Dirofilaria immitis, a filarioid nematode of dogs and other carnivores, is widespread in the USA and the world. Over 20 different mosquito species serve as intermediate hosts of D. immitis, but their contribution to transmission varies according to factors like host feeding patterns, geographic locations and climatic conditions. The yellow fever mosquito, Aedes aegypti, is a competent vector of D. immitis but is often dismissed as a vector of veterinary relevance given its anthropophilic feeding behavior. We evaluated the prevalence of D. immitis in pet dogs along the USA-Mexico border and assessed whether Ae. aegypti in the area are naturally infected with heartworm and are potentially acting as a vector.The canine heartworm D. immitis were tested using the DiroCHEK® Canine Heartworm Antigen Test Kit pre- and post-immune complex dissociations (ICD) and blood samples were tested using high-resolution melt (HRM) quantitative PCR (qPCR) and a probe-based qPCR. Additionally, mosquito specimens were collected and identified, and Ae. aegypti heads, abdomens and pools were tested using conventional PCR (cPCR) and HRM qPCR.A total of 200 whole blood samples collected from pet dogs in the Lower Rio Grande Valley in south Texas from 2016 to 2019 were included in this study. Canine serum samples for Ae. aegypti abdomens from blood-fed Ae. aeygpti tested, 20 (21.7%) from mosquitoes that fed on dogs and four (7%) from those that fed on humans tested positive for heartworm. Among Ae. aegypti heads from blood-fed Ae. aeygpti, two (1.1%) were positive based on cPCR and four (2.5%) were positive based on HRM qPCR. No D. immitis DNA was detected in the 208 pools of whole bodies  of Ae. aegypti gravid females.Overall, heartworm prevalence in dogs aged > 6 months was 40.8% (64/157) when the results from all testing modalities were considered. Heartworm antigen was detected in 33.5% and 40.7% of the dogs using DiroCHEK® pre- and post-ICD, respectively. By molecular screening, 20.1% of dogs tested positive with probe-based qPCR, while only one tested positive with HRM qPCR. Of the Ae. aegypti could be contributing to heartworm transmission in canine populations in this region.Our study highlights a high prevalence of heartworm in dogs in south Texas and provides evidence that  Aedes aegypti is an important arbovirus vector relevant to public health on the global scale, but it has not been implicated to date as being important to veterinary health due to its anthropophilic nature [Ae. aegypti to transmit animal pathogens, such as Dirofilaria immitis, the agent of dog heartworm, or allow for zoonotic bridge transmission of agents from animals to humans.c nature . Howeverc nature , there iD. immitis in the USA [D. immitis transmission are: (i) competent as biological vectors based on laboratory infection studies; (ii) mammalophilic and utilize dogs or other canines as hosts; and (iii) distributed in areas of high heartworm prevalence [Aedes vexans, A. trivittatus, A. punctipennis and Culex quinquefasciatus as important vectors [Aedes aegypti falls into this category, as it is used as a competent model of D. immitis transmission in the laboratory [Ae. aegypti infected with D. immitis [D. immitis DNA. Our recent study documented Ae. aegypti feeding more frequently on dogs than humans in south Texas [D. immitis in some contexts. In this area, Ae. aegypti meets the three criteria outlined above, implicating that it is potentially an important vector of D. immitis in south Texas, but no studies have been conducted on mosquito vectors of heartworm in the region.There are 23 known mosquito species or species complexes that are competent vectors of  vectors . Some moboratory –7 but isboratory . Studies immitis , 9, withDirofilaria immitis is a filarioid nematode that needs both a mosquito vector and a vertebrate host to complete its life-cycle. Microfilariae enter the mosquito during feeding on an infected dog and reach the head as third-stage larvae (L3) where they can be transmitted to new individuals [D. immitis, migrate and may develop into the adult stage in dogs and cats, which often develop clinical disease. Disease severity varies, with severe disease being characterized by exercise intolerance, coughing, hemoptysis, tachypnea, dyspnea and syncope, heart failure or death [D. immitis via a mosquito bite, but infection is usually asymptomatic [D. immitis, is one of the most ubiquitous parasitic diseases of dogs in the USA with more than 100,000 cases each year [ larvae L where thor death . Humans ptomatic . Canine ach year , 12. Theach year . In the ach year ,15.D. immitis in Texas [Ae. aegypti in the area is infected with heartworms, including disseminated infections that may support their role as a biological vector.From 2013 to 2016, canine heartworm incidence increased 17.9% in the southeastern USA, which is more than in other regions of the USA . Severalin Texas , althougin Texas  and inciin Texas . The objcolonias, which are predominantly Hispanic, low-income communities where one or more city infrastructure or service is missing  [Ae. aegypti larvae. A detailed description of these neighborhoods has been reported previously [Ae. aegypti [Samples of dog blood and mosquitoes were collected in the LRGV in south Texas along the USA-Mexico border in low- and middle-income communities, labeled communities A-V, located in Hidalgo and Cameron counties Fig. . Of theslection) . This laeviously , 20. Thieviously . The anncolonias in the LRGV from 2016 to 2019  pre- and post-heat treatment, according to the manufacturer’s instructions. For immune complex dissociations (ICD) by heat treatment, 500 µl of serum was heated to 103 °C for 10 min in a dry heat block , 25, folD. immitis DNA. Previously identified primers for a 115-bp section of the mitochondrial 12S ribosomal RNA gene found in filarial worms capable of detecting multiple species [2O and ≥ 1 positive controls of D. immitis DNA provided by the Texas A&M Parasitology Diagnostic Laboratory. We also used a simplex probe-based qPCR that targeted a 166-bp fragment of the cytochrome oxidase c subunit (cox1) gene of D. immitis based on a previously validated protocol [D. immitis provided by the Texas A&M Parasitology Diagnostic Laboratory. The non-template negative control consisted of nuclease-free water.A high-resolution melt (HRM) qPCR was used to test the extracted buffy coat DNA for  species  were use species . Each coprotocol  with theMosquito sample collection took place each month between September 2016 and December 2018 using baited BG-Sentinel 2 traps  and CDC Autocidal Gravid Ovitraps (AGO)   and werecox1 gene followed by Sanger sequencing [Ae. aegypti mosquitoes (n = 184) with identified bloodmeal hosts and with sufficient DNA were used. Briefly, mosquitoes were washed with 10% bleach to remove exogenous DNA, abdomens or heads were homogenized in 200 μl of lysis solution and DNA was extracted using the Kingfisher™ Flex Purification System and the MagMax Core Nucleic Acid Purification Kit (Thermo Fisher Scientific) per the manufacturer’s instructions.Species identifications based on morphology made under a dissecting microscope were confirmed with a molecular protocol based on a PCR using primers for the quencing . Methodsquencing . All femD. immitis DNA in mosquito samples: cPCR with Sanger sequencing and HRM qPCR. cPCR with Sanger sequencing was used to identify the presence filarial DNA as belonging to D. immitis, and HRM qPCR was used to detect small amounts of filarial DNA present in samples, with a positive read from one modality indicating a positive sample. Mosquito abdomens and heads were tested separately to distinguish D. immitis in the DNA of the bloodmeal (blood-engorged abdomen) versus disseminated L3 in the mouthpart tissues [cox1 gene that are specific to D. immitis for both types of PCR. cPCR followed by Sanger sequencing was used on DNA extracted from blood-engorged abdomens and heads. Heads only and pools of whole-bodied gravid females were tested with HRM qPCR given that the species identification results had already been confirmed using Sanger sequencing when testing the abdomens.We used two molecular screening methods to detect  tissues  for the D. immitis in dogs was calculated by categorizing as positive any dog that either tested positive by serum antigen and/or tested PCR-positive. Two prevalence values are reported, one for dogs of all ages and one for dogs of aged > 6 months. This distinction was made because the life-cycle of the parasite makes it difficult to detect infections in young dogs [Overall prevalence of ung dogs , 33 and D. immitis infection was found in sampled dogs of all ages: 33.5% (67/200) in pre-heat-treated samples and 40.7% (81/199) in post-heat-treated samples  dogs that tested positive. Of these 30 dog samples with evidence of D. immitis DNA in the blood (buffy coat), 27 also tested positive for heartworm infection by DiroCHEK® post-ICD treatment. Combining all diagnostic modalities, 39.3% (70/178) of dogs of all ages were positive for heartworm, and 40.8% (64/157) of dogs aged > 6 months were positive.A total of 200 dog serum samples were tested using the DiroCHEK® Heartworm Antigen Test Kit. A high prevalence of d 40.7% 8/199 in pD. immitis between sexes, with 67.7% (64/96) of males testing positive and 52.4% (43/82) of females testing positive  when dogs of all ages were considered. The number of positive dogs varied by age group , with dogs aged 6–12 months showing the highest level of prevalence followed by dogs aged between 1 and 5 years. However, the number of positive dogs did not significantly vary by breed group   with the blood host identified for D. immitis presence; 25 (13.6%) of these abdomens from mosquitoes with identified vertebrate bloodmeal tested positive for D. immitis DNA. Dirofilaria immitis was more commonly found  in the abdomens of mosquitoes that had fed on dogs  than in those that fed on cats, sheep, human and other vertebrates  . The obtained 25 sequences had 97–100% similarity to the reference D. immitis sequence .We used cPCR with Sanger sequencing to test abdomens of blood-fednce; 25 1.6% of thcolonias. Texas has been indicated to be an area of high canine heartworm incidence [D. immitis than our samples, with 19/33 (20.9%) dogs being positive [colonias, as low SES has been linked with higher prevalence of D. immitis in the state of North Carolina (USA) [Our findings suggest that there is a high prevalence of canine heartworm infection in dogs from south Texas along the USA-Mexico border, with an infection prevalence of 40.8% in dogs from diverse communities, most of which consist of low-income ncidence . In pet ncidence . These dncidence . Shelterpositive ; howeverpositive , 37. Howna (USA) , or by ona (USA) . Even fona (USA) . These sD. immitis worms but are less sensitive for male-only infection with ≤ 3 females [D. immitis [D. immitis microfilaria positive and NAD before and after ICD. However, to our knowledge, the sensitivity of probe-based qPCR and HRM qPCR for the detection of nematodes have not been compared, making it difficult to comment on the differences between the two types of qPCR used in the present study. The increased detection of antigen post-ICD was expected given the study population, which comprised dogs from an endemic area, many of which had no history of heartworm prevention. Also, antigen tests are recommended for dogs aged > 6–7 months [The American Heartworm Society recommends both antigen testing and microfilariae testing of all canine samples , but mic females . Moreove females . qPCR is immitis  and the  immitis , 43, 44. immitis , 45, sim7 months , 33. In 7 months . AccordiAe. aegypti abdomens with bloodmeal