Mice were fed for 16 weeks, and blood, liver, and heart samples were collected for biochemical, histological, and electron microscopic analysis. The physical factors indicated that mice fed on HFD gained more body weight than the C-ND group. Blood parameters do not show significant differences, but overall, the glucose and cholesterol concentrations were raised in the mice fed with a fat-rich diet, with the highest concentrations in the HFD-BG group. The mice fed with HFD-BG and HFD-O had more lipid droplets in the liver, compared to HFD-DG and C-ND.The intake of various types and amounts of dietary fats influences metabolic and cardiovascular health. Hence, this study evaluated the impact of routinely consumed Pakistani dietary fats on their cardiometabolic impact. For this, we made four groups of mice, each comprising 5 animals: (1) C-ND: Control mice on a normal diet, (2) HFD-DG: High-fat diet mice on a normal diet plus 10% ( Cardiovascular diseases (CVDs) upset the heart and blood vessels. The buildup of fats in the inner walls of arteries is associated with the development of CVD. The narrowing of arteries hinders blood flow, damaging vital organs, especially the heart and brain. Cardiovascular diseases mainly include coronary artery disease (CAD), stroke, peripheral arterial disease, and aortic disease .CVD afflicts people of all ethnicities, gender, ages, and socioeconomic levels. CVDs are the leading cause of death worldwide, accounting for more than 32% of total annual deaths in 2019, of which 85% were due to stroke and heart attack. CVD cases have increased substantially from 271 million in 1990 to 523 million in 2019. A surge in CVD-related deaths also reached 18.6 million in 2019, compared to 12.1 million in 1990 [According to the WHO report, Pakistan has the highest prevalence of cardiovascular diseases globally, with 19% of all deaths caused due to CVDs. One in four middle-aged adults has a CVD in Pakistan . The CVDThe likelihood of developing cardiovascular diseases depends on risk factors such as hypertension, diabetes, an unhealthy diet rich in fats , fatty liver disease, tobacco use, physical inactivity, and obesity. Obesity is an independent risk factor for developing cardiovascular diseases, leading to excessive fat storage in cells, such as adipocytes, hepatocytes, and muscle cells . The higA significant factor that has tremendously influenced cardiovascular health is poor choice and higher intake of dietary fats. Different investigations show that trans fatty acids (TFA) positively associate with CVD development . The conA diet enriched in saturated fatty acids (SFA) coupled with physical inactivity induces the buildup of fats and cholesterol in the arteries, which causes atherosclerosis that results in stroke, heart attack, and myocardial infarction (MI). Atherosclerosis is a complex multifactorial pathological condition characterized by fatty plaque deposition in the coronary arteries (arteries that supply blood to the heart tissues) . CholestMoreover, consuming a lipid-rich diet can be associated with excessively adding lipid droplets in the hepatocytes. The liver regulates lipid metabolism by incorporating lipids in bile and producing very low-density lipoproteins (VLDL) . HoweverThe dietary patterns differ with cultures worldwide and so does the risk of CVD in different populations. South Asian culture is enriched with cuisines that require fats  to be cooked and fried, making the consumption of fats higher than in western countries.Butter and desi ghee (homemade butter when heated up to remove water content) are saturated fats that are assumed to be safe, compared to industrially prepared cooking oil (unsaturated fats) or banaspati ghee (derived from the hydrogenation of plant-based saturated oils). Traditionally desi ghee has been considered a pure and beneficial source of fat, due to its direct production from milk, most often at home. Although expensive, desi ghee is highly used in rural areas of Pakistan, due to its perceived beneficial effects on health. No scientific study has been conducted before to determine the impact of desi ghee on human health, and very limited data is available on the effects of such diets at the tissue level.Henceforth, we conducted the present study in laboratory mice to investigate the effects of home-prepared, dairy-based desi ghee, industrial cooking oil, and banaspati ghee on cardiovascular health. Four groups of mice were fed with different fats for 16 weeks, and the effect of these fats on weight, serum lipid profile, and heart and liver histology were investigated.The study was conducted in Diabetes and Cardio-Metabolic Disorders laboratory at the National Institute for Biotechnology and Genetic Engineering (NIBGE) in Faisalabad, Pakistan. The institutional review committee approved the study for animal experimentation.w/w high fat diet, HFD) intervention study, female BALB/c mice were used, with 20 mice divided into 4 groups of 5 animals per group. All mice were kept in the NIBGE animal facility on 12 h light and dark cycles with the supply of food and ad libitum water and ambient temperature. Food was normal chow for control mice or with the addition of 10% (w/w) of a specific type of fat for the experimental group, as mentioned below.In this diet  desi ghee .HFD-DG: Mice on a normal diet with 10% (w/w) vegetable/plant-based oil.HFD-O: Mice on normal diet with 10% (w/w) banaspati ghee (hydrogenated vegetable oil).HFD-BG: Mice on a normal diet with 10%  were collected for further analysis, and each organ was weighed.For histology (light microscopy/transmission electron microscopy), whole heart and part of the liver were fixed in formalin. Electron microscopy processed the abdominal aorta and part of the liver for histological changes to be viewed.Clinically significant biochemical parameters, such as blood glucose, lipid profile , alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, albumin, creatinine, and uric acid, were measured for serum sample from each animal using commercially available kits on a semi-automated clinical chemistry analyser (Microlab300).Heart and liver tissues were processed for histological analysis. Samples were fixed in formalin. These paraffin-embedded tissues were cut into thin sections and analyzed under the microscope at 40× magnifications after staining with haematoxylin and eosin (H&E) stain. Image J software was used for the image analysis.Microscopic analysis of the mouse abdominal aorta and liver was performed. Tissues were dipped in 5% glutaraldehyde prepared in 0.2 M phosphate buffer at pH 6.8. A total of 1% osmium tetroxide was used for post-fixation at room temperature for 18 h; 15 min washing was performed with sterilized distilled water and then dehydrated with ethanol diluted at 30%, 50%, 70%, and 100%, as the ethanol does not give the proper fusion with spur resin, which is why the tissue was left in absolute acetone twice at 30 min as a transitional solvent. The ratio of resin-to-acetone was as follows 1:3 for 18 h, followed by 1:1 and 3:1 for 18 h each. The final 100% resin mixture was added to the samples and left overnight. The samples were poured into moulds and incubated at 60 °C for 48 h.w/v boric acid (H3BO3) for 10 min at 60 °C. Glass slide-mounted sections were examined with Nikon Light Microscope.The polymerized resin blocks were cropped and tackled with a fine scalpel blade before thin or semi-thin serial sections of 5 microns were cut with Ultra Microtome (RMC MT 7000). Staining was performed with 5% toluidine blue prepared in 1% Transmission electron microscopy, ultra-thin serial tissue sections of approximately 120 nm were placed on 300 mesh nickel grids and stained the grids with 5% uranyl acetate for 30 min. Double-stained the grids with lead citrate for 10 min in a NaOH chamber. Examine the grids under transmission electron microscope (JEOL JEM 1010) at 80 Kv.Continuous variables were expressed as mean ± standard deviation. Percentage increase or decrease was calculated by comparing the values of experimental groups  with the control group.The impact of three dietary fats was assessed on the body weight and biochemical parameters of experimental mice. While monitoring the body weight, it was observed that mice fed with HFD-O and HFD-BG gained substantial weight, relative to mice given HFD-DG and a normal diet. Mice fed with the HFD-O group gained maximum weight . A signiThe analysis of biochemical parameters in C-ND, HFD-O, HFD-DG, and HFD-BG mice showed that blood glucose levels were ≥30% higher in groups fed with a high-fat diet , as compared to control mice fed on a normal diet .Analysis of the lipid profile demonstrates that a high-fat diet caused an imbalance in the concentrations of specific lipid parameters. HFD-O and HFD-BG showed a 21% and 