Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and it is involved in several fundamental functions in the central and peripheral nervous systems, and in sensory organs. BDNF regulates the chemosensory systems of mammals and is consistently expressed in those organs. In zebrafish, the key role of BDNF in the biology of the hair cells of the inner ear and lateral line system has recently been demonstrated. However, only some information is available about its occurrence in the olfactory epithelium, taste buds, and cutaneous isolated chemosensory cells. Therefore, this study was undertaken to analyze the involvement of BDNF in the chemosensory organs of zebrafish during the larval and adult stages. To identify cells displaying BDNF, we compared the cellular pattern of BDNF-displaying cells with those immunoreactive for calretinin and S100 protein. Our results demonstrate the localization of BDNF in the sensory part of the olfactory epithelium, mainly in the ciliated olfactory sensory neurons in larvae and adult zebrafish. Intense immunoreaction for BDNF was also observed in the chemosensory cells of oral and cutaneous taste buds. Moreover, a subpopulation of olfactory sensory neurons and chemosensory cells of olfactory rosette and taste bud, respectively, showed marked immunopositivity for calcium-binding protein S100 and calretinin. These results demonstrate the possible role of BDNF in the development and maintenance of olfactory sensory neurons and sensory cells in the olfactory epithelium and taste organs of zebrafish during all stages of development. Brain-derived neurotrophic factor (BDNF) and its high-affinity signaling receptor TrkB are the most widely investigated parts of the neurotrophin family. Neurotrophins (NTs) are a family of growth factors promoting the development, survival, and phenotypic differentiation of discrete neuronal populations . FurtherZebrafish developed sensory mechanisms to detect and process essential signals for survival, feeding, and reproduction. Among them are senses of smell, taste, and the ICCC revealing chemical compounds and ions in water. The olfactory organ is localized in the dorsal part of the snout inside the olfactory cavity, connected with the aquatic environment formed by an olfactory epithelium organized in lamellae converging in a central raphe. The olfactory epithelium is a pseudostratified and columnar, and mainly constituted by bipolar neurons called olfactory sensory neurons (OSNs) and nonsensory cells  a. In zebIn zebrafish larvae, pro-BDNF was localized in a subpopulation of sensory cells in the olfactory epithelium with an elongated shape c,f. TherIn the olfactory rosette of adult zebrafish, a subpopulation of elongated sensory cells showed intense immunoreaction for BDNF b. The imUsing double immunofluorescence, these cells were also positive for calretinin a–c but nTaste buds are chemosensory organs for detecting and evaluating environmental chemical stimuli distributed on the outer surface of the skin, head, lips, and oral cavity a. MatureSingle and double immunofluorescence were carried out on serial sections to localize BDNF. Results showed positive immunoreaction for BDNF in the cytoplasm of sensory cells in the taste buds of larvae and adult zebrafish. a,d,g. MoIn oral taste buds, BDNF is localized in the Merkel cells and cytoplasm of a subpopulation of sensory cells that might be identified as light cells a,d,g. ThThe observed ICCCs are spindle-shaped and resemble taste-bud sensory cells, plentifully equipping the skin of the zebrafish lips. The ICCC shape closely resembles that of gustatory receptor cells, but these differentiated epithelial sensory cells are not grouped into organs. The ICCCs that we found were elongated, with their deep pole immediately above the basal layer of the epidermis and an extended apical part reaching the surface of the epithelium. We found the nucleus at a basal position. The aforementioned cells are immunoreactive to BDNF antibody both in immunoperoxidase and immunofluorescent methods a–d., and during the recent COVID-19 pandemic, the COVID-19 virus, and olfactory and gustatory dysfunction (OGD) were associated [In teleosts such as zebrafish, the chemosensory system consists of taste buds and the olfactory epithelium ,38. The sociated . The sensociated ,51,52. Dsociated . Environsociated ,55,56. Tsociated ,55,56. Tsociated  and mammsociated  are knowsociated ,60,61. Isociated ,58,62,63sociated ,15,64,65D. labrax [Calretinin was observed in the olfactory epithelium  and tast. labrax . These d. labrax  controll. labrax ,80,81,82. labrax ,83,84. O2+ BP by chemo and mechanosensory cells observed by Germanà et al. [Our data confirm the correlation between calcium-binding proteins and neurotrophins’ mode of action in sensory epithelia. Lastly, taste buds immunopositive to calretinin were observed in zebrafish larvae. In this larval stage, Calretinin immunopositivity is present only in the light cells identified by the presence of a single apical microvillus. In conclusion, the sense of taste and smell are fundamental in the life of vertebrates, including humans and teleosts. The regenerative abilities of the olfactory epithelia of zebrafish are known . Moreoveà et al.  could beà et al. ,87,88,89à et al. ,90, and à et al. .Further studies are needed to deepen the use of calcium-binding protein calretinin as a specific marker of ciliated cells of the olfactory epithelium and light cells of taste buds. Lastly, on the basis of the obtained results, we could assume that the zebrafish olfactory epithelium and taste buds represent an interesting model to investigate the involvement of growth factors in the chemosensory neurons that might be utilized in translational medicine. Studies are in progress in our laboratory targeted at creating mutant zebrafish for BDNF and TrkB to deeply analyze the functional activity of BDNF in the chemosensory organs of zebrafish.−1). The heads of adult zebrafish were quickly removed, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS)  0.1 m (pH = 7.4) for 12–18 h, dehydrated through graded ethanol series, and clarified in xylene for paraffin wax embedding. Zebrafish larvae were fixed as a whole body and processed as above for paraffin wax embedding. Included tissue samples were then cut into 7 μm thick serial sections and collected on gelatin-coated microscope slides.In this study, we used larvae (5 days postfertilization) and adult zebrafish. The fish were obtained from CISS , the experimental protocols in this study followed the principles outlined in the Declaration of Helsinki, and samples used in this review coming from previous experimentation were approved by Italian Ministry of Health . The fish at the above-mentioned stage were sacrificed with a lethal dose of tricaine methane sulfonate  at +4 °C for 1 h, dehydrated in graded ethanol, immersed in propylene oxide, and embedded in Durcupan . Ultrathin silver–golden sections were cut with a diamond knife on a Reichert Jung Ultracut E, placed on uncoated 200 mesh copper grids, contrasted with methanolic uranyl acetate, and lead citrate, and photographed with a JEOL JEM-100 SX transmission electron microscope at 80 kV.Samples were fixed by immersion in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at +4 °C, washed with 0.1 M phosphate buffer (pH 7.4), postfixed in 1% OsO2O2 (PBS) solution for 3 min to prevent the activity of endogenous peroxidase; then, to rinsed sections was added fetal bovine serum  for 30 min to avoid nonspecific binding, followed by incubation with the primary antibodies. Incubation was carried out overnight at 4 °C in a humid chamber with antibodies against pro-BDNF, BDNF, Calretinin, and S100 protein  containing 0.1% bovine serum albumin and 0.2% Triton-X 100 and incubated in 0.3% Htein see . These atein see . Both stMoreover, to better analyze the morphological details of the positive cells, indirect peroxidase immunoreactions were carried out as follows: after rinsing in Tris-HCl buffer  containing 0.1% bovine serum albumin and 0.2% Triton-X 100, the sections were then incubated overnight h at 4 °C with the antibody against BDNF see , at roomTo provide negative controls, representative sections were incubated with specifically preabsorbed antisera as described above. Under these conditions, no positive immunostaining was observed (data not shown).


