This study aimed to investigate the incidence rates and predictors of lower limb fractures in a general Japanese population.NIPPON DATA is a nationwide, long-term, prospective cohort study of individuals who participated in the National Cardiovascular Survey Japan and the National Nutrition Survey in 1990. Overall, 3,134 individuals  who participated in follow-up assessments in 1995, 2000, and/or 2006 were included in the present analysis. The outcomes of this study were lower limb fractures .2 in women and 22.9 kg/m2 in men. During a mean follow-up of 12.1 years, 271 total lower limb fractures were observed. In women, older age, lower BMI, and less intake of vegetables were associated with increased risks of proximal femur fractures. With regard to the outcome of total lower limb fractures, less intake of vegetables and regular exercise were significant predictors in women. Calcium intake was not significantly associated with proximal femur or total lower limb fractures. There were no significant predictors of proximal femur or total lower limb fractures in men, except for age.The mean age at baseline was 63.8 years in women and 63.1 years in men. The average body mass index (BMI) was 23.3 kg/mAging was a significant risk factor for proximal femur and total lower limb fractures in both men and women. With regard to modifiable risk factors, low BMI and low intake of vegetables were associated with increased risks of proximal femur and/or total lower limb fractures in the general population of Japanese women. Owing to the aging of society, the number of lower limb fractures  has been increasing in Japan –3. An inSeveral epidemiological studies have investigated the incidence rates and risk factors of lower limb fractures in Japan –3, 7 as The present study aimed to investigate the incidence rates and risk factors (including dietary factors) of lower limb fractures by location in a long-term prospective cohort study of general Japanese men and women recruited from all regions of Japan.NIPPON DATA 90  is a large-scale, population-based cohort study of individuals who participated in the 4th National Cardiovascular Survey Japan  and the In 1995, 2,140 elderly (≥65 years in 1995) participants, who lived in the area of the local health center that supported our survey, were invited to the “follow-up assessment 1995”. Among them, 31 refused, 59 relocated, and a total of 2,050 elderly participants (participation rate 95.8%) responded to the survey, including questionnaires on lower limb fractures, ADL, and instrumental ADL (IADL). In 2000, 2,114 elderly (≥65 years in 2000) participants with the same inclusion criteria were invited, and 1,940 (participation rate 91.8%) responded to the “follow-up assessment 2000”. In 2006, 2,114 elderly (≥65 years in 2000) participants with the same inclusion criteria were invited, and 1,817 (participation rate 81.1%) responded to the “follow-up assessment 2006”. Overall, 3,337 individuals participated in at least one of the three surveys . After excluding 203 participants , 3,134 participants  were included in the analysis.In NIPPON DATA90, participants’ consent was obtained using the opt-out approach. Verbal consent was obtained for participants in the follow-up assessments in 1995, 2000, and 2006. The institutional review board of Shiga University of Medical Science approved the NIPPON DATA 90 (R2005-021).The primary outcome of the present analysis was the time to first lower limb fracture. We used home-visit interviews of the participants and their family members (if available) by public health nurses to assess the outcome at each follow-up assessment ; if this was impractical, the questions were asked over the phone or questionnaires were mailed. At each follow-up assessment, detailed information on the history of lower limb fractures, including location  and onset date, was obtained. Only the first lower limb fracture in each participant was included in the analysis.2). Non-fasting blood samples were collected, and serum albumin levels were analyzed using an auto-analyzer . Smoking status was classified as non-smoker (never or ex-smoker) or current smoker. Alcohol use was categorized into non-drinkers (never or former drinkers) or current alcohol drinkers. Regular physical activity was defined as no exercise  or current exercise . We also used the data of the NNS conducted in 1990 [The region of residence was defined as Eastern Japan  and Western Japan . Body mass index (BMI) was calculated as weight divided by height squared  kcal. In men, the mean age was 63.1 (SD 8.1) years, mean BMI was 22.9 (SD 3.1) kg/m2, and mean total energy intake was 2273 (SD 468) kcal. Statistical differences were observed between men and women in total energy intake, smoking, and alcohol drinking status .In women, the mean age was 63.8 (SD 8.8) years, mean BMI was 23.3 (SD 3.4) kg/mDuring the mean follow-up period of 12.1 years, 281 lower limb fractures  were observed. The incidence rates of proximal femur fracture were 2.8 in women and 1.3 in men, those of other lower limb fracture were 4.6 in women and 2.3 in men, and those of total lower limb fracture were 7.6 in women and 3.7 in men. Incidence rates of lower limb fractures are shown by baseline age and sex .The incidence rates (per 1000 person-years) of proximal femur fractures was higher with older age in both women  and men . However, there were no clear associations between age and other lower limb fractures. When the proximal femur and other fractures were combined, a linear association between age and total lower limb fractures was observed only in women. Multivariable-adjusted HRs and 95% confidence intervals (95% CIs) for proximal femur fractures in women are shown in 2) had significantly higher risks of proximal femur fractures than the reference group  in Models 1, 3, 4, and 5. Furthermore, vegetable intake was associated with lower risks of proximal femur fractures in Model 5 . Calcium intake was not clearly associated with the risk of proximal femur fractures in Model 4. Among men, there were no significant predictors of proximal femur fractures, except for age, in Models 1 to 3 . Participants with low BMI . In women, regular exercise was also associated with an increased risk of total lower limb fractures (Models 4 and 5). Furthermore, vegetable intake was not significantly associated with total lower limb fractures in women in Model 5; however, relevant trends were also observed. Calcium intake was not clearly associated with the risk of total lower limb fractures in Model 4. Among men, there were no significant predictors of total lower limb fractures, except for age. In men, milk intake was associated with lower proximal femur fractures in Model 5.In the present long-term cohort study of a representative general Japanese population, aging was a significant risk factor for proximal femur and total lower limb fractures in both men and women. With regard to modifiable risk factors, low BMI and low vegetable intake were associated with increased risks of proximal femur fractures among women. Furthermore, less intake of vegetables and regular exercise were associated with an increased risk of total lower limb fractures. In contrast, there were no significant modifiable risk factors for proximal femur or total lower limb fractures in men. Calcium intake was not a significant predictor of total lower limb fractures in both men and women.The following content has been added to the discussion. Since no other studies of local residents throughout Japan were found, the incidence of peri-femoral fractures in men and women could not be compared with that reported in other studies. However, we were able to determine the incidence of proximal femur fractures diagnosed by an orthopedic surgeon nationwide or in certain areas in this study. Similarly, incidence increased with age; the incidence was high in all age groups in this study. However, some patients diagnosed by orthopedics may be admitted to a long-term care health facility. It is speculated that the difference in activity level is related to the difference in the incidence of proximal femur fractures between this study and other studies.2 was associated with increased risks of proximal femur fractures in women. Comparable findings were observed in a previous observational study [In the present analysis from NIPPON DATA 90, BMI <20 kg/mal study . The mecal study , 23. Theal study .In the present analysis, the risks of proximal femur fractures and any lower limb fractures were significantly lower in women with a higher intake of vegetables at baseline. Similar findings were reported in a few case-control studies that reported a link between vegetable intake and lower risks of forearm fractures as well as proximal femur fractures . A systeSeveral observational studies have reported the protective effects of regular physical activity on the prevention of lower limb fractures , 30. In Previous observational studies demonstrated the protective effects of dietary calcium intake and fractures , 31, whiIn the present analysis, incidence rates (per 1000 person-years) of proximal femur fractures were 2.8 in women and 1.3 in men. In a hospital-based study conducted in the Niigata Prefecture, Japan, incidence rates were comparable with those in the present analysis (3.1 in women and 0.9 in men) . In contThe strengths of this study include the study design (a long-term cohort study), generalizability of the findings (nationwide population-based study of Japanese), and detailed analysis of risk factors (including detailed dietary factors) of lower limb fractures by sex and fracture location. This study has several limitations. First, outcomes were defined based on information obtained from interviews with participants or their family members and were not confirmed by medical charts or imaging. Second, more detailed clinical information  was not available in the present study. Although we attempted to avoid memory bias by home-visit interviews of the participants and their family members (if available) three times during the follow-up period, the outcome might have been underreported in some participants. Third, information on some important risk factors of lower limb fractures  was not available in this study. Finally, we used only baseline diet factors, while food intake may change over time. This fact might have resulted in an underestimation of the association between diet factors and lower limb fractures.Aging was a significant risk factor for proximal femur and total lower limb fractures in both men and women. With regard to modifiable risk factors, low BMI, low intake of vegetables, and regular exercise were associated with increased risks of proximal femur and/or total lower limb fractures in a population-based long-term prospective cohort study of general Japanese women. Population strategies to reduce extreme thinness and increase the intake of vegetables among women appears to provide protection against the increasing burden of lower limb fractures in Japan.