22% increase in total cholesterol, compared to the C-ND group. However, in HFD-DG, the increase was only 4%, much less than the other fat-fed groups (HFD-O and HFD-BG). This can be linked to the fact that HFD-O and HFD-BG are prepared industrially with added preservatives. At the same time, HFD-DG is naturally derived from milk cream , without any further additives or preservatives.Similarly, the reduction in HDL-C (considered good cholesterol) concentration was nearly two-fold more in HFD-BG and HFD-O mice at 15% each, relative to HFD-DG-fed mice (at 8%). Moreover, there was a 4% increase in LDL-C in HFD-O and HFD-DG-fed mice, while an 11% increase was observed in HFD-BG-fed mice, compared to C-ND mice .Overall, the serum uric acid concentration was higher in mice fed a fat-rich diet. A substantial augmentation was witnessed in the HFD-BG group, which was 85% more than in C-ND. The oil group showed a 41% variation, and HFD-DG showed a 56% change, a moderate variation from HFD-O and HFD-BG .Heart and liver samples from control and high-fat diet mice were stained with hematoxylin and eosin. Results indicated no gross morphological change after 16 weeks in the heart —still, lHowever, L.D.s deposition was more in HFD-BG and HFD-O, relative to HFD-DG. All experimental mice, except C-ND, also observed apparent degeneration of cells, necrosis, and infiltration of inflammatory cells.Liver sections were processed for imaging by transmission electron microscopy to see any pathological changes in the cellular architecture. Accumulation of lipids in the C-ND group was found to be normal. At the same time, there was a considerable accumulation of lipid droplets in the mice on the HFD-O and HFD-BG diet, compared to the HFD-DG group mice . The current study is the first report from Pakistan assessing the impact of commonly used dietary fats  on cardiac health in the mouse model. Results of the biochemical analysis showed that the mice fed with oil and banaspati ghee gained more weight and showed a deranged lipid profile, compared to the controls. Uric acid was also higher in HFD-O and HFD-BG groups. Although the concentration of lipid profile, including total cholesterol, triglycerides, and LDL, was higher in the HFD-DG group than in control, this increase was less than in the other high-fat diet groups (HFD-O and HFD-BG). Histological analysis of liver sections also complemented these findings and showed a higher accumulation of lipid droplets in the HFD-O and HFD-BG groups. Although the amount of fat fed to the experimental mice was the same in all groups , the source of fat and its composition were different. Fats are the major component of the diet in our population, and the common sources are: (1) dairy (butter or desi ghee), (2) plant oils , and (3) banaspati ghee (obtained from plant oils after hydrogenation). Desi ghee (clarified butter or liquid butter) is more specifically used in Asian countries, especially Pakistan and India. Studies from these regions also complement the current study’s findings by showing that desi ghee helps reduce total cholesterol and LDL-C. Moreover, desi ghee helps attenuate LDL oxidation  and protFurthermore, repeated heating of oils deteriorate its beneficial effects by lipid oxidation and increases the chances of hypertension, vascular inflammation, and atherosclerosis . ChaturvGas chromatography (G.C.) coupled with a mass spectrometer detector showed that commonly used oil brands in Pakistan are rich in polyunsaturated fatty acids (PUFA). At the same time, banaspati ghee contains more saturated fatty acids and trans-unsaturated fatty acids. The percentage of monounsaturated fatty acid was almost similar in oil and banaspati ghee . Desi ghIn our society, where fat is considered an essential component of diet and foods are prepared with high amounts of fats, it is a common perspective that desi ghee is best among all other fat types because it is minimally processed and made at home without using any industrial procedures, while vegetable oil and banaspati ghee are prepared by industries by chemical processing of seeds or partial hydrogenation of vegetable oils and are, thus, considered less healthy. Research has shown that oil is superior to banaspati ghee, in terms of its health benefits, as it has less SFA and is rich in PUFA. However, the qualitative analysis of desi ghee, oil, and banaspati ghee by Raman spectroscopy showed the presence of distinctive isomers of conjugated linoleic acid in desi ghee, which was missing in banaspati ghee and oil . ScientiMoreover, desi ghee also contains phospholipids, especially cephalin, which act as an anti-oxidant, and the presence of short (12%) and medium chain fatty acids (20%) helps in its direct absorption to the liver and sustained supply of energy via β-oxidation and krebs cycle . The strOne of the major risk factors for CVD consequences is unhealthy dietary intake. The Pakistani diet has a predilection towards foods rich in carbohydrates and fats. Traditional cuisines are also cooked and fried in rich oil or Banaspati ghee containing trans-fatty acids . In addiThe current study was undertaken to evaluate the outcome of a high-fat diet, natural (desi ghee), and industrial sources (vegetable banaspati ghee and oil) in the mouse model by investigating blood chemistry and vital organs . Pronounced impacts of different dietary fats were observed on body weight, blood sugar, cholesterol, triglycerides, LDL-C, and HDL. Among diet intervention groups, HFD-oil and HFD-banaspati ghee were seen to increase mice’s body weight substantially, and their long-term use can lead to obesity. Obesity-driven metabolic syndromes are linked with the development of type 2 diabetes, established on insulin resistance and cardiovascular disease ,35.A Chinese study has reported a correlation between obesity and cardiovascular morbidities. The study demonstrates a high risk of cardiovascular morbidities with obesity, corroborating obesity as an independent risk factor of cardiovascular diseases . A similSerum cholesterol levels were 21% HFD-O and 22% HFD-BG, compared to HFD-DG (4%) and C-ND (7%). This high serum cholesterol, due to HFD-O and HFD-BG, can cause hypercholesterolemia, which causes the onset of obesity, insulin resistance, and decreased HDL levels. Dietary cholesterol is associated with reducing the fatty acid oxidation that increases hepatic and plasma triglyceride levels. Desi ghee can be a safer substitute, as the serum cholesterol level was nearly equal to the C-ND group. Studies have reported the association of the reduction in total cholesterol level with the reduced risk of atherosclerosis . Serum LDL-C levels were increased in all high-fat diet groups. In contrast, BG increase was 11%, compared to a 4% increase in groups fed with oil and DG. Our results are consistent with other studies , which dHistological analysis found no significant impact of a high-fat diet on heart tissues. This may be because the trial was conducted for only 16 weeks, which is not sufficiently long for the BALB/C mice to develop a fat reserve in heart tissues. However, lipid deposits were seen in the tissues of the liver. Electron microscopic analysis of hepatocytes revealed lipid droplet accumulation in all high-fat diet groups. L.D.s in the liver act as fat reserves, while more than 5% fat of liver weight is considered to be associated with non-alcoholic fatty liver disease (NAFLD) . Groups This trial comprised a brief period of 16 weeks, yet a considerable amount of lipid droplets were observed in the oil- and BG-fed groups. Thus, it can be inferred that prolonged intake of industrially sourced fat, both HFD-O and HFD-BG, can cause a buildup of fats in the liver. Evidence suggests a strong relationship between fatty liver with the onset of diabetes, cardiovascular diseases, and metabolic disorders ,42. A 6-Overall, we identified substantial differences in weight gain, serum cholesterol levels, and formation of lipid droplets in hepatocytes among high-fat diet groups. In contrast, the group fed with DG was observed to have lower levels than HFD-O and HFD-BG. No significant reduction in LDL-C levels in the mice fed with DG HDL-C was observed to decrease, which is an unexpected incidence, as a similar study conducted with butter intake found an increase in HDL-C levels . FurtherBased on these observations, we conclude that the long-term usage of desi ghee may pose less risk of obesity and obesity-related cardiovascular diseases in the animal models. Histological analysis of the hepatocytes indicates that desi ghee is a relatively safer option than industrially manufactured or hydrogenated oils. Studies with longer trials are recommended to precisely evaluate the effect of these fat sources on the progression of cardiovascular disorders.


