a beneficial effect on health status.Serum IgA and IgG are important immunoglobulins in humoral immunity . In partd et al.  reportedbacteria , 40. Alsbacteria . The mecbacteria . Cytokinbacteria . The probacteria . TNF- α bacteria . This coformance . TherefoE. coli inoculum used in the present study expresses LT and Stx2e, which could induce intestinal morphological lesions such as villus atrophy and leaky gut in weaned pigs [E. coli challenge also reduced the VH:CD ratio, indicating the deterioration of intestinal health. However, supplementation with a mixture of bitter citrus extract, thymol, and carvacrol improved VH, CD, and the VH:CD ratio in pigs compared to those in the PC treatment. Essential oils reduce the number of pathogenic bacteria in the gut through their antibacterial action [Intestinal morphology is commonly assessed by measuring VH, CD, and the VH:CD ratio . Intestined pigs , 48. Thened pigs , 50. In l action . It has l action . In addil action . TherefoE. coli improved intestinal permeability and mucosal damage caused by E. coli and improved tight junction integrity [E. coli-challenged pigs.The intestinal epithelia, especially the internal tight junctions such as occludin and CLDN1 between enterocytes, are critical in maintaining intestinal permeability . Entericntegrity . The dowE. coli, the addition of PFA alleviated the negative effects of E. coli and improved growth performance. When supplemented with thymol and carvacrol, the antibacterial and anti-inflammatory effects of thymol and carvacrol have been shown to prevent diarrhea. When a mixture of bitter citrus extract, thymol, and carvacrol was fed, immunity, intestinal morphology, and tight junction expression were all improved. Therefore, when a mixture of bitter citrus extract, thymol, and carvacrol is added, each PFA effect seems to be added and synergistic, and it is considered that this mixed additive is the most effective PFA among the five additives used in the study. However, it seems that additional studies are needed on the proper amount of each additive and the basic mechanism of PFA against PWD.In weaned pigs infected with


Diet may play an essential role in the aetiology of bladder cancer (BC). The B group complex vitamins involve diverse biological functions that could be influential in cancer prevention. The aim of the present study was to investigate the association between various components of the B group vitamin complex and BC risk.Dietary data were pooled from four cohort studies. Food item intake was converted to daily intakes of B group vitamins and pooled multivariate hazard ratios (HRs), with corresponding 95% confidence intervals (CIs), were obtained using Cox-regression models. Dose–response relationships were examined using a nonparametric test for trend.B1: 1.13, 95% CI: 1.00–1.20). In men, moderate intake of the vitamins B1, B2, energy-related vitamins and high intake of vitamin B1 were associated with an increased BC risk (HR (95% CI): 1.13 (1.02–1.26), 1.14 (1.02–1.26), 1.13 , respectively). In women, high intake of all vitamins and vitamin combinations, except for the entire complex, showed an inverse association (HR (95% CI): 0.80 (0.67–0.97), 0.83 (0.70–1.00); 0.77 (0.63–0.93), 0.73 (0.61–0.88), 0.82 (0.68–0.99), 0.79 (0.66–0.95), 0.80 (0.66–0.96), 0.74 (0.62–0.89), 0.76 (0.63–0.92), respectively). Dose–response analyses showed an increased BC risk for higher intake of vitamin B1 and B12.In total, 2915 BC cases and 530,012 non-cases were included in the analyses. The present study showed an increased BC risk for moderate intake of vitamin B1 (HROur findings highlight the importance of future research on the food sources of B group vitamins in the context of the overall and sex-stratified diet.The online version contains supplementary material available at 10.1007/s00394-022-02805-2. With an estimated 549,393 new cases and 199,922 deaths in 2018, bladder cancer (BC) is the tenth most common cancer worldwide  were also classified as NMIBC. MIBC included carcinomas that invaded into the detrusor muscle (stage T2), carcinomas that invaded into the peri-vesical tissue (stage T3), and carcinomas that invaded adjacent tissues and organs .In addition to information on dietary intake, the BLEND data also included study characteristics , participant demographics , BC pathology (MNIBC and MIBC), and smoking status (current/former/never and pack years), which were all measured at baseline .t test for continuous variables. To assess the association between dietary B group vitamins and BC risk, cox proportional hazard regression analysis was used to obtain hazard ratios (HRs) and corresponding 95% confidence intervals, stratified by study centre. Scaled Schoenfeld residuals were estimated for each covariate to test the proportional hazards assumption. In addition, the appropriateness of the use of the log-normal distribution was tested using a Wald test, and again no evidence of violation was found ; 2 [current heavy smokers (i.e. smoking more than 20 pack-years)]; 3 [current smokers (no information on pack-years)]; 4 [former light smokers (i.e. smokers who ceased smoking over 1 year prior and smoked less than 20 pack-years)]; 5 [former heavy smokers (i.e. smokers who ceased smoking over 1 year prior and smoked more than 20 pack-years)]; 6 [former smokers (smokers who ceased smoking over 1 year prior and no information on pack-years)])) ).p value for linear trend. To understand the relevance of the effect modification, the main interaction terms between B group vitamin consumption (low/moderate/high) and sex, smoking and water were added to the model. P interaction < 0.05 was considered statistically significant where upon all analyses were stratified for the covariate of interest. In addition, sensitivity analyses were performed in which BC cases diagnosed within the first 5 years after recruitment were excluded.The Wald-test was used to derive the Based on a priori hypothesis, additional supplemental analyses were performed by BC subtype (i.e. NMIBC and MIBC). In addition, dose–response analyses of vitamin intake (plotted on the x-axis) and HR  were performed using a