The effects of a HFD on the small intestine, the organ involved in the absorption of this class of nutrients, are still poorly investigated. (2) To address this aspect, we administered a combined HFD with sucrose  regimen (18 months) to mice and investigated the morphological and molecular changes that occurred in the wall of proximal tract of the small intestine compared to the intestine of mice fed with a standard diet (SD) . (3) We found an accumulation of lipid droplets in the mucosa of HFD w/Suc-fed mice that led to a disarrangement of mucosa architecture. Furthermore, we assessed the expression of several key players involved in lipid metabolism and inflammation, such as perilipin, leptin, leptin receptor, PI3K, p-mTOR, p-Akt, and TNF-α. All these molecules were increased in HFD mice compared to the SD group. We also evaluated anti-inflammatory molecules like adiponectin, adiponectin receptor, and PPAR-γ, and observed their significant reduction in the HFD w/Suc group compared to the control. Our data are in line with the knowledge that improper eating habits present a primary harmful assault on the bowel and the entire body’s health. (4) These results represent a promising starting point for future studies, helping to better understand the complex and not fully elucidated spectrum of intestinal alterations induced by the overconsumption of fat. The small intestine is actively involved in the metabolism of dietary fats, such as cholesteryl esters (CEs), phospholipids (PLs), and triglycerides (TGs); among these, approximately 90% of the total fat calories are provided by TGs ,2,3,4. TA surplus of TGs induces liver injury, such as nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and even cirrhosis and hepatocarcinoma ,16,17,18Although the intestinal tract is known to be directly involved in lipid metabolism, the mucosal alterations induced by an enriched fat diet are still limited ,21.Indeed, several studies have focused attention on the changes in intestinal microbiota and the exacerbation of several intestinal disorders , or colorectal cancer) ,23,24,25Lipid metabolism is regulated by several molecules, including peroxisome proliferator-activated receptor-γ (PPAR-γ), a master gene of adipogenesis and adipocyte differentiation that is also involved in the regulation of many processes, such as cell proliferation, differentiation, inflammation, and fibrosis ,31,32; fA HFD also increases the expression of adipokines involved in lipid metabolism, such as leptin, which are responsible for a downregulation of PPAR-γ and the upregulation of proinflammatory cytokines like TNF-α and IL-6 through the PI3K/mTOR pathway ,34,35,36In previous studies, we demonstrated that the abuse of an 18-month long chronic hyperlipidemic diet induced severe metabolic dysfunctions altering the mice’s body weight, liver weight/body weight ratio, serum parameters like ALT, AST, triglycerides, and cholesterol and prompted the liver parenchyma towards nonalcoholic steatohepatitis (NASH) and hepatocarcinoma ,18. On tHistological analyses by H&E staining showed a marked accumulation of lipid droplets in the mucosa of HFD w/Suc-fed mice, leading to a disruption of normal intestinal architecture A. Additionally, in the HFD w/Suc mice, a spread inflammatory infiltrate was found across the mucosa B, whereaTo highlight the dietary effects on intestinal epithelial barrier components, PAS staining elective for goblet cells was also undertaken A. The goFibrosis was absent in both the SD and HFD w/Suc mice .To confirm the presence of lipids, we performed immunohistochemistry analysis for perilipin 1, which is one of the CDL surface proteins A.HFD w/Suc mice showed increased perilipin expression compared to the control group, mainly localized on the rim of the lipid droplets present in the intestinal mucosa layer A. The inTo confirm that a HFD w/Suc leads to an overexpression of leptin, we assessed this molecule and its receptor .Immunostaining of leptin is noticeably increased in HFD w/Suc-fed mice compared to the SD group A,B. EvalFurthermore, we performed an immunohistochemistry evaluation of adiponectin, a molecule that generally counterbalances the activity of leptin A.The analysis revealed a significant reduction of adiponectin expression in the HFD w/Suc group compared to control mice A, as valSimilarly, adiponectin receptor immunopositivity was reduced in the HFD w/Suc group compared to the SD mice C,D.Immunohistochemistry of PPAR-γ showed a reduction of positivity in HFD w/Suc -fed mice compared to the SD group A, as conSince the increase of lipid droplets was associated with a concomitant increase of mucosal inflammatory infiltrate in HDF w/Suc mice, we performed immunohistochemistry analysis for the proinflammatory mediators PI3K, phosphorylated-mTOR (p-mTOR), and phosphorylated Akt (p-Akt)  37,38,3,338,39.Immunostaining of PI3K, p-mTOR, and p-Akt was increased in the HFD w/Suc group compared to the SD-fed mice A,C,E, whImmunohistochemistry A and semFinally, to investigate the integrity of the intestinal epithelium, we evaluated the expression of Zonulin-1, a key molecule in the formation and remodeling of tight junctions and the maintenance of the intestinal barrier. Immunohistochemical analysis showed a marked decrease in the expression of zonulin-1 in the epithelium of the group of mice receiving HFD w/Suc compared to control mice , supportDietary lipids are absorbed by the small intestine and, after their resynthesis, are transported to the endoplasmic reticulum where they are packaged in chylomicrons or temporarily stored in enterocytes in the form of CLDs as an energy reserve . HoweverLipid metabolism, as well as inflammation, are orchestrated by several molecules, including the adipokines leptin and adiponectin, which generally influence cellular behavior, acting in an opposite manner. In the physiological condition, leptin has a dual role, regulating food intake and body weight and inducing an inflammatory response, an action that is counterbalanced by adiponectin . HoweverIn this study we administrated a Surwit diet designed for diet-induced obesity studies, characterized by a combination of a high fat  and sucrose  content . This diWe demonstrate that the prolonged (up to 18 months) administration of a HFD w/Suc, leading to an accumulation of CLDs in the intestinal mucosa of the mice, is associated with relevant changes in the organ architecture, characterized by alterations of the typical villus conformation and increased inflammatory infiltrate in the lamina propria. Similar CLDs accumulation was reported in the intestine of animals but in different experimental conditions ,52. InteCLDs are characterized by a core of Triglycerides (TAGs) and cholesterol esters surrounded by a monolayer of PLs, cholesterol, and several proteins, including members of the perilipin family. Accordingly, the immunohistochemistry of perilipin highlighted a visible positivity in the rim of the lipid droplets in HFD w/Suc-fed mice, confirming the presence of adipose-like cells. We found a significant increase in leptin expression and a reduction of adiponectin in the HFD w/Suc group. Conversely, PPAR-γ positivity was more evident in SD mice compared to the HFD w/Suc group, where there was only a slight expression. PI3K, p-mTOR, and p-Akt, as well as TNF-α, were markedly increased in HFD-fed w/Suc mice compared to control mice receiving a SD, suggesting their role in the onset of the inflammatory process .Since similar alterations in the expression of adipokines, PPAR-γ, PI3K, p-mTOR, p-Akt, and TNF-α have been reported in experimental colitis and IBD, a hyperlipidic diet could have adverse effects on the course of IBD ,58,59,60There is evidence that the western, high-caloric and HFD influences IBD in patients and rodent models and it has been suggested that the deregulated production of adipokines, such as leptin and adiponectin, are involved in IBD pathogenesis ,64. Our data reflect the knowledge that leptin downregulates PPAR-γ expression on the one hand, while inducing PI3K/mTOR signaling on the other. In turn, these conditions lead to the secretion of proinflammatory cytokines that initiate and maintain the inflammatory process. Our data suggest that the abuse of a HFD w/Suc directly impairs the homeostasis of enterocytes and the integrity of the intestinal mucosa barrier. The local accumulation of lipid droplets can certainly interfere with the physiological functions of the intestinal mucosa. Currently, a crucial point is to clarify whether these alterations can be reversible or even promote a local chronic inflammation, as occurs in NASH. It could be hypothesized that a HFD w/Suc could cause not only hepatic steatosis but also an “intestinal steatosis”. Another important feature will be to verify whether the morphological and inflammatory intestinal changes are only related to the intake of lipids or whether they are also induced and maintained by specific alterations of the gut microbiome determined by the hyperlipidic diet ,66. The If confirmed by other studies, the metabolic and molecular alterations, as well as the alterations of the gut microbiome, could represent new therapeutic targets for the prevention and treatment of intestinal inflammatory disorders.This study employed paraffin-embedded bowel specimens harvested from our previous experimental procedures carried out at the University of L’Aquila, Department of Biotechnological and Applied Clinical Sciences . The project was approved by the Italian Ministry of Health and the internal Committee of the University of L’Aquila. All experiments complied with the governmental guidelines . All efforts were made to minimize animal suffering.Fifteen male C57BL/6 mice  were used in the study. All animals were housed in plastic cages and kept in a pathogen-free environment, at constant room temperature, with a 12 h/12 h light/dark cycle. We provided animals with 20 g dry weight of food, at 100 g animal/d, according to Animal Research Review Panel guidelines. The animals were housed two or three per cage to guarantee social behavior and equal access to food.n = 5; used as the control), HFD w/Suc (n = 10), and LF-HCD (n = 10).At 20 days of life, the mice were randomly divided into two groups: standard diet  was derived from hydrogenated coconut oil, while the fat source in the SD  was represented by ether extract. The carbohydrate source in control LF-HCD was sucrose (D12329 Research Diets). The detailed description of diet components is reported in All animals were sacrificed after 18 months of differentiated diets. Following laparotomy, the small intestine was identified and rapidly excised. The samples were then fixed in 4% buffered formaldehyde and embedded in paraffin for further evaluation.Small intestine fragments of all animals were washed and immediately placed in 4% buffered formalin in phosphate buffer saline (PBS) at pH 7.4 for 3 h at room temperature before being embedded in low-temperature fusion paraffin. Sections of 3 μm thickness were stained with hematoxylin and eosin (H&E) to highlight the degree of inflammation, Masson’s trichrome to detect the deposition of connective tissue and fibrosis, and periodic acid–Schiff (PAS) to assess changes in the amount of goblet cells.The stained sections were then observed under an Olympus BX51 Light Microscope .For immunohistochemical analysis, the samples were incubated for 40 min in methanol and then in 3% hydrogen peroxide solution for 5 min. The specimens were incubated overnight at 4 °C with the specific antibodies reported in Finally, the specimens were counterstained with Mayer’s hematoxylin, mounted, and observed under an Olympus BX51 light microscope . To control the specificity of the immunohistochemistry, all reactions included negative controls (sections were incubated omitting the primary antibody).Semi-quantitative comparison of immunohistochemical staining was measured using the ImageJ immunohistochemistry (IHC) profiler software plugin, a digital image analysis public domain software . Immunopt test was used for statistical analyses. Results were expressed as mean ± standard deviation; a p-value < 0.05 was considered statistically significant.The student’s