Subcortical ischemic vascular disease (SIVD) is a leading cause of vascular dementia. The present study tries to explore not only the gender-specific association between H-type hypertension and SIVD but also the indirect effects of H-type hypertension on cognition through the ischemic brain injury caused by SIVD.A total of 601 SIVD patients were included, comprising 322 males and 279 females. H-type hypertension was defined as hypertension accompanied with elevated serum total homocysteine (tHcy) level. The imaging manifestations of ischemic brain injury caused by SIVD were also evaluated, including white matter lesions (WML), lacunar infarction (LI) and brain atrophy (BA). Gender-specific subgroup analyses in association between H-type hypertension and SIVD were conducted, followed by a structural equation model based evaluation of the gender-specific mediating effects of SIVD on the relationship between H-type hypertension and cognition.For males, there was no noticeable difference in WML, LI and BA scores among control group, isolated hypertension group, isolated high tHcy group, and H-type hypertension group in most brain regions, but significant difference was found in all brain regions for females. Multiple regression analyses showed that H-type hypertension was significantly associated with WML, LI and BA for females, but not for males. For males, H-type hypertension mainly affected cognition through direct effect, while the H-type hypertension effect was mediated by ischemic brain injury caused by SIVD for females.H-type hypertension was more closely related to SIVD for females than males, suggesting a gender-specific difference in association patterns between H-type hypertension and cognition. Subcortical ischemic vascular disease (SIVD) is the most prevalent type of cerebral small vessel disease and a leading cause of vascular dementia . The maiA large number of studies suggest that the prognosis of patients with acute or chronic cerebrovascular diseases is closely related to hypertension . Serum tCognitive impairment is a core clinical feature of SIVD, resulting in a huge disease burden . Recent The aim of this study is to investigate gender differences in association between H-type hypertension and SIVD, and explore whether SIVD mediates the relationship between H-type hypertension and cognitive function, as well as related gender differences.A total of 601 SIVD patients who were consecutively admitted to the Department of Neurology, Second Affiliated Hospital of Guangzhou Medical University between June 2018 and April 2022 were eligible for inclusion. All patients met the brain imaging criteria proposed by Erkinjuntti T , and werThe following exclusion criteria were then applied: history of brain tumor, cerebral hemorrhage and brain trauma, presence of severe unrelated central nervous disease  and leukoencephalopathy of non-vascular origin, presence of physical reasons for failure to complete the questionnaire . The average age of these patients was 73.49 ± 8.60 years old, and there were more males (53.5%) than females (46.5%).Demographic information were collected from these patients, including age, gender, educational level, history of smoking and alcohol intake, history of hypertension, hypertension duration, hypertension drug taken, history of hyperhomocysteinemia, and history of coronary heart disease. Venous blood samples were collected from SIVD patients to detect the levels of plasma tHcy, glycosylated hemoglobin (HbA1c), total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL-C) and low density lipoprotein (LDL-C).Hypertension was defined as seated, resting systolic blood pressure (SBP) ≥140 mmHg or diastolic blood pressure (DBP) ≥90 mmHg or self-reported history of hypertension. High tHcy is defined as tHcy concentration ≥10μmol/L . H-type Neuropsychological examination was performed by an experienced neurological physician using the mini-mental state examination (MMSE). MMSE consisted of 5 items in its evaluation of orientation (10 points), memory registration (3 points), attention and calculation (5 points), recall (3 points) and language (9 points) . Total sPost hoc analyses were performed via the Student-Newman-Keuls multiple comparison test. Categorical variables were summarized as frequency and percentage and analyzed using the Chi-square test. Gender-specific subgroup analyses in association between H-type hypertension and SIVD were conducted. Multiple linear regression analyses were performed to study the gender-specific relationships between H-type hypertension, WML, LI, BA and cognitive function adjusted for demographics and the plasma biochemical parameters. Structural equation modeling (SEM) technique was employed to evaluate the gender-specific mediating effects of WML, LI, and BA on the relationship between H-type hypertension and cognitive function  for all SIVD patients was evaluated. The direct effect of H-type hypertension on cognitive function was insignificant for all SIVD patients . The ind2/df = 1.879/1.086, CFI = 0.985/0.999, TLI = 0.963/0.997, RMSEA = 0.052/0.018.The gender-specific mediating effects of WML, LI, and BA on the relationship between H-type hypertension and cognitive function were also examined. The direct standardized path coefficients were shown, respectively, for male (M) and female (F) in Our analyses showed a gender difference in association between H-type hypertension and SIVD. H-type hypertension was more closely related to SIVD for females than for males. In addition, we found difference in the association patterns between H-type hypertension and cognition for SIVD patients of different genders.This study found that, despite the proportion of male patients with H-type hypertension was higher than female patients with H-type hypertension, H-type hypertension brought a higher risk of ischemic brain injury in most brain regions to female SIVD patients, but it was not obvious for male. For males, there was no significant difference in WML, LI and BA scores among the control group, the isolated hypertension group, the isolated high tHcy group, and the H-type hypertension group, except for the scores of WML in frontal lobe, parietal-occipital lobe, basal ganglia area and whole brain, as well as the BA scores in frontal lobe, temporal lobe, and basal ganglia area. For females, WML, LI and BA scores were significantly different among the control group, the isolated hypertension group, the isolated high tHcy group, and the H-type hypertension group in all brain regions. Multiple regression models showed that H-type hypertension was significantly associated with WML, LI and BA for females, but not for males. Previous researches had shown that the prevalence of hypertension and tHcy level were higher in males than in females , the assOur results indicated that H-type hypertension, the combination of hypertension and high tHcy, presented a greater risk of SIVD than either of them alone. Hypertension has been widely proven to be associated with cerebrovascular diseases . The effWe also found that H-type hypertension was associated with VCI for both male and female patients with SIVD, but the pathways of association were different. VCI is considered to be the most common cognitive disorders for the elderly as it encompasses any degree of vascular-based cognitive decline . SIVD isTo the best of our knowledge, there is no published study on gender difference in the association between H-type hypertension and SIVD. This study with a relatively large sample set suggests that female is more vulnerable than male to the deleterious effects of H-type hypertension on SIVD. However, this study had several limitations. First, due to the cross-sectional design of our study, the causal relationship between H-type hypertension and SIVD as well as its cognitive impairment remains undetermined, which needs further study to explore. Secondly, our study was a single-center study despite a relatively large sample size, a homogeneous SIVD population, and a comprehensive evaluation of all components of MRI features. Our study results could not be easily generalized to all SIVD patients. Thirdly, instead of a volumetric measurement, a visual rating scale was employed to measure WML, which is a widely used and robust measurement tool in clinical practice. Fourthly, the lack of a group of patients with H-type hypertension but no SIVD prevented a deeper analysis of the effects of gender differences. Finally, in addition to the risk factor for SIVD and the role of H-type hypertension, future study may also consider the effect of protective factors in this population, such as the role of caffeine or acetyl-L-carnitine intake on mood and cognition .The results of this study provide valuable evidence for the prevention of the detrimental effect of H-type hypertension on SIVD and subsequent harm to cognition, and point out a new direction for both epidemic and mechanism research in related fields. The finding of gender differences in the association between H-type hypertension and SIVD was conducive to the development of precise prevention and treatment. The identification of key causes of cognitive decline was important to reduce the burden of SIVD patients and their families. Future longitudinal studies with larger sample sets are expected to verify the results of this study and find out specific mechanism.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of Guangzhou Medical University (ID: 202107013). The patients/participants provided their written informed consent to participate in this study.JW undertook the statistical analysis and wrote the first draft of the manuscript. W-WZ conceptualized and designed the study. Y-XX and J-QL made contributions to interpretation of data. All authors contributed to the article and approved the submitted version.