identified from Ae. aegypti that fed on dogs, 21.7% contained D. immitis DNA, suggesting that Ae. aegypti in these communities are frequently exposed to microfilaremic dogs. Some mosquitoes that fed on other vertebrates also tested positive for D. immitis, which may be explained by infections in other hosts (e.g. cats) and/or by a previous feeding on dogs that was not the most recent bloodmeal . We also found four Ae. aegypti heads positive for D. immitis DNA out of the 157 tested, a rare observation which has only been documented for wild populations in the state of Florida (USA) [D. immitis, move to the mosquito head and enter the salivary gland; saliva, hemolymph and L3 move to the skin then enter the host body through the mosquito bite wound [D. immitis DNA in mosquito heads represents the presence of L3, providing evidence of the suitability of the Ae. aegypti as a biological vector. The prior findings of Ae. aegypti being a competent vector of D. immitis based on laboratory studies [Ae. aegypti likely contributes to canine heartworm transmission in south Texas. Indeed, potential transmission has been assumed of a mosquito species if a positive result was found in a competent species [Ae. aegypti can lead to destruction of the excretory system and therefore mortality, a phenomenon that may be occurring in Ae. aegypti in the LRGV as no gravid females tested positive for D. immitis DNA [D. immitis to dogs is rarely measured, little is known on the role of Ae. aegypti contribution to transmission. In an earlier study, Paras et al. concluded that a mosquito can be considered to be a relevant vector based on its abundance and infection prevalence, reporting Ae. albopictus as an important vector based on a prevalence of approximately 15% calculated based on pool testing [Aedes albopictus [Culix erraticus [Ae. vexans [Ae. aegypti has a sufficiently high prevalence to be a vector of D. immitis.We found that of the da (USA) . L3, thete wound . Therefo studies , 7 combi species . In theiitis DNA . As the  testing . Still, bopictus , 0.7% inrraticus , 2.3% in. vexans ) has beeAe. aegypti fed 50% of the time on dogs, we also documented 31% feeding on humans and 12% feeding on cats [Ae. aegypti to also contribute as a bridge vector species, allowing spill-over transmission of D. immitis from dogs to humans. Human infections with D. immitis have been reported in North and South America, Europe, Asia and Australia, although most cases are asymptomatic [D. immitis infection in a human patient has been reported in Texas [Ae. aegypti in many regions of the southern USA [While our previous study documented that these populations of ptomatic . When syptomatic . Infectiptomatic . In the ptomatic . At leasin Texas . Infectiin Texas  and is iin Texas . It shouhern USA , 19, 54D. immitis infection in owned dogs in low- and middle-income communities in the LRGV, Texas, was high (40.8%). As prevalence estimates varied based on the diagnostic modality used, our results further support the use of integrated immunodiagnostic and molecular tests to determine heartworm prevalence. The finding of D. immitis DNA in the heads of Ae. aegypti provides evidence that this mosquito could be contributing to heartworm transmission in canine populations and potential spill-over to human populations. However, further study is needed to inform the relative role of Ae. aegypti and other mosquito species in transmitting D. immitis in the region.In the present study, the prevalence of


Background: Spontaneous simultaneous multiple intra-cerebral hemorrhages (SMICHs) and its occurrences in different territories of arterial disposition has been viewed as uncommon clinical occurrences, since the pathophysiological and predisposing factors as mechanisms aren’t vividly defined. This research primarily aims for demographic stratification and dichotomization pertaining to risk factors, etiological classifications, anatomical distributions and outcome analysis by focusing on management strategies and pertinent stroke care.Methods: 40 patients presenting to the College of Medical Sciences, Chitwan, Nepal in the last two years were included in the study. The patients with two or more spontaneous SMICHs with affected arterial territories with similar tomographic density based profiling were chosen as samples. Regression analysis was chosen to test three hypotheses.Results: Among our study cohorts, cortical and cortical territory (60%) was the major anatomical patterns of involvement. A conservative approach was undertaken in nine patients (22.5%), whereas surgical intervention was needed in five others (12.5%). A total of 14(35%) patients leaving against medical advice and a further seven (17.5%) patients were referred for adjuvant oncologic care. Mortality was observed among five (12.5%) patients. Hypertension was seen as a significant variable in its pathogenesis. Male patients were more affected. Age groups comprising 36-45years and 56-65 years were involved in 32.5% and 30% of cases respectively.Conclusion: This study proves the need for a national stroke data bank pertaining to spontaneous SMICHs. This will help foster effective patient education during preoperative counseling; as well as formatting a management algorithm combating them. Spontaneous simultaneous multiple intra-cerebral hemorrhages (SMICHs) are characterized by intracerebral hemorrhages within the two-or-more distinct and non-contiguous intracranial vascular territories visualized in the initial radiological imaging  of spontaneous SMICHs patients;–To analyze pivotal factors that impact spontaneous SMICHs patients significantly, namely: hypertension, diabetes, etiological classifications, anatomical distributions and outcome analysis.The methodology for the research was focused to spontaneous SMICHs patients from the hospital medical record section of the College of Medical Sciences, in Bharatpur, Chitwan, Nepal, presenting between January 2019 to January 2021 (time we started recording and compiling data of patients with spontaneous SMICHs). The outcomes were compiled to study the patterns through frequencies measures and analysis. The approval for the research conduction was obtained by the Institutional Review Committee (COMSTH-IRC/2021–56). Written informed consent was obtained from all the participants or their next of kin  at the time of their admissions.The research mainly focused upon the spontaneous SMICHs patients as the independent variable and how it impacts the respondents under varied dependent variables like: age, gender and hypertension.40 patients from the College of Medical Sciences, Chitwan, Nepal with spontaneous SMICHs were included in this study. Data was retrieved from the medical record section of the hospital pertaining to patients presenting with spontaneous SMICHs. The study size was arrived through convenience sampling.Inclusion criteriaAll patients presenting with spontaneous SMICHs.Exclusion criteria include:History of previous strokes;Patients with recurrent strokes;Failure to obtain consent for participation in the study.The minimal necessary sampling for research has been estimated by Fischer’s formula  as2 x p x q/d2N=zWhere:z represents a value of1.96 at the confidence-interval of 95%,p represents SSMICH prevalence as 6%,q as 1-p andd represents the margin-of-error as 10%.The final result through sampling estimation was calculated as 21.66 and the sample-size of 40 patients was finalized for the research purpose.Spontaneous SMICHs is the presence of two-or-more ICHs that affects different vascular territories with similar tomographic density-profiles. All findings were validated with radiologists.All data was taken from patient medical records.Etiologic classification for the cause of ICH was stratified as per the SMASH-U classification into hypertensive angiopathy, cerebral amyloid angiopathy, structural vascular abnormalities, medication related, other systemic causes, and undefined causes respectively  sequences sparing the basal ganglion and the thalamus.7.Tumor associated strokes was considered among patients with proven primary lesions with a histological diagnosis following either the therapeutic hematoma evacuation or minimally invasive biopsies.All patients were thoroughly evaluated for all possible etiological factors including cerebral angiography (including the venogram) and MRI stroke protocol (T1/T2/FLAIR/DWI/SWI/MR angiography) whenever feasible. The biopsy of the hematoma was sent for every operative case so as to aid in the definitive cause of the SMICHs. The images of the varied etiological causes of SMICH are demonstrated in theThe regression through analysis of variance (ANOVA) testing was adopted for examining the hypotheses formed as part of this research. Three hypotheses were tested and are analyzed in the following sections. The pertinent study variables of all 40 patients were retrieved and there were no missing data.The patients’ demographics and clinico-radiological based outcomes are provided inAmong the cohort of 40 patients, the most common age group with 13(32.5%) patients was the 36–45 age group followed by 12(30%) in the 56–65 age group. The male to female gender ratio was 25:15 (62.5% vs. 37.5%).The incidence of spontaneous SMICHs from our study (40 cases) within the specified duration of two years  was 16%. The majority (75%) of the cohort had hypertension as the risk factor. Merely 10% of the respondents were affected by diabetes. The vast majority (97.5%) of patients lacked adherence to drug compliance. Cortical and cortical territory (60%) was the major anatomical patterns of involvement. Mortality was observed in 5 (12.5%) patients, and 14 (35%) patients left against medical advice. Surgery was required in 12% of the cohorts who presented with cortical, subcortical and cerebellar bleeds and had clinical features of cerebral herniation. Surgery was aimed to remove the clinically significant hematomas only. One mortality (20%) was observed among the individuals who underwent operative intervention. Ther overall mortality in the study was 12%.Age and spontaneous SMICHs. Age related to spontaneous SMICHs was verified against the spontaneous SMICHs patients in the first hypothesis through the regression technique. FromThe regression equation used in analyzing the association between spontaneous SMICHs and age in patients was calculated through:Spontaneous SMICHs = 3.692 + (-0.846 * AGE)This equation is made from the beta values obtained from the aboveThe outcome is negative denoting that the hypothesis is invalid where age doesn’t impact the likelihood of spontaneous SMICHs.Gender and spontaneous SMICHs. Gender was verified against the spontaneous SMICHs patients in the second hypothesis through the regression technique. FromSpontaneous SMICHs = 0.718 + (0.641 * GENDER)This equation is made from the beta values obtained from the aboveThe outcome denotes that the hypothesis is invalid wherein gender doesn’t impact spontaneous SMICHs patients.Hypertension and spontaneous SMICHs. Hypertension as a factor affecting spontaneous SMICHs was verified, against the spontaneous SMICHs patients in the first hypothesis through the regression technique. FromSpontaneous SMICHs = .718 + (.641 * Hypertension)This equation is made from the beta values obtained from the aboveThe outcome denotes that the hypothesis is valid where hypertension impacts spontaneous SMICHs patients.While comparing to the cohorts of 85 patients with single spontaneous intra-cerebral hemorrhage (ICH) that underwent operative intervention in our institution during the same interval, the male: female ratio was 1.66:1 compared to 4.3:1 for spontaneous SMICHs (The incidence of spontaneous SMICHs from our study (40 cases) within the specified duration of two years  in our institution was 16%. In a study byContrary to single ICH, spontaneous SMICHs significantly showed lobar territorial preponderance  results, large hematoma and concurrent ventricular extension to be prognostic markers determining 90-day mortality   can be another limiting issue in this observational study.This is one of the first observational studies pertaining to spontaneous SMICHs to be carried out within our subcontinent. This study proves the need for a national stroke data bank pertaining to spontaneous SMICHs. This will help foster effective patient education during preoperative counseling; as well as formatting a management algorithm combating them. The authors have revised the article suitably. The 16% incidence rate of SMICH is considerable. SMICH has been found increasingly in younger age males over cortical areas with non-compliance on anti hypertensive drugs and required more surgical intervention than isolated bleed. These facts are to be highlighted in conclusion.