nonparametric test for trend.All statistical analyses were performed using Stata14 .Baseline characteristics of the study population are described in Table B1: 1.10, 95% CI: 1.00–1.20) . In addition, for vitamin B1 also high intake was related to an increased BC risk  , except for the entire B group vitamin complex    Table .B3: 0.78, 95% CI: 0.63–0.97, HRB6: 0.79, 95% CI: 0.63–0.98) and for high intakes of vitamin B1, B3, B6, B9, vitamins related to energy metabolism, oxidative stress reduction and DNA stability   Table .Overall, no significant association was observed between dietary vitamin B intake and NNMIBC risk Table .Table 5HB12: 0.89, 95% CI: 0.80–1.00)  .p = 0.03) and a decreased risk for higher intake of vitamin B12 (p = 0.002). No other compound or combination showed a significant association with BC risk.Overall dose–response curves are shown in Fig. p = 0.009) and B3 (p = 0.02) p = 0.02), B6 (p = 0.005), B9 (p = 0.03), B12 (p = 0.0001) and for the vitamins related to the reduction of oxidative stress (p = 0.02)  (Supplementary Table 2). Meat has previously been associated with an increased BC risk due to its pro-carcinogenic components [Vitamin B2 is a coenzyme for many metabolic processes in the body , 41. Altmponents –50, whicmeat-B3, men: 17.83 mg, SD = 15.71, Mmeat-B3 women: 14.54 mg, SD = 13.44, t = 78.49, p < 0.001) (Supplementary Table 2). However, adjustment for the main food sources did not significantly change the findings. Therefore, future research is needed to clarify this gender difference. When analyzing the effect of the vitamins B1, B2 and B3 (i.e. the vitamins related to energy metabolism) together, moderate intake among men showed an increased risk on BC and high intake a decreased risk among women.Vitamin B3 plays a role in energy metabolism, which is essential to maintain cellular metabolism and respiration  and it iOxidative stress is an imbalance in the cell which leads to DNA damage , therebyB9, men: 194.27 µg, SD = 128.66, MB9 women: 182.12 µg, SD = 96.28, t = 37.76, p < 0.001).Vitamin B9 plays a pivotal role in cell metabolism which is necessary for DNA synthesis and repair, as well as for methylation . PreviouB12, meat men: 3.86 µg, SD = 4.30, MB12 meat women: 3.35 µg, SD = 3.85, t = 43.03, p < 0.001) (Supplementary Table 2), it might be argued that the observed effect in men are caused by the earlier mentioned negative effects of other (micro)nutrients in meat [Vitamin B12 is essential for the reduction of DNA damage . Experim in meat . HoweverAlthough BLEND is one of the largest known pooled cohort studies investigating the association between dietary B group vitamin intake and the risk of developing BC, allowing for detailed analyses with enough statistical power, it has several limitations. First, limited information was available for possible BC risk factors, such as body mass index, physical activity, socioeconomic status, and occupational exposures. Nevertheless, current literature shows only a small proportion of BC cases can be attributed to these factors , 64–66. A second limitation arises from the use of FFQs, which could lead to recall bias, systematic and random error when estimating vitamin intake. However, since the dietary intake of all included studies were validated, recall bias has likely only played a minor role. In addition, measurement error could be negligible, considering our large sample size.Thirdly, although people are less likely to change their dietary habits at an older age, most of the included studies only measured their participants at baseline and we were, therefore, unable to take possible changes of dietary habits over time into account. This could have led to misclassification of long-term exposure . HoweverFourthly, most of the included studies did not provide information on supplement use. Therefore, we were unable to take supplemental vitamin intake into account, which may have led to an underestimation of the true effect of B group vitamins.Fifthly, a single database was used for the conversion of food into nutrient intake. Since the food composition of similar food items and the food fortification may differ between different countries, the use of country specific food composition tables might be more accurate. Previous studies, however, showed that the use of a common food composition database advantages over the use of country-specific food composition databases in that errors are consistent between the countries . In addiBesides, results obtained from cohort studies on diet and cancer risk cannot always rule out the possibility of reversed causality. Since there is no evidence that people are likely to alter their diet in the period before BC diagnosis, we decided to not exclude study participants who received a BC diagnosis within a short period of follow-up.p value adjustments should have been applied. However, it previously has been argued that the use of Bonferroni p value adjustments is impractical and likely too conservative when testing a priori hypotheses [n = 10) the significance level would have been 0.005. In that case, most of the observed associations between the vitamin B intake and BC risk would still be statistically significant.Finally, in view of multiple testing, it could indeed be debated whether, for instance, Bonferroni potheses . Since wThe present study showed a slight increased BC risk for moderate intake of vitamin B1. In men, moderate intake of the vitamins B1, B2 and the vitamins related to energy metabolism and high intake of vitamin B1 were found to be associated with an increased BC risk. In women, high intake of all vitamins and vitamin combinations, except for the entire complex, showed to have a protective effect. These findings may be helpful for informing BC prevention strategies. It should be noted, however, that dietary recommendations on vitamin B intake carefully consider the food sources from which this nutrient is retrieved. In addition, future studies should focus on nutritional patterns and look deeper into B group vitamins’ interactions with other nutrients.Supplementary file1 (PDF 67 KB)Below is the link to the electronic supplementary material.