Currently, there is no effective tool for predicting the risk of nonventilator hospital-acquired pneumonia (NV-HAP) in older hospitalized patients. The current study aimed to develop and validate a simple nomogram and a dynamic web-based calculator for predicting the risk of NV-HAP among older hospitalized patients.A retrospective evaluation was conducted on 15,420 consecutive older hospitalized patients admitted to a tertiary hospital in China between September 2017 and June 2020. The patients were randomly divided into training  and validation (n = 4624) cohorts at a ratio of 7:3. Predictors of NV-HAP were screened using the least absolute shrinkage and selection operator method and multivariate logistic regression. The identified predictors were integrated to construct a nomogram using R software. Furthermore, the optimum cut-off value for the clinical application of the model was calculated using the Youden index. The concordance index (C-index), GiViTI calibration belts, and decision curve were analysed to validate the discrimination, calibration, and clinical utility of the model, respectively. Finally, a dynamic web-based calculator was developed to facilitate utilization of the nomogram.P = 0.694) and validation (P = 0.614) cohorts did not cross the diagonal bisector line, suggesting that the prediction model had good discrimination and calibration. Furthermore, the optimal cut-off values for the training and validation cohorts were 1.58 and 1.74%, respectively. Analysis of the decision curve showed that the nomogram had good clinical value when the threshold likelihood was between 0 and 49%.Predictors included in the nomogram were the Charlson comorbidity index, NRS-2002, enteral tube feeding, Barthel Index, use of sedatives, use of NSAIDs, use of inhaled steroids, and "time at risk". The C-index of the nomogram for the training and validation cohorts was 0.813 and 0.821, respectively. The 95% CI region of the GiViTI calibration belt in the training (The developed nomogram can be used to predict the risk of NV-HAP among older hospitalized patients. It can, therefore, help healthcare providers initiate targeted medical interventions in a timely manner for high-risk groups.The online version contains supplementary material available at 10.1186/s12890-022-01941-z. Hospital-acquired pneumonia (HAP) is a common healthcare-associated infection (HAI) that affects approximately 1% of all hospitalized patients . NonventTo date, few risk prediction tools for NV-HAP in older hospitalized patients have been developed. A nomogram is a visualization method for complex mathematical models that combines multiple risk factors. This method provides accurate and individualized risk estimates for patients and presents them intuitively , 13. TheThe current study adopted a retrospective cohort study design. A total of 15,420 consecutive older hospitalized patients admitted to Wenzhou People's Hospital  between September 2017 and June 2020 were recruited. Patients with a stay length of < 48 h and those who had received mechanical ventilation during hospitalization were excluded from the study. In the case of repeated admissions for a patient, only the first admission was considered. The current study was approved by the Ethics Review Committee of Wenzhou People's Hospital (IRB no: WRY2018070). The same committee waived the requirement for informed consent due to the retrospective nature of the study.The following candidate predictors were collected: demographic data , Nutritional Risk Screening 2002 (NRS-2002), Barthel Index, Morse Fall Scale, other nosocomial infections, season of admission, Charlson comorbidity index (CCI), and admission category. Furthermore, the patient's personal history , comorbidities (chronic obstructive pulmonary disease (COPD), swallow disability, stroke, diabetes mellitus, peptic ulcer disease, moderate or severe renal disease, liver disease, congestive heart failure, and solid tumor), clinical procedures , in-hospital medications , systemic steroids, inhaled steroids, and anticoagulant) and laboratory values  were collected. The burden of comorbid diseases was described using the CCI . The NRSTo determine the effect of time on NV-HAP, the "time at risk" was also included as a potential predictor. For patients with NV-HAP, "time at risk" was calculated as the NV-HAP diagnosis date minus the admission date. For patients without NV-HAP, the "time at risk" was determined as the total length of stay. The variable "time at risk" was categorized into three subgroups based on tertile points, ≤ 6, 7–11, and ≥ 12 days. Laboratory data were collected from the first-time examinations at admission. During the "time at risk" period, clinical procedures, other nosocomial infections, and in-hospital medications were also collected.The data on NV-HAP were drawn from the Xinglin system, which is a real-time nosocomial infection surveillance system . The sysFor nosocomial infection cases, clinicians made an initial diagnosis, which was then confirmed by a senior infection control practitioner. In case of a discrepant diagnosis between the two sides of the diagnosis, a joint discussion was held to reach a consensus. NV-HAP was defined according to the 2018 version of the Chinese guidelines for diagnosing and treating hospital-acquired pneumonia and ventilator-associated pneumonia in adults . The defThe least absolute shrinkage and selection operator (LASSO) method was used to select optimal predictive features from the aforementioned 38 candidate predictors in the training cohort . The LASThe performance of the model in predicting NV-HAP risk was expressed in terms of discrimination and calibration. Discrimination of the prediction model refers to the ability to distinguish between individuals developing and not developing the outcome (NV-HAP) . The conP value > 0.05 in the calibration test demonstrated that there was no evidence of poor fit in the nomogram.The calibration of a prediction model refers to testing the agreement between the predicted and actual observed risk . The GiVPt represents the threshold probability at which the expected benefit of intervention-all-patients is equal to the expected benefit of intervention-none.Decision curve analysis (DCA) was performed to assess the clinical utility of the developed nomogram . DecisioIn addition, the highest Youden index was used to obtain the best cut-off value for clinical application (sensitivity + specificity − 1).IQR). Categorical variables were expressed as numbers (%) and compared using the chi-square (and Fisher's exact) test. Patients with missing data were excluded from this study because the proportion of cases with missing data was minimal (< 5%), and it was considered to be missing at random [https://www.R-project.org). A two-tailed P value of ≤ 0.05 was considered to be statistically significant.The present study was reported as per the Transparent Reporting of a multivariable prediction model for Individual Prognosis or Diagnosis (TRIPOD) statement . All thet random , 30. AllP = 0.507). The baseline characteristics of patients in the training and validation cohorts are presented in Table A total of 15,420 participants were included in the final analysis . For instance, using the dynamic nomogram for NV-HAP, the probability of NV-HAP in an older hospitalized patient with CCI = 8, NRS-2002 = 3, no enteral tube feeding, Barthel Index = 95 (slight dependency), use of sedatives, use of NSAIDs, use of inhaled steroids, and "time at risk" = 7 days was estimated to be 15.6%    is a major safety issue among hospitalized patients and leads to high treatment costs, mortality rates, and longer hospital stays. Therefore, it is important to develop risk prediction models that can accurately identify high-risk groups to facilitate the early implementation of preventive interventions. Researchers have developed tools for measuring the risk of NV-HAP. For instance, Evans  developeLASSO and multivariate logistic regression analyses were conducted to identify the key risk factors for NV-HAP in older hospitalized patients. Consequently, CCI, NRS-2002, enteral tube feeding, Barthel Index, use of sedatives, use of NSAIDs, use of inhaled steroids, and "time at risk" were identified as the major predictors and were used to develop a predictive nomogram. The constructed nomogram showed good discrimination and strong concordance performance in predicting NV-HAP. Similarly, the results of the DCA plots revealed that the nomogram was more effective in predicting NV-HAP than intervention-all-patients or intervention-none strategies. This implied that the developed nomogram can be implemented in clinical practice.It was also found that patients with 12 or more days of "time at risk" experienced a nearly fourfold increased risk of NV-HAP compared with those who had a "time at risk" of less than six days. A longer "time at risk" usually means a longer period of bed rest and hospitalization. Previous studies have shown that hospital stay duration is an independent predictor of healthcare-acquired infections , 34. TheThe Barthel Index, the CCI, and the NRS-2002 were used to assess performance in daily living activities, the burden of comorbid diseases, and nutritional status, respectively. These assessment tools were found to be independent predictors of NV-HAP. A lower Barthel Index indicates poor performance in daily activities, which may reduce respiratory secretion clearance and contribute to the development of NV-HAP . MalnutrCurrently, NV-HAP is diagnosed using imaging examinations and clinical feature changes , 43. HowThis study has some limitations. First, the study adopted a retrospective design and was performed in a single center, which limits the generalizability of the findings. Furthermore, although internal validation was conducted to ensure the robustness of the developed nomogram, external validation could not be conducted. Therefore, further research is needed to validate the nomogram using an external cohort. Second, the study selected a few readily available risk factors and medical interventions, leaving out others such as the risk across the continuum of care and indicators of blood gas analysis . Third, In conclusion, a novel nomogram combining CCI, NRS-2002, enteral tube feeding, Barthel Index, use of sedatives, use of NSAIDs, use of inhaled steroids, and "time at risk" was constructed in the present study. The nomogram showed good discrimination and calibration and hence can help healthcare workers estimate the risk of NV-HAP disease in older hospitalized patients. This will promote the implementation of early intervention for high-risk groups. However, there is a need to conduct external validation of the proposed nomogram in larger populations.Additional file 1. Fig.S1. Flow chart of the study population. Fig. S2. Screenshot of the online tool used for the prediction of NV-HAP risk. Table. S1 Diagnostic efficacy of the nomogram model for estimating the risk of NV-HAP.


