There is no sufficient evidence on the effectiveness of acupuncture for neuropathic pain. This protocol describes a study that aims to evaluate the effectiveness and safety of electroacupuncture combined with conventional medicine for patients with intractable neuropathic pain, when compared with conventional medicine alone.This study is a prospective, open-labeled, randomized, cross-over clinical trial. A total of 40 patients with neuropathic pain who had a numeric rating scale (NRS) score of over 4 despite receiving conventional treatment for more than 3 months will be enrolled. Participants will receive conventional treatment for neuropathic pain (treatment C) or treatment C combined with 12 additional sessions of electroacupuncture treatment (treatment A) for 6 weeks. Participants will be randomly assigned to 1 of the 2 sequence groups (AC and CA group) with a 1:1 allocation. The differences of responder in the composite efficacy outcomes, which consist of the NRS, Brief Pain Inventory-Short Form (BPI-SF) pain subscale, and global assessment at 6 weeks after randomization will be examined as the primary outcome. Secondary outcomes include differences in the NRS, the Short-Form McGill Pain Questionnaire, BPI-SF, Fatigue Severity Scale, Hospital Anxiety and Depression Scale, Medical Outcomes Study Sleep Scale, global assessment, EQ-5D, and incremental cost-effective ratio at 6 and 15 weeks after randomization. Adverse events, vital signs, and physical examinations will be recorded to evaluate safety.The study protocol for this trial will provide up-to-date evidence on the effectiveness and safety of electroacupuncture for patients with intractable neuropathic pain. The results will be disseminated through a peer-reviewed journal and conference presentations.https://cris.nih.go.kr/cris/search/search_result_st01_kren.jsp?seq=13410& ltype=&rtype=Clinical Research Information Service, ID: KCT0003615. Registered on March 12, 2019.  This could easily progress to chronic and refractory pain, which presents a great challenge in terms of management. Conventional pharmacological treatments for neuropathic pain include antidepressants and antiepileptics; however, the number of patients who achieve satisfactory pain relief is low, and is only 10% to 25% more than patients receiving placebo. Moreover, high comorbidity rates accompanied by chronic neuropathic pain, such as insomnia, anxiety, or depression, further makes the treatment challenging, and patients seek alternative treatment strategies including acupuncture treatment.Neuropathic pain develops after injury to the nervous system, and is often accompanied by maladaptive changes in the somatosensory nervous system. or inactivation of the myofascial trigger point. Acupuncture analgesia can also be induced segmentally by the gate-control theory of pain. Needling of some acupuncture points located at the extremities can generally reduce pain through descending inhibitory pain control by serotonin and noradrenaline.,9 Recently, clinical evidence on acupuncture for chronic pain, which is similar to nociceptive pain, has been well-established through many systematic reviews–12 and clinical guidelines.,14 A meta-analysis assessing 20,827 individual patient data from 39 trials suggested that acupuncture is an effective treatment option for chronic pain, including nonspecific musculoskeletal pain, osteoarthritis, headache, or shoulder pain, and that its effects persist over time, with only a small decrease at 1 year. The guidelines published by the American College of Physicians recommend that acupuncture should initially be selected for patients with chronic low back pain. However, when focusing on neuropathic pain such as diabetic neuropathy, chemotherapy-induced peripheral neuropathy, postherpetic neuralgia, or failed back surgery syndrome, there is no sufficient evidence on the effectiveness of acupuncture.Some studies have proposed the mechanisms of acupuncture for pain reduction. The local analgesic effects of acupuncture are mediated by adenosine A1 receptors explored effectiveness of electroacupuncture (EA) for patients with moderate to severe neuropathic pain despite already receiving conventional oral medications, and assessed the feasibility of a large-scale trial. The results showed that EA significantly decreased the intensity of pain in neuropathic patients, especially burning sensations, electric shock-like pain, and mechanical hyperalgesia. Furthermore, the number of acupuncture sessions was a significant factor for favorable results of reducing pain intensity. Based on our previous trial, we designed a large-scale, prospective, open-labeled, randomized, cross-over clinical trial to evaluate the effectiveness and safety of 12 sessions of EA for the treatment of neuropathic pain.Our pilot study22.1The aim of this study is to determine the effectiveness and safety of EA treatment for neuropathic pain when added to conventional neuropathic pain pharmacotherapies compared with no EA treatment. The pain intensity, function, quality of life, global assessment, and adverse events will also be assessed.2.2This study is a prospective, open-labeled, randomized, cross-over clinical trial conducted at Seoul National University Hospital (SNUH) Pain Center and Kyung Hee University Korean