IntroductionCryptosporidium spp. plays an essential role in causing diarrhea, malnutrition, and poor cognitive function, especially in children. This study was conducted to identify the frequency of Cryptosporidium cases and other parasitic agents. Gastrointestinal parasitic infections are one of the global health concerns in developing countries like Bangladesh. Among them, MethodsCryptosporidium spp. A polymerase chain reaction (PCR) was performed to determine the SSU rRNA and gp60 gene of Cryptosporidium. A cross-sectional observational study was conducted among 219 hospitalized children with diarrhea. The conventional microscopic technique was applied for parasitic detection. Particular staining (modified Ziehl-Neelsen) procedure was performed to identify oocysts of ResultsGiardia duodenalis (2.3%), ova of Ascaris lumbricoides , Trichuris trichiura (0.5%), and both A. lumbricoides and T. trichiura (0.9%) were identified in samples through wet mount preparation. The distribution of Cryptosporidium spp. as detected by the staining method and nested PCR was 1.4% and 4.1%, respectively. Cysts of ConclusionCryptosporidium infection are unsafe water, lack of regular hand washing, and insufficiency of exclusive breastfeeding. This study reports, presumably for the first time, the detection of Cryptosporidium oocysts in Chattogram metropolitan city of Bangladesh.Factors independently associated with  Diarrhea is the passage of three or more liquid or loose stools per day or if the individual experiences more frequent passage than usual -3. It isA single episode of moderate-to-severe diarrhea has significant repercussions on mortality and linear growth among survivors, facilitating the hazards of growth retardation, ill health, and cognitive impairment in the pediatric community -19. DiarEtiological agents of diarrheaShigella, nontyphoidal Salmonella (NTS), Diarrhoeagenic Escherichia coli (DEC), Vibrio cholerae, Campylobacter, and Yersinia spp. [Cryptosporidium spp., Giardia duodenalis,Entamoeba histolytica, Blastocystis hominis, Dientamoeba fragilis, Ascaris lumbricoides, Trichuris trichiura, Ancylostoma duodenale, and Necator americanus [The rotavirus is the most prevalent among numerous viral, bacterial, and parasitic agents causing pediatric diarrhea ,30. Somenia spp. ,31,32. Ania spp. -35, inteericanus -38.Cryptosporidium as a protozoan diarrheal agentCryptosporidium is second only to rotavirus as the leading cause of moderate-to-severe diarrhea [Cryptosporidium-ascribable cases is about 2.9-4.7 million in children under two years, the sub-Saharan African and South Asian territories face tremendous challenges in combating this infection [An intracellular apicomplexan protozoan parasite belonging to the genus diarrhea -41. The diarrhea ,42,43. Anfection . Cryptosporidium, the ubiquitous coccidian parasite, has over 40 established species, and of these, 20 species and subtypes account for the vast majority of human gastrointestinal infections worldwide. Additionally, C. hominis and C. parvum are the leading culprits causing cryptosporidiosis globally [Cryptosporidium spp. has a low infective threshold with a robust oocyst that survives adequately in moist, ambient environments and is resistant to many commonly available disinfectants such as chlorine [Various species of this parasite have been identified and distinguished regarding host range and public health concerns . Thus, Cglobally , 46. Trachlorine ,47.Effects of CryptosporidiosisCryptosporidium infection. However, in AIDS patients, the gastrointestinal parasite distribution is more complex and extensive [Diarrhea associated with cryptosporidiosis has been linked to three fundamental mechanisms ,48,49: ixtensive ,57.Cryptosporidium in its "Neglected Disease Initiative 2004" by WHO, it's one of the significant causes of diarrhea in children [The infection ranges from asymptomatic, self-limiting diarrhea to chronic -60. The children . Severalchildren . Recent children ,65.Studies on CryptosporidiumCryptosporidium is more prevalent in developing countries than in developed ones [Cryptosporidium, enterotoxigenic E. coli, and Shigella [Epidemiologic studies have demonstrated that ped ones ,40,42,66ped ones . A meta-ped ones . The sigShigella .Cryptosporidium infection was widespread (77%). The study also highlights a close correlation between poverty and stunted growth during the first two years of life [Cryptosporidium oocyst was detected in 9.2% of cases, where the infection was highest among children aged less than two. Moreover, that study also observed the infection decreases with age [Cryptosporidium, where these protozoan oocysts were detected in 2.2-7.7% of cases [Cryptosporidium oocysts were detected in 3.8-8% of pediatric and immunocompromised patients [In a longitudinal cohort study on 392 Bangladeshi slum-dwelling children (in the first two years of life) performed from 2008 to 2014,  of life . A survewith age . In a prof cases . In Tehrof cases . Anotherpatients .Diagnostic modalitiesThe diagnostic procedure comprises the microscopic examination of the fecal sample by wet mount and staining by modified Ziehl-Neelsen (mZN) staining  or auramCryptosporidium are documented targeting its species [Cryptosporidium outer wall protein (COWP), 70-kDa heat shock protein (HSP 70), thrombospondin-related adhesive protein (TRAP-C2), dihydrofolate reductase (DHFR), and actin genes [SSU rRNA and gp60 are the most common genetic markers for Cryptosporidium species identification and subtype determination [SSU rRNA gene is considered extensively used in genotypic differentiation between infections belonging to both humans and animals. In addition, the 60-kDa glycoprotein (gp60) gene possesses highly variable regions that permit many intraspecies sequence heterogeneity. These sites are crucial to determining many C. parvum and C. hominis subspecies [Various genes of  species , such asin genes ,79-81. Smination -84. The bspecies ,57. The SSU rRNA and the gp60 gene. The study would help the clinician diagnose the case properly by exploring the actual etiology of protozoa-related diarrheal illness and indirectly minimizing the empirical use of antibiotics to treat parasitic diarrhea, thus reducing the multi-drug resistant problem in Bangladesh. In the current study, microscopy by wet mount preparation, concentration technique , and staining procedure (mZN) were applied to all samples. Subsequently, nested PCR was performed targeting Objectives of the studyCryptosporidium microscopically by mZN staining