Background and objectiveOver the past few decades, new infectious diseases have emerged, and these have played a key role in changing behavior and lifestyle in all age groups. More recently, with the emergence of the coronavirus disease 2019 (COVID-19) pandemic, governments around the world have made unprecedented efforts to contain the epidemic by implementing quarantine measures, social distancing, and isolating infected individuals. Social behavioral adaptations  impact children's and adolescents' lifestyle activities and lead to increased incidence of psychosocial problems, worsening of preexisting mental illness, and fears of infection, uncertainty, isolation, and stress. In light of this, this study aimed to assess the impact of COVID-19 on the behaviors and lifestyles of the children and adolescent population of Pakistan.MethodologyA cross-sectional study was conducted involving 323 children and adolescents by targeting parents of children and adolescents in the age group of 4-18 years living in Pakistan. The study was conducted from April 2021 to September 2021. A well-designed structured questionnaire was used to collect data about the sociodemographic profile, attitudes, and behavioral factors impacted by COVID-19 in children and adolescents. SPSS Statistics version 23  was used to enter and analyze data.ResultsParents or caregivers of a total of 189 male and 134 female children aged between four and 18 years took part in this study. During COVID-19, the consumption of fast food and fried foods by children and adolescents increased significantly. In this study, out of 323 participants, almost all  had increased their screen time significantly. Nearly half of the total individuals experienced the feeling of depression and loneliness during the pandemic. Additionally, some children and adolescents felt fearful when leaving home. COVID-19 lockdowns have led to many changes in children's and adolescents' lifestyle habits. They reduced physical contact with others due to the fear of transmission of COVID-19. Based on our findings, the pandemic and its containment strategies have adversely affected the behaviors, lifestyles, and attitudes of children and adolescents.ConclusionGovernments around the world have imposed social distancing during the COVID-19 pandemic, leading to adverse short-term and long-term negative mental health issues such as unhappiness, fear, worry, irritability, depressive symptoms, anxiety, and post-traumatic stress disorder (PTSD). Interventions are needed to focus on building resilience in children and adolescents, addressing their fears and concerns through better communication, encouraging routine and physical activity, and taking measures to alleviate loneliness. Human behavior represents the latent and expressive capacity of various physiological, psychological, and social activities at all stages of human life; however, the earlier the habits are formed, the more likely they are to take root and flourish . Over thBy the end of April 2020, an estimated 1.5 billion children (aged 5-12 years) and adolescents (aged 13-17 years) worldwide had switched to distance learning following school closures. School closures and additional social behavioral adaptations  impacted children's and adolescents' lifestyles and activities . PrelimiA study in China found that only 17% of children achieved adequate levels of physical activity, and 66% of them were rated "inactive" during the pandemic. Canadian children reported a similar decline in physical activity . Obese cThe two key pillars of human civilization - social interaction and structured schedules - have been distorted by the pandemic, which has led to significant psychological effects on children and adolescents. The increased incidence of psychosocial problems, worsening of preexisting mental illnesses, and fears of infection, uncertainty, isolation, stress, and mass panic have all significantly increased due to the ongoing pandemic .A web-based survey was conducted targeting the parents of children and adolescents aged 4-18 years. The data were collected from April 2021 to September 2021. An informed written consent form was developed in the English language describing the objectives of this research, and IRB approval from the Ethical Review Board of General Hospital, Lahore (00/89/20) was obtained before the data collection.A descriptive cross-sectional study design was adopted for this research. A nonprobability, convenience sampling method was used for data collection. The Raosoft sample size calculator was used to estimate the sample size; maintaining a 5% margin of error, a 95% confidence level, and a prevalence of 31.3%, the minimum sample size of 250 was determined. We, however, conducted our study on 323 children. Parents of children and adolescents (aged 4-18 years) living in Pakistan presenting at the speech therapy clinic of our hospital were included in the study, while those who denied consent were excluded.A standardized electronic questionnaire including queries about the sociodemographic profile, changes in eating style and behavior, changes in physical activities and screen time, lifestyle changes, and disturbances in sleep and mental health were used to collect data through a web-based survey using Google Forms. Data were entered into the SPSS Statistics version 23 . The data were coded and refined for further analysis. Results were presented in the form of frequency tables and bar/pie charts of different variables representing the research topic, and quantitative values were presented as means and standard deviations.Sociodemographic factorsParents of 323 children participated in this online survey. The mean age of the children was 11.2 years, with a standard deviation of ±4.2. Among them, 38.4% were between the ages of three to nine years and the rest were between the ages of 10-19; 41.5% (n=134) were females and 58.5% (n=189) were males; 58.2% (n=188) were dwelling in the metropolitan city while 41.8% (n=135) lived in small cities. The demographic factors are presented in Table Changes in eating styles and behaviorsDuring COVID-19, the consumption of fast food and fried foods by children and adolescents has increased significantly. According to this survey, during COVID-19, a higher consumption level of fizzy drinks has been observed. Our findings are presented in Table Changes in physical activities and screen timeIn this study, out of 323 participants, almost all  had increased their screen time significantly. Similarly, among the 323 individuals, 52 (16.1%) spent less than two hours in front of the screen, 112 (34.7%) spent two to five hours, 93 (28.8%) spent five to eight hours, and 66 (20.4%) spent more than eight hours /movie time and increased their social media usage during the COVID-19 lockdown Table .These results indicate that COVID-19 had an impact on the lifestyle behaviors and attitudes of children and adolescents. The pandemic and the subsequent containment strategies driven by governments around the world have adversely affected the behavioral health of children and adolescents. Further studies are however needed to study the nature of these impacts in a detailed manner.This study explored the experiences of children and adolescents during the COVID-19 pandemic and its impact on lifestyle attitudes and behaviors in this age group. The study gathered its findings primarily through observations by parents and caregivers. The major alterations that we observed included changes in eating behavior, physical activity, and screen time; we also noted changes in sleep, mental health, and lifestyle attitudes. A Brazilian study on food habits during the pandemic showed continued worsening in dietary patterns, with reduced intake of fruits and vegetables and increased consumption of sweets and fast food . AnotherCOVID-19 restrictions appear to have had a great impact on physical activity and behavior in children and adolescents. In an Italian study, during COVID-19 isolation, the proportion of low-active individuals increased to 39.62%, while the proportions of moderately active and highly active individuals were 29.75% and 30.63%, respectively . A CanadA Chinese survey of children and adolescents  reported that leisure screen time significantly increased compared to that before the COVID-19 lockdown. The total screen time increased by approximately 30 hours/week, and the time spent in the long term (≥2 hours/day) increased by 23.6% . In anotParents reported higher levels of family stress, depression, and anxiety due to social isolation. These findings are relevant to healthy development, as adverse psychological experiences in childhood are associated with an increased risk of anxiety later in life. The public health concern is that these short-term behavioral changes in response to COVID-19 may become entrenched. In a survey conducted in Portugal, Spain, and Italy, more than half of the participants felt bored (52.2%) and a third felt lonely (37.7%), particularly among Portuguese and Italian children . AnotherA Portuguese study showed that most patients (69.6%) reported difficulty sleeping, with waking up frequently being the most common problem during the COVID-19 lockdown . InteresThe rapid spread of COVID-19 to almost all regions of the world has brought about enormous health-related, environmental, economic, and social challenges. Social distancing, surgical masks, hand washing, and other precautions are considered the only way to fight the spread of the virus. All these factors can lead to changes in an individual's lifestyle. In a study conducted in Brazil, results showed that only 3.0% of the total participants reported not practicing physical and social distancing during the study period . In anotLikewise, in a survey conducted in Hong Kong, 74.2%, 72.7%, and 59.7% of 1,501 respondents said they avoided going out, going to crowded places, and attending social gatherings of more than four people, respectively . In anotIn another study conducted in Iran, the overall frequency of use of masks was 45.6% and the proportion of women using masks was significantly higher than that of men . SimilarLimitationsLike all observational studies, our study also had some limitations. Firstly, due to a strict lockdown that was in place, the study questionnaire was distributed via online platforms, including social media. The availability and use of the internet vary with different sociodemographic indicators. Therefore, this could be a hindrance to the precision of the results, especially when the ability to judge the changes in the behavior of their children also varies with the different sociodemographic settings. Secondly, our sample size of 323 also presents a challenge in adequately predicting the public perception at large and generalizing the results.Our study, however, has been successful in addressing the specific concerns that family physicians, pediatricians, and psychiatrists would face in the future in the aftermath of the pandemic. It could also provide a framework through which more studies can be conducted to devise a strategy to cope with an upcoming "behavioral" pandemic in the future.The COVID-19 pandemic has had a major effect on the health and lifestyle behaviors of both adolescents and children. Children are less affected by COVID-19 directly but suffer indirect, potentially long-term health consequences due to changes in lifestyle, eating behavior, physical activity, sleep patterns, screen time, and mental well-being. Due to an unbalanced diet, children are at a higher risk of both obesity and malnutrition. Governments around the world have imposed social distancing, leading to adverse short-term and long-term negative mental health issues such as unhappiness, fear, worry, irritability, depressive symptoms, anxiety, and post-traumatic stress disorder (PTSD).Parents must take care of their mental health and coping strategies and develop positive mental attitudes to support children and adolescents through this pandemic. Interventions should focus on building resilience in children and adolescents, addressing their fears and concerns through better communication, encouraging routine and physical activity, and taking measures to alleviate loneliness. Even with social distancing, social interaction is important; video conferencing, phone calls, or real-time texting may be worth considering, possibly on a daily basis.We believe our findings on the impact of the COVID-19 pandemic on the lifestyles and behaviors of children and adolescents will encourage parents, healthcare professionals, and policymakers to put appropriate measures in place to counter them. Further studies should be conducted to investigate the nature of these impacts and to draw long-term strategies to cope with their consequences and promote physical and mental health in this age group.