Medicine Hospital (KHUKMH) in South Korea.2.2.1A total of 40 patients with chronic neuropathic pain who did not achieve satisfactory pain relief despite receiving conventional treatment for more than 3 months will be recruited in SNUH Pain Center. The expected recruitment period is from December 2019 to May 2021. Participants will be recruited through advertising on the bulletin boards of the hospital, internet homepages of the hospital, public institutions, and through the media.2.2.2This study is a cross-over trial that aims to compare conventional medical treatment (treatment C) with conventional medical treatment and an add-on EA treatment (treatment A) for patients with intractable neuropathic pain. A cross-over design was planned in which participants will be randomly assigned to 2 groups (AC and CA groups) according to the 2 treatment sequences.Screening will be conducted after receiving written informed consent from the participant at SNUH Pain Center Visit 1). They will then be randomly assigned to 1 of the 2 sequence groups: the AC group and CA group (Visit 2). The first treatment period will be conducted for 6 weeks. In the case of “treatment A”, during the treatment period, a total of 12 EA sessions will be performed for 6 weeks at KHUKMH. After the first treatment period, evaluations will continue at SNUH Pain Center (Visit 3). After a washout period of 2 weeks, participants will undergo a pain evaluation period for 7 days (Visit 4). If the average numeric rating scale (NRS) pain score, which is self-recorded in a diary, is 4 points or more during this period, patients will proceed to the second treatment period. If it is less than 4 points, the same “treatment C” will be maintained. The second treatment period will also be conducted for 6 weeks, as in the first treatment period, followed by a final evaluation (Visit 5). Figure . They wi2.32.3.1Participants who meet the following conditions will be included:1.Patients aged 19 years and over at screening.2.Patients who have peripheral neuropathic pain and satisfy criteria A) and B), and have criterion C) or D) identified on examination, depending on the conditions.A)Have a confirmed medical history of diagnosis for neuropathic pain according to clinical findings.B)Symptoms, history, and physical examination of patients suggest peripheral neuropathic pain.C)Electromyography/nerve conduction study or evoked potential test suggest peripheral neuropathy.D)Ultrasonographic examination at screening reveals lesions that indicate peripheral entrapment syndrome or peripheral neuropathy.3.Have unsatisfactory pain relief despite appropriate conservative treatment for more than 3 months before screening.4.Have a score ≥13 on the Korean version of the PainDETECT Questionnaire (PD-Q) at screening.5.Have an average score ≥4 on the NRS measured by a pain diary during the 7-day screening period before randomization.6.No evidence of disease recurrence in cancer survivors.7.Patients who can voluntarily consent to the study, understand the process of the study, and adequately write a reportable questionnaire.2.3.2Participants will be excluded if any of the following conditions are satisfied:1.Have other severe pain not related to the target disease that could cause confusion during pain assessment at screening or randomization.2.Use prohibited concomitant therapies within 7 days prior to screening, or change to concomitant medications with restricted usage within 14 days prior to screening.3.Have a clinically significant unstable nervous system, ophthalmological disease, hepatobiliary disease, respiratory disease, blood disease, cardiovascular disease, or mental disease within 12 months before screening.4.Used EA treatment at the affected area within 1 month before screening.5.Are pregnant, breastfeeding, or expecting a pregnancy during the study period.6.Have implanted medical devices such as spinal cord stimulators, implantable drug delivery systems, pacemakers, automatic defibrillators, aneurysm clips, vena cava clips, and skull plates.7.Have brain damage, symptomatic brain metastases, or epilepsy.8.Have abnormal skin conditions that would prevent proper application of the EA treatment.9.Are participating in other interventional clinical trials within 30 days before screening or present.10.Have severe bleeding tendency: low doses of oral aspirin and a general dose of antiplatelet/anticoagulant medication generally do not limit study participation, but only when apparent risk is predicted by the investigators judgment.11.Use immunosuppressive drugs.2.4The randomization table was generated using SAS Version 9.2  to assign patients to the AC group or CA group in a 1:1 ratio by the block randomization method. After obtaining informed consent, participants who satisfy the inclusion/exclusion criteria will be allocated on a web-based basis according to this randomization table. A random allocation table will be created at the Medical Research Collaborating Center (MRCC) of SNUH, and will be operated through web random allocation. The outcome assessors, data managers, and statisticians will be blinded to the allocation.2.5 which was developed through a consensus of acupuncture specialists based on western medical acupuncture techniques as well as traditional meridian theory. EA treatment