technique, iii) detection of the presence of Cryptosporidium spp. by nested PCR, and iv) comparison of the result of microscopy with that of PCR.The objectives of the study include i) identification of intestinal parasites microscopically by wet mount preparation, ii) identification of https://www.preprints.org/manuscript/202203.0108/v1).This paper was previously posted to the Preprints preprint server on March 7, 2022 , Chattogram, Bangladesh, from July 2019 to June 2020. The study population included indoor and outdoor pediatric diarrheal patients of Chittagong Medical College Hospital and Chattogram Maa-O-Shishu Hospital Medical College, Chattogram, Bangladesh.The study received ethical approval from the Institutional Review Board of Chittagong Medical College Hospital (CMCH), Chattogram, Bangladesh . The research method was strictly aligned with the Declaration of Helsinki. Participation and reporting were hinged on consent forms signed by patients/guardians of the pediatric diarrheic patients (children up to 18 years) before administering the questionnaire. The respondents were informed correctly using the participant's information sheet about their rights and all the relevant aspects of the study, including its aim and interview procedure. No patient was older than 18 years.Sample size calculation and techniqueas a nonprobability, purposive type of sampling. n= z2pq/d2==123.2. The n is the sample size. z is the confidence interval (95%) z=1.96. p is the pre-estimated prevalence of 9.2% obtained from a study performed by Ahmed et al. conducted in Dhaka, Bangladesh . d is a Eligibility criteriaThis study was conducted on both inpatients and outpatients. Bangladeshi pediatric patients up to 18 years) suffering from acute watery and persistent diarrhea (≥14 days) irrespective of their social status ,86,87, w8 years sPreparation of questionnaireA questionnaire was prepared and modified from the study conducted by Tombang et al. in Cameroon . The queSample collectionAfter taking consent, the patient's history details, including demographic information and clinical findings, were recorded in a predesigned case record form. Then stool sample was collected in a clean, leak-proof, wide-mouth container appropriately labeled with the patient's name, age, time of collection, and identification number. It was then transported to the Department of Microbiology, and some portions of each sample were transferred to the Eppendorf tube and refrigerated at -80ºC to perform the molecular method . The remLaboratory procedureCryptosporidium oocysts were identified as red, small (4-6 µm), characteristically round or slightly ovoid, and acid-fast oocysts against a blue background [All concentrated samples were subjected to wet mount preparation by both saline and iodine preparation , respectively, on a glass slide and employing a coverslip over the smear) ,94 and sckground .Procedure of PCRProcess of DNA ExtractionSSU rRNA and gp60Gene [SSU rRNA gene, and the second pair of primers were used for second-round PCR to amplify a 240 bp sequence. For the gp60 gene, first set primers  and the procedure was performed at room temperature (20-25°C). The purified DNA in the tube was preserved at -20°C for further use. Primers used for gp60Gene ,97 are grs Table  were usePreparation of Reaction MixtureSterile micro-centrifuge tubes (1.5 ml) were taken and labeled with the date and identification number. Primer tubes were centrifuged for a few seconds. Then it was vortexed for 15 seconds and diluted with nuclease-free water to make a 1:10 dilution. For each sample, a total of 20 µl of the mixture was prepared by mixing 10 µl of master mix , 1 µl forward primer, 1 µl of reverse primer, 2 µl DNA template, and 6 µl of nuclease-free water.Cyclic Condition used for Nested PCR [sted PCR SSU rRNA Gene of Cryptosporidium Species: In the first round PCR, initial denaturation at 94°C for five minutes was followed by 30 cycles of denaturation at 94°C for 45 seconds, primer annealing at 450C for two minutes, extension at 72°C for 1.5 minutes, and a final extension at 72°C for 10 minutes. In the second round PCR, initial denaturation at 94°C for five minutes was followed by 35 cycles of denaturation at 940C for 30 seconds, primer annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 10 minutes. This is shown in Table gp60 Gene of Cryptosporidium Species: In the first round PCR,initial denaturation at 94° C for five minutes was followed by 35 cycles of denaturation at 94° C for 30 seconds, primer annealing at 55°C for 45 seconds, extension at 72°C for one minutes, and a final extension of 72°C for 10 minutes. In the second round PCR, initial denaturation at 94° C for five minutes was followed by 30 cycles of denaturation at 94° C for 30 seconds, primer annealing at 55°C for 30 seconds, extension at 72° C for 30 seconds, and a final extension at 72° C for 10 minutes. This is shown in Table Cryptosporidium spp. when PCR product of 240 bp could be detected for SSU rRNA gene. Samples were detected as PCR positive for Cryptosporidium spp. when 350 bp could be seen for the gp60 gene. The flowchart of nested PCR is given in Figure In the nested PCR, the amplified product of the first round of PCR is used as the template. The second set of primers was added to the reaction mixture in the second amplification (nested PCR). The PCR reactions were conducted in a thermal cycler . The amplicon size was determined by comparing the position of the amplicon concerning that of a 100 bp DNA ladder loaded in the adjacent well and simultaneously electrophoresis. Samples were scored as PCR positive for Data analysisCryptosporidium infestation. The regression model was adjusted by age and gender.A predesigned questionnaire systematically recorded all the relevant information (history), socio-demographic history, clinical findings, and laboratory findings of every case. The data were analyzed using IBM SPSS Statistics for Windows, Version 25.0  and Stata Statistical Software: Release 15 . Statistical analysis was done by standard statistical procedure; Microsoft Excel  was used to prepare graphs and charts. A p-value <0.05 was considered significant. The multivariate logistic regression model was used to see the association between independent factors and Distribution of the study population according to their age and sexCryptosporidium. The majority of the study population belonged to the age group of 1-5 years (47%), followed by the <1 year age group (30.1%)  samples. Moreover, other parasites like helminthic eggs were found in