Music influences many physiological parameters, including some cardiovascular (CV) control indices. The complexity and heterogeneity of musical stimuli, the integrated response within the brain and the limited availability of quantitative methods for non-invasive assessment of the autonomic function are the main reasons for the scarcity of studies about the impact of music on CV control. This study aims to investigate the effects of listening to algorithmic music on the CV regulation of healthy subjects by means of the spectral analysis of heart period, approximated as the time distance between two consecutive R-wave peaks (RR), and systolic arterial pressure (SAP) variability. We studied 10 healthy volunteers  both while supine (REST) and during passive orthostatism (TILT). Activating and relaxing algorithmic music tracks were used to produce possible contrasting effects. At baseline, the group featured normal indices of CV sympathovagal modulation both at REST and during TILT. Compared to baseline, at REST, listening to both musical stimuli did not affect time and frequency domain markers of both SAP and RR, except for a significant increase in mean RR. A physiological TILT response was maintained while listening to both musical tracks in terms of time and frequency domain markers, compared to baseline, an increase in mean RR was again observed. In healthy subjects featuring a normal CV neural profile at baseline, algorithmic music reduced the heart rate, a potentially favorable effect. The innovative music approach of this study encourages further research, as in the presence of several diseases, such as ischemic heart disease, hypertension, and heart failure, a standardized musical stimulation could play a therapeutic role. The therapeutic use of music in its different forms is the subject of several studies in clinical and non-clinical fields ,6,7,8,9.In the present protocol, music was created with the specific goal of producing contrasting effects (activation/excitement versus relaxation/quiescence), based on specific sound parameters . AutonomTen healthy volunteers  were enrolled at IRCCS Istituti Clinici Scientifici Maugeri in Milan. The sample size was derived from previous studies assessing CV indices in young healthy subjects during rest and orthostasis ,17. At ttempo, musical values and melodic characteristics of the songs were predefined. For the activating algorithmic music track , respiration  and non-invasive beat-to-beat AP, via a photopletismographic device . The arm was fixed at the level of the heart to optimize the AP measurements. Signals were sampled at 500 Hz. The timeline of the protocol is shown in After 10 min in supine rest to allow stabilization, the signals were continuously acquired during basal conditions (B), i.e., without any stimulus, but the orthostatic challenge, listening to Track1 and listening to Track2. The two sessions were randomized. Each session lasted 12 min: 6 min with the subject lying supine (REST) and 6 min during head-up tilt test at 70° (TILT). A recovery period of 10 min was performed in the supine position without any interference between one session and another, in order to restore baseline signal conditions.This study was conducted in accordance with the rules of the Declaration of Helsinki of 1995, revised in 2013, and was approved by the local Ethics Committee . Each enrolled subject signed a written informed consent.The CV neural control was assessed using the analysis of the RR interval variability, the AP variability and the cardiac baroreflex sensitivity (cBRS).The RR interval was approximated as the temporal distance between two consecutive R-wave peaks detected on the ECG signal. An automated algorithm detected the QRS complexes imposing a threshold on the first derivative of the ECG signal and fixed the R peaks by means of parabolic interpolation. From the AP signal, systolic AP (SAP) and diastolic AP (DAP) beat-to-beat time series were derived. SAP values were derived as the maximum of the AP signal inside each considered RR interval. DAP values were derived as the minimum of the AP signal inside each considered RR interval. The detection of R peaks, DAP and SAP values were visually checked and manually corrected in case of misdetection. In presence of artifacts or ectopic beats, parabolic interpolation was performed, paying attention to never exceed 5% of the total time series.RR, μSAP, μDAP, respectively) were calculated and expressed in ms and mmHg. The variance of RR and SAP series were calculated as well  and expressed in ms2 and mmHg2. Parametric power spectral analysis was performed on the RR and SAP time series to assess the CV neural control. The RR and SAP series were modelized by an autoregressive model, whose order was chosen according to Akaike criteria and decomposed into power spectral components. The power spectral components were classified as low frequency (LF) or high frequency (HF), if their central frequency dropped in the LF (0.04–0.15 Hz) or HF (0.15–0.4 Hz) band, respectively [a,RR and HFa,RR, respectively, and expressed in ms2. LFa,RR and HFa,RR were also expressed in normalized units and labeled as LFnu,RR and HFnu,RR, respectively. LFnu,RR and HFnu,RR were obtained by dividing LFa,RR and HFa,RR, respectively, by the total variance diminished by the power of the very low frequency band (below 0.4 Hz) and then multiplying the results by 100. HFa,RR and HFnu,RR were considered as markers of the vagal modulation directed to the sinus node [a,RR and LFnu,RR as indices providing some information about the sympathetic modulation directed to the sinus node [a,RR and HFa,RR was also calculated and labeled as LF/HF. As to the SAP series, the sum of the absolute power of the LF power spectral components was labeled as LFSAP, expressed in mmHg2, and taken as an index of the sympathetic modulation directed to the vessels [For each experimental session , selections of 300 consecutive and synchronized values of RR, SAP and DAP time series were selected at REST and TILT for further analysis, in accordance with the rules for short-term variability analysis . After lectively . The sumnus node , while Lnus node ,22. The We estimated the cBRS by means of the sequence method ,24. The All RR and SAP variability parameters are summarized in p < 0.05 was considered as significant. Data are expressed as mean ± standard deviation. The statistical analysis was carried out using the statistical program Sigmaplot .Two-way repeated measures analysis via Wilcoxon signed rank test was applied to test the difference between the CV control indices in the two experimental conditions  during the three different experimental sessions . The level of significance of each pairwise comparison was lowered according to the total number of tests  to deal with the multiple comparison issue. A All the enrolled subjects completed the protocol without symptoms. .A representative example of RR and SAP series for each experimental phase is shown in Figure 2The results of the RR variability analysis for each experimental session  are summarized in RR (corresponding to a HR of 71 ± 8 bpm at REST and 85 ± 10 bpm during TILT), as well as normal indices of cardiac sympathovagal balance, in both REST and TILT. Physiologically, from REST to TILT, μRR decreased, the indices of vagal modulation directed to the sinus node, i.e., HFa,RR and HFnu,RR, also decreased, while the indices of cardiac sympathetic activity, i.e., LFa,RR and LFnu,RR, increased. Accordingly, the LF/HF was 2.90 ± 1.15 and 5.39 ± 5.82 in REST and TILT, respectively.In B, all subjects were characterized by normal μRR at REST, (corresponding to a HR of 71 ± 8 bpm at REST and 67 ± 8 bpm during Track1 and 69 ± 9 bpm during Track2), without modifying σ2RR, the sympathetic or vagal indices, including the LF/HF (2.47 ± 2.07 for Track1 and 5.42 ± 6.75 for Track2). Of notice, also the respiratory rate remained unchanged . There were no differences between each TILT phase in the three experimental conditions.Similar to B, TILT during Track1 and Track2 induced a decrease of μSAP and μDAP within normal range in B, both at REST and TILT.SAP, an index of the sympathetic modulation directed to the vessels, increased during TILT, while σ2SAP remained unchanged, as it occurs in normal subjects. Compared to B, both Track1 and Track2 had no effects on μSAP, μDAP, σ2SAP, and LFSAP. Similarly, TILT induced an increase of LFSAP, during both Track1 and Track2, while μSAP, μDAP, and σ2SAP remained unchanged.LFFinally, the analysis of the three different recovery periods did not show any significant result.In the present study, a group of healthy subjects underwent an experimental protocol to evaluate their CV neural profile at baseline and in response to algorithmic music listening, consisting of an activating and a relaxing song in a randomized order.The striking finding is that, compared to baseline, listening to both algorithmic music tracks, composed with activating or relaxing purposes, induced a significant decrease of HR at REST, without affecting AP, breathing rate, and the indices of CV neural modulation. In several studies, it has been reported that music listening leading to emotional arousal is associated with higher HR than listening to tranquilizing music ,26,27. IOur results also showed that passive orthostatic challenge had a dominant effect on both musical stimuli, as during TILT no differences between Track1 and Track2 were found. In other words, the orthostatic stimulus elicited a physiological response regardless of the music listening.Indeed, the physiological response to TILT consisted of an increase in HR, together with the expected rise of sympathetic modulation directed to the heart and vessels, and withdrawn of vagal modulation directed to the sinus node ,17,21,22Therefore, in these healthy subjects featuring a normal CV neural profile at baseline, musical stimulation, either Track1 or Track2, reduced HR, a potentially favorable effect, independently from breathing rate and sympathovagal balance.In fact, it is well known that a physiological lower HR in healthy populations is associated with lower CV and all-cause mortality ,29,30. SfI) in sinoatrial nodal tissue without affecting cardiac inotropy or systemic vascular resistance can be recommended in chronic coronary syndromes and heart failure [Pharmacological lowering of HR has long been recommended and employed with a cardioprotective aim in several conditions ,36. Howe failure . In thisFurthermore, additional different effects of music listening are conceivable in the case of patients with abnormal CV nervous control. In fact, it is known that in pathological conditions, some interventions may restore some sympathovagal control ,40, thatInterestingly, in the present study, algorithmic music had no effects on the respiratory rate, thus the HR reduction was independent, unlike what has been demonstrated with the rhythmic activity of music or other practices ,43. FromIt remains to be established which mechanisms may intervene in reducing HR during listening to algorithmic music. It has been previously reported a decrease of HR in young healthy subjects during physiological conditions without changes in cardiac ANS . An endoRegardless of the underlying factors, mean HR, and even more nocturnal HR, is associated with increased mortality in several cardiac conditions, and algorithmic music may exert favorable preventive and therapeutic effects.This study analyzed the acute effect of music listening for a short lapse of time. Chronic and/or repeated exposure to music listening might produce more noticeable effects, perhaps highlighting the differences between activating and relaxing stimuli.The sample size was limited; therefore, it prevents us from having strong results. For the same reason, we did not analyze the data along the lines of gender and sex. However, as these variables are known to influence the CV responses to external stimuli ,47, futuIt is widely accepted that music has therapeutic effects in many neurodegenerative and psychiatric diseases and stress-related disorders ,8. EffecIn this study, on a small group of healthy subjects, both the relaxing and activating algorithmic music decreased the HR, without supposedly changing the cardiac sympathovagal balance, arterial blood pressure, vascular sympathetic modulation, baroreflex sensitivity, and the response to the orthostatic challenge, that were all physiological at baseline in this study population. Importantly, no kind of music, neither the activating nor the relaxing one, produced negative effects. Still, some music-induced effects on an already normal ANS might be difficult to detect. Otherwise, different results could be found in patients, in which music could determine a rebalancing of an altered cardiovascular neural profile, for example in the elderly, in neurodegenerative and CV diseases ,48. In oIn this perspective, this study represents an absolute novelty introducing an innovative and standardized music approach based on tailored musical patterns with therapeutic goals. This approach creates considerable potential in the use of musical listening aimed at the care and health of individuals and opens the door to further studies on different categories of patients.