Apis mellifera is highly appreciated worldwide because of its products, but also as it is a pollinator of crops and wild plants. The beehive is vulnerable to infections due to arthropods, fungi, protozoa, bacteria and/or viruses that manage to by-pass the individual and social immune mechanisms of bees. Due to the close proximity of bees in the beehive and their foraging habits, infections easily spread within and between beehives. Moreover, international trade of bees has caused the global spread of infections, several of which result in significant losses for apiculture. Only in a few cases can infections be diagnosed with the naked eye, by direct observation of the pathogen in the case of some arthropods, or by pathogen-associated distinctive traits. Development of molecular methods based on the amplification and analysis of one or more genes or genomic segments has brought significant progress to the study of bee pathogens, allowing for: (i) the precise and sensitive identification of the infectious agent; (ii) the analysis of co-infections; (iii) the description of novel species; (iv) associations between geno- and pheno-types and (v) population structure studies. Sequencing of bee pathogen genomes has allowed for the identification of new molecular targets and the development of specific genotypification strategies.The honeybee  Apis mellifera) is of enormous economic and cultural relevance to human society. On the one hand, its service as a pollinator of crops is indispensable to procuring food production worldwide, and on the other hand, it provides subsistence for beekeepers and farmers, and apicultural activity is associated with important social and cultural values. In addition, honey commercialization and export is a source of income for several countries around the world, and constitutes a natural food with many potential health qualities . O. OP. lar by qPCR ,291.M. plutonius DNA detection protocols are particularly useful for the screening of large numbers of samples: (i) a LAMP protocol targeting the gyrB (DNA gyrase subunit B) gene, and (ii) a colorimetric nanoparticle-based detection method. The first method has the advantages of showing a 10 times improved detection limit compared to a seminested PCR based on the 16S rRNA gene, and it does not require special equipment to analyze the results, while the second is characterized by its speed and low cost [In addition to the above-mentioned PCR-based diagnostics, two alternative low cost ,293,294.M. plutonius strains pertaining to CC12 comprise “atypical strains” with less demanding cultivation requirements than those of STs of “typical strains” that pertain to CC3 and CC13. In contrast, strains of CC12 display a higher virulence than those of CC3, while strains of CC13 are avirulent [napA (Na+/H+ antiporter) gene for identification of typical strains (CC3 and CC13) and the Fur (ferric uptake-regulator family) gene, for the detection of atypical strains (CC12), is available  and Iflaviridae , while some are unclassified . Recent advances in metagenomics have allowed us to identify new viruses in bees and other pollinators; including bee macula-like virus (BeeMLV) from the Tymoviridae family, Osmia cornuta nudivirus (OcNV) from the Nudaviridae family, Apis rhabdovirus (ARV-1), A. mellifera iflavirus, and A. mellifera feranovirus, among others [Most bee viruses belong to the families g others ,330.A. mellifera filamentous virus (AmFV) [Most genomes of bee viruses are composed of positive-sense single-strand RNA (+ssRNA). RNA viruses have high mutation rates and often form diverse populations of variants or quasi-species . In conts (AmFV)  and OcNVs (AmFV) .Since bee viruses do not replicate efficiently in cell culture, the effect of viral replication in the host cell cannot be assessed. Thus, infections by bee viruses are classified as overt and covert infections according to the presence or absence of clinical signs, respectively. Covert infection appears when a high replication level of the viral agent is reached in affected tissues or organs, and favors horizontal transmission. In overt infections, on the other hand, vertical transmission is the preferred route of virus spread, and the infection typically persists in the population for many generations. The latter is the most common way viruses infect colonies, but as stated before, under adverse conditions, overt infections may turn into covert infections. In addition, different viral pathogens can infect colonies simultaneously and interact with each other, increasing their pathogenic effect. Diagnosis of bee viruses by conventional methodologies is complicated due to the lack of appropriate cultures, common clinical signs of different viral infections, and the high frequency of multiple infections in the individual bee and/or colony. Thus, detection of bee viruses is mainly carried out by molecular diagnostics, focusing on the amplification of a particular specific sequence region of the viral genome. Nucleic acid amplification by conventional RT-PCR has been widely used for the detection of many bee viruses . MoleculOf note, LAMP has been demonstrated to be a rapid, cost-effective, and robust technology that can be used with crude extracts, avoiding time-consuming purification of nucleic acids. For detection of bee viruses, LAMP assays have only been developed for country-specific SBV outbreaks. The developed LAMP assays target the polymerase or viral protein gene of SBV with a similar analytical sensitivity to RT-qPCRs (reviewed in ).In recent years, RT-qPCR assays have become the preferred technique for bee virus detection due to their high sensitivity and the possibility of multiplexing, quantification and high-throughput processing ,336. MorThe most commonly detected viruses associated with economic losses worldwide are ABPV, BQCV, CBPV, DWV, KBV, IAPV and SBV. Studies describing the presence, abundance or prevalence of these viruses utilize either end-point RT-PCR or RT-qPCR assays, and there is an enormous number of specific primers available designed for this purpose. In the context of this review, we will focus on newly developed molecular approaches and on recently reported field studies applying previously developed assays. In Iflavirus, (+)ssRNA. Three variants of DWV have been described, comprising DWV-A, DWV-B , and DWV-C. DWV-C variants group all the sequences resulting from recombination between DWV-A and DWV-B [The deformed-wing virus is one of the main viruses affecting the honeybee. As mentioned above, DWV belongs to the genus nd DWV-B ,340,341.V. destructor functions as a highly efficient vector within the honeybee colony [A. mellifera . Virus infection is associated with the appearance of bees with deformities in the wings and results in a shortened life expectancy, manifested at the colony level by a progressive decrease in the size of the population [DWV spreads via different transmission routes and e colony ,342. Thipulation ,344.The most common method for DWV detection is RT-PCR. A variety of primers have been described that amplify different regions of the virus genome encoding structural and non-structural proteins. Most of the amplification primers target the RNA-dependent RNA polymerase gene (RdRp) ,342,345.Recently, a real-time, micro-scale, chip-based PCR system allowing for rapid detection of viral RNA was described. This methodology amplifies a RdRp region through the use of DNA chips, a technique that identifies a specific nucleotide sequence through DNA hybridization . On the The advancements in sequencing methodologies have provided the possibility of implementing the analysis of complete DWV genomes in such a way that recombination points between the DWV-A and DWV-B variants can be determined, circulating strains originating in different regions can be characterized, and molecular differences between covertly circulating DWV strains or highly virulent viruses can be identified ,354,355.Another virus closely related to DWV is the Kakugo virus (KV), with which it shares 96% nucleotide identity. Both are often considered to be variants of a single species complex ,356. As Iflavirus; its detection by molecular assays such as RT-PCR or RT-qPCR uses specific regions encoding capsid proteins or non-structural proteins such as helicase and RdRp [A. mellifera colonies [Sacbrood virus (SBV) also belongs to and RdRp ,362,363.colonies ,363,364.Iflavirus first discovered in the predatory social wasp. In a recent study testing for the presence of MV by RT-qPCR, 43% of honeybee colonies (n = 69) resulted positive; MV replication could be confirmed in A. mellifera and Vespidae species [Moku virus (MV) is an  species .Sinaivirus) that affect honeybees and were recently detected through metagenomics studies [V. destructor [Varroa infestation levels are not associated, suggesting that this mite is not a vector of LSV [Lake Sinai viruses (LSV) are (+)ssRNA viruses  is a bipartite, (+)ss RNA virus. Although it was the first bee virus isolated and described from honeybees , its famV. destructor could be determined by Celle et al. [Detection and quantification of CBPV based on RT-qPCR assays have been reported. Blanchard et al.  developee et al. ; howeverDicistroviridae (+ssRNA), these viruses normally persist as covert infections within the colony, and are detectable by RT-PCR assays [The acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), and Israeli acute paralysis virus (IAPV), form the ABPV-KBV-IAPV complex. Infection by each of the three viruses shows a similar pathology at the level of the individual bee and the colony. Like most members of R assays .Since ABPV, KBV and IAPV are genetically very similar, there is a potential risk of misdiagnosis including false-positive and false-negative detection, and strain misassignment . To avoiV. destructor is an effective vector of IAPV [IAPV is one of the most studied viruses because it can infect every member of the colony and its detection strongly correlates with colony collapse disorder (CCD) ,381,382. of IAPV .IAPV covert infection induces a broad spectrum of clinical signs including paralysis and body trembling. However, whether the neuron system is the preferred tropism of the virus has not yet been resolved . In a stDicistroviridae and has two ORFs each encoding for a polyprotein, and each preceded by an internal ribosomal entry site (IRES) [IAPV is a positive single-stranded RNA virus that was first described as a distinct pathogen from KBV and ABPV based on its genome sequence in Israel in 2004 . IAPV bee (IRES) . In a phe (IRES)  reportedSeveral RT-PCRs assays have been developed to detect IAPV ,381,383;Tai Truong et al.  developeDistrictoviridae family and has a similar genome structure to ABPV and KBV. Transmission is driven mainly by nurse honeybees that horizontally transfer BQCV from infected cells to healthy larvae in brood food, but queens may also vertically transmit it to eggs. It has been shown that BQCV is associated with the infection of the microsporidian N. apis [Orf2 and the 5′UTR region are most frequently chosen as targets for nucleic acid amplification [One of the most prevalent honeybee viruses is the black queen cell virus, which affects developing queen larvae and pupae during the capped-cell stage. BQCV belongs to the  N. apis . This vification ,389,390.Most studies on bee viruses analyze the presence of several, including DWV, BQCV, ABPV, CBPV, IAPV and SBV. In early developments, Benjeddou et al.  applied V. destructor, Nosema spp., and a diverse set of honeybee viruses. All viral agents were detected by RT-qPCR using either previously reported primers sets [Moharrami and Modirrousta  describeers sets ,389,392 ers sets .In a prospective study, Runcket et al.  investigSimilar results were reported by Cavigli et al. , who shoIn order to identify synergistic interactions between pathogens and/or parasites that may be involved in colony death, D’Alvise et al.  studied V. destructor. Detection of all viruses was performed by RT-qPCR using primers developed by Locke et al. [V. destructor.Molineri et al.  comparede et al. . Five viSeveral multiplex PCR assays have been designed for honeybee viruses ,402,403.