some samples. Ova of Ascaris lumbricoides, Trichuris trichiura, and mixed infection were detected in three (1.4%), one (0.5%), and two (0.9%), respectively.All fecal samples were first examined for direct microscopic examination by saline and iodine preparation. Table Identification of Cryptosporidium by mZN StainingCryptosporidium oocysts  samples  and gp60 genes (350 bp) are shown in Figure Nested PCR detected s Figure . The banCryptosporidium spp. was more in the 1-5 years age group, and male predominance was observed, but both were statistically non-significant. Table  Cryptosporidium. Here the Chi-square test was done, and it was found to be statistically significant (p<0.001). The sensitivity and specificity of Cryptosporidium Spp. microscopy was found to be 33.3% and 100%, respectively, taking the PCR method as the gold standard.The prevalence of Cryptosporidium infectionMultivariate logistic regression to determine the independent factors associated with Cryptosporidium infestation, OR= 3.91; 95% CI ; p=0.049, lack of regular hand washing had a chance of being infected with Cryptosporidium infestation by 9.68 times higher compared to traditional hand washing, OR= 9.68; 95% CI  and insufficiency of exclusive breastfeeding have a risk to infected with Cryptosporidium infestation by 3.82 times higher compared to feed breast milk exclusively, OR=3.82; p=0.049. But other factors like female gender , rural residence , and H/O having a pet  have no significant association with the infection. Most of the study population (64.8%) drink safe water, and 70.3% use regular hand washing.Table Cryptosporidium spp. and other parasites are liable for diarrhea, especially among children in developing countries [Intestinal parasites such as ountries . Though ountries ,100-105.Giardia duodenalis positive by wet mount preparation among 219 samples. The prevalence of Giardia duodenalis infection among 0-15 years Portuguese children was 1.9%, as estimated by direct microscopic examination. Nevertheless, when monoclonal ELISA techniques were utilized, the rate increased to 6.8% [Giardia duodenalis infestation was found in the younger group (0-5 years), and there was no difference observed between the sexes [Giardia cysts were found in 4.14% of cases [Giardia duodenalis positive samples were found in 6.01% of cases among school-going children [Giardia cysts were found in 5% of cases by wet mount [Giardia was detected in 15.5% by immediate wet mount preparation [In this study, 2.3% cases were found  to 6.8% . Furtherhe sexes . A studyof cases . In a slchildren . In Sikket mount . About 8et mount . Anotherparation , much hiAscaris lumbricoides, Trichuris trichiura, and mixed infection were detected in three (1.4%), one (0.5%), and two (0.9%), respectively. A study reported 0.9% Trichuris trichiura [Ascaris lumbricoides were found in hospitalized pediatric diarrheic children (<5 years), according to our study. Another study demonstrated 0.6% of Trichuris trichiura ova, but Ascaris lumbricoides was 9% [Ascaris lumbricoides and Trichuris trichiura ova as 1.5-8.2% and 0.8-0.9%, respectively [In wet mount film, the current study showed that ova of richiura , but no s was 9% . Other sectively ,113. In Cryptosporidium was found positive in 1.4% of diarrheal samples by mZN staining. A surveillance study on children under 12 years old in Tehran was done where the prevalence rate of the protozoa was 1.19% [Cryptosporidium oocyst [Cryptosporidium spp. from outpatients of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR'B) of 4.96% [Cryptosporidium infection was 13.4% in microscopy (mZN staining), having the highest detection rate in the 31-60 months group [Age and sex-specific vulnerability are essential for the prevalence of diarrheal illness, where various factors can contribute to this. These may include biochemical factors like hormones, enzymes, specific proteins, genetic or immunologic factors, food habits, culture, etc. . Cryptosas 1.19% . Anotherm oocyst . A studyof 4.96% . In Malaof 4.96% , 116. Inhs group , which iIn this study, there were some limitations. There was low trophozoite and/or cyst and oocyst detection because of the empirical use of antimicrobials, especially antiprotozoal and anthelminthic. These medications are often self-medicated or given by non-graduate medical practitioners. Additionally, this study could not collect samples from the same patient on multiple occasions. Moreover, microscopic findings depend on individual expertise. Direct microscopic examination after mZN staining of Cryptosporidium relies on morphologic recognition of small-sized oocysts that may be scanty in number, which may be challenging to visualize. Again, they could be inconsistently stained, and misdiagnosis may happen. Therefore, this method is impractical to standardize as it is influenced by the microscopist's individual skills of the microscopist involved . MoreoveCryptosporidium spp. was found positive in 4.1% of samples by nested PCR. In one study, the prevalence of Cryptosporidium was 4.8% among children below five years of age with diarrhea [Cryptosporidium positive cases in 6.3% of cases [diarrhea . A longiof cases . A prospof cases . Anotherof cases . Improveof cases . Anotherof cases ,57.Cryptosporidium were detected in the same age group (one to five years) 7/103 (6.79%), followed by <1-year age group 2/66 (3.03%), which was not statistically significant according to the p-value (p=0.376). Moreover, we revealed from our study that all Cryptosporidium-positive cases were children <3 years old. In another study, it was seen that the highest prevalence of Cryptosporidium infection was in the age group of three to four years (14.3%), followed by <1-year age group (4.5%) [Cryptosporidium infection was predominant among children <5 years (22%) [Moreover, the study was performed in urban rather than rural or slum areas. PCR is considered a more sensitive and specific diagnostic method, but some limitations remain such as PCR bands were not confirmed by sequencing because of structural and financial difficulty. There is a list of PCR inhibitors in stool samples, namely lipids, hemoglobin, bilirubin, bile salts, polysaccharides from mucous, bacteria, and food degradation products, which can also affect the result of amplification. Commercial kits, including extraction columns, have been used to purify the DNA to minimize the effect of DNA inhibitors. Despite using such a kit, false-negative results may happen ,121. Thep (4.5%) . A studyrs (22%) . Most inrs (22%) .Children (1-5 