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NeurovascularI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Authors have studied 40 patients over a period of two years with spontaneous simultaneous multiple intra-cerebral hemorrhages (SMICHs). Being not so common, this entity demands more elaborate study.It is suggested to add “spontaneous” to SMICHs to differentiate from traumatic conditions.The formula for calculating sample size needs to be formatted to convey the correct method of identifying minimal necessary sample size.It is pertinent to mention how the authors classified patients with mixed etiologies or when two or more factors exist without fulfilling the definition individually.To identify the etiological factors, what were all investigations carried on each patient?Table 1 when mentioned in the text, is presented with a reference to figshare dataset. These two are however are different. This confuses the reader. It would be good to mention the dataset separately as done in the "data availability" section.Minor corrections like adjusting font size and style are required to allow proper spacing between the words and letters.Besides the above, it would have been great if the authors compared SMICHs with single ICH for the variables they have studied, from the available dataset. The incidence of SMICH among all ICH could also have been calculated in Nepalese cohort.The fact that various researchers have found varied anatomical distribution of SMICHs demonstrate no territorial predilection for SMICHs.Results do not talk about who needed surgical treatment for SMICH and their outcome. However these datasets are discussed in Discussion. Overall the authors need to be commended to have properly conducted and presented nicely the findings of this little researched condition. The present study evaluates the association of age, gender and presence of hypertension with SMICHs. Authors by using SMASH-U classification have simplified as well as standardized the etiological classification of ICH in this study.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NeurovascularI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This study described the clinical features of simultaneous multiple intracerebral hemorrhages in a neurosurgical center in Nepal. The study was well designed and the data acquisition and analysis are both reasonable. The limitation, such as the sample size may influence the results, could be emphasized at the end of the manuscript thus to avoid an inadequate citing of the results in any other scenarios.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:TBII confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


The spleen of the patients was decreased and showed a predominance of red pulp with a high concentration of CD68+ and stabilin-1+ cells. The white pulp contained many medium and small follicles and a lower concentration of CD68+ and stabilin-1+ cells, which was comparable to that in the infarct area of the myocardium. The concentration of CD68+ and stabilin-1+ cells increased in the myocardium in the late period of MI, but did not show any dynamics in the spleen. A high number of CD68+ cells in the red pulp and reduced concentration of stabilin-1+ cells in the white pulp were associated with unfavorable post-infarction outcomes. These fundamental findings could be a basis for the development of new personalized therapeutic and diagnostic approaches for the treatment of MI and its complications.Myocardial ischemia triggers neurohumoral activation of the cardiosplenic axis. In rodents, adverse outcomes occur upon prolonged entrance of mononuclear cells from the spleen into myocardial tissue. The purpose of this study is to assess the features of spleen structure in patients with fatal myocardial infarction (MI), the dynamics of macrophage infiltration of the spleen and its relationship with cardiac macrophage infiltration and unfavorable outcomes. Using immunohistochemistry techniques, we analyzed the macrophage infiltration of the spleen and myocardium sections collected from patients ( Myocardial infarction (MI) is followed by the development of post-infarction heart failure (HF) and an unfavorable prognosis for one in every six patients . Post-inThe involvement of the innate immune system in the development of adverse LVR has been one of the promising areas in cardiology and has been intensively studied over the past decade . MonocytThe purpose of this study is to assess the features of spleen structure in patients with fatal MI, and to evaluate the dynamics of macrophage infiltration of the spleen and its relationship with cardiac macrophage infiltration and unfavorable outcomes.n = 30) during autopsy. In addition, we analyzed myocardium fragments both from the infarction area (IA), peri-infarction area (peri-IA), and non-infarct area. These materials also were taken during autopsy and used for histological examination. The criteria for exclusion were MI types II–V, infectious complications , oncological diseases, and severe valve diseases [The criterion for inclusion in this study was fatal MI type 1. We analyzed spleen fragments obtained from a study group of patients . Paraffin blocks had been archived for 6 years.Autopsy was done during 24 h after registration of the patient’s death. We fixed the material in 10% buffered formalin during the day. The material for histological examination was prepared by the standard method using a Thermo Scientific Excelsior AS histological wiring machine , then it was embedded in paraffin using a Tissue-TekMicroscopic characteristics of the spleen were assessed using one section with a thickness of 5–6 μm cut with the HM 355S rotary microtome from each block  and stained with hematoxylin and eosin  using Leica ST5010 Autostainer XL for histological staining of tissue samples . Cover slipping was performed using a ClearVue machine . Histological preparations were made in accordance with the manual for the operation of automatic machines provided by equipment suppliers.Two independent researchers performed morphological examination of the spleen via routine light microscopy using an Axio Imager M2 microscope . An Axiocam 506 color camera  was used to obtain microphotographs of histological preparations. In the first stage, the macroscopic characteristics of the study sample were assessed. The following parameters were evaluated: organ dimensions ; the consistency of the organ ; the state of the capsule ; the state of the parenchyma across the section . The data were obtained retrospectively by analyzing the case histories and autopsy reports of deceased patients.The second stage involved assessment of microscopic characteristics, which included analysis of the state of the capsule  and trabeculae ; determination of the white pulp (WP)/red pulp (RP) ratio; as well as characterization of WP follicles , and the state of the central arteries of follicles  . The stuFor immunohistochemical studies, microtome sections of the spleen and myocardium were prepared using an HM 355S rotary microtome . From each block, 10 sections of spleen fragments and 20 sections of the myocardium were obtained. The material was applied to glasses coated with L-polylysine (two sections per glass). Two independent researchers investigated the macrophage infiltration of the spleen and myocardium via immunohistochemical studies conducted using an automatic immunostainer . Macrophage immunophenotyping was performed using mouse monoclonal antibodies for the common macrophage marker CD68 , and antibodies for the M2 macrophage marker synthesized in the Laboratory of Innate Immunity and Immunological Tolerance (University of Heidelberg) against stabilin-1 (dilution 1:1000) .The studied markers were visualized using the HRP-DAB system . Immunohistochemical staining was performed in accordance with the standard protocol . Two indThe patients were divided into 2 groups according to the time of death. The first group included patients who died within the first 3 days after the onset of MI, while the second group included patients with fatal outcome within 4 to 28 days after MI. We have already presented clinical and anamnestic data on these groups of patients . It shou2 (Pearson’s test) and Fisher’s tests were used for comparison of categorical indicators. Correlations between the concentration of cells and clinical and anamnestic data were identified using Spearman’s correlation coefficient. A value of p = 0.05 was used to confirm the significance of the hypotheses considered. The multivariate logistic regression model included the numbers of the studied cells CD68+ and stabilin-1+; in both the myocardium and the splenic RP and WP, as well as the presence of LV aneurysm associated with the number of these cells. The model was used to determine the cell phenotype, and its localization correlated with the formation of LV aneurysm.We used the software STATISTICA 12.0 for the analysis of the data. A Shapiro–Wilk test was used for testing the normality of quantitative data. The quantitative indicators that did not have a normal distribution were described by using the median (Me) and interquartile range , except for age, which was described using the mean (M) and standard deviation (SD). Categorical indicators were described using frequencies and percentages. The Mann–Whitney test was used for comparison of the quantitative indicators in the groups, while χIn the study sample, 87% of the patients had ST-segment elevation MI upon admission to the hospital, and in 65% of the cases, it had occurred for the first time. More than one third of the examined patients had a lethal outcome during the first day after the onset of the disease. n = 9) was greater than N, while in 14% (n = 4), it corresponded to N, and in 57% (n = 17), it was less than N. The spleen width was less than N in 42% (n = 12) of the cases, and in other patients, the spleen thickness slightly exceeded N. The thickness was within the normal range in 86% (n = 26), and was reduced in 14% (n = 4). The mass in 71% (n = 21) of the cases was less than N, and in 29%, it exceeded N. Interestingly, in patients with reduced spleen mass, the spleen length was also reduced.The spleen length in patients with fatal MI and its average thickness were slightly less than the norm (N). However, the analysis showed that the spleen length in 29% of cases . However, the percentage of CD68+ cells in the RP of the spleen  showed a direct correlation with these indicators. All the cells analyzed in both the spleen and the myocardium, were used for the multivariate analysis model to assess the cell type associated with the formation of LV aneurysm (p = 0.04).In addition, the percentage of stabilin-1+ cells in the WP of the spleen correlated with the presence of LV aneurysm and preinfarction angina  and its relationship with pro-inflammatory remodeling of circulating leukocytes. They also observed high prognostic value of increased spleen activity after MI and the development of subsequent cardiovascular events . HoweverFurthermore, we assessed the size and the micro- and macroscopic characteristics of the spleen in patients with fatal MI. It was found that indicators such as the length and mass of the spleen were reduced in most patients. These data are contradictory since similar data previously obtained in an experiment on rodents were attributed to the depleted pool of macrophages during MI . Yet theChronic venous congestion is known to develop in patients with CHF, in both the pulmonary and systemic circulation; thus accompanied by characteristic morphological changes in the organs, including primarily atrophy and replacement of the parenchyma by connective tissue. This remodeling ultimately decreases the parenchyma volume, and reduces the size and mass of the organs. To understand how these changes reflect the activation of the cardiosplenic axis under ischemic conditions, they should be analyzed in patients who survived after MI.The analysis of the microscopic characteristics revealed a predominance of the RP performing mainly a filtration function, which may indicate excessive venous blood storage in the spleen . The quaWe assessed the percentage of CD68+ and stabilin-1+ cells when analyzing macrophage infiltration of the spleen and myocardium. The CD68+ marker is most frequently used in oncohematology and is involved in the detection of the general population of macrophages, but it has also been reported to have a significant role in cardiology ,28. StabIn 1987, Buckley revealed different types of macrophages and dendritic cells in the spleen . HoweverDuring experimental MI, about 40% of splenic monocytes involved in healing of the damaged area of the myocardium were found in the focus of inflammation . The numInterestingly, the increase of the macrophage concentration in the infarcted myocardium, and in the later period of MI in patients with a fatal outcome, did not reduce the number of these cells in the spleen. Our data partly correspond to previously published results  and indiAnalysis of the relationship between the macrophage pools in the spleen and myocardium showed that these indicators correlate only in the early period of MI. This is probably due to the highest monocyte migration from the spleen to the myocardium occurring in only the first 24 h. A high percentage of CD68+ cells in the IA of myocardium associated with a lower percentage of stabilin-1+ cells in the splenic RP and characterizes the early period of MI, which involves active inflammation. A high concentration of CD68+ cells in the splenic RP was due to the formation of LV aneurysm, as was the low concentration of stabilin-1+ cells in the WP to a lesser extent. This can be promoted by persistent active inflammation and decreased regenerative activity in the myocardium.Undoubtedly, the study had a number of limitations. The study sample was limited in volume, and the ratio of splenic/myocardial macrophages at a given time period was not a static indicator. Therefore, a promising direction for future research is the changes in the amount of cells with these phenotypes in vivo. This is supported by the ability of the spleen to change structure quickly depending on the functional state of the body, and autolytic processes begin immediately after death .Our data provide insight on changes in macrophage infiltration of the spleen under ischemic conditions and their relationship with the alterations of the macrophage infiltration of the heart, as well as the development of an adverse outcome in MI patients. Such information could shed light on the mechanisms of formation and progression of the inflammatory response in the myocardium. This could enhance the development of treatment methods for local control of the intensity and duration of the response. There has been no success in attempts to influence the inflammatory response after MI through available therapeutic strategies to suppress inflammation. The current therapeutic modes have shown no beneficial effect, and there is no prognostic evidence of improved outcome of the disease in this patient cohort. Such difficulties are most likely due to the insufficient knowledge about the mechanisms of cell-stromal interactions, as well as the molecular genetic features of the regenerative processes in patients with MI. These could become a potential target for personalized and controlled therapy aimed at activating and controlling the intensity of pro- and anti-inflammatory responses during development of post-infarction HF, as well as regulating their balance.The spleen of the patients with fatal MI was decreased and showed a predominance of red pulp with a high concentration of CD68+ and stabilin-1+ cells. The white pulp contained many medium and small follicles, and a lower concentration of CD68+ and stabilin-1+ cells, which was comparable to that in the infarct area of the myocardium. The concentration of CD68+ and stabilin-1+ cells increased in the myocardium in the late period of MI, but did not show any dynamics in the spleen. A high number of CD68+ cells in the red pulp and reduced concentration of stabilin-1+ cells in the white pulp were associated with unfavorable post-infarction outcomes. These fundamental findings could be a basis for the development of new personalized therapeutic and diagnostic approaches for the treatment of MI and its complications.


Colorectal cancer (CRC) ranks as the second among the causes of tumor death worldwide, with an estimation of 1.9 million new cases in 2020 and more than 900,000 deaths. This rate might increase by 60% over the next 10 years. These data are unacceptable considering that CRC could be successfully treated if diagnosed in the early stages. A high-fat diet promotes the hepatic synthesis of bile acids (BAs) increasing their delivery to the colonic lumen and numerous scientific reports correlate BAs, especially secondary BAs, with CRC incidence. We reviewed the physicochemical and biological characteristics of BAs, focusing on the major pathways involved in CRC risk and progression. We specifically pointed out the role of BAs as signaling molecules and the tangled relationships among their nuclear and membrane receptors with the big bang of molecular and cellular events that trigger CRC occurrence. Colorectal cancer (CRC) ranks as the fourth most common cancer and the second cause of cancer in men and the third in women. The International Agency for Research on Cancer (IARC) estimated 1.9 million new cases in 2020, with more than 900,000 deaths, and this rate might increase by 60% over the next 10 years [This rapid growth may reflect both the increased lifespan, as well as genetic predisposition, such as positive family history  and other genetic syndromes, such as Lynch and Gardner syndromes, and familial adenomatous polyposis (FAP) , as wellThe cancer gradual progression offers an opportunity to detect it before the malignant changes; studies have shown that over 90% of all CRC results from colon–rectal adenomas and just the metastatic phases are related with a high mortality rate and a 5-year survival of below 10% .The CRC pathogenesis is a multi-step process that involves a series of events from adenoma to carcinoma with the histological transition of the normal mucosal epithelium. The stepwise accumulation of genetic and epigenetic alteration leads to a generalized disorder of stem and epithelial cells replication and differentiation, thus causing aberrant proliferation of cells within crypts that may progress to becoming adenomas with different grades of dysplasia and may even become more invasive leading to carcinoma .There are several mechanisms underlying tumorigenic development, such as the aberrant activation of oncogenes and the inactivation of tumor suppressors. In the hereditary CRC forms, there has been frequently reports of a mutation in the adenomatous polyposis coli (APC) gene, which plays a key role in the Wnt/B-catenin signaling pathway, and a mutation in the family of mismatch repair genes (MMR), which preserves genomic integrity. Instead, in sporadic CRC, which accounts for nearly 85% of all CRC cases, it has been identified a plethora of mutations, which can lead the oncogenic events. Signaling pathways, such as Wnt/β catenin, the mitogen-activated protein kinase (MAPK), the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT), the transforming growth factor-β (TGF-β), and Notch, have been frequently reported to be dysregulated in the CRC ,7. TheseThere is a strict correlation between an unhealthy diet, dysbiosis, bile acids (Bas) synthesis and metabolism, and CRC; indeed, epidemiological studies showed that over 90% of gastric and colonic cancers could be imputable to diet . Bas hepBAs are cholesterol end-product derivatives with detergent-like properties; a high-fat consumption increases their synthesis into the liver and their delivery to the large bowel where they are metabolized by the microbiota into secondary Bas, which are products with potential inflammatory and tumorigenic activity. Moreover, different transport rates in jejunum and ileum have been reported .When BAs are at high concentrations, they self-aggregate to form micelles that could cause cell membrane injury resulting in the loss of the intestinal epithelium integrity. This damage consequently induces repair mechanisms involving inflammatory processes and hyperproliferation of undifferentiated cells . These rDuring the past years, the role of BAs, not only as detergents for dietary lipids and vitamin absorption but also as signaling molecules that regulate the activity of lipid and glucose metabolic pathways, has been well established . SeveralHerein, the role of BAs as signaling molecules involved in principal mechanisms underlined the CRC initiation and progression is discussed. We reviewed in deep the physicochemical and biological characteristics of BAs, focusing on the concept of detergency and lipophilicity that are often confused and misinterpreted. We described the major signaling pathways dysregulated in CRC risk and progression, specifically pointing out the role of BAs as signaling molecules and the tangled relationships among their nuclear and membrane receptors with the big bang of molecular and cellular events that trigger CRC occurrence.The purpose of this literature review is to satisfy the following research questions: (i) What are the physicochemical properties of BAs? (ii) What are their main functions in our body? (iii) Is there an association between dysregulated BAs production/excretion and CRC? (iv) Which are the most relevant signaling pathways involved in CRC onset? (v) Is there any overlapping changes in the BA composition in people suffering from CRC? (vi) Which are the BA receptors, and what is their role in modulating the most relevant signaling pathways involved in CRC onset?Discussing BAs synthesis and metabolism, physicochemical properties and BA receptors in the gut;Including findings from human and animal studies (if relevant) with the support of preclinical data related to the role of BAs in CRC onset;Published in peer-reviewed journals;Available in full-text;Written in English.Published within the time frame of 2002–2022. Papers published before 2002 were included if they were relevant in the field.The literature search was run on the electronic databases Scopus, PubMed, Google Scholar, and Web