® probes with different fluorophores. The main reason for multiplexing is to save cost and time; however, multiplex qPCR is less sensitive than uniplex qPCR and more complex to optimize. Multiplexing is far more effective through a microarray. Numerous honeybee microarrays have been designed, including honeybee immune gene-pathogen arrays [Real-time qPCR can also be multiplexed, usually for the simultaneous amplification of a target and internal reference standards, by using TaqMann arrays ,379 and n arrays . For resn arrays .®) for quantifying ABPV, BQCV, DWV, VDV1 and SBV VLs in naturally infected honeybees with and without clinical signs. After analyzing the VL distribution in both groups, it was possible to define a threshold that allowed for differentiating between covert and overt infections.Real-time PCR assays allow for the quantification of viral load (VL) and determination of thresholds at which clinical signs are observed, so that VL levels can be compared between different seasons or between healthy and diseased bees. Correspondingly, the VLs of six honeybee viruses  have been determined and compared in adult honeybees with and without clinical signs. Two newly developed RT-qPCR assays for the detection of LSV3 and SBV were applied in this investigation. Importantly, statistically significant differences between the VL of positive samples were identified between healthy and clinically affected honeybees for ABPV, CBPV, DWV, and SBV, while for BQCV and LSV3, no statistical differences were observed . MoreoveV. destructor-parasitized pupae of known patrilines were determined at different levels of mite infestation. The results showed different patterns of intra-colonial variation for both variants of DWV, suggesting more complex spatiotemporal dynamics of bee virus infections than was previously thought [The pattern of infection of DWV-A and B variants was analyzed at the intra-colonial level by quantification of bee virus RNA in pupae and larvae using RT-qPCR. To this end, VLs from  thought .The development and optimization of high-throughput sequencing technologies make it possible to sequence the genetic material of the entire community of viruses present within a host, without any prior knowledge of viral genome sequences. This approach is known as viral metagenomic analysis. Metagenomics allows for a better characterization of viral diversity and accelerates the identification of novel viruses affecting honeybees and other pollinators ,411,412.Tymoviridae family, A. mellifera flavivirus (AFV) and A. mellifera nora virus 1 (ANV) [Rhabdoviridae: A. mellifera rhabdovirus-1 (ARV-1) [A. mellifera rhabdovirus-2 (ARV-2) [Recent studies have reported the identification of additional honeybee-infecting (+)ssRNA viruses including the bee macula-like virus (BeeMLV) of the  1 (ANV) . In addi (ARV-1)  and A. m (ARV-2) .Secoviridae, ; (ii) a novel (+)ssRNA virus within the family Nodaviridae, (iii) a tymo-like virus, (iv) A. mellifera Rhabdovirus 1 (ARV-1) (family Rhabdoviridae), (v) a partiti-like virus (family Partitiviridae), and (vi) a circo-like virus (family Circoviridae)Galbraith et al.  performeA. m. ligustica, A. m. syriaca, A. m. intermissa, and A. cerana and their respective V. destructor mites, were analyzed by RNA metagenomics to compare the composition of their viral populations. In this study, two novel viruses: Varroa orthomyxovirus-1 (VOV-1) in A. mellifera and V. destructor, and a Hubei like-virga virus-14 homolog in V. destructor were reported, as well as some recently described viruses: ARV-1, BRV-1, VDV-2, and VDV-3. by [Viromes from the Western honeybee subspecies DV-3. by . It can In this article, we have presented an overview of available molecular diagnostics and genotyping tools for the detection and analysis of the main pathogens of the Western honeybee. This overview should assist in the selection of adequate tools for planned research and inform on a potential lack of molecular tools that would be important to be developed.T. mercedesae), and (vi) eradication programs or rapid and cost-efficient screening of hive products for pathogen contamination .The available molecular tools are indispensable for: (i) in-depth investigations of still-understudied or emerging bee pathogens, (ii) detection of as-yet-unknown or cryptic species and elucidation of their strain composition, (iii) studies of coinfection with particular bee pathogens that synergistically increase pathogenicity, (iv) worldwide surveillance of the most virulent pathogens, (v) tracing the source of outbreaks, (v) a rapid response to emerging pathogens (e.g., Furthermore, the presented information is aimed at facilitating an integrated view on the particularities of each pathogen and pathogenic group and interacting effects between them, as well as their commonalities. On the other hand, it allows for comparing and contrasting the molecular diagnostic approaches used to study these pathogens and pathogenic groups.Many of the pathogenic agents dealt with in this review have been introduced into the managed population of the Western honeybee through man-made upheavals or by trading of bees. As evolutionary adaptation of honeybees to introduced alien pathogens is an extremely slow and gradual process, human intervention is needed to prevent their further distribution and to minimize their damage on the honeybee population. The availability of reliable diagnostic methods for the sensitive and specific detection of bee pathogens is the first step needed in the efficient implementation of control measures.


Pulmonary embolism (PE) is a common disease with an annual incidence rate ranging from 39–115 per 100,000 inhabitants. It is one of the leading causes of cardiovascular mortality in the USA and Europe. While the clinical presentation and severity may vary, it is a life-threatening condition in its most severe form, defined as high-risk or massive PE. Therapeutic options in high-risk PE are limited. Current guidelines recommend the use of systemic thrombolytic therapy as first-line therapy (Level Ib). However, this treatment has important drawbacks including bleeding complications, limited efficacy in patients with recurrent PE or cardiac arrest, and formal contraindications. In this context, the use of venoarterial extracorporeal membrane oxygenation (VA-ECMO) in the management of high-risk PE has increased worldwide in the last decade. Strategies, including VA-ECMO as a stand-alone therapy or as a bridge to alternative reperfusion therapies, are associated with acceptable outcomes, especially if implemented before cardiac arrest. Nonetheless, the level of evidence supporting ECMO and alternative reperfusion therapies is low. The optimal management of high-risk PE patients will remain controversial until the realization of a prospective randomized trial comparing those cited strategies to systemic thrombolysis. Pulmonary embolism (PE) is a common disease with an annual incidence rate ranging from 39–115 per 100,000 population  and it iTherapeutic options in high-risk PE are limited. Current European guidelines recommend the use of systemic thrombolytic therapy as the first-line therapy (Level Ib) . HoweverHigh-risk PE is defined as acute PE with hemodynamic instability, characterized either as persistent hypotension (systolic blood pressure less than 90 mmHg for more than 15 min without signs of organ hypoperfusion), shock (systolic blood pressure less than 90 mmHg with signs of organ hypoperfusion), or cardiac arrest . To signWhich patients? Thrombolysis is an established treatment in patients presenting an obstructive shock. It has been used in severe PE for more than 50 years. According to the 2019 guidelines of the European Society of Cardiology, systemic thrombolysis is recommended as the first-line therapy in high-risk PE (Level I) .,40.39,40VA-ECMO and catheter-guided thrombolysis. Data regarding the use of catheter-directed thrombolysis associated with ECMO in PE patients are scarce (three studies with 36 patients). As with surgical embolectomy, pre-emptive VA-ECMO could be implemented in the presence of cardiogenic shock, to stabilize the patient and bridge them safely to that procedure. Georges et al. reported a mortality of 25% among 16 patients with combined procedures whereas it was 100% among patients with systemic thrombolysis only [sis only . FurtherVA-ECMO and long-term outcomes. Few studies have investigated the long-term outcome of patients requiring ECMO for high-risk PE. Stadlbauer et al. analyzed the data of 119 high-risk PE patients supported by VA-ECMO of whom 67% had ECMO during or after cardiopulmonary resuscitation (CPR) [on (CPR) . The oveE-CPR in PE: is it too late? As in many interventions, the timing of ECMO onset is of utmost importance. Indeed, survival rates were constantly significantly higher when ECMO was implanted for PE-associated cardiogenic/obstructive shock compared to during E-CPR [ng E-CPR ,53,54,55ng E-CPR . Similarng E-CPR . A recenng E-CPR . Among 3ng E-CPR . Nonetheng E-CPR . Severalng E-CPR ,34,45.A future RCT in the context of PE-related cardiac arrest is unlikely. Data from registries with a large sample size could offer a glimpse into this important clinical question. In this regard, Hobohm et al. investigated the outcomes of patients with PE deteriorating to cardiac arrest over 14 years . Among 1Altogether, those results suggest that E-CPR