years) develop a habit of putting unwashed hands, toys, and other objects inside their mouths. Moreover, their compromised immunological status and poor hygiene practices are susceptible to intestinal infections, especially during this period ,123. TheCryptosporidium Spp. Infection was similar to Hawash et al. [Cryptosporidium microscopy was 33.3%, and specificity was 100% in the present study. Two other studies found similar validity [In this study, male predominance was observed in the case of h et al. , but theh et al. ,130. Thivalidity ,132.Cryptosporidium remains one of the common causes of spreading cryptosporidiosis [Cryptosporidium infection that is statistically significant , which agrees with another study conducted in Pakistan [Cryptosporidium oocysts are highly resistant to numerous disinfectant agents [Globally, foodborne cryptosporidiosis has been recognized as the main reason for such infestation -135. Addridiosis -138. Thiridiosis ,140. Theridiosis . United ridiosis . The resPakistan . Anothert agents -145 and t agents . In addit agents ,147,148.Another salient factor behind protozoal diarrhea is inadequate hygiene practices like handwashing ,149-151.Cryptosporidium infection [Undoubtedly, breastfeeding has a widespread impact on children's health conditions, and there remains a significant correlation between breastfeeding and diarrhea -160. Ournfection ,161. It nfection ,161,162.nfection ,163,164.nfection -167, we nfection ,168. Thenfection ,169,170.The present research can be brought into play that the PCR approach demonstrated better results than the traditional microscopic procedure as it yields more pragmatic outcomes. This procedure also denotes that it can adequately diagnose the parasitic causes of diarrhea among children. Consequently, it brings considerable advantages to the treatment of pediatric diarrheal patients in Bangladesh and other low and middle-income countries (LMICs). As a result, irrational prescribing and self-medication of antimicrobials hopefully be minimized. Thereby, it is expected that the rate of antimicrobial resistance will be cut down. Moreover, this research provides scientific data to detect other protozoal parasitic diarrhea through mentioned methods. Additionally, this study enables information regarding the prevalence of protozoan causes of diarrhea in the locality or region of Bangladesh.Cryptosporidium spp.; oocyst was successfully proved positive by molecular assay (PCR), which recommends the practice of PCR approaches and conventional microscopic procedures to overthrow the practice the diagnostic drawbacks. So, the staining method should be combined with wet mount preparation as a routine examination for stool to detect and diagnose the parasite effectively. Moreover, molecular analysis is also recommended where facilities are available. This would help to diagnose the protozoa precisely. Further molecular analysis like DNA sequencing can be done, which will provide us with specific information about the species and genotypes, hence, lending the researcher a transparent idea about the epidemiology to identify risk factors, mode of transmission, pathogenicity, and genetic diversity, etc.The samples were identified positively by mZN staining for Cryptosporidium spp. mZN is much more cost-effective in considering those countries that have poor budgetary allocation and access to healthcare. Finally, the practice of regular hand washing and consumption of boiled water is an essential factor in minimizing parasitic infection, including cryptosporidiosisParasites are one of the critical causes of pediatric diarrheic disease in the Bangladeshi community. mZN staining should be introduced as a routine-based detection method for


P. aeruginosa was determined by Minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC), Growth curves, alkaline phosphatase (AKP) activity, crystal violet method, scanning electron microscopy (SEM), membrane potential, PI, cell contents leakage. Results showed that the antibacterial activity of FCs increased after esterification, and the antibacterial activity significantly increased and then decreased with the extension of the alkyl chain of the FCs. Hexyl ferulate (FC6) showed the best antibacterial activities against E. coli and P. aeruginosa . And Propyl ferulate (FC3) and FC6 showed the best antibacterial activities S. aureus and B. subtilis . In addition, the growth, AKP activity, bacterial biofilm, bacterial cell morphology, membrane potential and cell contents leakage of P. aeruginosa after different FCs were investigated, which found that FCs could damage the cell wall of P. aeruginosa and showed different effects on the P. aeruginosa cell biofilm. FC6 showed the best inhibition on the biofilm formation of P. aeruginosa cells, which caused the surface of P. aeruginosa cells to be rough and wrinkled. Some P. aeruginosa cells showed aggregation and adhesion, even rupture. The membrane hyperpolarization was obvious, which appeared as holes, leading to cell contents leakage (protein and nucleic acid). All these results concluded that the antibacterial activities FCs against foodborne pathogens depended on different fatty alcohol esterification of FA. FC6 showed the best inhibition on P. aeruginosa due to its effect on P. aeruginosa cell walls and biofilms and the leak of the cell contents. This study provides more practical methods and a theoretical basis for giving full play to the bacteriostatic effect of plant FA.Ferulic acid (FA) is a natural antibacterial agent rich in plants, FA has excellent antioxidant and antibacterial properties. However, because of its short alkane chain and large polarity, FA is difficult to penetrate the soluble lipid bilayer in the biofilm to enter the cell to play an inhibitory role, limiting its biological activity. To improve the antibacterial activity of FA, with the catalytic condition of Novozym 435, four alkyl ferulic acid esters (FCs) with different alkyl chain lengths were obtained by fatty alcohols (including 1-propanol (C3), 1-hexanol (C6), nonanol (C9), and lauryl alcohol (C12)) modification. The effect of FCs on  Globall10H10O4) is a natural antibacterial agent rich in plants such as coffee, rice husk, vanilla bean, wheat bran and rice bran. As a phenolic acid, FA has been proven to have various biological activities that can reduce the risk of serious diseases such as diabetes, cholesterol, heart disease and cancer, and it has excellent antioxidant and antibacterial properties  and their mode of action were investigated.However, because of its short alkane chain and large polarity, FA is difficult to penetrate the soluble lipid bilayer in the biofilm to enter the cell to play an inhibitory role, limiting its biological activity . It was 2.2.1.Ferulic acid, 1-propanol (C3), 1-hexanol (C6), nonanol (C9), and lauryl alcohol (C12) with >98% purity were purchased from Aladdin Biochemical Technology Co., LTD. . Novozym 435 (A Candida antarctica lipase B immobilized on acrylic resin) was purchased from Novozymes Biotechnology LTD. (Denmark). 