Until recently, the incidence of COVID-19 was primarily estimated using molecular diagnostic methods. However, the number of cases is vastly underreported using these methods. Seroprevalence studies estimate cumulative infection incidences and allow monitoring of transmission dynamics, and the presence of neutralizing antibodies in the population. In February 2020, the Mexican Social Security Institute began conducting anonymous unrelated sampling of residual sera from specimens across the country, excluding patients with fever within the previous two weeks and/or patients with an acute respiratory infection. Sampling was carried out weekly and began 17 days before Mexico’s first officially confirmed case. The 24,273 sera obtained were analyzed by chemiluminescent-linked immunosorbent assay (CLIA) IgG S1/S2 and, later, positive cases using this technique were also analyzed to determine the rate of neutralization using the enzyme-linked immunosorbent assay (ELISA). We identified 40 CLIA IgG positive cases before the first official report of SARS-CoV-2 infection in Mexico. The national seroprevalence was 3.5% in February and 33.5% in December. Neutralizing activity among IgG positives patients during overall study period was 86.1%. The extent of the SARS-CoV-2 infection in Mexico is 21 times higher than that reported by molecular techniques. Although the general population is still far from achieving herd immunity, epidemiological indicators should be re-estimated based on serological studies of this type. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can produce a wide spectrum of symptoms and disease severity, from mild and even asymptomatic cases to potentially fatal severe acute respiratory syndrome ,2.According to the World Health Organization (WHO), through 31 December 2020, there were 83,955,204 SARS-CoV-2 infections and 1,820,400 deaths associated with COVID-19. The countries most significantly affected to date are as follows: The United States , India , and Brazil  [Since the publication of the first genome, diagnostic techniques based on molecular methods have been developed . These sBeyond this approach, there are serological techniques based on technologies such as enzyme-linked immunosorbent assay (ELISA), lateral flow, chemiluminescence microparticle immunoassay (CIMA), and chemiluminescent-linked immunosorbent assay (CLIA) that can determine both the presence of IgA, IgM, and IgG antibodies  proteins of SARS-CoV-2, respectively), and their neutralizing capacities ,8.As recommended by the WHO , this stGiven the imminent introduction of SARS-CoV-2 to Mexico, beginning on 10 February 2020 (two weeks before the first case was detected), the Mexican Institute of Social Security (IMSS)  began gathering an anonymous unrelated sampling (AHS) of residual patient sera. To maintain a population-based focus, specimens were collected from 34 clinical laboratories  and 34 blood banks , across all 32 of the United Mexican States.Sampling was carried out weekly for 44 weeks . Each CL was asked to provide 20 samples of 1 mL of surplus sera, corresponding to two samples from each of the following age groups: 0–20 years, 21–40 years, 41–60 years, 61–80 years, and 81–100 years.The samples could come from any outpatient collection, excluding patients with fever within the previous two weeks and/or patients with an acute respiratory infection, because the majority of individuals infected with SARS-CoV-2 develop symptoms within a period of 14 days  and patiTo focus on a more limited age range, the BBs were asked for eight weekly samples, six from each of the following age groups: 21–40 years and 41–60 years. However, unfortunately there is not the desired representativeness for each state according to its population, so when grouping by geographic region there is a greater representativeness of the samples by age group to carry out statistical analyzes. The inclusion criteria were the same as those used in BBs to guarantee donation safety .The samples were deidentified, retaining only information on location, age group, and sex. This protocol was approved by the scientific, ethics, and biosafety committees of the IMSS National Scientific Research Commission (R-785-2020-60).Samples were sent by participating CLs and BBs to the Central Epidemiology Laboratory (CEL), in triple packaging conditions for category B biological samples, following the International Air Transport Association (IATA) recommendations . When saDetermination of IgG antibodies against the S1 and S2 antigens of the SARS-CoV-2 virus by CLIA.Determination of IgG antibodies was made from 200 µL of serum using the LIAISON SARS-CoV-2 S1/S2 IgG kit , following the manufacturer’s instructions. This protocol was authorized for emergency use by the U.S. Food and Drug Administration and registration by the Federal Commission for Protection from Sanitary Risk (COFEPRIS).Negative and positive controls were used to validate the results. Cutoff values were as follows: negative <12 AU/mL, indeterminate ≥12 to ≤15 AU/mL, and positive >15 AU/mL. The assays specificity ranged between 97–98.5% [Determination of neutralizing activity of sera was carried out using the SARS-CoV-2 Surrogate Virus Neutralization Test ELISA Kit , which was validated by Tan and colleagues , with a The tests were carried out in all IgG positive’s samples, following the manufacturer’s recommendation. Negative and positive controls were used to validate the results, with cutoff values as follows: negative <20% inhibition and positive >20% inhibition.p values < 0.05 were considered statistically significant. Student’s t and one-factor ANOVA were used to compare continuous variables; Mann–Whitney U and Kruskal–Wallis H were used when data did not meet the criteria of normality or homoscedasticity. Analyses were performed using IBM SPSS Statistics®  and graphs were generated with GraphPad Prism® , and Microsoft® Excel® .Descriptive statistics were used to express the seroprevalence values, which were defined as the proportion of the total number of positive cases among the total number of serological samples analyzed per epidemiological week, age group, sex, or region. In all the calculated proportions, the 95.0% confidence intervals (CI) were also obtained. The chi-square test for homogeneity and independence, and Fisher’s exact probability test were used to compare categorical variables. Results are presented for five age groups  and the country’s 32 states are divided into five regions: (one) Central (C): Mexico City, State of Mexico, Morelos, Puebla, Tlaxcala, Hidalgo, and Guerrero; (two) Northeast (NE): Durango, Coahuila, Nuevo León, San Luis Potosí, and Tamaulipas; (three) Northwest (NW): Baja California, Baja California Sur, Chihuahua, Sonora, and Sinaloa; (four) West (W): Aguascalientes, Colima, Guanajuato, Jalisco, Michoacán, Nayarit, Querétaro, and Zacatecas; and (five) Southeast (SE): Veracruz, Chiapas, Oaxaca, Campeche, Quintana Roo, Tabasco, and Yucatán.The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. All authors had full access to all the data in the study and the corresponding author had final responsibility for the decision to submit for publication.From February to December 2020, 24,273 serum samples were gathered from across the country. This period allowed assessment of the first and second wave of infections in Mexico. Collection started two weeks before the first confirmed case and ended in the second descending phase of the epidemic curve . Weekly distribution of all the analyzed samples and their results are presented in p < 0.01). Individual results show differences between BB and CL over several weeks, as shown in To identify potential selection biases, the results from both the overall seroprevalences and those for each of the 44 weeks were compared between CLs and BBs. To our surprise, even including criteria to unlink samples from a suspected case, the cumulative seroprevalence differ significantly  , positive IgG anti S1/S2 cases of SARS-CoV-2 were identified. From the 1057 samples collected prior to the first case officially detected by the Mexican epidemiological surveillance system on 27 February 2020 , 40 were positive. Another 54 positive samples were detected through the day before community transmission was declared in Mexico (23 March 2020) a–d. PresThe dynamics of infection dissemination across Mexico were evaluated using the accumulated cases within states during the 44 weeks  . The staAlthough seroprevalence in Mexico increased consistently over time during these weeks of the pandemic, a different pattern was noted with respect to neutralizing antibodies. With few exceptions, the samples with IgG antibodies against S1/S2 did not show neutralizing power until epidemiological week 20 .p < 0.05) (When comparing double-positive patients (IgG+/RBD+) with those who were neutralization negative, despite having IgG antibodies (IgG+/RBD–), a higher concentration of IgG antibodies was observed (x = 106.5 AU/mL) for double positives ( < 0.05) .p < 0.05)  a, with m < 0.05) b–c.The seroprevalence by region and epidemiological week is presented in The prevalence of antibodies against SARS-CoV-2 in Mexico during December was 33.5% (CI 32.2–34.7). At that time, NW was the most affected region, with 40.7% (CI 36.9–44.5) of the population already having IgG antibodies against S1/S2, followed by NE , and SE . However, through December (the study cutoff date), the C , and W  regions appeared least affected and consequently may be the most vulnerable in the future.The age groups with the lowest percentage of those with the presence of IgG antibodies were 81–100 years  and those 61–80 years . The highest seroprevalence rates were found among the group 21–40 years and 41–60 