in the context of massive PE seems to be associated with a higher survival rate compared to systemic thrombolysis. Its use should be discussed on a case-to-case basis, as pre-ECMO thrombolysis, especially in the context of cardiac arrest, appears to be associated with severe complications and worse outcomes. Additional investigations are required to clarify the optimal sequence and timing of therapies in this highly selected population.What are the latest recommendations? While the physiological rationale is appealing, the use of VA-ECMO in the setting of high-risk PE is not supported by any RCTs and remains poorly defined. In this context, the 2019 ESC guidelines stated “the use of VA-ECMO as a stand-alone technique with anticoagulation is controversial” and is “of no clear clinical benefit unless combined with surgical embolectomy”. Finally, the European guidelines recommend that ECMO “may be considered in combination with alternative reperfusion strategy, in patients with PE and refractory circulatory collapse or cardiac arrest”  [Level C) .VA-ECMO and PE: key points for implantation. Several key practical points should be discussed regarding the management of PE patients requiring ECLS. In the context of cardiogenic shock and contraindication to thrombolysis, ECLS should be implanted under local anesthesia and procedural sedation in a spontaneously breathing patient. Indeed, mechanical ventilation in the context of acute RV failure should be avoided at all costs as an additional increase in the RV afterload may precipitate circulatory collapse. In addition, after failed thrombolysis, hemorrhagic complications must be anticipated accordingly when VA-ECMO is used as a rescue therapy. In centers where intensivists or cardiologists carry out the procedure, we believe that vascular or cardiac surgeons should be available during the cannulation to face potential catastrophic complications associated with canulation and thrombolysis-induced coagulopathy. A practical algorithm for ECMO-decision and care management of high-risk pulmonary embolism is proposed in More than 60 years after the first trial on systemic thrombolysis, the optimal management of high-risk PE remains controversial. The latest guidelines pointed out that thrombolysis is still the treatment with the highest level of evidence, which is factually correct. Nonetheless, the latest trial supporting this strategy was conducted in 1995 and only included eight patients. In addition, meta-analyses of RCTs supporting the efficacy of thrombolysis included only a minority of high-risk PE patients.There is a growing body of evidence suggesting there is room for other strategies in the algorithm management of this population. VA-ECMO support appears suitable either to reverse the hemodynamic impairment and bridge patients to recovery or to further reperfusion therapies. Surgical embolectomy seems to be associated with a higher survival rate than catheter-guided thrombolysis, especially when combined with VA-ECMO. However, to date, there is no RCT comparing any of these strategies with systemic thrombolysis. It may be time to conduct this trial to optimize the treatment and prognosis of those patients. Beyond short-term mortality, the safety of these strategies should be carefully assessed.As highlighted in this narrative review, one of the main limiting factors of such RCTs will be the sample size, as studies published from high-volume tertiary centers and often conducted over more than 10 years, rarely included more than 100 patients . An inteHigh-risk PE is a life-threatening condition. Compared to other cardiovascular diseases, research has been limited and physicians have a limited therapeutic arsenal at their disposal. The main standard treatment is still systemic thrombolysis. However, the use of VA-ECMO has increased worldwide in the last decades. In case of thrombolysis contraindications or failure, using VA-ECMO as a stand-alone therapy or as a bridge to alternative reperfusion strategies seems wise and is associated with acceptable outcomes. Further studies, especially RCTs are urgently needed to better define the optimal care for this highly selected population.


Deep learning-based automatic classification of breast tumors using parametric imaging techniques from ultrasound (US) B-mode images is still an exciting research area. The Rician inverse Gaussian (RiIG) distribution is currently emerging as an appropriate example of statistical modeling. This study presents a new approach of correlated-weighted contourlet-transformed RiIG (CWCtr-RiIG) and curvelet-transformed RiIG (CWCrv-RiIG) image-based deep convolutional neural network (CNN) architecture for breast tumor classification from B-mode ultrasound images. A comparative study with other statistical models, such as Nakagami and normal inverse Gaussian (NIG) distributions, is also experienced here. The weighted entitled here is for weighting the contourlet and curvelet sub-band coefficient images by correlation with their corresponding RiIG statistically modeled images. By taking into account three freely accessible datasets , it is demonstrated that the proposed approach can provide more than 98 percent accuracy, sensitivity, specificity, NPV, and PPV values using the CWCtr-RiIG images. On the same datasets, the suggested method offers superior classification performance to several other existing strategies. For both industrialized and developing nations, female breast cancer is a very pressing issue. According to a current report from the American Cancer Society’s Cancer Statistics Center, there are expected to be 1,918,030 new cases of breast cancer in 2022, while there will be only 609,360 cancer deaths in the country overall, with 290,560 of those cases (or roughly 48 percent) being breast cancer cases [Among the other imaging modalities, such as mammography and MRIs, one of the most promising techniques is breast ultrasonography (US) imaging for classifying breast tumors. Numerous studies have been conducted and are continually being conducted to increase the precision of classifying benign from malignant breast tumors automatically. The depth-to-width ratio, the normalized radial gradient, and the autocorrelation feature were three computer-extracted characteristics that were combined in 2002 by K. Horsch et al.  The clasAnother multi-resolution transform domain name contourlet transform-based technique is depicted in . PyramidContrary to the contourlet transform, the DWT has poor directional selectivity in two dimensions and is unable to give a wide range of directions. However, the curvelet transform also has a variety of directions. Breast tumor classification using a local binary pattern, the curvelet transform-based feature extraction method, is presented in  and repoStatistical modeling is another imaging technique where various features are extracted from the statistical models, such as Gaussian or Nakagami images obtained from the original B-mode images, which is called parametric imaging ,22, and In this study, it is demonstrated that an extremely successful model is the Rician inverse Gaussian (RiIG) distribution, which comprises the statistics of the contourlet and curvelet coefficient images . It has The features recovered from the database US B-mode images, parametric (P) images, contourlet-converted (C) images, contourlet parametric (CP) images, and weighted contourlet parametric (WCP) images, have previously been demonstrated to have the highest level of accuracy . So, onlThe appropriateness of the Rician inverse Gaussian (RiIG) distribution for statistical modeling of both contoured and curvelet-transformed breast ultrasound images is demonstrated in this study.To assess the feasibility of correlated-weighted contourlet- and curvelet-transformed parametric (CWCtrP and CWCrvP) images in classifying breast tumors employing three distinct publicly available datasets, a new investigation is conducted.A new correlated-weighted contourlet-transformed RiIG (CWCtr-RiIG) and curvelet-transformed RiIG (CWCrv-RiIG) image-based deep CNN architecture is proposed.The following list summarizes this work’s significant contributions: https://data.mendeley.com/datasets/wmy84gzngw/), accessed on 28 February 2020, includes work by Rodrigues et al. [http://www2.docm.mmu.ac.uk/STAFF/m.yap/dataset.php), accessed on 21 April 2020 [https://scholar.cu.edu.eg/?q=afahmy/pages/dataset), accessed on 13 December 2020 [This study examined 996 clinical cases in 1193 US images; Database-I (Mendeley Dataset) provided 250 of these cases, Database-II (Dataset UDIAT) provided 163, and Database-III ((Dataset BUSI)) provided 647. The Database-I, which is available at  domain includes only horizontal, vertical, and diagonal dimensions, it offers limited dimensional information. The contourlet transform, on the other hand, supports a wide range of arbitrary forms and contours that are not restricted to three dimensions. The normalized B-mode images are transformed using the contourlet transform to use a filter bank to separate the directed and multiscale decompositions , as illuThe best sparse representation of objects with edges and contours is provided by the curvelet transform. In contrast to the isotropic components of wavelets, the needle-shaped elements of the curvelet transform have extremely high directional sensitivity and anisotropy. Later, the second-generation curvelet transform was demonstrated to be a very effective tool for a variety of applications, including partial differential equations, seismic data exploration, image processing, and fluid dynamics (PDEs). Periodization was used to treat image borders in earlier iterations of the transform previous version. The data have been properly arranged in this case, and the discrete cosine domain will be tiled instead of the discrete Fourier domain, which is a significant change. A discrete filter bank structure, called smooth images with piecewise smooth contours, can be handled via contourlets. Structures resembling curvelets in the continuous domain can be coupled to this discrete transform. As a result, the contourlet transform can be considered a discrete version of a certain curvelet transform. Rician inverse Gaussian (RiIG) imageα and β affect the distribution’s steepness and skewness respectively; β < 0 indicates a distribution that is skewed to the left, and β > 0 indicates one that is slanted to the right, whereas δ is the dispersion parameter. The value of Eltoft et al.  introducα and β, the distribution becomes steeper and skews to the right, respectively. On the other hand, as β decreases, it skews to the left. In addition, as δ) map is created by processing the contourlet and curvelet coefficient images via a square sliding window, which results in the contourlet and curvelet