4 Å molecular sieve and n-hexane (moisture ≤0.02%) were purchased from Guangfu Fine Chemical Research Institute . The alkaline phosphatase (AKP) kit and BCA protein quantitative kit were purchased from Dalian Meilun Biotechnology Co., LTD. . P-Iodonitrotetrazolium Violet (INT) was purchased from Macklin Biochemical Co., LTD. . 2.5% glutaraldehyde was purchased from Leagene Biotechnology Co., LTD. . Rhodamine 123 was purchased from Biotopped Life sciences. . Propidium iodide (PI) was purchased from Saiguo Biotechnology Co., LTD. . Ferulic acid, 1-propanol, 1-hexanol, nonanol, lauryl alcohol, Novozym435 and other organic solvents were treated with a 4 Å molecular sieve for at least 24 h before use.2.2.Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923 and Bacillus subtilis were gift samples from the Institute of Microbiology Heilongjiang Academy of Sciences. The strains preserved in the tube were removed from the −80°C refrigerator and thawed overnight in a 4°C refrigerator. All strains were activated by Tryptone Soy broth (TSB) medium  at 37°C in an orbital air shaking bath (180 rpm) for 24 h and maintained on TSA slants at 4°C.2.3.In this experiment, four fatty alcohols  were selected for esterification with FA to obtain four kinds of FCs with different alkane group lengths. The method described by 2.4.1H-NMR determination and 15 mg for 13C-NMR determination. After the FCs were dissolved in 0.6 ml deuterium chloroform (CDCl13), the solution was quickly transferred to a clean and dry nuclear magnetic tube  for determination. The propyl ferulate (FC3) hexyl ferulate (FC6)nonyl ferulate (FC9) Lauryl ferulate (FC12) of 1H NMR and 13C NMR were all 400 MHz and 100 MHz, respectively . FCs were weighted with 4 mg for ectively .Fourier Transform high-resolution mass spectrometry  was used to analyze FCs molecular weight. Five hundred micro liter of methanol was added to a 1.5 mL centrifuge tube, and a trace of the tested sample was dipped into a capillary tube and then dissolved in chromatograde methanol. Methanol solution containing FCs was extracted with an injection needle and injected into the pump for determination. The ESI-MS(m/z) of FC3, FC6, FC9 and FC12 were 237.1[M + H] + ，279.2[M + H]+, 321.2 [M + H]+, 363.3[M + H]+, respectively. The NMR results of FC3, FC6, FC9 and FC12 were shown in 2.5.P. aeruginosa were inoculated in TSB medium and cultured at 37°C for 14–16 h. The culture solution was centrifuged, and the collected bacteria were washed with sterile 0.01 mol/l PBS (pH 7.4). The cultures obtained were diluted with TSB to obtain a 0.5 MCF (1 × 108 CFU/ml) and diluted 10 times.The method described by Ninety six-well microplates were filled with 160 μL TSB medium, and then 20 μL of diluted bacterial strains and 20 μL of each FCs concentration were added to the wells. Two controls were used in two columns that contained 160 μL TBS medium and 20 μL of diluted bacterial strains: one with 20 μl of sterile distilled water and another with 20 μl of Anhydrous ethanol. Tests were carried out in triplicate. After being cultured at 37°C for 14–16 h, 10 μL 0.4% (w/v) INT was added to the wells as an indicator and cultured at 37°C for 30 min in the dark. The colorless tetrazolium salt acts as an electron acceptor and is reduced to a red-colored formazan product by biologically active organisms. When bacterial growth was inhibited, the solution in the well remained colorless after incubation with INT. When bacterial growth was inhibited (no evidence of growth), MIC values were recorded as the minimal concentration of FCs concentration. For MBC determination, 50 μl from each well without INT was spread on TSA agar plates and incubated at 37°C for 24–48 h. At least two duplicate tests were performed with each strain and antibacterial compound.2.6.P. aeruginosa were inoculated in TSB medium and cultured at 37°C for 14–16 h in an orbital air shaking bath (180 rpm). The culture solution was centrifuged, and the collected bacteria were washed with sterile 0.01 mol/l PBS (pH 7.4). The cultures obtained were diluted with TSB to obtain a 0.5 MCF (1 × 108 CFU/ml) and diluted 100 times. FA, FC3, FC6, FC9 and FC12 were added, respectively, until the final concentration was 0.4 mg/ml, and no drug was added as control. The absorbance values were measured every 2 h at 600 nm after 24 h culture at 37°C. The growth curve was plotted with the culture time as the abscissa and the OD600 value as the ordinate.The method described by 2.7.The method described by 2.8.The method described by i and E0 are absorbance values treated with or without FCs, respectively.where E2.9.The method described by 2.10.This step was performed by slightly modifying the method of 2.11.2.11.1.The method described by 2.11.2.FA, FC3, FC6, FC9, and FC12 were, respectively, added into the bacterial suspension (As described in 2.6) to reach the final concentration of 0.4 mg/ml, with no drug added as the control. The supernatant was cultured at 37°C and 160 rpm for 2, 4, 6, and 8 h, filtered by 0.22 μm microporous membrane. The protein content was determined by BCA protein quantitative kit.2.11.3.The method described by 2.12.p < 0.05).All experiments were performed in triplicate. Data were presented as mean ± SD. Data were analyzed for analysis of variance (ANOVA) by SPSS software to determine the least significant differences   . In this3.2.P. aerugino as the indicator pathogen, the antibacterial activity of 0.4 mg/ml FA, FC3, FC6, FC9 and FC12, respectively, on the growth were investigated, and the results are shown in P. aeruginosa of the control group entered the logarithmic growth stage after culture for 4 h, and showed an increasing trend to the stable stage until 16 h. In the concentration of 0.4 mg/ml FC6 (1 × MIC) treatment group, the growth of the P. aeruginosa was completely suppressed—the growth of P. aeruginosa treated with FC3 was inhibited, and the logarithmic growth period was prolonged. However, FC9 and FC12 showed insignificant growth inhibition on P. aeruginosa. The effect of FCs on the