The subcarinal lymph node metastasis rate in patients with esophageal cancer is high. Patients with mid-thoracic esophageal cancers that infiltrate beyond the esophageal muscle layer with subcarinal lymph node size > 1.0 cm should undergo lymph node resection, despite increased operation time, incidence of postoperative pulmonary infection, hospitalization time, and total postoperative cost; lymph node resection may improve the 5-year survival rate.We investigated the value of subcarinal lymph node dissection in esophageal cancer surgery. Altogether, 240 patients with esophageal cancer admitted to our department between June 2012 and January 2016 were prospectively assigned to an experimental group  and a control group . The number of subcarinal lymph nodes and the rate of subcarinal lymph node metastasis were determined, and the factors influencing subcarinal lymph node metastasis were analyzed using logistic regression in the experimental group. The operation time, postoperative complications, intraoperative blood loss, postoperative hospital stay, total postoperative hospital cost, and 5-year survival rate were compared between the 2 groups. In the experimental group, an average of 6.03 subcarinal lymph nodes were dissected, and the lymph node metastasis rate was 18.33%. The subcarinal lymph node metastasis rate in the experimental group was related to the size of the subcarinal lymph nodes, depth of tumor invasion, and tumor location. The 5-year survival rate was higher in the experimental group than in the control group (44.2% vs 30.0%,  Similar to other gastrointestinal malignancies, lymph node metastasis is an important pathway for distant metastasis of esophageal cancer. Surgical treatment of esophageal cancer requires not only complete resection of the diseased esophagus but also systematic resection. Mediastinal lymph nodes are routinely resected, and the subcarinal lymph nodes represent the extent of mediastinal lymph node resection. Therefore, the subcarinal lymph nodes are within the scope of routine dissection. However, subcarinal lymphadenopathy may also be caused by benign chest diseases such as lung infection and silicosis. Moreover, excessive subcarinal lymph node dissection increases surgical trauma and pulmonary infection and reduces the short-term quality of life of patients postoperatively. Dissection of subcarinal lymph nodes has always been problematic for esophageal surgeons. Therefore, this study aimed to investigate subcarinal lymph node dissection to understand its value in esophageal cancer surgery.Esophageal cancer and esophagogastric junction cancer are among the most common digestive tract malignancies, accounting for 310,400 new cases worldwide yearly, Furthermore, 82, 99, and 49 were well-differentiated, moderately differentiated, and poorly differentiated cancers, respectively. According to stage, 64, 76, 82, and 18 cases were in stages I, II, III, and IV, respectively. Tumor length was ≤3 cm, 3 to 5 cm, and >5 cm in 53, 45, and 22 cases, respectively. The study protocol was approved by the Ethics Committee of our hospital . All patients enrolled in the study provided written informed consent.In total, 240 patients  with esophageal and esophagogastric junction cancer who received surgery in our department from June 2012 to January 2015 were selected. Of all cases, 25, 145, and 54 were localized in the upper, middle, and lower thoracic esophageal junction, respectively, and 16 in the esophagogastric junction. In the postoperative pathological diagnosis, all esophageal carcinomas were squamous cell carcinomas, and all esophagogastric junction carcinomas were adenocarcinomas. In this study population, 53, 10, 48, 9, 120, 59, 44, and 17 tumors were in stages Tis/T1, T2, T3, T4, N0, N1, N2, and N3, respectively, based on the 2009 International Union Against Cancer and American Joint Committee on Cancer standards for pathological grading and staging.We prospectively assigned patients into an experimental group  and a control group .The inclusion criteria were pathological diagnosis of malignant tumor by electronic gastroscope biopsy with clinicopathological stage cT1-3N0-3M0; computed tomography (CT) revealing no metastasis; no radiotherapy or chemotherapy before operation; and complete postoperative follow-up treatment.The exclusion criteria were small cell carcinoma upon preoperative electronic gastroscopic biopsy; preoperative severe cardiopulmonary disease or inability to tolerate surgery; concurrent presence of other malignant tumors or previous history of malignant tumors; preoperative radiotherapy and/or chemotherapy; and incomplete postoperative follow-up data.Thoracic and upper thoracic esophageal cancers were treated using thoracic laparoscopy combined with lower 3-field radical esophagectomy, and lower thoracic esophageal and esophagogastric junction cancers were treated using right thoracoabdominal esophagectomy.In the experimental group, lymph nodes around the bilateral recurrent laryngeal nerve, middle esophagus, lower esophagus, and below the carina were dissected. In the control group, lymph nodes around the bilateral recurrent laryngeal nerve, middle esophagus, and lower esophagus were dissected. The postoperative management was the same for all patients. All patients who required postoperative radiotherapy and/or chemotherapy received the same regimen.The number of lymph nodes dissected and the rate of lymph node metastasis in the 2 groups of patients were documented. The number of subcarinal lymph nodes dissected and the rate of lymph node metastasis in the experimental group were determined. The operation time, intraoperative blood loss, incidence of postoperative complications, postoperative hospitalization time, total postoperative hospitalization costs, mortality, and postoperative 5-year survival rates of the 2 groups were compared.Follow-up was once every 3 months for 1 year postoperatively; once every 6 months for 1 to 2 years postoperatively; and once a year for 3 to 5 years postoperatively. Detailed medical history; physical examination; investigations for the full set of tumor markers; direct enhanced CT of the brain, neck, chest, and upper abdomen; and whole-body bone imaging were checked during follow-up. Follow-up endpoint was death.P < .05.The SPSS version 25.0 software  was used for statistical analysis; measured data are expressed as mean ± standard deviation, and the comparison between the 2 groups was performed using the t-test. Enumerated data were compared between the groups using the chi-square test and are presented as frequencies (percentages). Logistic regression analysis was used for the multivariate analysis. Statistical significance was set at No significant differences were noted in sex, age, tumor location, tumor length, postoperative pathological staging, or postoperative tumor differentiation between the 2 groups. All patients in the 2 groups successfully completed the operation, and no deaths occurred during or immediately after the operation Table .A total of 7916 lymph nodes were dissected in both groups . In the experimental group, 724 subcarinal lymph nodes were dissected .P = .04). The intraoperative blood loss was 230.71 ± 107.893 mL, which was higher than that of the control group, 202.13 ± 84.875 mL . The incidence of postoperative pulmonary infective complications was 40.00%, which was 25.83% higher than that of the control group . The postoperative hospital stay and total postoperative cost were higher in the experimental group than in the control group    were lost to follow-up. The 5-year survival rate in the experimental group was 44.2%, which was significantly higher than that in the control group (30.0%) , which may be related to the fact that most of the