parametric (CP) image. For the Nakagami image, parameter (δ) was examined utilizing the contourlet and curvelet sub-band efficient images with a sliding window of 13 × 13 pixels. The size of the sliding window that is being used should be larger than the speckle and be able to discern different local structure differences in malignancies. The new pixel added to the window’s center at each point when the window was moved across the entirety of the contourlet and curvelet sub-band efficient pictures in steps of 1 pixel was designated as the local RiIG parameter (δ). The map of RiIG parameter values produced by this technique is known as the RiIG parametric image. With the relevant figures and a percentile probability plot (pp-plot), the RiIG statistical model is already proven to be preferable to the Nakagami statistical model [It is seen that, with an increase in shown in , where tshown in ,39,40 obal model . In thisThe CP images are linked with the appropriate contourlet and curvelet sub-band coefficient images to produce the CWCtr-RiIG and CWCrv-RiIG images. By executing correlation operations with the corresponding contourlet and curvelet sub-bands, the CP images’ parameter values are all weighted. The term “correlated-weighted contourlet- or curvelet-transformed RiIG” might be used to describe these images. The proposed correlated-weighted statistically modeled contourlet and curvelet coefficient image-based classification schemes are illustrated in Regarding contourlet transform domain and curvelet transform domain, when applied to the CNN network, those 0-to-255-pixel value images are normalized and variations will be less because the pixel values such as 255 and higher than 200 will be converted to pixel value 1, thus having fewer variations. On the other hand, the CWCtr-RiIG and CWCrv-RiIG image pixel values are −1 to 1, having more variations when normalized in a deep CNN network, which will be an impact on feature extraction. The suggested classification schemes evaluate the classification performance on correlated-weighted parametric versions of contoured and curvelet-transformed images for both schemes. The findings are displayed in Applying this combined activation function, the accuracy, sensitivity (true positive rate), and specificity (true negative rate) is attained at 98.95%, 98.9%, and 99%, respectively, with an F1 score of 0.99, which means that although the accuracy is not changed, the F1 score slightly increases. In the deep learning-based classification task, each value after a point with a high accuracy (such as 98%) is significant. The best classification accuracy is demonstrated in the previous section using the deep CNN classifier with the RiIG-based CWCtr-RiIG pictures. In this paper, a novel approach to breast tumor classification is presented, employing RiIG statistically modeled correlated-weighted contourlet- and curvelet-transformed RiIG images in a deep CNN architecture. In the first approach, the RiIG statistically modeled CWCtr-RiIG and CWCrv-RiIG images are classified by deep CNN fully connected classifiers. In the second approach, the RiIG statistically modeled CWCtr-RiIG and CWCrv-RiIG images are classified by deep CNN-SVM, KNN, and random forest machine learning classifiers. It is demonstrated that a high level of accuracy can be attained by using the deep CNN fully connected classifier. Second, a brand new, specially created deep CNN architecture is suggested for classifying CWCtr-RiIG and CWCrv-RiIG images of breast tumors since it performs more accurately. Additionally, the suggested deep CNN design can use the loss function to provide extremely high levels of sensitivity, specificity, NPV, and PPV values, combining SoftMax and sigmoid activation functions. On benchmark publicly available datasets, both algorithms show superior classification performance to the state-of-the-art techniques. Additionally, the RiIG distribution is a distribution that is very well-suited for simulating the characteristics of the contourlet and curvelet transform coefficients of breast tumor images obtained in B-mode ultrasound. By applying the transformer model-based approach and including additional datasets, there is room for improvement.


Science 2019; 363(6431):1093-97], or protecting proteins, e.g. preventing stress-induced damage of the translation termination factor Sup35 . Correspondingly, they play fundamental roles at cellular and organismal levels. However, if the regulatory mechanisms of these assemblies are error-prone, aggregation-related diseases ensue. These ideas inform us that 1) protein structure, 2) nearby physical interactors, and 3) cellular regulatory elements overall serve as the molecular determinants of protein assemblies. The contributing articles in this Research Topic explore protein structure-function, structure-aggregation, and aggregation-disease relationships.Protein structure determines its biological function. Conceptually, protein structure can be classified as folded or “natively-unfolded”. While the defined fold of the former dictates a unique function, the latter also called “intrinsically-disordered”, allows polypeptides to engage in a multitude of interactions. Structural features determine also the self-assembly propensity of a protein. Beyond the intrinsic structural aspect, a protein’s tendency to form higher-order assemblies can be influenced by interacting proteinaceous/non-proteinaceous biomolecules. The material properties of assemblies that are not membrane-enclosed range from liquid to gel-like biomolecular condensates to solid-like aggregates. These assemblies can serve to fulfill various biological functions, including fostering a reaction, for instance, increasing the activity of actin regulatory proteins [Angelotti) is a comprehensive review that explores the Yip (Ypt-interacting protein) family proteins, their binding partners, and cellular functions that mostly encompass membrane shaping. The article epitomizes classic examples of dealing with and debating topology-to-function links of proteins.“Exploring the eukaryotic Yip and REEP/Yop superfamily of membrane-shaping adapter proteins (MSAPs): A cacophony or harmony of structure and function?”, by  disaggregase system from E. coli on aggregates with a particular focus on aggregate internal structure. The authors show that aggregates form particles resistant to chaperone-mediated disassembly over time. This transitioning of assemblies into more solid-like states has been observed for a number of phase-separating and aggregating proteins and can be associated with a variety of age-related diseases. For example, the nucleating core of the Alzheimer’s disease-related amyloid-beta 42 peptide assembles through a liquid-like state into the characteristic amyloid cross-ß structure. The study by Gordon-Kim et al. about “Polyanion order controls liquid-to-solid phase transition in peptide/nucleic acid co-assembly” demonstrates how nucleic acid structures can externally template this liquid-to-solid transition. The authors propose mechanisms for templating events in condensation and amyloid nucleation/propagation events that might be applicable to other assemblies.Various mechanisms monitor and regulate the formation, properties, dynamics, and dissolution of biomolecular condensates and aggregates/amyloids, such as posttranslational modifications, and the protein quality control system, consisting of molecular chaperones and the protein degradation machinery. The article “The Impact of Hidden Structure on Aggregate Disassembly by Molecular Chaperones” by Dinamarca et al. “Synaptic and functional alterations in the development of mutant huntingtin expressing hiPSC-derived neurons” illustrates how hereditary disease mutations influence early development processes of the individual and the way this might impact the progression of diseases thought to be age-related.Huntington’s disease is another well-characterized neurodegenerative protein misfolding disorder, caused by pathogenic polyQ expansion in the N-terminal huntingtin (HTT) protein that drives the protein to misfold and aggregate into amyloid fibrils. Many protein misfolding diseases are associated with the occurrence of missense mutations. The paper from Yoo et al. discuss in “Polymorphism in alpha-synuclein oligomers and its implications in toxicity under disease conditions” how the different shapes of oligomers could differentially contribute to neurotoxicity.Parkinson’s disease, yet another widely studied neurodegenerative disorder, is characterized by the intracellular deposition of amyloid fibrils in the brain tissues. It is proposed that the prefibrillar oligomeric states and not the mature amyloid fibrils contribute primarily to disease progression, thus being the cytotoxic species. Curto et al. summarizes the evidence about how amyloid structures formed by microorganisms could cross-seed the amyloid conformations of human proteins and contribute to the onset of diseases. They also highlight the manipulation of the microbiome as a therapeutic strategy.In addition to genetic and internal regulatory mechanisms, several extrinsic factors might modulate the formation of amyloid states and, ultimately, cross-seeding events. The review “Microbiome Impact on Amyloidogenesis” by authors Patel et al. in “Principles and functions of condensate modifying drugs” where they showcase different categories of condensate modifiers or c-mods.Biomolecular condensates are further gaining center stage as potential drug targets. Many condensate-forming proteins are enriched in intrinsically disordered regions (IDRs) which are often necessary to build up the network of sustaining condensates. However, their unique properties make them also prone to aggregate under non-physiologically settings. Identifying condensates as drug targets holds the potential of impacting these otherwise undruggable IDRs. The current efforts are summarized by Angelotti) and self-assembly including the functional role of solid-like assemblies . Other studies here concentrate on the factors including microbiota affecting amyloidogenesis , developmental outcomes of aggregation-prone protein mutations , proteotoxicity exerted by oligomers , and naturally-occurring aggregation modulators like nucleic acid  or chaperones , which help tune the state of these assemblies. These key molecular insights along with a report on potential alleviating strategies against aggregation disorders  make this a well-rounded compilation of complementary and unique articles. We believe this topic would be of interest to biophysicists, evolutionary biologists, material scientists, and cell biologists alike.Overall, in this issue, we present a bouquet of novel research plus timely review articles highlighting protein structure-function connection (