of Science for peer-reviewed research articles until June 2022 published in English . The seaBAs are the end products of the catabolism of cholesterol, exclusively produced in hepatocytes. They are the major constituents of bile, a duodenal fluid, acting both as physiological detergents with phospholipids (at an mM micellar concentration) essential to dietary lipid absorption  and as sConcerning their chemical structure, common BAs have 24 carbon atoms and, for this reason, are abbreviated as C24 BAs. The steroid nucleus is composed of four fused carbon rings consisting of three 6-carbon rings and one 5-carbon ring  Figure . The traThere are two major pathways for BAs biosynthesis: a classic or “neutral” pathway and an alternative or “acidic” pathway . In the The alternative (acidic) pathway starts with hydroxylation at C-27, catalyzed by CYP27A1, which synthesizes oxidized sterols, followed by oxysterol hydroxylase (CYP7B1), a microsomal enzyme specific for the synthesis of CDCA. In humans, the acidic pathway contributes only a small fraction 3–18%) of total newly synthesized BAs but may be quantitatively important in BAs biosynthesis in patients with liver diseases and neonates. Notably, the liver is the only organ where the classic BAs synthesis pathway takes place while the alternative pathway can occur in various body parts besides the liver, such as in macrophages, brain, adrenal glands, and other peripheral tissues [% of tota2+ precipitation and passive absorption, and makes them resistant to pancreatic carboxypeptidases cleavage [The final step of BAs synthesis occurs in peroxisomes and involves the conjugation (amidation) of the terminal side-chain carboxylic acid with the amino acids glycine and/or taurine, usually in a ratio of 3:1, respectively, in humans, carried out by the bile acid-coenzyme A (CoA) synthase  and bile acid-CoA: amino acid N-acyltransferase (BAAT)  . The concleavage , and thuAfter each meal, the gallbladder evacuates its contents in response to the cholecystokinin (CCK) hormone, promoting the release of BAs in the small intestine thus enables the emulsification and absorption of lipids and liposoluble vitamins . After aOnce reaching the liver again, conjugated BAs are actively transferred from the hepatocytes into the bile, inducing bile flow and cholesterol and phospholipids release. This recovery process is also known as enterohepatic circulation (EHC) . TherefoAlthough ileal BAs absorption is a very efficient process (~95%), approximately 400–800 mg of BA salts escape the daily EHC . The BAsThe major BA modifications in the human colon include the first deconjugation, then 7α-dehydroxylation limited to the unconjugated BAs, and the oxidation/epimerization of hydroxyl groups at C-3, C-7, and C-12. Primarily, the conjugated ones are hydrolyzed by bile salt hydrolases (BSH), a cytoplasmic enzyme from the microbiota, such as Bacteroides, Clostridium, Lactobacillus, Bifidobacterium, and Listeria, which can catalyze the hydrolysis of a conjugated BAs into its unconjugated form and free amino acid moieties . ConjugaThus, the secondary BAs, DCA and LCA, formed from CA and CDCA, respectively, by anaerobic gut bacteria, can then be passively absorbed from the large bowel, and mostly secreted in the feces .In humans and other species with gallbladders, BAs have been discovered to cross the entire EHC  approximately 12 times/day, while animals without gallbladders  maintain a much smaller BAs pool, yet they cycle it more often (approximately 33 times/day). Around 1% of the BAs pool passes into the large intestine and is lost with each cycle via fecal excretion; only about 12% of the pool requires a replacement each day by new bile acid synthesis in the liver, approximately 500–600 mg/day .The most important physicochemical properties involved in BAs metabolism and toxicity are lipophilicity, i.e., the tendency to distribute in a lipid domain from an aqueous solution described by the octanol/water partition coefficient (LogP) and the detergency, i.e., their self-aggregation to form micelles also with phospholipids and, therefore, acting as a natural surfactant toward ordered lipid structures and cells described by the critical micellar concentration (CMC). To note, BAs that tend to stay more in the non-polar phase (1-octanol) are defined as hydrophobic . The two properties are not correlated since some lipophilic BAs are not detergent and vice versa since the detergency is related to a proper hydrophilic/hydrophobic balance, which drives the formation of the micelles via BA back-to-back interactions of the hydrophobic side C; any reThe detergency behaves quite differently, and the more detergent BAs are the DCA and CDCA followed by CA. UDCA, despite a similar hydrophobicity with DCA and CDCA because of a hydroxyl in 7 β positions oriented toward the hydrophobic side, is much less detergent and, therefore, safer toward membrane damage as the opposite occurs for CDCA and DCA. Surprisingly, UDCA acts as protector against membrane damage induced by CDCA and DCA. Conjugation slightly increases the detergency as the CMC is reduced in respect to the corresponding unconjugated BAs, and this is apparently in contrast with the fact that they are less lipophilic, but the side chain modification mainly acts to the hydrophobicity due to the amide bond but does not affect the self-association process. To summarize, BAs could be hydrophobic but poor detergents, and this implies that the molecule could cross membranes without major damage but just accumulate in the lipid domain. Other BAs did not come across as membranes being hydrophilic, but at a given concentration (above CMC) became cytotoxic, since they promote the solubilization of the main lipid constituents of plasma membranes.LCA and its conjugates behave quite differently from the other BAs; the water solubility of LCA is less than 0.1 µM and does not reach a concentration to form micelles even when ionized. LCA with a very high LogP could accumulate in cells and precipitate. GLCA has a higher solubility than unconjugated LCA (around 40 uM) but is still lower than that needed to self-aggregate. For these compounds, only hydrophobicity plays a physio-pathological role ,20.Another important physicochemical property is the acidity constant pKa. The unconjugated BAs independently from the steroid hydroxyl groups have a pKa value of five, and this means that at a physiological pH they are fully ionized. In addition, the glycine-conjugated BAs present a lower pKa of 3.9 and are, therefore, ionized even at a lower pH with respect to unconjugated BAs while the taurine-conjugated BAs are always ionized independently from the pH of the medium . All theThe 3-, 7- or 12-hydroxyl groups of BAs can undergo oxidation through the activity of hydroxysteroid dehydrogenases (HSDHs) of intestinal bacteria, such as Bacteroides, Eubacterium, Clostridium, Escherichia, Eggerthella, Peptostreptococcus and Ruminococcus, leading to numerous oxo-BAs structures with a different arrangement in position and orientation of hydroxyl groups. Epimerization of BAs hydroxyl groups is a reversible conversion in stereochemistry from the α to the β configuration (or vice versa), with the generation of a stable oxo bile acid intermediate . For insConsequently, the microbial metabolism of the BAs results in a raised variety and a more hydrophobic BAs pool, and for this reason, a variation of the intestinal microbiota composition is reflected in the BAs biological modification . The rolDCA levels can differ from 1% to over 50% as the human liver is unable to 7a-hydroxylate DCA to the primary bile acid CA, thus it accumulates in the bile acid pool (LCA to a much lesser extent) as a result of passive absorption through the colonic mucosa . As the In the intestine, the largest barrier against the external environment is represented by the epithelial surface. In most adult mammals, epithelial cells are renewed every 2 to 5 days to counteract intestinal damage; it is the fastest self-renewing tissue in mammals . RenewalCRC is a multifactorial disease involving genetic, environmental, and lifestyle risk factors . AlthougThe dysregulation of several signaling pathways involved in fundamental cellular processes, including cell proliferation, differentiation, angiogenesis, apoptosis, and survival,  are reported as key factors in CRC onset. Various genes, associated with the signaling pathways previously mentioned, have been frequently reported to be dysregulated due to mutations or the altered function of their products in the CRC, such as APC, RAS, and RAF ,51. MoreVogelstein and coworkers firstly proposed a multistep genetic model that has come to serve as a paradigm for CRC . The WntMoreover, the activation of the K-Ras/RAF/MAPK pathway due to additional mutations results in the growth of a small adenoma to a clinically significant size (>1 cm) . The furThe epidermal growth factor receptor (EGFR) pathway has also been associated with the CRC progression since the binding of its ligand EGF stimulates downstream signaling cascades, such as MAPK, PI3K/Akt, p53, and signal transducer and activator of transcription (Stat)-3 signaling pathways , which aThe oncogenic role of PI3K/Akt pathways is also linked to crosstalk with two other pathways. Firstly, PI3K up-regulates β-catenin signaling. Indeed, it may inactivate Glycogen synthase kinase 3 β (GSK3B), thus blocking the formation of β-catenin-destroying-complex and promoting the β-catenin accumulation, triggering the Wnt downstream signaling and resulting in the promotion of neoplastic events . SecondlIt has been observed that constitutive NF-κB activation also occurs in long-term inflammation  patients), which induces the expression of cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and other chemokines, supporting the inflammation-related tissue damage. Therefore, NF-κB may also contribute to the development of CRC by sustaining the ongoing inflammatory process in the intestinal tissue . It has In 1971, Judah Folkman brought the concept that tumor growth beyond 2 mm is critically linked to angiogenesis, since it stimulates tumor development by providing nutrients and oxygen and also supplies growth factors that support tumor cell proliferation . VasculaNotch signaling plays a significant regulatory role in angiogenesis; unlike the VEGF antibodies, blocking the Notch–Dll4 axis increases the vascularization of the tumor . HoweverMoreover, the activation of Notch signaling appears to be an early event in CRC development, since the process of ISC differentiation in the crypts is regulated at least in part by Notch receptors other than TGF-β signaling ,81. NotcAll these mentioned pathways represent fundamental paths that regulate development and may drive oncogenesis events. Several studies suggest that they are closely interconnected and understanding how they are interrelated is critical to the development of successful targeted therapies.As already mentioned, sustained inflammation triggers the pathogenesis of CRC due to barrier disruption, and bacterial translocation resulting in inflammation and neoplastic transformation of colonic epithelial cells  predispoStudies back to the 1970s have highlighted that the risk for developing CRC was much higher among Japanese people who switched from a low-fiber to a high-fat ‘Western’ diet after immigration to the United States ,87. SincAlexi N Archambault et. al. recently reported a risk prediction model for early onset CRC using 13 population-based studies (3486 cases and 3890 controls). This model incorporates a novel aggregate environmental risk score (ERS) and a recently expanded polygenic risk score (PRS) and allows for identifying individuals