will be performed by Korean medicine doctors who graduated after 6 years of Korean medical college education and have at least 8 years of clinical experience. A total of 12 acupuncture sessions for 6 weeks will be performed using 0.25 × 40 mm disposable sterile acupuncture needles , which will be retained for 15 minutes. Up to 20 acupuncture points will be chosen by the practitioner based on traditional Asian medicine and western medical perspectives,17 for local, segmental, and general stimulation. Local stimulation is defined insertion of the needle at a classical acupuncture point, tender point, or trigger point near the painful area. Segmental stimulation is insertion of the needle around area innervated by the same meridian or spinal nerve as that of the painful region. If direct needling to the local and segmental points exacerbates the pain, perisegmental points that are close to the affected segment, such as those above and below the area, or those on the opposite side of the equivalent contralateral segment will be used. Distal points which are located in the extremities for general stimulation will be chosen to treat the comorbidities of chronic pain according to traditional theory, such as insomnia , depression/anxiety , or fatigue .The EA treatment was modified from our pilot study,Electrical stimulation will be delivered using an EA device  with 2 Hz , 2 to 6 Hz , 120 Hz , or 2 to 120 Hz   approximately 80% of the maximum intensity.2.6Existing oral and transdermal drugs  should be taken at the existing dose from screening to the end of the study. Depending on the pain intensity, additional prescription of oral acetaminophen is allowed as a rescue medicine for analgesic purposes. If acetaminophen is used, the participant should record the dose of acetaminophen and date in the patient diary. Anticonvulsants and antidepressants, which were being used to control symptoms of existing neuropathic pain, are also taken at the maintenance dose during the study period. Only when there are safety problems with the use of these drugs, dose reduction or discontinuation of these drugs will be permitted if necessary.Subjects who received interventional therapies such as nerve block, iontophoresis, laser therapy, spinal cord irritation, and transcutaneous electrical nerve stimulation have a minimum washout period of 7 days prior to their first visit. These therapies are prohibited in the study period. Other forms of pain relief therapy for neuralgia such as psychotherapy and physiotherapy can be continued from 14 days prior to the first visit if the frequency of therapy does not change; however, these are not allowed to start during the study period.2.72.7.1The proportion of participants who achieved improvement, as measured by the predefined composite efficacy outcome (CEO)  at 6 weeks after randomization, will be used as the primary outcome measurement.2.7.2Secondary outcomes include the differences in NRS score, the short-form McGill Pain Questionnaire, the BPI-SF, the Fatigue Severity Scale, the Hospital Anxiety and Depression Scale, the Medical Outcomes Study Sleep Scale, PGIC, EQ-5D-3L, PD-Q, Patient Satisfaction Scale, and incremental cost-effective ratio at 6 and 15 weeks after randomization will be measured.2.7.3Safety will be examined on every visit by measuring vital signs, performing physical examinations, and recording adverse events (AEs). For vital signs, the degree of change after the treatment period compared to before the treatment period will be summarized as mean, standard deviation (SD), median, minimum, and maximum value. The results of the physical examination will be summarized by the frequency and ratio. AEs will be summarized by the number and ratio of patients who experience AEs and serious AEs. The human chorionic gonadotropin urine test will only be performed in fertile women at the first and fifth visits.2.8This study was developed according to the following hypotheses:0: p1 = p2H1: p1 ≠ p2Hp1 = proportion of participants in the acupuncture group who achieved improvement, as measured by the predefined CEO after 6 weeks from baseline.p2 = proportion of participants in the control group who achieved improvement, as measured by the predefined CEO after 6 weeks from baseline. the patients who achieved significant improvement was 44%, defined as a decrease in NRS, a reduction in PIS of BPI-SF scores of more than 20% from baseline, and change to “minimally improved”, “much improved”, or “very much improved” status, as measured by PGIC. Though there was no control group in the pilot study, we assumed that the conservative estimated proportion in the placebo group will be 10%. Assuming that the period and carryover effect does not exist, the difference in proportions of the 2 groups is 0.34 and within-subject SD is 0.34. Sixteen participants are required for each sequence group with a two-sided significance level of 5% (α = 0.05) and 90% power (1-β = 0.9). Consequently, a total of 40 patients (20 in each group) will be included, accounting for a potential 20% drop-out rate.Based on our pilot study,2.9The analysis set will consist of a modified intention-to-treatment (ITT) analysis set, a per protocol (PP) set, and a safety set. The modified ITT set will include the participants who were randomized, received