years, with prevalence rates of 37.7% (35.4–40.0) and 34.0% (31.8–36.2), respectively .For positive samples to IgG antibodies, their neutralizing capacity was also determined by inhibition ELISA. This technique allows for evaluation of the neutralizing capacity in the absence of a biosafety level three laboratory. The results show that adults aged 21–40 years and those aged 41–60 years had the highest percentage of neutralizing antibodies, while old people aged 81–100 years had the lowest prevalence of these antibodies .Over the past year, different strategies have been implemented around the world to mitigate the coronavirus pandemic. Early detection, which allows timely isolation, has been a primary strategy. In this regard, molecular techniques for screening are valuable, as they allow identification of positive cases during the early stages of infection. Other strategies include isolating infected individuals, contact tracing, determining the number of positives, and counting deaths. Serological data are thus essential, as a complement to the regular epidemiological surveillance system, to allow proper estimates of the extent of the infection, the accumulated incidence, and the cases that could be asymptomatic.Herein, we used two serological techniques, to determine both the extent and dynamics of the SARS-CoV-2 infection in Mexico—using the CLIA technique—and the presence of neutralizing antibodies by ELISA.Our data show that before the first case was detected by the country’s epidemiological surveillance system, there were already positive cases within the population . The posUnlike other seroprevalence studies recently published ,21,22, hAs with transmission dynamics, for seroprevalence at the national level, our study provides a far more time-extensive analysis, making it possible to compare our data with those reported by others. Until March 2021, only two published study had evaluated the seroprevalence of SARS-CoV-2 in Mexico. One of these was carried out in Veracruz City from 1 June to 31 July 2020, with 2174 individuals aged 18 years or older; among the 1141 (52.5%) who were asymptomatic, prevalence was 21.3% . In the It must be taken into consideration that the seroprevalence calculated in this study, in each epidemiological week, could even be higher, since the quantitative technique used had the limitation of measuring only the IgG antibodies, which means that some samples could still be positive to other type of antibodies, like IgM and/or IgA.In all the studies carried out in Mexico ,26, inclAmong the first national-level SARS-CoV-2 antibody seroprevalence studies was that by Havers and colleagues, covering 16,025 residents of 10 cities in the United States (March–May 2020). Their proportion with the presence of antibodies varied from 1.0–6.9%. Compared with our national data during the same period , 2.9% seroprevalence was registered, with the NW region showing the highest seroprevalence (4.1%). Interestingly, this region includes states that border the United States, indicating probable mobility of Mexicans working in border cities as a cause of viral spread across these areas .Recently, seropositivity to SARS-CoV-2 in the United States through September 2020 varied in the range of <1.0–23.0%, depending on the state, with antibodies against SARS-CoV-2 detected among less than 10.0% of the North American population . CompareAlthough the factors that may confer protection remain unknown, if we extrapolate from other viral infections, neutralizing antibodies could correlate with the development of immunity; were this the case, 30.6% of Mexicans would have developed immunity in December 2020. It has been estimated that to achieve group immunity in the case of SARS-CoV-2 infection, 60.0–70.0% of the population is required to be immune ,30. ThusBased on these new data, the mortality rate in the country should be reviewed and recalculated, since, with official data, the mortality rate in Mexico is one of the highest in the world (10.5%) . LikewisUnlike the findings by Bajema and colleagues , the higThough no significant sex variation was observed in seroprevalence, variation was noted between age groups. The portion of the population that has been most exposed is those who are most economically active  . This maHerein, we have identified that formation of neutralizing antibodies is related to the total amount of IgG antibodies and age, as described by Premkumar and colleagues and Weisberg and colleagues, respectively ,32. As sThe present work was limited insofar as our samples were remnants from CLs and BBs, rather than population-based sampling. However, the strength of this study is that it offers a dynamic, temporal view of the pandemic’s spread in Mexico. This initial report is being made within the context of the ongoing pandemic; we will continue both sample collection and seroprevalence analyses in the coming months.The actual extent of SARS-CoV-2 infection in Mexico has been far greater than that reported by molecular techniques. Though we are still far from achieving group protection, calculations of all epidemiological indicators of disease burden and mortality should be recalculated based on serological studies of this type.


CXCs are important genes that regulate inflammation and tumor metastasis. While there are many studies with a focus on individual CXCs, few present a pan-cancer analysis of the whole CXC family. Our results indicate that CXCs are a potential therapeutic target in a variety of tumors and a potential prognostic marker that could improve the survival of cancer patients and the accuracy of prognosis. Meanwhile, we found that CXCs may be involved in diseases caused by intestinal flora.Background: CXCs are important genes that regulate inflammation and tumor metastasis. However, the expression level, prognosis value, and immune infiltration of CXCs in cancers are not clear. Methods: Multiple online datasets were used to analyze the expression, prognosis, and immune regulation of CXCs in this study. Network analysis of the Amadis database and GEO dataset was used to analyze the regulation of intestinal flora on the expression of CXCs. A mouse model was used to verify the fact that intestinal bacterial dysregulation can affect the expression of CXCs. Results: In the three cancers, multiple datasets verified the fact that the mRNA expression of this family was significantly different; the mRNA levels of CXCL3, 8, 9, 10, 14, and 17 were significantly correlated with the prognosis of three cancers. CXCs were correlated with six types of immuno-infiltrating cells in three cancers. Immunohistochemistry of clinical samples confirmed that the expression of CXCL8 and 10 was higher in three cancer tissues. Animal experiments have shown that intestinal flora dysregulation can affect CXCL8 and 10 expressions. Conclusion: Our results further elucidate the function of CXCs in cancers and provide new insights into the prognosis and immune infiltration of breast, colon, and pancreatic cancers, and they suggest that intestinal flora may influence disease progression through CXCs. Cancer is a major worldwide public health problem, causing an estimated total of 9 million deaths in 2016 . Although significant survival benefits have been achieved in recent years because of early detection, screening, and treatment methods, improving overall survival (OS) remains a challenge in the clinic. Tumor metastasis is an important factor that correlates with a poor prognosis of cancer. Metastasis depends on the tumor microenvironment, which is a complex system composed of tumor cells, lymphocytes, cancer-associated fibroblasts, and other cells. These cells secrete cytokines, including CXCs, which promote the growth of tumor cells and tumor angiogenesis as well as contribute to tumor metastasis [CXC chemokines (C corresponds to cysteine and X denotes any amino acid) are a subfamily of the chemokines. CXC chemokines comprise CXCL1–17 and are divided into two structurally distinct groups according to with or without a three-amino-acid sequence, glutamic acid–leucine–arginine (ELR), in their primary structure . In geneBreast cancer, colon cancer, and pancreatic cancer are malignant tumors that easily metastasize and progress, and they are characterized by poor prognosis and high mortality. Previous researches have demonstrated that aberrant expression of some members of the CXC family is associated with tumor development in breast, colorectal, and pancreatic cancer. For instance, a high level of CXCL1 in ER-negative breast cancer cells stimulates invasion , and up-Fusobacterium nucleatum, and human diseases (including inflammatory bowel disease and colon cancer). To validate this association, an azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated cancer (CAC) mouse model was used. Our results proved that intestinal bacterial dysregulation could affect the expression of CXCL8 and 10.In this study, we used data from several large public databases to perform comprehensive bioinformatics analysis of various aspects of CXC functions in multiple cancers, including expression, prognosis, immune infiltration, genetic changes, and functional analysis. We collected clinical samples and found that CXCs were differentially expressed between tumor tissues and normal tissues. With the aid of network analysis tools of the Amadis database, we found that there could be an association between CXCL8, This study provides guidance for the clinical treatment of three cancers and reveals the interaction between flora and colon cancer. https://www.oncomine.org, accessed on 11 February 2021) is a database for sequencing data analysis [t-test. Cut-off of p value and fold change were as follows: p value: 0.05; fold change: 2.0; gene