growth curve of P. aeruginosa further verified the antibacterial activity of FCs against P. aeruginosa. P. aeruginosa (G-) and S. aureus (G+) were more sensitive to FC6, with MIC values of 0.4 mg/ml. E. coli and L. monocytogenes, and the results showed that C6 had the best bacteriostatic activity, which was consistent with the results of this study. The possible explanations are as follows: the antibacterial activity of FCs is closely related to the species of bacteria and the length of the carbon chain of FCs (E. coli(G-) (MIC = 0.5 mg/mL), P. aerugino(G-) (G-) (MIC = 0.4 mg/ml), S. aureus(G+)(MIC = 0.4 mg/mL) was more sensitive to FC6 than B. subtilis(G+)(MIC = 1.1 mg/mL). E. coli, P. aerugino) than Gram-positive bacteria.Using n of FCs . We also3.3.P. aerugino after being treated by antibacterial agents FA, FC3, FC6, FC9 and FC12. After FA and different FCs were treated with the same inhibitory concentration of P. aerugino, AKP activity increased with the extension of culture time. It should be noted that FC6-treated P. aerugino rapidly increased AKP activity within 2 h and showed the largest AKP activity at 8 h, with an activity of 2.66 U/gprot. Based on these results, it was speculated that FCs increased the permeability of the cell wall and damaged the integrity of the cell wall, thus allowing AKP to leak into the cell culture medium. This observation agrees with a recent review by P. aerugino cell wall, resulting in content leakage. Bearing in mind that the threats posed by this and other foodborne pathogens are still major threats to public health, it will be interesting to investigate synergistic antibacterial effects in the future.Cell walls can effectively prevent foreign substances, such as antibiotics and bacteriostatic agents, from entering cells. AKP exists between the cell membrane and the cell wall and plays an important role in the metabolism of substances in organisms. Therefore, the integrity of the cell wall can be reflected by detecting AKP outside the cell . Figure 3.4.P. aerugino biofilm at the same concentration. Floating motion plays an important role in the pathogenic ability of P. aerugino, which can initiate the adhesion of bacteria to the adhesive surface and contribute to the formation of P. aerugino biofilm. Therefore, these partial results suggest that the inhibition of this motion may be one of the reasons for the reduction of biofilm formation(p-coumaric acid (p-CA) against pathogenic Salmonella enteritidis by binding to proteins involved in flagella motility. In addition to maintaining high antibiofilm effectiveness in simulated conditions, both phenolics were forwarded as candidates for modifying this notable pathogen’s phenotypic and molecular structures. This report supports our observation that further studies will prove valuable in enhancing the pathogen-inhibiting applications of FA.The inhibition of FA, FC3, FC6, FC9, and FC12 on FA biofilm formation was evaluated by crystal violet staining. The results are shown in formation. Further3.5.P. aerugino cell wall and membrane, the morphological changes of P. aerugino cells treated with an antibacterial agent for 2 h were observed by scanning electron microscopy. As shown in P. aerugino cells in the control group  with 1 × MIC concentration to deform the bacterial cells and gather them together in large numbers, causing damage to the bacterial membrane. This agrees with the current study’s findings, and thus future experiments may need to investigate if there is a molecular basis for these processes, as previously posited by In order to further observe the effects of FA, FC3, FC6, FC9 and FC12 on the integrity of ol group  were com and FC9 , C, E, t by FC12  showed n by FC12  showed r3.6.P. aerugino. As shown in P. aerugino, and FC3 and FC6 have significant effects on the membrane potential of P. aerugino. Control group’s fluorescence intensity of 340.33 ± 18.77 on average, after FC3 and FC6 treatment, and the fluorescence intensity increased to 453.67 ± 10.41 and 564.33 ± 14.01 respectively, compared with the control group, with a growth of 33 and 66% respectively, compared with the control group, both were extremely significant differences. The results showed that FC6 had more serious effects on P. aeruginosa. Membrane potential is the potential difference between the inside and outside of a biological cell membrane, and maintaining normal membrane potential is of great significance for ATP production and cell function maintenance . Rhodami3.7.3.7.1.P. aerugino membrane by damaging its integrity and preventing further disease spread. These observations were similar to earlier findings that FA and other phenolics have growing bactericidal effects against foodborne pathogens . As shown in P. aerugino, the leakage of nucleic acid was significantly higher than that of the control group and other FCs, and the absorbance was 2.48 times that of the control group. Combined with P. aerugino cells to varying degrees, indicating that FCs change the permeability and integrity of cell membrane, which is consistent with the above results of cell integrity. P. aerugino cells, resulting in nucleic acid and protein leakage.Nucleic acid, protein, Na4.E. coli, P. aerugino, S. aureus, and B. subtilis) showed that after esterification, the antibacterial activity increased, but with the extension of the alkyl chain of antibacterial compounds, the antibacterial activity increased first and then decreased. FCs can destroy the cell wall of Pseudomonas aeruginosa and affect the biofilm of P. aerugino cells to varying degrees. Among them, FC6 inhibits the formation of the biofilm of P. aerugino cells the most, making the surface of P. aerugino cells rough and wrinkled, and showing aggregation and adhesion. Some cells even rupture. The integrity of the cell membrane is damaged, and the contents of the cell  are leaked. The effect of FCs on the biofilm of P. aerugino cells further indicated that the antibacterial activity of FA increased after esterification, but the antibacterial activity increased first and then decreased with the extension of the alkyl chain of antibacterial compounds. However, the application of FCs in the food system needs further research.Antimicrobial resistance posed by foodborne pathogens makes them a constant threat to global public safety, prompting studies into novel ways of overcoming this challenge. In this study, MIC and MBC values of the 4 pathogenic bacteria.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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