at differential relative and absolute risk for early onset CRC ; recentlHigh-fat consumption stimulates the hepatic synthesis of BAs and their delivery to the colon, where they are metabolized by the microbiota into products with potential inflammatory and tumorigenic activity. BAs can also modify microbiota composition, leading to dysbiosis, thanks to their detergent properties , thus prSome epidemiological studies have shown an association between high levels of fecal and serum BAs and CRC , above aIn 1940, DCA was firstly shown to be a carcinogen that induced CRC development in mice ; it is cmin/+ mice [Moreover, the hydrophobicity and detergency of DCA perturbate cell membranes by activating protein kinase C (PKC) and NADPH oxidase, which promote reactive oxygen species (ROS) and reactive nitrogen species (RNS) accumulation, causing oxidative stress that damages the DNA, disrupting the base excision repair pathway and able to induce NF-κB activation ,97. DCA n/+ mice . Moreoven/+ mice  discoverDespite the numerous studies that attributed the tumor-promoting function of BAs to DCA, studies performed in both human and animal models have also revealed that the dysregulation of CDCA with similar hydrophobicity and detergency, and its taurine-conjugated form (T-CDCA) synthesis, can lead to cancer-promoting effects in the liver and a consequent aberrant BAs metabolism . Indeed,These findings are not surprising, since the recent discovery that BAs are endogenous ligands of different receptors has given some mechanistic insight into the role of BAs in the regulation of gene transcription and signaling pathways , even ifAs mentioned above, the digestive role of BAs in the intestine is to emulsify dietary lipids and cholesterol to facilitate their absorption. However, during the last two decades, extensive research has supported interesting evidence, showing that BAs not only are crucial in the digestive process but also play multiple important physiological functions besides their function as detergents.The intestine is provided with complex bile acid-sensing processes that allow sophisticated coordination of different intestinal actions and regulate the crosstalk between the gut and other body organs. Seminal work by numerous investigators proposed the roles of BAs as signaling molecules, which can trigger cellular signaling pathways through the activation of specific receptors that modulate biological processes at low concentrations. BAs can activate both nuclear receptors and plasma membrane-associated receptors to elicit their physiological effects in the intestine .Two major classes of intestinal receptors for BAs have been discovered in the gut: three nuclear receptors (NRs), farnesoid X receptor (FXR), pregnane X receptor (PXR)/steroid and xenobiotic receptor (SXR), vitamin D receptor (VDR), and the membrane Gαs protein-coupled receptor (GPCR) TGR5 ,104,105.The nuclear BAs receptors play an important function in protecting against carcinogenic effects due to BAs themselves by activating transcriptional programs aimed at coordinating the maintenance of BAs uptake, detoxification, and basolateral secretion ,107. In FXR is highly expressed in the liver and intestine (mainly in the ileum) and its activation occurs in the presence of free and conjugated BAs. In vitro studies have consistently shown a rank order for the affinity of BAs to bind FXR: CDCA > DCA > LCA > CA (CDCA: EC50 =~10 μM) . BrieflyFXR regulates not only the BAs pool but also BAs uptake and export systems by inducing some binding proteins and transporters, i.e., IBABP and OSTα/OSTβ or by inhibiting other ones, such as NTCP and ASBT ; so, anymin/+) or those treated with the colon chemical carcinogen azoxymethane (AOM)/dextran sulfate sodium (DSS), FXR loss promotes intestinal tumor progression and an increased adenoma size, which leads to early mortality, while the transgenic overexpression of FXR in gut cells reduces tumor growth and development [5+ ISCs proliferation and curtails CRC progression in an APCmin/+ mouse model. In line with these observations, Farhana et al. revealed that DCA is able to induce ISCs proliferation by modulating muscarinic 3 receptors (M3R) and Wnt/β-catenin signaling, presumably acting through FXR [Given the central role of FXR in the preservation of BAs concentrations within a physiological range, which prevents BA-induced cytotoxicity,, the loss of FXR would be expected to be related to a pro-tumorigenic phenotype . De Gottelopment ,118. It ough FXR .Taken together, these data suggest that the loss of FXR expression promotes the transcriptional activity of β-catenin, whereas FXR activation results in the opposite effect in both human colon cancer mouse models and cell lines. Nevertheless, FXR functions as a tumor suppressor in CRC, at least in part by antagonizing Wnt/β-catenin signaling ,120.Notably, in colon cancer cells, the constitutive activation of FXR can suppress colonic epithelium proliferation and induce the expression of a pro-apoptotic network of genes  while repressing anti-apoptotic genes, such as Bcl-2 , showingThe process behind the silencing of FXR in CRC may lie in its DNA methylation caused by mutated APC that, in turn, leads to reduced expression of SHP and IBABP and induced cyclooxygenase-2 (COX-2) and c-Myc  and alsoK-Ras is a frequently mutated oncogene in CRC; its diverse mutations promote the activation of the MAPK pathway and cause spontaneous tumor development , and theThere is a mutual antagonism between FXR and NF-κβ too because not only does FXR promote inflammation inhibition but also, in different model systems, its activation is inhibited by pro-inflammatory stimuli (TNF-α and IL-1β) . FirstlyFXR is also involved in modulating other CRC-related pathways; for instance, the knockdown of FXR induces the expression of several EMT markers, such as vimentin, snail, slug, fibronectin, and MMP-9, while suppressing E-cadherin and zonula occludens-1 (ZO-1) . On the FXR also takes part in the regulation of antibacterial defense by controlling the expression of inducible nitric oxide synthase (iNOS), IL-18, angiogenin1 (ANG1), and carbonic anhydrase 12 (CAR12) , thus inAll these data suggest there is an inverse correlation between the FXR expression levels and CRC progression and malignancy. So, the FXR gene is supposed to have some characteristics of a tumor suppressor in colon cancer; in this context, the restoration of basal FXR expression might slow or prevent the progression of colon cancer allowing it to be a potential prognostic and target for the treatment of CRC.Recently, it has been discovered that not just FXR, but also the nuclear BAs receptor vitamin D receptor (VDR), the rodent pregnane X receptor (PXR), and its human homolog steroid and xenobiotic receptor (SXR) play an important role in protecting against carcinogenic effects of BAs by activating transcriptional programs aimed at coordinating the control of BAs uptake, detoxification, and basolateral secretion . VDR andVitamin D deficiency is recognized as a general risk factor for CRC; when vitamin D binds to VDR it behaves as a transcription factor for many genes, exerting profound antimitogenic and differentiating effects on many normal and malignant cells, including colon cancer cells ,135. ConThe protection given by VDR activation may become compromised when the detoxification pathway is overwhelmed  or under pathologies of vitamin D deficiency  . Indeed,Pirc/+ rats, VDR loss did not enhance tumor multiplicity, growth, or progression in the colon, thus supporting previous findings that vitamin D itself does not play a role in colon cancer development or progression. To sum up, there is not a clear association between vitamin D and colon cancer risk; human studies are needed to better define the link between Vitamin D, VDR, and colon cancer.Recently, the strength of the causal link between vitamin D and colon cancer has been called into question; He et al. recently reported that in over 10,000 colorectal cancer cases and 30,000 controls there was no evidence for a relationship between 25(OH)D3 levels and colorectal cancer risk . Irving Likewise, the human xenobiotic receptor SXR and its rodent homolog PXR are nuclear BAs receptors expressed in the intestine and liver that when activated induce a response that detoxifies BAs . SXR proAltogether, these results reveal a complex network of interactions, suggesting that the PXR/SXR pathway promotes the expression of detoxification genes, thereby leading to enhanced drug resistance, but, on the other hand, their induction gives an anti-proliferative effect in colon cancer cells.In summary, FXR is downregulated in both mouse and human models of CRC when the tissues are progressing from normal intestinal epithelia to dysplastic lesions, indicating a therapeutic and/or diagnostic potential for these transcription factors in CRC. However, the role of VDR and PXR/SXR in CRC is still controversial, and more studies are necessary to clarify their activities in colon cancer cells.The BA receptor TGR5, discovered later than FXR, was first recognized as a regulator of energy and bile acids homeostasis, as well as glucose metabolism . It is aDifferent conjugated and unconjugated BAs are ligands for TGR5. In vitro studies have highlighted that the unconjugated bile acid rank order for TGR5 activation is: LCA > DCA > CDCA > CA > UDCA (EC50 of LCA: 35 ± 5 nM) . TGR5 mRDespite its physiological role in integrating intestinal homeostasis, intestinal barrier integrity and glucose metabolism are well defined; TGR5 also seems to be involved in other cell signaling pathways, such as NF-κB, the central transcriptional regulator of inflammatory and immune responses, Akt, and ERK. Indeed, mice lacking TGR5 develop altered colonic histopathology, which results in severe alteration in the distribution and maturation of mucous cells and the disruption of the molecular architecture of colonic tight junctions . Since tContinuous inflammation was involved in the CRC pathogenesis due to barrier disruption, and bacterial translocation resulted in the inflammation and neoplastic transformation of colonic epithelial cells . TGR5 acThe ability of TGR5 to lower the levels of pro-inflammatory cytokines in intestinal cells and macrophages has opened new insights into the modulatory role of BAs in diseases where inflammatory processes play a central role, such as CRC; however, its involvement in tumors has to be clarified since TGR5 seems to have different actions in different cell and tissue background.For instance, Guo et al. (2015) and Chen et al. (2012) observed that TGR5 acts as a suppressor of cancer cells migration and proliferation in gastric  and liveMany reports have shown the Stat3 activation in various tumor cell lines, including colon cancer cells ; howeverSo, TGR5 activation likely promotes intestinal healing via a dual mechanism encompassing an anti-inflammatory activity and a pro-regenerative program in epithelial cells and could be further explored in CRC clinical settings.Sphingosine-1-phosphate receptor 2 (S1PR2 or S1P2) is a G protein-coupled receptor for sphingosine-1-phosphate (S1P) expressed in the ileum and colon, activated also by glycine and taurine conjugated primary bile acids . In normS1PR2 activated by TCA stimulates the sphingosine kinase (SphK2) expression and activity by increasing the conversion of sphingosine into S1P, leading to increased lipid and sterol metabolism in the liver . Thus, cTo date, however, we have only a few studies investigating the potential therapeutic effects of S1PR2 in CRC, even if preliminary data identify it as a promising candidate tumor suppressor gene in CRC.Since the CRC incidence and mortality rates