rank: 10%.The Oncomine database , a database focusing on the tumor microenvironment (TME), provides single-cell level cell-type annotation [Tumor Immune Single-cell Hub  is a web-based tool to deliver fast and customizable functionalities based on 9736 tumors and 8587 normal samples from GTEx database and The Cancer Genome Atlas  [t-test, and p < 0.05 was considered statically significant.Gene Expression Profiling Interactive Analysis  . In our https://www.kmplot.com, accessed on 5 March 2021) is a database that contains gene expression data and survival information of breast cancer patients. The prognostic value of mRNA expression was analyzed using this database [p value. Only the JeSet best probe set was selected.Kaplan–Meier plotter  is a tool for interactively exploring survival correlations, which contains survival data for 21 cancer studies performed by TCGA. The PDAC and COAD patients were divided into two groups; we assessed the OS of these groups by using Kaplan–Meier plots and log-rank p value; and the cut-off criterion was log-rank p value < 0.05. OncoLnc  is a dataset that provides the transcription factor (TF) of target genes and the regulatory network between them. It includes 8444 TF–target regulatory relationships of 800 human TFs and 6552 TF–target regulatory relationships of 828 mouse TFs. These data are derived from 11,237 PubMed articles that describe small-scale experimental studies of transcriptional regulations [TRRUST v2  is a database providing available resources of human gene expression profile datasets, which are associated with TF knockdown/knockout. The database annotates TFs and their targets in a tissue/cell-type-specific way [KnockTF , an open source platform, can predict and validate miRNA-binding sites of genes from humans, mice, rats, dogs, and cows. The core of miRWalk is the TarPmiR (random-forest-based approach) that can predict miRNA target sites of the transcript sequence. MiRWalk 2.0 , a comprehensive database, provides analysis and visualization functions to process multi-tumor genomics data [CBioPortal  collects and integrates PPI (protein–protein interaction) data from public sources and predicts potential functions [STRING 11.0 Dataset  [GO and KEGG analyses of 66 proteins interacting with CXCs found in the STRING database were performed using the Database for Annotation, Visualization, and Integrated Discovery  . GO analhttps://cistrome.shinyapps.io/timer/, accessed on 10 March 2021) is a database that focuses on analyzing tumor-infiltrating immune cells throughout 32 kinds of malignancies from TCGA [The Tumor Immune Estimation Resource .Amadis is a database that provides experimentally supported microbiota–disease associations . With aiFusobacterium nucleatum (F. nucleatum) strain ATCC 25586, which was purchased from American Type Culture Collection , was cultured in brain heart infusion (BHI) broth at 37 °C under anaerobic conditions.The animal experiments obtained permission through the Animal Ethics and Welfare Committee (AEWC) of the First Affiliated Hospital of Harbin Medical University. C57BL/6J wild-type (WT) mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. . Six- to eight-week-old female C57BL/6J mice aged 6–8 weeks were housed in standard specific pathogen-free conditions. F. nucleatum (1 × 109 CFU) by gavage from a fortnight before AOM injection until sacrifice. During the DSS intervention, F. nucleatum administration was suspended. The negative control mice were gavage-fed with PBS only. Intragastric gavage administration was carefully carried out with the animal immobilized, using a gavage needle appropriate for mice. Before bacterial intragastric administration, mice were fed with broad-spectrum antibiotics (BSA) in the drinking water for 5 days to ensure the consistency of regular microbiota and facilitate F. nucleatum colonization. The DAI (disease activity index) and body weight were observed daily.The mice were injected with a single intraperitoneal(i.p.) injection of the AOM (10 mg/kg). One week later, they were given three cycles of 2% DSS treatment (1 week per cycle). The mice were treated with Western blots were performed according to standard protocols. A 12% SDS-PAGE gel was used to separate total proteins extracted from mice colon tissue. Then, proteins were transferred onto polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies for CXCL8 (Novus) and CXCL10 (Affinity) overnight at 4 °C after blocking with 5% non-fat dry milk in PBST. Anti-GAPDH  was used as the control. Each experiment has been repeated at least three times.® Mouse CXCL10 ELISA Kit .The mouse blood samples were centrifuged, serum was collected, and immediately cryopreserved in liquid nitrogen. According to the manufacturer’s instructions, the quantification of serum cytokine was carried out using the QuantibodyCancer tissue samples and paracancerous tissue samples were collected from the First Affiliated Hospital of Harbin Medical University. The studies obtained permission through the Ethics Committee of the First Affiliated Hospital of Harbin Medical University. Written informed consents were signed by patients/participants to participate in this study.For histologic evaluation, formalin-fixed colon tissue sections were embedded in paraffin and cut (5 μm) for H&E staining or immunohistochemistry (IHC). For IHC assays, we deparaffinized the paraffin sections, inactivated endogenous enzymes, and thermally repaired antigens. These sections were stained with CXCL8 (Novus) and 10 (Affinity) antibodies, followed by a corresponding secondary antibody and a Streptavidin Biotin Complex kit . Stained slides were scanned by KFBIO. SlideViewer and quantified by Image-pro-plus software.p < 0.05 was considered statistically significant.All data were analyzed using SPSS 22.0 software  by ordinary one-way analysis of variances with Tukey’s multiple comparisons. https://www.ncbi.nlm.nih.gov/geo, accessed on 15 March 2021) datasets. We conducted a comprehensive analysis of CXCs in eight steps . The Curtis dataset of invasive ductal breast cancer showed significant differences in the expression of CXCL2, 8, 9, 10, 12, and 14 (fold change > 2) . Colon cWe used the TISCH database to analyze subpopulation distribution  of 16 geAccording to the number of cells detected in the dataset and the expression of CXC in each dataset, the breast cancer dataset BRCA_GSE114727_inDrop, colon cancer dataset CRC_GSE146771_10X, and pancreatic cancer dataset PAAD_CRA001160 were selected for further analysis.Using the GEPIA dataset, we compared the expression of mRNAs in three types of cancer tissues versus normal tissues. The results showed that the expression of CXCL1, 2, 3, 12, and 14 in BRCA was lower in tumor tissues, and the expression of CXCL9, 10, 11, and 13 was higher than its expression in normal tissues; in COAD, the expression of CXCL12, 13 and 14 was lower than that in normal tissues, and the expression of CXCL1, 2, 3, 5, 8, 9, 10 and 11 was higher than that in normal tissues; in PDAC, the expression of CXCs was significantly higher than that in normal tissues, except CXCL2, 7, 11 and 12 .In addition, we detected expression differences of CXCRs (CXC receptor) in three cancers. The results showed that there was no significant difference between normal tissue and cancer tissue, except for the fact that CXCR4 and 6 were less expressed in pancreatic cancer tissue .We also analyzed the expression of CXCs in three types of cancers at various stages. The TISCH database was used to analyze the relationship between CXC expression and staging in different subsets of cells. In BRCA, the staging differences in CXCL1, 2, 5, 8, 12, and 14 were statistically significant A. Among We examined CXCL protein levels through IHC and found that the protein expressions of CXCL8 and 10 were statistically significantly up-regulated in human breast cancer, pancreatic cancer, and colon cancer tissues versus the corresponding normal samples .We further made the survival analysis of CXCs in three cancers. A public dataset was used to analyze the associations between CXC mRNA levels and the survival time of breast cancer patients using the Kaplan–Meier mapping tool. The public dataset OncoLnc was used to analyze the associations between CXC mRNA levels and the survival of patients with colon and pancreatic cancer.p < 0.05) (p < 0.05) (p < 0.05)  ( < 0.05) A. In col < 0.05) B. In pat < 0.05) C.Because of the significant differences in the expression of CXCs in the cancer tissues versus normal tissues, we used the TRRUST database and KnockTF database to identify possible TFs and regulatory relationships between CXCs and TF.We determined that the key TFs of the CXC family include RELA, NFKB1, and SP1  predict experim TFs inclFusobacterium nucleatum, and human diseases (including inflammatory bowel disease (IBD) and colon cancer)  A.F. nucleatum. Differential expression of CXCL8 and significant differential expression of TFs  in HT-29 cell lines treated with or without p < 0.05, log2FC > 2)  A,B. Anal2FC > 2)  (predict2FC > 2) .F. nucleatum’s regulatory role in the expression of CXCs .We have explored the co-expression relationships of CXCs. In BRCA samples, there were significant positive correlations between the expression of CXCL1 and that of CXCL2, 3, 5, 6, and 8. These correlations were also found in the expression of CXCL2 and CXCL3, 5 and 6. and in the expression of CXCL3 and CXCL5, 6 and 8. It is also found that CXCL5, 6, and 9 were positively correlated with CXCL10, 11, and 13, and so is the expression of CXCL10 with the expression of CXCL11 and 13; similarly, the expression of CXCL11 was found to be positively correlated with the expression of CXCL13 . Positive correlations were also found between CXCL6 and