acupuncture or conventional treatments, and have at least 1 effect assessment after treatment. The PP set will include only participants who received at least 1 assessment and completed the study protocol without major deviation. The safety set will include any participants who received at least 1 acupuncture or conventional treatment after randomization. The modified ITT analysis will be the main analysis of this study.t test or a Wilcoxon rank sum test will be performed for continuous data, and a Chi-Squared test or Fisher exact test will be performed for categorical data.For the descriptive analysis, Student For a confirmatory analysis, the primary endpoint is the proportion of participants who show improvement, as measured by the predefined CEO at 6 weeks after randomization. The carry-over effect will be checked with a linear mixed model, and if the carry-over effect is not significant, the improvement frequency and ratio at 6 weeks for each treatment period in each sequence group will be summarized. The difference of the overall improvement rate (δ) between the groups will be estimated, and the null hypothesis will be rejected as follows:σd: variance of the difference of the improvement rate between the groups within the subjectn: number of participants in each groupIf the carry-over effect is significant, only data from treatment period A will be used and tested using the Chi-Squared test or Fisher exact test. Additionally, the carry-over effect, period effect, and sequence effect will be corrected using a generalized linear mixed model, and then the additional effects of acupuncture will be examined. At this time, the random effect, sequence, treatment period, and acupuncture treatment will be considered as fixed effects, and carry-over effect and period effect will be tested in terms of sequence order and treatment period, respectively. The term with a level of significance greater than 0.3 will be excluded from the model and the add-on effect of acupuncture treatment will be identified.For secondary outcome measurements, continuous data will be summarized as mean, SD, median, and minimum/maximum value for each treatment period in each group, and compared between the 2 groups using a linear mixed model. Categorical data will be summarized as frequency and ratio for each treatment period in each group, and compared between the 2 groups using a generalized linear mixed model.Statistical analyses will be performed by a statistician in the MRCC blinded to the group allocation using SAS Version 9.2 .2.10The independent researcher will conduct regular monitoring to ensure quality control of the data. The researcher will monitor the written informed consent forms, compliance of protocol, and data documents during the study period. Monitoring also check for AEs.2.11,19Participants will be asked to report any AEs including those induced by EA throughout the study period at each visit. The expected AEs related with EA treatment include treatment-related pain, bruising, bleeding, or infection at the local site, nausea, dizziness, or anxiety.If AEs occur during the clinical trial, researchers will investigate the severity of symptoms, relevance to the intervention, action related to the intervention, treatments for AEs, and the result and seriousness of AEs. In the event of serious complications, researchers will provide adequate treatment to patients and terminate the EA treatment early if the patients find it difficult to continue the study. After that, a report will be prepared and submitted to the institutional review board (IRB).2.12The study was designed in accordance with the Helsinki Declaration to protect the participants and was approved by the IRB of the SNUH (H-1810-075-979) and the KHUKMH (KOMCIRB 2018-12-002). Only participants who voluntarily provide written consent after receiving a full description of the study prior to screening will be included. Participants are informed of the potential benefits, risks, alternatives, and responsibilities during the study by the researcher throughout the consent process. The results will be disseminated through a peer-reviewed journal and conference presentations.3This is the study protocol for a prospective, open-labeled, randomized, cross-over clinical trial for patients with intractable neuropathic pain. This study aims to evaluate the effectiveness and safety of EA combined with conventional treatment compared to conventional treatment alone after 6 weeks of treatment. which showed that the protocol was feasible and acceptable for a large scale trial considering the low dropout and high compliance rate. The study identified the appropriate dose of EA treatment, which was 12 sessions, once or twice a week, for 8 weeks. EA twice a week during the first 4 weeks led to an almost 50% reduction in pain intensity from baseline; however, EA once a week for the following 4 weeks did not have a clinically meaningful analgesic effect. Therefore, we will administer a total of 12 sessions of EA treatment twice a week for 6 weeks.This confirmatory trial was designed after adapting the results of our previous pilot trial affective dimensions, overall quality of life, and pain intensity were also improved after EA treatment. Since accompanying symptoms such as insomnia, anxiety, and