esophageal cancers in this group were squamous cell carcinomas, and most patients visited a doctor when they had difficulty swallowing, which suggested that the disease was in the middle or late stages.For esophageal cancer and esophagogastric junction cancer, the distant metastasis routes include lymphatic and hematogenous metastasis, with lymphatic metastasis as the most common route. Cancer cells first enter the submucosal lymph nodes of the esophagus and pass through the muscle layer into the regional lymph nodes that drain the tumor site; moreover, the affected lymph nodes drain the neck, chest, and abdomen. Furthermore, lymph node metastasis is mostly longitudinal Paroxysmal bronchial smooth muscle contraction occurs, producing an irritating cough. Concurrently, an imbalance of cholinergic receptors can cause the accumulation of alveolar secretions, leading to pulmonary complications such as pulmonary infection. Patients with preoperative chronic bronchial disease could experience respiratory failure and even acute respiratory distress syndrome, which is life-threatening during the procedure. Additionally, bronchial artery damage and bronchial heat stimulation can cause bronchial mucosal ischemia, which negatively affects bronchial mucociliary function, causing difficulty in sputum removal, further aggravating pulmonary infection, increasing operation time, intraoperative blood loss, length of postoperative hospital stay, and total cost of postoperative hospital stay.In this study, the pulmonary-related complications in the experimental group were 40%, which was significantly higher than that of the control group; this may be due to damage to the medial border of the left and right main bronchi, left and right bronchial arteries below the carina, and pulmonary plexus branch of the vagus nerve during the dissection of the lymph nodes in this region. Additionally, when the electrocoagulation hook or ultrasonic scalpel is used to resect the lymph nodes in this area, the left and right bronchi may be thermally stimulated, which may lead to an increase in postoperative respiratory secretions. have revealed that the size of the subcarinal lymph nodes cannot be used as a basis for judging whether the cancer has metastasized to the lymph nodes in the region, and that it is not necessary to routinely dissect the regional lymph nodes. After frozen section to confirm metastasis, the lymph nodes may then be resected. Our findings may be related to the fact that most patients in the experimental group in this study were in the middle and late stages of the pathology.Subcarinal lymphadenopathy is not specific to malignancies. Long-term smoking, silicosis, chronic lung disease, and advanced age can lead to enlarged subcarinal lymph nodes. In this study, preoperative chest CT revealed a metastasis rate of esophageal cancer in patients with subcarinal lymph node diameters <1.0 and ≥1.0 cm of 5.41% and 39.13%, respectively. Therefore, when the subcarinal lymph node diameter exceeds 1.0 cm, routine dissection of the lymph nodes in this area is required. However, Shibamoto et alIn this study, the subcarinal lymph node metastasis rates of upper, middle, and lower thoracic esophageal, and esophagogastric junction cancers in the experimental group were 7.14%, 38.00%, 1.45%, and 10.00%, respectively. The subcarinal lymph node metastasis rate was significantly higher in esophageal cancer in the mid-thoracic region than in cancers in other parts of the esophagus, which may be related to the fact that mid-thoracic esophageal cancer is the closest to the subcarinal lymph node and is more likely to metastasize to the regional lymph nodes. Furthermore, some studies have demonstrated tumor invasion depth to be an independent risk factor for subcarinal lymph node metastasis. In this study, the logistic regression analysis indicated that Tis/T1 stage esophageal cancer was less likely to cause subcarinal lymph node metastasis. Once the tumor infiltrates beyond the muscle layer, it can invade lymphatic vessels and reach the regional lymph nodes. The cancer may then metastasize to the subcarinal lymph nodes, and the deeper the infiltration, the greater the chance of subcarinal lymph node metastasis.The subcarinal lymph node metastasis rates of esophageal cancer of tumor length ≤3 cm, 3 to 5 cm, and >5 cm were 18.87%, 8.89%, and 36.36%, respectively, in the experimental group. Therefore, the tumor length is an important factor in lymph node metastasis. It has been suggested that the length of the esophageal tumor is related to subcarinal lymph node metastasis. This is because the longer the esophageal tumor invading the surrounding tissues of the esophagus, the greater likelihood that invasion of the lymphatic vessels in the subcarinal area will occur, resulting in subcarinal lymph node metastasis; this is consistent with the findings of Feng.In the experimental group, the subcarinal lymph node metastasis rates of well-differentiated, moderately differentiated, and poorly differentiated esophageal cancers were 8.13%, 8.02%, and 6.29%, respectively, and the difference was not statistically significant. This suggests that subcarinal lymph node metastasis was not associated with the degree of tumor differentiation because the degree of tumor differentiation only reflects the degree of malignancy of the tumor, and whether subcarinal lymph node metastasis occurs is mainly related to whether the cancer cells invade the lymphatic vessels in this area. Therefore, tumors that are less differentiated do not have a greater chance of invading lymphatic vessels in the region.The 5-year follow-up rate was 92.5%; 18 patients had anastomotic recurrence, 10 had liver metastasis, 7 had lung metastasis, and 4 had bone metastasis. The 5-year survival rate of the experimental group was 44.2%, which was significantly higher than that of the control group (30.0%). Subcarinal lymph node dissection may improve the 5-year survival rate of patients with esophageal cancer.This was a single-center study that had a relatively small number of enrolled cases and a short-term follow-up. Thus, the results need to be further validated. The next step is to extend this research to other hospitals and perform a statistical analysis of the 10-year postsurgical survival rate.In conclusion, the subcarinal lymph node metastasis rate in patients with esophageal cancer was high, and the metastasis rate was related to the size of the subcarinal lymph node, depth of tumor invasion, tumor location, and tumor pathological stage. For patients with mid-thoracic esophageal cancers infiltrating beyond the adventitia of the esophagus and those with subcarinal lymph node size >1.0 cm, routine lymph node dissection in this area should be performed; although this may prolong the operation time, increase the incidence of postoperative pulmonary infection, prolong the hospitalization time, and increase the total postoperative cost, it may improve the 5-year survival rate.We thank Huang Guozhong  for his assistance with editing this manuscript.Study conception and design: Bindong Xu and Hao Chen. Data collection: Bindong Xu, Hao Chen, Qiang Zhang, and Pengfei Chen. Analysis and interpretation of results: Bindong Xu, Hao Chen, Qiuxia Liu, Mingyu Chen and Qiang Zhang. Manuscript preparation: Bindong Xu and Hao Chen. All authors reviewed the results and approved the final version of the manuscript.Conceptualization: Bindong Xu, Hao Chen, Qiang Zhang, Mingyu Chen.Data curation: Bindong Xu, Qiang Zhang, Qiu Xia.Formal analysis: Bindong Xu, Hao Chen, Pengfei Chen, Qiu Xia.Investigation: Bindong Xu, Mingyu Chen.Resources: Hao Chen, Pengfei Chen.Software: Qiang Zhang, Pengfei Chen.Writing – original draft: Bindong Xu.Writing – review & editing: Bindong Xu.