CXCL8; CXCL8 and CXCL9, 10; and so were CXCL9 and CXCL10, 11, 12, and 13. It is also found that CXCL10 was positively correlated with CXCL12 and 13; and that CXCL12 was positively correlated with CXCL13 .In COAD samples, there are similarities in the correlations and also distinct differences. The expression of CXCL1 was found to be highly correlated with the expression CXCL2 and 9; however, CXCL4 was negatively correlated with CXCL12 and 13   C–E.With the above co-expression analysis results, we found that the co-expression of CXCs may be related to the chromosomal localization of genes and transcription factors. CXCL1, 2, 3, 5, 6, 7, and 8 are located at 4q12-13. In BRCA samples, the expression of CXCL1 was highly correlated with CXCL2, 3, 5, 6, and 8. In PDAC samples, the expression of CXCL1 was positively correlated with CXCL2, 3, 6, and 8. Meanwhile, according to the analysis of transcription factors, the co-expressed genes may be regulated by the same one or more transcription factors, as NFκB and RELA may be responsible for multiple CXCs, including CXCL1, 2, 5, 8, 10, and 12.The CXC family performed functions by binding to receptors, so it is important to analyze the relation between CXCs and proteins interacting with CXCs. We analyzed 50 proteins interacting with CXCs using the String database. As a result, 66 nodes and 1498 edges were obtained in the PPI network, and a network map was constructed using Cytoscape A.Additionally, the functions of CXCs and their 50 interacting proteins were analyzed using the DAVID database by GO and KEGG enrichment analysis. The results presented the top 10 highly enriched biological processes pathways include chemokine-mediated signaling pathways, inflammatory responses, chemotaxis, immune responses, G protein-coupled receptor signaling pathways, cell chemotaxis, and other biological processes, suggesting that CXCs in cancer are involved in chemotaxis and function in the inflammatory response B. The exIt is known that CXCL1, 2, 3, 5, 6, 7, and 8 are bound to CXCR1 and 2; CXCL9, 10, and 11 are bound to CXCR3. Correlation analysis was performed for CXCs sharing the same receptor and their targets. All correlation coefficients between CXCR and CXCs were not significant (R < 0.8), suggesting that there was no strong correlation between CXCs and CXCR expressed in colon cancer .Salmonella infection; hsa05321: inflammatory bowel disease (IBD); and hsa05134: Legionnaires’ disease  > 0.5; all pairs were positively correlated. As shown, for these 24 pairs of data, we mainly focused on the association between CXCL9, 10, and 13 and infiltrating immune cells A,B,C. OtWe used the TISCH database to analyze the distribution of CXCL9, 10, and 13 cells in each subgroup of three types of cancer. It was found that CXCL9 and 10 were essentially enriched in mononuclear/macrophage subsets among the three cancers. CXCL13 is enriched in fibroblasts and CD8 + T cells in breast cancer, CD8 + Tex and CD4 + Tconv cells in colon cancer, and plasma in pancreatic cancer D,E,F.We also analyzed the distribution of CXCR in colon cancer. The results showed that CXCR1 mainly expresses in NK cells. CXCR2 mainly expresses in neutrophils and monocytes/macrophages. CXCR3 is widely distributed in Treg, Tprolif, CD8T, CD8Tex, and CD4Tconv cells. CXCR4 is widely distributed in T cells, such as Treg, Tprolif, and CD8T, as well as in NK cells and B cells. CXCR5 is mainly distributed in B cells. CXCR6 is mainly distributed in NK cells and T cells, such as Treg, Tprolif, and CD8T . These rThe imbalance of CXC expression has a considerable impact on tumorigenesis, tumoral cell proliferation, apoptosis, and tumor metastasis. Intercellular communications between stromal cells and tumor cells affect the expression of CXCs in various types of cells, thus regulating tumor metastasis and invasion. Some studies have already shown correlations between CXCs and the tumor microenvironment, suggesting that CXCs can regulate tumor progression and immunotherapy. Our previous studies have shown that the protection of colorectal cancer cells from radiotherapy by CXCL12/CXCR4 is mediated by survivin in colorectal cancer . The appWe initially investigated the expression of CXC chemokines and their relationships with pathological stages of the tumors. We found that nine genes were differentially expressed in breast cancer versus normal tissues . Additionally, we demonstrated that the expression of CXCL1, 2, 5, 8, 12, 13, and 14 was closely associated with the stage of breast cancer. Similarly, 11 genes were differentially expressed in colon cancer . The development of tumors was associated with an increase in the expression of CXCL9, 10, and 11. The results on pancreatic cancer data showed that 12 genes were up-regulated . The expression of CXCL1, 3, 5, and 8 were associated with the stages of pancreatic cancer. These data suggest that differentially expressed CXC chemokines may play important roles in these three types of tumors.Analysis using large groups of patients with breast, colon, or pancreatic cancer in the K–M plotter database indicated that a number of CXC family members were significantly associated with survival and had specific associations. In patients with colon cancer, the survival time of patients with higher levels of expression of CXCL1, 3, 8, 10, and 14 was longer than that of patients with lower expression. In pancreatic cancer patients, the survival time of patients with higher levels of expression of CXCL5, 8, 9, 10, 11, and 17 was remarkably shortened. In breast cancer, the groups with higher expression of mRNAs of CXCL2, 6, 9, 10, 12, 13, and 14 and the groups with lower expression of CXCL3, 8, and 17 had significantly better overall survival (OS).Fusobacterium nucleatum, and human diseases (including inflammatory bowel disease and colon cancer) was found. We investigated the results of sequencing obtained after coculture of Fusobacterium nucleatum with colon cancer cell lines and determined that the expression of CXCL8 was significantly increased, and the expression of CXCL10 was decreased in the HT29 cell line cocultured with Fusobacterium nucleatum. Fusobacterium nucleatum may change tumor proliferation, invasion, metastasis, and drug resistance by increasing the expression of CXCL8 and reducing the expression of CXCL10, thus affecting the prognosis of patients. Combining these data with the data on differential expression of miRNAs in SW480 cells cocultured with Fusobacterium nucleatum indicated changes in the expression levels of transcription factors related to CXCL8 and 10, transcriptional suppressors, and miRNAs acting on the corresponding 3’-UTR of mRNAs. At present, there are no reports on the influence of the intestinal flora on the prognosis of colon cancer patients and the progression of colon cancer mediated by the expression of chemokines. Our analysis demonstrated that Fusobacterium nucleatum might influence the changes in the chemokine family members at the transcriptional and posttranscriptional levels and, thus, influence the development and progression of colon cancer and the prognosis. Although the expression of CXCL8 and 10 increased significantly, their role in the process of F. nucleatum aggravating CAC remains unclear. Future experimental verification of the mechanism may identify new pathogenic pathways and therapeutic targets.There are contradictory evidences in the role of CXCL8 in the development and progression of colon cancer. High amounts of serum CXCL8 prevent liver metastasis of CRC and are correlated with good favorable prognostic outcomes . In contCo-expression analysis revealed that the co-expression of CXCs might be related to their chromosomal locations, and this co-expression might be regulated by transcription factors. Interestingly, in the analysis of the expression of CXCRs, we found that there was no significant difference in the expression of CXCRs in tumor tissue and normal tissue, and there was no diagnostic or prognostic value of CXCRs. Through the single-cell sequencing database data, we found that CXCRs are mostly distributed on the surface of immune cells, which may indicate that CXCs play a role in recruiting immune cells by binding to receptors on the surface of immune cells.Our study has certain limitations. The results at the transcriptional level can reflect the immune status; however, this analysis cannot reflect the overall changes. Independent cohort studies should be performed to verify our results. The CXC family members may play a dual role in disease progression. Increased expression of CXCs in tumor tissues may promote carcinogenesis and regulate the tumor microenvironment; however, in some tumors, high expression of the CXC family members may suggest a better overall survival time. Most of the results were predicted by bioinformatics analysis, and, as a result, further experiments in vitro or in vivo are needed to demonstrate the associations between these factors.In this study, we systematically analyzed the expression and prognostic value of CXCs in a variety of tumors and provided a thorough evaluation of the heterogeneity and complexity of the molecular and biological characteristics of the tumors. High expression of certain CXCs can be used as a molecular marker to identify tumor patients in high-risk groups. This is the first study to propose the theory that intestinal flora may influence disease by influencing the transcriptional changes in CXCs, thus providing a direction for further research. Our results indicate that CXCs are a potential therapeutic target in a variety of tumors and a potential prognostic marker to improve the survival of cancer patients and accuracy of prognosis, and they may be involved in diseases caused by intestinal flora.