are expected to grow in the near future, understanding the molecular and cellular mechanisms underlying CRC becomes pivotal. The evidence reviewed here indicates that perturbations of BAs synthesis and composition are related to colorectal oncogenic signals, even if a clear link between BAs receptors, located in the gut, and CRC has not yet been fully elucidated. Diet and the consequent gut microbiota profile are most likely the key environmental drivers that mediate and confer BAs-associated tumorigenic activity. Indeed, a high-fat diet-associated BAs level impact gut microbiota composition favoring increased levels of 7α-dehydroxylation bacteria, which stimulate secondary BA formation with tumor-promoting activity.Further studies are required to demonstrate or exclude a causal correlation between BAs receptor levels in the gut and CRC onset. This would allow researchers to better understand which patients would obtain not just the greatest preventative benefit from reducing their secondary BAs levels, with diet and/or with specific drugs, but also to define subjects with high-CRC risk for whom finding biomarkers, such as BAs receptors profile in the gut, is an urgent clinical need, especially for recurrences. Unfortunately, the overall 5-year survival rate of CRC is approximately 60% and after curative treatment, 30% to 40% of the patients develop recurrent disease .Right now, BAs profile analysis and the characterization of microbe–host interactions implicated in bile acid metabolism can be considered diagnostic biomarkers for CRC prevention. However, it is not yet possible to conclude whether a dysregulated intestinal BA receptors profile or their derived signaling pathways detrimentally affect the risk of developing CRC.The application of specific drugs, probiotics, and their products, as well as lifestyle changes may become effective interventions. We look forward to furthering research and exploration to provide further mechanisms for the occurrence and development of CRC and new diagnostic directions in the near future.


The impact of digital devices and the Internet has generated various changes at social, political, and economic levels, the repercussion of which is a great challenge characterized by the changing and globalized nature of today's society. This demands the development of new skills and new learning models in relation to information and communication technologies. Universities must respond to these social demands in the training of their future professionals. This paper aims to analyze the empirical evidence provided by international studies in the last eleven years, related to the digital literacy of university students, including those pursuing degrees related to the field of education. Our findings highlight the fact that the digital literacy that is offered in universities to graduate/postgraduate students, in addition to treating digital literacy as a central theme, also focuses on perceived and developed self-efficacy. This is done by strengthening competencies related to digital writing and reading, the use of databases, the digital design of content and materials, and the skills to edit, publish or share them on the web, or applications aimed at treating digital literacy as emerging pedagogies and educational innovation. Secondly, we found studies related to digital competencies and use of the Internet, social networks, web 2.0, or the treatment of digital risks and their relationship with digital literacy. Thirdly, we found works that, in addition to focusing on digital literacy, also focused on different psychological constructs such as motivation, commitment, attitudes, or satisfaction.Systematic review registration:https://www.scopus.com/home.uri; https://www.recursoscientificos.fecyt.es/. The concept of digital literacy (DL) appears for the first time in the works of Zurkowski , for whoIn recent years, society has undergone enormous changes with the digitalization of many of its spheres at the information level, the communication level, the level of knowledge acquisition, the level of the establishment of social relations, and even the level of leisure. Thus, our habits and means of accessing, managing, and transforming information have also changed  is composed of four components: basic digital skills , gender, and personal digital resources.The systematic review is composed of four phases, following the model of Miller et al.  and ScotPHASE 1: Search terms: In this phase, we developed a schematic of search terms from Web of Science and Scopus databases. We also accessed the databases to locate specific studies that were referenced in the publications that we found in the databases during our initial search. The schematic of terms and thematic axes that were used as a starting point for scanning both databases for anything related to the descriptor “digital” and the descriptor “literacy” is presented in ad hoc” instruments to measure digital competence.PHASE 2: Selection process based on inclusion and exclusion criteria. The following selection criteria were applied: year of publication between 2010 and 2021, availability of full text, and language of publication in English, Portuguese, or Spanish. Once the first results were obtained, they were selected based on title, abstract, and the use of standardized instruments in their methodology. We rejected the studies that used “In addition, the selection indicators provided by Cooper and Hedges  and CoopPHASE 3: Analysis of methodological quality and indicators based on scientific evidence. Following Torgerson  and RiskPHASE 4: Reliability and outcomes. Reliability was established for both the selection criteria and the coding criteria during each phase, to evidence the replicability of the results. In addition, the results entailed a qualitative analysis of the selected studies, the central arguments, and the evidence provided in a modulated way to address the research questions.Therefore, the procedure to be followed was documented and charted according to the PRISMA statement  with instructions on the development of the activities, in which the students were provided with what to do and the steps to follow. In this case, the development scenario was both online and face-to-face, based on different activities given through workshops or seminars for their development.It should also be noted that in those investigations in which the intervention itself required a specific application or program, the same was used, specifically, and even the intervention had a specific scenario since it was carried out in person in specialized laboratories where experts and specific material was available for this purpose. As an example of these specific materials, in our results, we found the use of the Photo Story 3, Dashboard, and Wikipedia, as well as the EMODO program or the SELI platform , the Digital Literacy Scale, or the E-Learning Attitudes Scale.In the case of scales, we found two papers that employed a Likert-scale elaborated Some of the studies we reviewed used semi-structured interviews as a means of monitoring and providing feedback to the students As for the sequence through which the different interventions were developed, we found two types—first, those that divided the contents in time, as is the case of the work of Kajee and Balfour , who covIn the investigation by Elliott et al. , the intIn the study by Ball , in the Hamutoglu et al.  organizead hoc created applications as examples).In the work of Gabriele et al. , the expWe also found interventions that had very short time durations, but provide daily detail of the contents and interventions. Similarly, Alfonzo and Batson  dedicateIn the research by Istenic et al. , the intIn some interventions, the sequence through which they are developed is the very development of the subject of the degree course from which they are implemented, as is the case of the work of Gill et al. .In the work of Carl and Strydom , studentIn one work we found narrative was used as a technique so that the students could later present their work, analyze it in groups, rework it and present it again to their classmates. Kuhn , PequeñoAdopting a novel consultative approach, Botturi  co-desigThe limitations of our search are listed in Analyzing the interventions themselves, we identified a few limitations, especially in those studies that neither indicates the tasks, record the entire process, or lack key information to replicate the intervention. In some studies, key information relating to the person carrying out the intervention was missing, particularly on whether they had the specific training for this purpose. Another limitation that was identified was that very few evaluation strategies were in place to evaluate the interventions see .Similarly, gaps were found regarding ethical controls, where in some studies the main limitation was that ethical controls were non-existent or not specified , and in its academic aspect , and research work focused on measuring the competencies of these devices , gender, and personal digital resources. In terms of applications for educational practice, these results do not indicate that university students are competent in terms of digital literacy, although they demonstrate some competencies like online information search, information evaluation, information processing, information communication, and dissemination skills , which are: alignment of theory, findings, reliability and validity, descriptive details of participants, and the study, sample, and consistency of findings and conclusions with the data , Universidad de Almería (Spain), Universidad de Salamanca (Spain), Instituto Politécnico de Coimbra and NICSH .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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cefiderocol (CFDC) is a siderophore cephalosporin with activity against a wide variety of Gram-negative bacteria, including carbapenem-resistant Enterobacterales and non-fermenters. We evaluated the In vitro susceptibility of cefiderocol and comparator agents to DTR isolatesSusceptibility testing was performed by broth microdilution according to CLSI guidelines. All antibiotics were tested in cation-adjusted Mueller-Hinton broth (CAMHB) except for CFDC, for which iron-depleted CAMHB was used. Susceptibility rate (%) was determined according to CLSI breakpoints, and DTR pathogens were defined as being resistant to cefepime, ceftazidime ceftriaxone , imipenem, meropenem, ciprofloxacin and levofloxacin according to CLSI/FDA breakpoints.Pseudomonas aeruginosa, and 586 Acinetobacter calcoaceticus-baumannii complex (ACB) clinical isolates from the United States, 50 Enterobacterales (0.6%), 36 P. aeruginosa (1.6%) and 114 ACB (19.5%) isolates showed a DTR phenotype, respectively. CFDC demonstrated its potent in vitro activity against these DTR isolates with MIC90 of ≤4 μg/mL with a susceptibility rate of ≥92% except for DTR ACB based on FDA breakpoint (75.4%). In contrast, MIC90s of β-lactam/β-lactamase inhibitor combination drugs showed lower activity (Table).Among a total of 8,328 Enterobacterales, 2,241 in vitro activity against DTR isolates of Enterobacterales, P. aeruginosa and ACB, indicating cefiderocol has high potential for treating infections caused by these difficult-to-treat strains.Cefiderocol demonstrated potent Yoshinori Yamano, PhD, Shionogi: Employee Miki Takemura, n/a, Shionogi: Employee Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support Christine M. Slover, PharmD, Shionogi: Employee Christopher M. Longshaw, PhD, Shionogi: Employee Roger Echols, MD, Shionogi: Advisor/Consultant Roger Echols, MD, Shionogi: Advisor/Consultant Roger Echols, MD, Shionogi: Advisor/Consultant.