This study suggests that the rs671 polymorphism modifies the association between DBs and BMI independently of drinking habits, providing evidence for the potential use of rs671 polymorphism information for precision nutrition with East Asians.The rs671 polymorphism, unique to East Asians, is well known to change the sensitivity to alcohol. Moreover, this polymorphism is associated not only with alcohol intake but also with several dietary behaviors (DBs), chronic diseases, and BMI, but the triadic association among the rs671 genotype, DBs, and BMI is unclear. This study included 12,271 Japanese subjects and aimed to observe this three-way association using the rs671 polymorphism, data of 56 DBs, and BMI. All analyses were stratified by participant sex. First, linear regression analyses resulted in significant associations between 18 and 21 DBs and BMI in males and females, respectively. Next, genetic heterogeneity was observed in all sub-groups via interaction analysis of the rs671 genotype stratified by drinking habits. Finally, we observed the characteristics of BMI-related DBs based on the rs671 genotype via stepwise regression analyses stratified by the rs671 genotype and drinking habits. Notably, positive associations were observed between  The research field of precision nutrition aims to understand the complex interplay among many factors, such as genetics, metabolism, the gut microbiota, food environments, and their impact on health ,2. This ALDH2) gene, an East Asian-specific polymorphism, is well known to change sensitivity to alcohol [The rs671 polymorphism of the aldehyde dehydrogenase 2  and Genequest Inc. . The participants were 18 years of age or older and were asked to complete an Internet-based questionnaire that included socio-demographic factors, lifestyle habits, and medical history at the time of registration. All participants provided written informed consent for the general use of their genotype and questionnaire data. After informing the participants of the purpose of this study, we provided the opportunity to opt out. This study was conducted under the principles expressed in the Declaration of Helsinki after gaining approval from the Genequest ethics committee.We previously reported that the 12q24 locus, which is located in the rs671 polymorphism, is associated with several DBs ,9. Accorp-values (<1 × 10−6), or low minor allele frequencies (<0.01) and performed principal component analysis using Eigensoft.We used 296,675 markers included in both genotyping platforms in this QC protocol. In addition, we excluded participants with inconsistent sex data between genotypes and the questionnaire, closely related pairs among the participants , and those with estimated non-Japanese ancestry using PLINK (version 190b3.42)  and EigeParticipant age, sex, BMI, and a total of 56 DB items  were obtained from a web-based questionnaire. In addition, participants responded to 5–8 items based on the frequency of their monthly food or beverage intake. Categorical terms of the DBs, intake frequency, and scoring are shown in We performed linear regression analysis of the association between 56 DBs and BMI with an adjustment for age and population stratification  using PLINK 1.9. Additionally, we carried out the linear regression analysis of the association between the rs671 polymorphism and 56 DBs with an adjustment for age and population stratification with or without alcohol intake frequency and alcohol consumption as covariates. Furthermore, we performed linear regression analysis of the associations between 56 DBs and BMI stratified by the rs671 genotypes and drinking habits using R v3.2.1 . We defip trend < 0.001; p interaction < 0.001; Characteristics of our participants are shown in p < 8.9 × 10−4, after Bonferroni correction) associated with BMI in males and females, respectively (p < 8.9 × 10−4) associated with 11 and seven DBs in males and females, respectively (p < 8.9 × 10−4) associated with the rs671 polymorphism  across all analysis groups  [Lactobacillus to the AA type of mice would decrease body weight and white fat mass. However, the present results differed from this expectation. Possible reasons for this include differences between humans and mice and differences in the food environment, such as the induction of obesity via the administration of a high-fat diet in mice. Alternatively, Lactobacillus products might only be a proxy for other diets or participants with a higher BMI might have consumed the Lactobacillus beverage for its anti-obesity effects. Further studies, including more detailed diet surveys and gut microbiota analyses based on large populations, are needed to elucidate these causes.In non-drinkers, the characteristics of the association between DBs and BMI differed among genotypes of the rs671 polymorphism. Notably, for the AA type in both sexes, positive associations were found between the frequency of drinking  effects ,38,39. Yxamined) . From thIt is worth mentioning that there was a female-specific negative association between the intake frequencies of soy products  and BMI. Genistein, which is one of the isoflavones that is abundant in soybeans, has a reported estrogen-like effect ,42,43. BThere are several limitations to this study. First, this study was a questionnaire-based food intake frequency (FFQ) survey. Thus, energy adjustment and nutrient estimation were not possible. In addition, we could not determine what was consumed with the food being studied, such as sugar and milk in coffee. Furthermore, our previous results of GWASs to examine the association between genetic loci and DBs ,7,8 was In conclusion, we show that the rs671 polymorphism modifies associations between DBs and BMI. Moreover, these modifications have characteristics that depend on sex and drinking habits. These findings provide evidence to support the possibility of using rs671 polymorphism information for precision nutrition for East Asians.


The deterioration of plant mineral nutrition during drought is a significant factor in the negative influence of drought on plant performance. We aimed to study the effects of seasonal and multiyear water shortages on nutrient supply and demand in Scots pine and Norway spruce. We studied pine and spruce trees naturally grown in the Bryansk region (Russia). The dynamics of several nutrients  in wood, needles, and bark of current-year twigs and the dynamics of the available pools of these elements at different soil depths were analysed. To assess the physiological consequences of changes in element concentrations, lipid peroxidation products and photosynthetic pigments were measured in the needles. Water shortage increased the wood concentrations of all elements except for Mn. In pine, this increase was mainly due to seasonal water deficit, whereas in spruce, multiyear differences in water supply were more important. This increased availability of nutrients was not observed in soil-based analyses. In needles, quite similar patterns of changes were found between species, with Mg increasing almost twofold and Fe and Mn decreasing under water shortage, whereas the remainder of the elements did not change much under differing water supplies. Neither the concentrations of photosynthetic pigments nor the contents of lipid peroxidation products correlated with element dynamics in needles. In summary, water shortage increased the availability of all elements except Mn for the plant; however, needle element contents were regulated independently of element availability for plants. In recent years, the mutual effects of plant mineral nutrition and plant performance under drought have begun to receive increased attention ,2,3.Water deficit can influence plant mineral nutrition on multiple levels. Drought influences all three ways of nutrient acquisition by roots, namely, diffusion, mass flow, and root interception . TranspiMultiple recent studies have focused on the impacts of drought on plant mineral nutrition, with controversial results. Drought-stressed seedlings of several drought-sensitive pine species were proposed to face an “isohydric trap”—a feedback cycle of nutrient deficit in leaves that exacerbates the negative drought effect on the plant’s performance . The usePinus sylvestris L.) and Norway spruce (Picea abies (L.) H. Karst) as well as the effects of water deficiency on the available pools of mineral elements in soils under the plants. We studied trees grown under natural conditions . DroCompared with the almost uniform effect of decreased water supply on element concentrations in the wood, relatively few changes in the content of available elements were found in the soil analysis. In some cases, these changes coincided with the element dynamics in the wood; for example, the K accumulation in pine wood A coincidThe leaf is the major sink for mineral nutrients, and both the deficiency and excessive content of mineral elements can be detrimental to physiological processes in leaf tissues. As a result, leaf nutrient concentrations can be maintained strictly even under substantial drought-induced nutrient shortages , being oa and b and the Mg content in needles. Mg is required for chlorophyll biosynthesis, and decreased Chl content as well as an increased Car/Chls ratio are observed in Mg-deficient plants, leading to leaf chlorosis [The first group of elements includes Mg in both species. The concentration of Mg increased in wood, needles, and bark in a coordinated way, mainly in response to the rainless period , Table 2hlorosis . TherefoThe second group of elements was the most represented and included K, Ca, P, and Zn in both species and Cu in pine. The concentrations of these elements were quite stable in needles and did not change much in bark despite the substantial increase in wood under conditions of water shortage. Excessive nutrients can be actively transported to the other plant parts by the phloem; by this transport, it is possible to maintain the total needle concentration of elements on an unchanged level despite their increased supply from the xylem. However, calcium has rather low phloem mobility  and was Picea abies, the concentration ratios of Fe and Mn between young and old needles were very close to their phloem mobility ratios, which implies that Fe and Mn remobilization is indeed restricted by their phloem mobility [Pseudotsuga menziesii demonstrated that Mn can be transported by the phloem, albeit at a quite slow rate, and that this transport is important for maintaining needle Mn concentrations under increased Mn uptake by roots [The third group of elements includes Fe and Mn, which decreased prominently and uniformly in needles of