Nonalcoholic steatohepatitis (NASH) is a prevalent disease related to lipid metabolism disorder and oxidative stress. Lipid hydroperoxidation is known to be a critical driving force of various disorders and diseases. However, the combination of both intact and hydroperoxidized lipids in NASH has not yet been studied. In this work, the liver and kidney samples from NASH-model mice were comprehensively investigated by using the LC/MS-based lipidomic analysis. As a result, triglycerides showed the amount accumulation and the profile alteration for the intact lipids in the NASH group, while phosphatidylethanolamines, lysophosphatidylethanolamines, plasmalogens, and cardiolipins largely depleted, suggesting biomembrane damage and mitochondria dysfunction. Notably, the lipid hydroperoxide species of triglyceride and phosphatidylcholine exhibited a significant elevation in both the liver and the kidney of the NASH group and showed considerable diagnostic ability. Furthermore, the relationship was revealed between the lipid metabolism disturbance and the lipid hydroperoxide accumulation, which played a key role in the vicious circle of NASH. The present study suggested that the omics approach to the lipid hydroperoxide profile might be the potential diagnostic marker of NASH and other oxidative stress-related diseases, as well as the evaluative treatment index of antioxidants. Nonalcoholic fatty liver disease (NAFLD) is a prevalent noninfectious chronic liver disease with an estimated global occurrence of about 20–30%, becoming one of the most important causes of liver disease worldwide ,2,3. Thiβ-oxidation of lipids, causing abnormal lipid accumulation, and eventually worsening the oxidative stress-antioxidation balance [Oxidative stress has been recognized as one of the key factors in NASH development, which originates from hyperleptinemia-induced oxidation of hepatic lipids and leads to the lipotoxicity of organs and release of the reactive oxygen species (ROS) . At the  balance . Therefo balance . On the  balance ,12. The  balance , though  balance ,14. Impo balance , and theTherefore, in the present work, the NASH model mice were used to conduct a comprehensive lipidomic analysis covering both intact lipids and lipid hydroperoxides in the liver and kidney tissues using liquid chromatography-mass spectrometry (LC/MS). The diversity and featured differences were revealed between the normal and the NASH model groups with organ specificity. Furthermore, the usefulness of lipid hydroperoxides as a feasible biomarker for NASH diagnosis was evaluated. The correlation between lipid hydroperoxide accumulation and intact lipid dysregulation was also discussed.w/v) and stored at −80 °C for no more than two weeks before use.For the authentic internal standards (IS), triacylglycerol (TG) 11:0/11:0/11:0 and free fatty acid (FFA) 17:0 were obtained from Sigma-Aldrich , and phosphatidylcholine (PC) 13:0/13:0, phosphatidylethanolamine (PE) 15:0/15:0, phosphatidylinositol (PI) 8:0/8:0, lysophosphatidylcholine (LPC) 15:0, lysophosphatidylethanolamine (LPE) 13:0, and lysophosphatidylinositol (LPI) 13:0 were purchased from Avanti Polar Lipids . Cardiolipin (CL) 15:0/15:0/15:0/15:0 was synthesized in our laboratory previously . Other cn = 8 for each) were fasting for 12 h before being anesthetized and sacrificed. The model was considered established successfully based on the physiological, histological, and blood data, which were reported in our previous work [All animal protocols conformed to the Public Health Service Guide for Care and Use of Laboratory Animals, which were approved by (1) Institutional Animal Care and Use Committee at Hokkaido University  and (2) the ethical committee at the Faculty of Health Sciences, Hokkaido University . The study design complied with the associated guidelines and 3R  principle. In detail, the NASH model in C57BL/6J mice was induced with a high-fat diet and oxidized low-density lipoprotein injection as previously described ,18. The v/v, with IS). The extraction was performed twice, and the combined extract was dried under vacuum. The obtained total lipids were dissolved in methanol and centrifuged to remove any insoluble material prior to analysis. The sample pretreatment was completed within 1 h to avoid lipid auto-oxidation or degradation.Total lipids from tissue samples were extracted according to Folch’s method  as previ1 scan mode, Fourier transform (FT); resolution power, 60,000; scan range, m/z 150–1100 under positive mode and m/z 220–1650 for negative mode; and MS2 scan mode: Ion-trap (IT), using collision-induced dissociation (CID) by data-dependent acquisition, with the normalized collision energy set at 35% and the isolation width set at 2 Da. The sample injection sequence was randomized to minimize the running time-induced variation.The lipidomic analysis and semi-quantitation were carried out on a Shimadzu Prominence HPLC  and an LTQ Orbitrap mass spectrometer  operated in both electrospray ionization (ESI) positive and negative ionization modes, which was accorded to our previous studies ,20. In b1 and 0.5 Da for MS2. The identification of intact and hydroperoxidized lipids was accorded to the retention behavior in reversed-phase LC, the HR-MS1, and the MS2 fragments, with the help of comparison with LIPIDMAPS  as well as our in-house library [The raw data were processed by using the workstation Xcalibur 2.3 . The mass tolerance was 5.0 ppm for HR-MS library ,20. The analyte = Amount IS × Peak area Analyte / Peak area ISAmount t-test were calculated using SPSS 24 , of which the differences were considered significant at p < 0.05. Principal component analysis (PCA) was performed by JMP pro 16 , and orthogonal partial least squares discriminant analysis (OPLS-DA) was processed using SIMCA-P 14.1 . The diagnostic performance was evaluated based on the receiver operating characteristic (ROC) using Prism 8.0 . The correlations between variables were assessed by using R 4.0 , and the lipidomic network was visualized using Cytoscape 3.8 .All the data were expressed as the mean and standard deviation (SD). Two-tailed Student’s +, for PC and LPC), deprotonated , ammoniated , or acetate  ion peaks on HR-MS, which were further identified according to the fragmentation of MS/MS  and ethanolamine (PlsEtn) types, as well as CL A. SpecifMoreover, PCA was performed using all the intact lipid species as variables , of whicp-value < 0.01 and |log2(fold change)| > 1.5) were found in the NASH liver, and 57 species were found in the NASH kidney, respectively. Notably, the alteration for the NASH model of intact lipids differentiated between liver and kidney: in the liver, 21 species increased, and 30 species decreased; on the other hand, in the kidney, only 6 lipids increased, while the other 51 species, as most of the differences, showed significant decreasing.The volcano plots have been performed to explore the most significant changes of all the intact lipid species in the liver and the kidney samples between normal and NASH groups A, in whiAbout one-third of the significantly altered lipid species in both liver and kidney belonged to TG; however, the detailed alterations differed between the two organs. In the NASH model mice, TG was found significantly accumulated in the liver samples (823.8 ± 155.9 nmol/g), accounting for 1.5-fold of the normal (554.5 ± 58.4 nmol/g) B, which To achieve more insightful discrimination of lipid metabolic impairments in the NASH mouse model, the OPLS-DA was accomplished for liver and kidney samples. Regardless of liver or kidney, the normal and the NASH samples were clearly separated into two clusters in the scatter plots A,D, suggp < 0.05 for all), which was also similar in the liver. As the main biomembrane constituent, PE is functionally associated with various cellular processes, such as oxidative phosphorylation, membrane fusion [p < 0.001 for both LPE18:0 and LPE20:4). Similar findings of the decreased LPE were reported previously in serum and liver from NASH and NAFLD patients [Among the top varied lipids in the NASH kidney samples, all the PE species were found to contain PUFA , which was in agreement with the renal TG profile alteration, as mentioned above. These renal PE were decreased by 24.1–73.1% in the NASH group  and 43.0% (43.5 nmol/g vs. 24.8 nmol/g) reduction, respectively, compared with the normal groups . These mp < 0.001 for all of them).Besides the intact lipid species, the hydroperoxidized lipids were also measured, including the hydroperoxides of TG (TGOOH) and PC (PCOOH) in our study; 7 TGOOH and 9 PCOOH species were detected in the liver, and 9 TGOOH and 8 PCOOH species were detected in the kidney. The comparison of their content between normal and NASH is shown in p values less than 0.05. Interestingly, for the liver, total TGOOH  showed stronger predictive ability than PCOOH , while for the kidney, PCOOH  was more reliable than TGOOH . The possible explanation might be due to the characteristic changes of specific intact lipid classes: TGOOH originates from the excessive intact TG stored in lipid droplets, which was attacked by the produced ROS under oxidative stress; while PCOOH as the hydroperoxidation products of the major constitutes of biomembrane, PC, are more closely involved in the damage and loss of the phospholipids. Nevertheless, the detailed differences of their production mechanisms and their interactions remain to be explored.Considering that these lipid hydroperoxides are supposed to be the key intermediates playing a role in the pathogenesis of NASH and its relevant complications, we subsequently evaluated the diagnostic powers of total TGOOH and total PCOOH as potential NASH markers B,D,F,H. For clinical descriptions and evaluations of NASH, the commonly used approaches include body examinations, blood tests, liver biopsy, and others. Therefore, we investigated the possible usefulness of the hepatic and renal lipid hydroperoxides as a diagnostic standard of NASH. The correlations between the organ lipid hydroperoxides obtained in this study, and the physiological, histological, and blood data obtained in our previous study , were caIn order to obtain a global view on the relationships between all these intact and hydroperoxidized lipid species in the NASH model mice, we constructed the correlation-based dependency network for the liver and the kidney A. In botTherefore, the putative processes of oxidative stress and lipid dysregulation in NASH were proposed as the “vicious circle” shown in Considering the central role of oxidative stress and peroxidation of the lipids, we proposed that the lipid hydroperoxide levels might be potentially designated as the diagnostic standards of NAFLD/NASH and other oxidative stress-related chronic metabolic diseases. The quantitative monitoring of the lipid hydroperoxides was also hopeful to be the evaluation index for the effects of antioxidants as a therapeutic strategy. Thus, the validation of the lipid hydroperoxide profile analysis by performing the antioxidant treatment trials  required further investigation. Another limitation of this work was that the sample size was relatively small, which limited the generalizability of these findings. Future studies need to develop the quantitative determination of these lipid hydroperoxides and combine their data with multidimensional biological, clinical, and pathological indexes in the larger cohorts. Nevertheless, the comprehensive assessment of both the intact and the hydroperoxidized lipid profiles took an exploratory step for understanding the key roles of oxidative stress-involved lipid species, particularly lipid hydroperoxides, in the mechanisms of NAFLD/NASH.In conclusion, the current LC/MS-based lipidomic study on NASH model mice proved that oxidative stress played a crucial role in lipid dysregulation and peroxidation at the molecular level. Lipid hydroperoxides, being involved in the vicious circle of the lipid abnormal metabolism and energy production disturbance, showed the potential to be the possible diagnostic markers and the evaluation indexes for the effects of the antioxidants. As crucial metabolism organs, both liver and kidney expressed oxidative stress-involved lipidome dysregulation and, at the same time, suggested organ-specific characteristics. Our data provided a better understanding of oxidative stress in NASH development from the view of lipid metabolism, providing direct evidence for the oxidative damage-caused lipid hydroperoxidation in NASH.