To systematically evaluate the guidelines for the diagnosis and treatment of radioactive enteritis, compare their differences and reasons and provide some reference for updating them.This study used guidelines related to radiation enteritis by searching a database. Four independent reviewers used the AGREE II evaluation tool to evaluate the quality of the included guidelines, collate their main recommendations, and analyze the highest evidence supporting the main recommendations.Six diagnostic and therapeutic guidelines for radiation enteritis were included in this study, one of which, the American Society for Gastrointestinal Endoscopy guidelines, had an overall score of over 60%, which is worthy of clinical recommendation. In the diagnosis and treatment of radioactive rectal injury, the recommendations for hemorrhagic endoscopic treatment are mature and mainly include (I) argon plasma coagulation; (II) formalin treatment; (III) bipolar electrocoagulation; (IV) heater probe; (V) radiofrequency ablation; and (VI) cryoablation.The methodological quality of radioactive enteritis guidelines is unequal; even in the same guidelines, different domains have a large difference. For radioactive rectal damage diagnosis, a type of endoscopic treatment recommendation is more mature, but the overall diagnosis and treatment of radioactive enteritis still lacks high-quality research evidence.The online version contains supplementary material available at 10.1186/s13014-023-02204-9. This article reviews the diagnosis and treatment guidelines for radiation enteritis in order to promote further update of the guidelines.The online version contains supplementary material available at 10.1186/s13014-023-02204-9. Radiation enteritis refers to the intestinal radiation damage caused by radiotherapy in patients with pelvic malignancies such as bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer and rectal cancer. According to the onset time, course and location of the disease, it can be divided into acute radiation enteritis, acute radiation proctitis, chronic radiation enteritis, and chronic radiation proctitis . In the Obviously, this situation has also been considered important by different experts in various countries. In recent years, many guidelines on how to treat radiation enteritis have been formulated , 2, 6–9,This study comprehensively evaluated and analyzed the guidelines for the diagnosis and treatment of radioactive enteritis by using the AGREE tool. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocols (PRISMA-P) statement .In this study, the OVID, Web of Science, ScienceDirect, PubMed, CBM, CNKI and other databases were searched, and at the same time, the official website of the Gastrointestinal Diseases Association, American Gastroenterology Association (AGA) and related website of the guidelines of Yimai Tong were searched without language restrictions. Considering the time limit of evidence, we only included guidelines from 2011 to 2021. Considering the existence of multiple translations of the word, we used variations of the guidelines for radioactive enteritis to make the retrieval comprehensive using the following search terms:, “radiation enteritis”, “radiation enterocolitis”, “guide”, “guideline”, manual”, “guidance”, “recommendation”, and “consensus”, which were used in our study. At the same time, the references of the included guidelines related to radioactive enteritis were manually retrieved in this study.A series of inclusion and exclusion criteria were established in the selection of the literature in this study. The inclusion criteria were as follows: (I) the study population was patients with radiation enteritis; (II) the full text was available online; and (III) the guide was the latest version. The exclusion criteria for the guidelines were as follows: (I) guidelines that were not closely related to radioactive damage; (II) duplicate reports; (III) unavailable interpretation of the guidelines; and (IV) full texts were not available. The literature was selected by two authors independently according to the above inclusion and exclusion criteria using EndNote (X9). The guidelines with high relevance to this study were selected by reading their abstracts and titles. When two authors had disputes over the selection of the guidelines, the third author participated in the selection and further discussed the selection of the guidelines. At the same time, basic information such as the title of the guide, the year of publication, the first author and the main content of the guide was extracted.We evaluated the quality of the selected guidelines by using the latest version of the AGREE II Tool (2017 version) . The AGRIn this study, standardized scores for each domain were calculated using descriptive statistical analysis, expressed as percentages and presented in tabular form as averages and ranges. We used two-way analysis of variance to calculate the intragroup correlation coefficients (ICCs) to test whether the scores of the four evaluators were consistent. An ICC value greater than 80% was considered to indicate good agreement among the four evaluators. The statistical software used in this study was SPSS Version 26.0 .We consulted guidelines with a relatively high AGREE II score to extract and analyze significant recommendations related to the treatment of radioactive enteritis to further obtain and analyze the highest level of evidence supporting these recommendations and the highest evidence currently available in the search database. The level of recommendation was determined by reclassifying this evidence using the Oxford Centre for Evidence-Based Medicine (OCEBM) grading system  Guideline authors should use systematic retrieval methods for evidence retrieval when writing guidelines, and display the diagnosis and treatment methods of radioactive enteritis as comprehensively as possible, instead of being limited to a certain classification of radioactive enteritis, to facilitate the reference of the users of the guideline. (II) If conditions permit, experiments with larger sample sizes should be carried out to improve quality. (III) Any recommendation should provide a detailed source of evidence so that guide users can review it at their discretion and decide whether to use the guidelines or not. (IV) Guide writers should be familiar with guide evaluation tools, such as the AGREE II tool. (V) The tool manual should be provided for users to view quickly. (VI) Taking into account the opinions of the people to whom the guidelines are applied will go a long way in improving the quality of the guidelines. (VII) Inviting a third party to review the guide will greatly improve its reliability. (VIII) More consideration should be given to the hindrance and facilitation factors of guidelines in the writing process. (IX) Rather than treating radiation proctitis and enteritis, it is better to use appropriate modern radiotherapy techniques to minimize the radiation dose to the rectum and intestines, such as intensity regulation and rectal septal hydrogel . These mEvery study has its advantages and limitations, and our study is no exception. The strengths of our study are as follows: (I) we attempted to review guidelines and recommendations independently and objectively; (II) to make it as convenient as possible for users, such as clinicians, to make a better choice of treatment options, we listed various recommendations; and (III) as much as possible, the retrieved literature was collated and compared to provide an improvement direction for guideline makers in the future. There are also some limitations in our study: (I) the languages used in the literature included only English and Chinese, which cannot fully represent global research results; (II) the guideline evaluation tool we used can only evaluate the guideline formulation method and cannot represent the therapeutic effect of the proposed recommendation itself, and the scoring is somewhat subjective; (III) the selection of retrieval words may have led to incomplete retrieval; (IV) clinical success was broadly defined as 10% improvement or normalization of hemorrhage- stopping hemoglobin in accordance with guidelines; and  bleedingThis study found that the differences in the methodological quality of the guidelines for radioactive enteritis, even within the same guideline, were pronounced between different domains, especially in the scope and purpose of stakeholder rigor. When applied, there was much dissent, and the opinions varied. The high consistency of the recommendations was due to the argon ion coagulation technique . Other treatments, such as radiofrequency ablation and cryoablation, also need more experimental data to prove their safety and efficacy. It is hoped that these observations will be taken into account when new guidelines are developed.Additional file1. Table S1: Levels of evidence and grades of the recommendations based on the Oxford Centre for Evidence-Based Medicine.