both species under water shortage. The Fe concentrations could be considered optimal in spruce and optimal or suboptimal in pine . Togethemobility . On the by roots .2O2 in the photosynthesis electron transport chain is often increased, leading to Fe- and Mn-catalysed generation of highly reactive hydroxyl radicals [Fe and Mn were significantly correlated in both pine and spruce needles . One posradicals . Therefo-3; forest type, bilberry-spruce forest; and soil type, slightly soddy sandy mesopodzol with a ferruginous illuvial horizon on fluvioglacial sand. Based on the living ground cover species, this site is mesohydrophilous [Site I . Mean age of adult trees, 70 years; productivity class, I; relative density, 0.8; stand composition, 10 spruce; forest site type, Bophilous , and it -2; forest type, cowberry pine forest; and soil type, medium soddy sandy mesopodzol on fluvioglacial sand, with quartz-glauconitic sand containing phosphorites underneath. Based on the living ground cover species, this site is mesophilic [Site II . Mean age of adult trees, 70 years; productivity class, I; relative density, 0.9; stand composition, 8.7 pine, 1.3 spruce + birch; forest site type, Asophilic , and it -1; forest type, cowberry-pine forest; and soil type, slightly soddy sandy cryptopodzol on fluvioglacial sands. Based on the living ground cover species, this site is mesoxerophilous [Site III . Mean age of adult trees, 60 years; productivity class, I; relative density, 0.5; stand composition, 10 pine; forest site type, Aophilous , and it Investigations were performed in native undisturbed pine and spruce stands of productivity class I in the Bryansk region (Russia). Based on the annual growth data, 3 experimental sites were selected for this study :-Site I In total, for each species, one normal (N) and one arid (A) site were studied; see  for a moPlant and soil samples were collected in 2018. The year 2018 was relatively hot and dry compared with both climatological reference normals (1961–1990 and 1981–2010), and August was the driest month of the vegetation season, with a rainless period occurring from 2 August to 29 August  and spruce (10–25 years) around the perimeter of the tree crown. For sampling, 8 plants of each species were chosen in the normal and arid sites, with each plant taken as a biological replicate. Twigs were excised, wrapped in plastic bags, placed on ice, transported to the laboratory within 12 h, and used for the determination of nutrient contents in current-year wood, needles, and bark.Soil samples were collected from depths of 5–15 cm, 25–35 cm, and 60–70 cm on 3 August. During sample collection, the roots of pine and spruce undergrowth were found to have penetrated the soil profile to depths lower than 60–70 cm. Therefore, during soil sampling on 24 August, samples were also collected from depths of 110–120 cm. For each site and depth, 4 soil samples were taken as biological replicates. The soil samples were placed in hermetically sealed plastic containers and transported to the laboratory for the determination of soil pH and available pools of mineral nutrients. The collected soil samples were air-dried and then sieved to obtain fractions <2 mm in size. Before the analysis, a small portion of each soil sample was oven-dried at 105 °C to correct the dry weight results .v/v) suspension of soil in a 0.01 M CaCl2 solution (pH in CaCl2) [The soil pH was measured in a 1:5 (n CaCl2) .w/v) extractions with 0.01 M CaCl2, in which 2.5 g of soil and 25 mL of solution were used [w/v) extractions with 1 M CH3COONH4, in which 1 g of soil and 5 mL of solution were used [2SO4, 0.85 mM (NH4)6Mo7O24, and 43 mM ascorbic acid. After colour development incubation for 3.5 h at 25 °C, the optical density was measured at 825 nm. To construct the calibration curve, KH2PO4 was used.The available pools of K, Mg, Fe, Mn, Zn, and Cu were determined in duplicate by 1:10 ) [The collected twigs were washed in 10 mM aqueous solutions of EDTA·Na1 (v/v)) , after wThe contents of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) were determined spectrophotometrically, with maximum optical absorption at 586 nm, by measuring the product that formed during the reaction using the selective reagent 1-methyl-2-phenylindole  in accordance with . To consa, b, and carotenoid contents were determined using the Lichtenthaler method [The chlorophyll r method . The samr method .p < 0.05). To calculate the strength and significance of correlations between variables, Pearson’s correlation was used. The Pearson’s correlation coefficients presented in the text are significant at p < 0.05. The values presented in the figures are the arithmetic means, and the values presented in the tables are arithmetic means ± standard errors.Each tree was treated as a biological replicate; therefore, there were 8 biological replicates for the plant-based analyses of nutrient contents in needles, bark, and wood and of the contents of pigments and lipid peroxidation products in needles. For soil analyses, there were 4 biological replicates. The data were statistically analysed using SigmaPlot 12.3 . To separate the effects of site and sampling date, two-way ANOVA was used  under water shortage. In pine, this increase was mainly due to seasonal water deficit after the rainless period, whereas in spruce, multiyear differences in water supply between normal and arid plots were more important. This increase was not revealed by soil analyses, indicating the limited ability of soil-based methods to assess changes in nutrient availability for trees under water shortage. We demonstrated that the influence of water shortage on tree nutrient status can differ substantially depending on the analysed organs or tissues, with the nutrient status of needles being the least affected and that of wood being the most affected by water shortage. This indicates the necessity of simultaneous analysis of wood and leaf element concentrations to better understand the influence of water shortage on tree nutrient supply, in contrast to the most common approach, where physiological conclusions are made based on either the leaf or wood element composition. Only three elements, namely, Mg, Mn, and Fe, changed consistently in the needles of both species in response to the decreased water supply. However, no strong correlations of these elements with the contents of photosynthetic pigments and lipid peroxidation products were found, thus suggesting that these changes in nutrient concentrations neither maintained the functioning of the photosynthetic apparatus nor decreased the risk of oxidative damage under water stress. Therefore, the physiological importance of strict maintenance of Mg, Mn, and Fe concentrations in needles remains to be identified.


Compared to the placebo, the caffeine chewing gum supplementation significantly improved the accuracy of the attack . However, the ingestion of caffeinated chewing gum had no effect on the remaining other performance tests (p from 0.12 to 1.00). A caffeine-containing chewing gum with a dose of ~3 mg/kg BM effectively improved the attack’s accuracy in volleyball players. However, this effect was not observed in better results in jumping, running, and other skill-based volleyball tests.No previous study analyzed the effect of caffeinated chewing gum on volleyball-specific skills and physical performance in volleyball players. Twelve volleyball players participated ina randomized, crossover, and double-blind experiment after ingestion of (a) ~3.2 ± 0.4 mg/kg of body mass (BM) of caffeine via chewing gum or (b) non-caffeinated chewing gum (placebo) and performed: (a) a countermovement jump, (b) a squat jump, (c), an attack jump, (d) a block jump, (e) 5 and 10 m sprints, (f) a modified agility  Volleyball is a physically demanding team sport with technical and tactical requirements . This spCaffeine is considered the most popular psychoactive drug used by the majority of the general population in the world . Due to Previous studies have shown that caffeine has a positive effect on various types of exercise, such as intensive efforts of short duration , resistaIn most of the studies and real sports scenarios, caffeine is typically consumed 45–60 min before exercise in the form of capsules, coffee, or an energy drink . HoweverTherefore, the aim of this study was to determine the effect of caffeinated chewing gum in a dose of ~3 mg/kg BM on volleyball-specific skills and physical performance in volleyball players. To the best of our knowledge, this is the first study to evaluate the effects of caffeinated chewing gum on both numerous performance tests and specialized volleyball skills tests.t-test, difference between two dependent means” were assumed as a statistical test ; the statistical power was 0.8, the significance level was 0.05, and the effect size was 1.22 based on previous study [The study included 12 healthy volleyball players, of which nine are men and three are women  = 24.4 ± 1.7; habitual caffeine intake = 2.7 ± 2.2 mg/kg/day) with at least 5 years of volleyball training experience (9 ± 4 years). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders; (b) minimum 5 years of experience in volleyball training; (c) no medication nor dietary supplements used within the previous month which could potentially affect the study outcomes ; (d) self-described good health status; (e) the same phase of the menstrual cycle in both trials  . On thist-test, (g) an attack and service speed test, and (h)a spike and serve accuracy test. At the end of each trial, players were asked about possible side effects from caffeine ingestion and how they perceived improvement in performance during the study.A randomized, crossover, double-blind design was used to evaluate the effects of the caffeinated chewing gum. Randomization was performed by a research team member who was not involved in the data collection. Therefore, when assigned to intervene, participants and investigators were blinded on each trial. Each player took part in one familiarization session and two identical experimental sessions with an interval of 72 h between trials to ensure complete recovery ,30 and sAfter the warm-up, players performed the CMJ, SJ, attack jump, and block jump. Players performed 3 jumps in each trial, with 1 minute’s rest between them. The technique of making individual jumps was as follows. The CMJ was performed from a standing position with a straight torso, knees fully extended with hands on hips, and feet shoulder-width apart. Players descended to their usual squat depth and jumped for maximum height, and landed in an athletic position . The SJ t-test) was used to determine the speed with direction changes. The modified t-test , which was used to record the time of running tests in other authors’ research [The running time was assessed with 5 m and 10 m straight sprints, and a simple running agility test  and at a constant net height of 2.24 m for women and 2.43 m for men. A modified test designed to standardize skill assessment for junior volleyball players was used to assess the accuracy of the attack . The couAll of the above tests are designed to assess the physical performance of volleyball players and volleyball-specific skills. For this purpose, both jumping tests and running tests were used, as well as tests assessing the speed of the ball and accuracy in the performance of two elements of the volleyball game, i.e., attack and serve.After the experimental trials were completed, the players answered questions related to their feelings about the possible side effects of using caffeine. Players were asked about the possible occurrence of side effects: (a) increased urine output, (b) tachycardia and heart palpitations, (c) anxiety or nervousness, (d) headache, (e) gastrointestinal problems, (f) increased vigor activeness, (g) perception of performance improvement. They were permitted to cite others issue not listed ,43. Addit-tests. The relative CAF–PLAC effect was calculated through effect sizes (Cohen’s d). Parametric effect sizes (ES) were defined as large for d > 0.8, moderate for d between 0.8 and 0.5, and as small for d < 0.5 [p < 0.05.All calculations were performed using Statistica 13.3 and presented as means with standard deviations (± SD). The Shapiro–Wilk test was used in order to verify the normality of the data. The differences between the CAF vs. PLAC were identified using paired sample  d < 0.5 . Statistt-test showed no significant differences in the CM, SJ, attack jump, and block jump height between CAF and PLAC conditions . Caffeine-containing chewing gum did not improve other skill-based , and physical  tests evaluated in the current study. This may suggest that caffeine in a dose of ~3 mg/kg BM via chewing gum has minor effect on physical performance and volleyball skills-based tests among volleyball players, at least with mild-moderate caffeine consumption levels.The results of the presented study showed no significant effect of caffeine on the height of both CMJ and SJ, as well as in volleyball-specific jumping techniques. Interestingly, the vast majority of studies conducted so far have shown an improvement in the height of CMJ and SJ after caffeine supplementation in team sports including basketball ,46, soccSince jumping is considered a crucial aspect of volleyball, it is necessary to analyze the effect of caffeine on volleyball-specific movements, such as attack and block jumps . The rest-test. In addition, it was found that the ball’s velocity is significant in the context of the effectiveness of the attack and serve [Volleyball players change direction frequently during training and game . Therefond serve . The resnd serve ,61, inclnd serve ,16. Intend serve ,16,50,61nd serve  comparednd serve  showed tnd serve ,63,64 alnd serve .In addition to evaluating the physical tests, this study aimed to assess the effect of caffeinated chewing gum on volleyball skills, which translates into points in match conditions. For this purpose, the players performed specialized tests to check the accuracy and precision of their attacks and services. To the best of the authors’ knowledge, this is the first study analyzing the effect of caffeine supplementation on skill-based volleyball performance. The results of the current study showed that caffeinated chewing gum improved the number of points obtained during the accuracy attack test. Although the results during the accuracy service test did not reach a significant improvement, a positive trend (ES = 0.57) in caffeine condition has been observed. It is worth noting that the accuracy assessed in the current study may demand maintaining vigilance, alertness, and related parameters. It has been shown that low doses of caffeine  improve vigilance, alertness, mood, and cognitive processes during exercise, but do not alter the peripheral whole-body responses to exercise . TherefoThe results of the current study showed no side effects when caffeinated chewing gum was used. Thus, it confirmed that using caffeine via chewing gum reduces the risk of possible side effects compared to other forms of this supplement ,65. HoweBeyond the present study’s strengths, its limitations should also be considered. The current study did not include any biochemical analyzes. Only one caffeine dose of caffeine  was assessed; thus it is not known whether higher doses would improve analyzed outcomes. In addition, the experimental dose of caffeine was similar to the daily caffeine intake of the athletes, and it is possible that higher caffeine doses may provide performance benefits. Future studies should be conducted with a larger number of participants and involve a comparable number of men and women. Lastly, it is also worth considering to reduce the number of tests in future studies to exclude possible fatigue effects during following performance tests.The results of the present study indicated that the acute intake of caffeine (~3 mg/kg BM) via chewing gum significantly improves the results of attack accuracy in volleyball players. However, this supplement did not improve the results in jumping tests, running tests, and other volleyball specific-skills, including speed tests and accuracy during the volleyball, serve. Additionally, caffeine supplemented in such a dose and form minimizes the risk of side effects. From a practical viewpoint, the benefits of caffeinated chewing gum seem to be minor in comparison to caffeine capsules or energy drinks, at least in athletes habituated to caffeine consumption.


No significant association was found between maternal age, BMI, parity, level of education, place of residence, source of water used, and smoking with the levels of PFOA and PFOS in the human milk. Additionally, the consumption of cereals at least twice per week was significantly associated with higher levels of PFOS. These findings call for actions to improve the local environmental and agricultural practices, and the regulations and standards for inspecting imported food. It is important to highlight that the benefits of breastfeeding outweigh the reported contamination with PFOS and PFOA in our study.Human milk is the primary source of nutrition for infants in their first year of life. Its potential contamination with perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), a group of toxic man-made chemicals, is a health concern that may threatens infants’ health. Our study aims to assess the levels of PFOA and PFOS in the breast milk of Lebanese lactating mothers and the maternal factors associated with their presence. High-performance liquid chromatography (HPLC) coupled with a Micromass Quattro micro API triple quadrupole mass spectrometer was used to detect the level of contamination in 57 collected human milk samples. PFOA and PFOS were present in 82.5% and 85.7% of the samples, respectively, while PFOA levels ranged between 120 and 247 pg/mL with a median of 147 pg/mL, and those of PFOS ranged between 12 and 86 pg/mL with a median of 27.5 pg/mL. The median contamination for PFOA exceeded the threshold set by the European Food Safety Authority (EFSA) (60 pg/mL); however, that of PFOS was below the threshold (73 pg/mL). The consumption of bread, pasta, meat, and chicken more than twice per week and that of white tubers and roots at least once per week was significantly associated with higher levels of PFOA ( Per- and polyfluoroalkyl substances (PFAS) are a complex group of anthropogenic chemicals containing more than 9000 distinct compounds ,2. ThesePerfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are the most studied compounds among all PFAS. Over the last few decades, they have received global attention due to their widespread use, high stability, and toxicity ,9. SeverAccording to , it is eDue to their extensive use and bioaccumulation properties, several studies have shown that PFOA and PFOS are now present in the serum of humans worldwide . Human eIn addition, it was reported that human exposure to PFAS begins during gestation and continues during breastfeeding, leading to severe health outcomes that are mainly associated with immunotoxicity, hepatotoxicity, neurobehavioral toxicity, reproductive toxicity, lung toxicity, and hormonal effects ,21,22.Human milk is the best source of nutrition for infants providing them with the optimum quality and quantity of nutrients to grow. In addition, it offers unique immunological benefits, decreasing their risk of acute illnesses and chronic diseases later in life . AccordiIt is still unclear how PFOA and PFOS are transferred from the mother’s blood to her breast milk. However, it has been shown that these perfluorinated substances are bound to proteins in maternal blood . PreviouVarious factors have been linked to PFAS in human milk, including maternal age, socioeconomic status, education level, parity, infant sex, and maternal diet . MoreoveInfants’ exposure to PFAS has become a public health concern since it has been linked to adverse health outcomes that are more likely to occur due to their low weight, immature immune system, and low detoxification capacity ,35,36,37Due to the toxicological concerns over PFOS and PFOA, the US Environmental Protection Agency (EPA) reported in 2006 that these contaminants and their precursors should be eliminated by 2015 . In 2009The European Food Safety Authority (EFSA) identified PFAS as emerging toxicants in the food chain. It established tolerable daily intakes (TDI) of 150 ng/kg BW/day and 1500 ng/kg BW/day for PFOS and PFOA, respectively . HoweverRegarding human milk, the EFSA set critical values for these contaminants based on the association found between the concentration of PFAS in the plasma and the amount of antibodies against diphtheria and tetanus in one-year-old infants . The thrIn Lebanon, food safety issues are of public health concern ,50,51. ASocio-demographic characteristics were gathered from participating breastfeeding mothers using a culturally tailored questionnaire. In addition, a semi-quantitative food frequency questionnaire (FFQ) was used to get information regarding eating habits of the lactating mothers for different food groups . The FFQ was based on guidelines of the WHO , and theA convenient sample of 57 samples of human milk were obtained from breastfeeding mothers across Lebanon between November 2015 and December 2016. Samples (10–75 mL) were collected in sterile plastic containers, transported in an ice box to the laboratory, where they were stored in the freezer at −20 °C until analysis. Before approaching the participants, Institutional Review Board approval was obtained from the Lebanese American University. Furthermore, a letter was received from the Ministry of Public Health in Lebanon to support the recruitment of eligible participants from primary care centers and hospitals. All participants obtained written and signed informed consent before data collection. The latter was conducted following the World Health Organization protocol . To be e4OH in methanol, then by 6 mL of methanol in addition to 6 mL of Milli-Q water, following a rate of 1 drop/s. We ensured the cartridges were preserved from drying during the preconditioning and sample-loading. The cartridges were washed right after passing the sample with 6 mL of Milli-Q water and 6 mL of 25 mM sodium acetate/acetic acid buffer (pH 4). Then, they were cleaned by passing 6 mL of 40% methanol in Milli-Q water. Afterwards, the cartridges were dried for 10 min under vacuum (−70 kPa) to get rid of the residual water before eluting with 6 mL of 0.1% NH4OH in methanol at 1 drop/s. The eluate was concentrated under nitrogen to near dryness, then reconstituted to 200 uL using 0.1% NH4OH in methanol.As per , solid-pPFCs in breast milk were identified and quantified using high-performance liquid chromatography (2695 separation module) coupled with a Micromass Quattro micro API triple quadrupole mass spectrometer from . Then, 10 μL of the extract was injected onto a 2.1 mm × 100 mm, 3.5 μm Xterra MS C18 column  at a flow rate of 0.3 ml/min. The mobile phase was 2 mM ammonium acetate/methanol at a flow rate of 300 μL/min. The gradient begun at 10% methanol, amplified to 99% methanol at 12 min, and was kept for 3 min before switching back to 10% methanol and holding for 3 min for a total run time of 18 min. The API was executed in electrospray negative ionization mode with a source temperature of 100 °C, desolvation temperature of 250 °C, desolvation gas flow of 500 L/h, and cone gas flow of 200 L/h. The target compounds were identified using multiple reaction monitoring. Nine target compounds were spiked to determine matrix-spike recoveries of individual PFCs via the analytical procedure in cow’s milk (3% fat content) and breast milk. PFCs were spiked at 80, 200, and 600 pg/mL in cow’s milk (n) 6 for each spiking level) procured from a local retailer. PFCs were spiked at 200 pg/mL in breast milk (n) 6). The samples were extracted and then analyzed by following the procedure described earlier. Recoveries of PFCs spiked in the cow’s milk were between 86 ± 9% (mean ± standard deviation SD) and 104 ± 3. Recoveries of PFCs spiked in the breast milk were 75 ± 14% to 100 ± 11 for all PFCs, except for LPFBS where the recoveries were 68 ± 35%. Recoveries of the two 13C-labeled internal standards, spiked into all samples before extraction, were between 72 ± 11% and 80 ± 7%. Procedural blanks (n = 6) were made by substituting 10 mL of Milli-Q water for the milk. This was followed by a complete analytical procedure. The average contamination level in procedural blanks was 80 pg/mL for PFOA and <10 pg/mL for the other PFCs. Quantification was conducted using linear regressions  obtained from an eight-point calibration standard, which was prepared in methanol at concentrations between 20 pg/mL and 600 pg/mL. The limit of quantitation (LOQ) was 10 times the standard deviation (SD) of the highest concentration found in the blanks. In comparison, the limit of detection (LOD) was determined as three times the (SD).2 tests were used to assess the difference in PFOA contamination in terms of consumption of the different food sources such as bread and pasta, cereals, white tubers and roots, potatoes, dry beans, canned beans, fresh vegetables and fruits, dried fruits, meats and chicken, eggs, fish low in mercury and high in mercury, pasteurized milk, sweets, yogurt and labneh, white cheeses, nuts and seeds, spices and condiments, herbal infusions, soft and caffeinated drinks. Second, the human milk samples were divided into two groups for PFOS contamination. Group one included the samples with PFOS levels below the threshold of 73 pg/mL, whereas group two included those with PFOS greater than 73 pg/mL. Considering only 6 participants had PFOS values above the threshold, the analysis was conducted on the non-parametric variable without categorization. Mann Whitney U was conducted to assess the difference in PFOS contamination between the two consumption levels of the previously mentioned food sources.STATA V13 was used for the statistical analysis. To summarize the study variables and to detect out-of-range values, descriptive analysis was used. Using frequencies and percentages, categorical variables were described, while means and standard deviations were used in order to represent continuous variables. Shapiro–Wilk was used to assess data normality. Median and Interquartile ranges (IQR) were used to describe the non-parametric variables. Spearman’s rank correlations coefficient (rho) was used to measure the association between the non-parametric variables PFOA, PFOS, age, and BMI. First, the collected human milk samples were divided into two groups for PFOA contamination. Group one included the samples with PFOA levels below the threshold of 60 pg/mL, whereas group two included those with PFOA levels greater than 60 pg/mL. None of the samples had a PFOA level of 60 pg/mL. χDemographic characteristics of the participants are shown in p = 0.017 and 87% vs. 50% p = 0.009, respectively). As for the white tubers and roots, at least once weekly consumption was associated with PFOA contamination (86.4% vs. 50% p = 0.010). There was no association between PFOA and other food sources, as indicated in PFOA contamination was detected in 47 (83%) human milk samples. Among the latter, the median PFOA was 147 (IQR 120–274). There was no difference in PFOA contamination in human milk by any socio-demographic variables. Females who consumed bread/pasta and meat/chicken more than twice per week had PFOA levels greater than 60 pg/mL in their breast milk versus those who consumed it less frequently (84.4% vs. 50% p = 0.217). Cereal consumption at least twice per week was associated with greater PFOS contamination in breast milk compared to those who consumed it less than twice per week .PFOS contamination was detected in six (10.71%) breast milk samples. The median PFOS level was 22 (IQR 13–48.5) among all tested samples. There was no difference in PFOS contamination in human milk across the governorates . Concerning PFOS, it was reported in 86% of the samples ranging between 12 and 86 pg/mL, with a median of 27.5 pg/mL, below the threshold set by the EFSA for PFOS in human milk (73 pg/mL).Compared to the literature, the levels of PFOA in the collected human milk samples in Lebanon were higher than that reported in a neighboring country, Jordan, where the level of contamination ranged between 24–1220 pg/mL with a median of 82.5 pg/mL, compared to a median of 147 pg/mL in Lebanon. However, our samples were less contaminated with PFOS than in Jordan, with a median of 50 pg/mL compared to 27.5 pg/mL in our study . SimilarHaving the highest contamination level in our samples for PFOA and the lowest for PFOS, compared to the literature, can be explained by a recent report in Lebanon that showed legal actions being taken to control the use of PFOS in the industry. At the same time, other PFAS substances, which include PFOA, are still poorly regulated . AccordiThere was no statistical significance between maternal age and human milk contamination with PFOA and PFOS, which is inconsistent with the literature. In Jordan, it was shown that the average contamination levels of PFOA and PFOS were significantly higher in the breast milk of older women . This wa2), this might have affected the results. Our findings are consistent with other studies in France and eastern Slovakia, where no significant association was reported  re rep = 0.man milk .To conclude, our study is the first in Lebanon to assess the presence of PFOS and PFOA in the breast milk of Lebanese mothers and study the factors associated with their contamination. Additionally, using the ICP-MS technique to assess the levels of these contaminants in the human milk samples is a strength of the study. However, some limitations should be carefully considered when interpreting our results. The main limitation is that our sample is small and might not be representative, affecting the generalizability of our results. Additionally, our study might have a self-reporting bias, where mothers self-reported their dietary intake, smoking status, and alcohol consumption. Future studies must evaluate the circulating/serum PFOS and PFOA levels to be coupled with the levels in human milk.Our results suggest the need for further investigations with a larger and more representative sample to examine the sources of contamination in Lebanon , and assHuman milk is the ideal source of nutrition for infants, essential for their proper growth and development. Milk contamination with PFOA and PFOS poses a serious health threat, hindering the growth of infants, and increasing their risk of chronic diseases later in life. Therefore, infants’ exposure to PFOA and PFOS through human milk is a global health concern. Our study has shown that 86% and 83% of our samples were contaminated with PFOS, and PFOA, respectively. Since human milk is the primary source of infant nutrition, it is essential to ensure its safety by minimizing the mother’s and baby’s exposure to these environmental contaminants, and maximizing the benefits of lactation. Nevertheless, it is important to highlight that exclusive breastfeeding protects nursing infants from exposure to various contaminants, and thus the benefits of breastfeeding compensate for the potential risks. In addition, we should acknowledge that human milk is just one of many routes of exposure of infants to these harmful contaminants. Raising awareness about the importance of choosing healthy and safe food, and avoiding contaminated foods and water, is essential to ensure the health of our infants in the country. Our findings strongly suggest the need for an integrated collaboration between different public health stakeholders to protect the Lebanese population from these toxic chemicals that have lifelong health consequences. It is important to highlight that the benefits of breastfeeding outweigh the reported contamination with PFOS and PFOA in our study.