depression, which are major comorbidities of chronic pain can affect pain intensity,,20 it is necessary to identify the degree to which the analgesic effect of EA treatment correlates with the effect of these symptoms. Therefore, we measured the effect of EA treatment on these comorbidities with validated scales.In our pilot study, Therefore, integrating the assessment of pain intensity and functional disability is recommended for a trial assessing chronic pain, although there are some methodological disadvantages. Based on the results of our previous study, we defined the CEO to include pain intensity, various aspects of pain, and global assessment of the patient, and adapted it for this confirmatory trial.Some studies have suggested that pain intensity alone could not assess the value of interventions and overall experience of patients with chronic pain, and the functional aspect and quality of life should also be comprehensively considered.To summarize, using data from our pilot study, this confirmatory trial was adapted with several improvements, and will assess the effectiveness and safety of EA in patients with refractory neuropathic pain.We would like to thank all members of the research team  at KHUKMH and SNUH pain center.Conceptualization: Seunghoon Lee, Jee Youn Moon.Funding acquisition: Seunghoon Lee, Jee Youn Moon.Methodology and project administration: Seunghoon Lee, Jee Youn Moon, Sang Hoon Lee.Writing – original draft: Seunghoon Lee, Suji Lee.Writing – review & editing: Jee Youn Moon, Chang-Soon Lee, Yongjae Yoo, Sang Hoon Lee.


Air pollution is killing close to 5 million people a year, and harming billions more. Air pollution levels remain extremely high in many parts of the world, and air pollution-associated premature deaths have been reported for urbanized areas, particularly linked to the presence of airborne nano-sized and ultrafine particles.To date, most of the research studies did focus on the adverse effects of air pollution on the human cardiovascular and respiratory systems. Although the skin is in direct contact with air pollutants, their damaging effects on the skin are still under investigation. Epidemiological data suggested a correlation between exposure to air pollutants and aggravation of symptoms of chronic immunological skin diseases. In this study, a systematic literature review was conducted to understand the current knowledge on the effects of airborne particulate matter on human skin. It aims at providing a deeper understanding of the interactions between air pollutants and skin to further assess their potential risks for human health.Particulate matter was shown to induce a skin barrier dysfunction and provoke the formation of reactive oxygen species through direct and indirect mechanisms, leading to oxidative stress and induced activation of the inflammatory cascade in human skin. Moreover, a positive correlation was reported between extrinsic aging and atopic eczema relative risk with increasing particulate matter exposure. Traffic≤ 10 μm) .10 fraction consists primarily of crustal materials, sea salt, biological factors, including bacteria and fragments of pollen, and are generated by mechanical processes rather than combustion. On the other hand, PM2.5 and PM0.1 are predominantly produced by combustion processes and consist primarily of metals, hydrocarbons, and secondary particles formed by chemical reactions with gaseous compounds in the atmosphere [0.1 with the greater surface area can carry a large amount of adsorbed pollutants, oxygen gases, organic compounds, and transition metals [The PMmosphere , 8. The n metals . An examn metals , as medin metals  and incrn metals .Long-term effects of air pollution on the pulmonary and cardiovascular system have been extensively studied, and include a strong correlation between different levels of air pollution and mortality, exacerbation of asthma, chronic bronchitis, respiratory tract infections, ischemic heart disease and stroke . AlthougThis review highlights the current understanding of the impact of airborne PM on the skin and the underlying physiological mechanisms that are affected. Furthermore, it provides an in-depth overview of in vivo and in vitro studies of airborne PM exposure to skin and lists the biomarkers of interest and findings. The following search engines and databases, PubMed, ScienceDirect, Google Scholar, and Web of Science, were used to identify peer-reviewed research articles and reviews on the effects of airborne PM on skin. From among 540 articles identified, 75 articles were included for further consideration after manual screening of the articles. The articles that were excluded either did not contain data or were classified as editorials or conference abstracts. Finally, articles in languages other than English or German were also omitted.Mammalian skin is the largest organ in the human body and can be divided into two major layers, the epidermis and the dermis Fig. . The derThe layer above the dermis, the epidermis, is divided into four to five layers and contains mainly keratinocytes . The lowExposure to airborne PM is associated with several skin diseases and disorders and has been extensively reviewed elsewhere , 37–39.In healthy skin, exposure to urban PM has been associated with accelerated extrinsic aging –42, its 10 resulted in a 52% increase in the relative risk of non-melanoma skin cancer [2.5 [In Germany, a higher incidence of skin melanoma among women was reported for women living in rural areas compared to women living in non-rural areas . In anotn cancer . The gencer [2.5 .10 and PM2.5-exposure and several skin diseases [2.5-exposure of younger people , which was not the case for PM10 exposure. However, with an odds ratio of 1.05 and the small quantity of observational, cohort studies, and individual studies, the statistical power of this meta-analysis is limited. Several new studies have been published that were not included in this meta-analysis [10 and sulfur dioxide (SO2)) was reported. Contrary to the small-scale exposure assessments (PM2.5), where in most of the studies (23 out of 30), a significant association between AE and traffic-related emissions was determined. Additionally, in all of the panel studies, there was a strong correlation between the AE symptom severity and outdoor pollution. Interestingly, an association between maternal exposure to traffic-related pollution and AE incidence in the offspring was found. From the prospective birth cohort study, Cohort for Childhood Origin of Asthma and Allergic Diseases (COCOA) [10 and PM2.5 and transepidermal water loss (TEWL), which is a measure for skin barrier dysfunction [10, PM2.5, and PM0.1 have been extensively studied for their ability to enhance Th2-related immune responses, their role as an immune adjuvant, and their effects on the development of atopic disease [The chronic relapsing inflammatory skin disease, atopic eczema (AE), is characterized by skin barrier defects, intense pruritus, and immunological dysregulation –51. Thisdiseases . It repoanalysis –60. Moreanalysis . No suff (COCOA) , 468 onefunction . The efffunction . A posit disease –67.Dong and co-workers reviewed reports on PM exposure and skin inflammation in China and concluded that, indeed, PM emissions aggravate the symptoms of inflammatory skin diseases like AE . The afoThe exact mechanisms through which PM can aggravate AE symptoms are yet to be further investigated. Additionally, determining the synergistic effects of different air pollutants while taking solar radiation into account can be challenging and is often not addressed when studying the effect of a single pollutant on the skin . In a stThus far, the studies mostly investigated the link between AE and PM. However, it is hypothesized that patients with other inflammatory skin diseases with an impaired barrier function, such as the rare genetic skin disease Netherton syndrome  and psorThe effects of airborne PM on the skin and underlying mechanisms will be critically evaluated, i.e., by reviewing studies on exposure of in vitro human two-dimensional (2D) and three-dimensional (3D) models, and finally in vivo animal models. The studies used either standard reference materials or in-house diesel emission and urban particulate matter extracts as exposure constituents.As aforementioned, the composition of PM is complex and includes metals, organic compounds , inorganic carbonaceous material, sulfate, and nitrate, among others. To assess their health effects, different approaches have been applied, such as epidemiological studies, controlled exposure studies to a defined source, or collection of particles on filters which can then be used for further experiments. It is important to add that the thorough characterization of the particles, including particle size and chemical analysis of the adsorbed chemicals onto the particles to correlate observed effects, is challenging but highly relevant .2.5. Besides using certified standards, diesel particulate extracts from several different diesel engine emissions collected on filters have been extracted and tested on skin models. Additionally, NIST offers two airborne PM standards named SRM® 1649b and SRM® 1648a that have been collected in the Washington, DC area and the St. Louis, MO area, respectively, between 1976 and 1977. Other standards for fine atmospheric PM are SRM® 2787  and SRM® 2786  collected in Prague, the Czech Republic in 2005.The most common particles used in in vitro and in vivo studies to simulate the effects of airborne PM on the skin are Standard Reference Materials® available from the National Institute of Standards and Technology (NIST) . NIST of10-like urban dust without  or with PAHs (ERM-CZ100). The National Institute for Environmental Studies (NIES) in Japan offers PM10-like urban dust certified reference material (CRM) no. 28 that have been collected from a central ventilating system in a building in Beijing, China [The Joint Research Centre (JRC) of the European Commission produces European Reference Materials (ERM) that are used to simulate airborne PM . JRC is g, China .2.5. Others reported usage of concentrated ambient air particles (CAPs), which are airborne particles that are concentrated to PM2.5 for skin model exposure.Several studies reported self-collection of airborne PM or PM from seasonal dust storms in Asia and West Africa, e.g., on building rooftops in Asia, using an in-house filtering system, yielding PMThe main in vivo approaches to study skin responses to either drug permeation or toxicological outcome are either rodents  or pigs . Rodent Animal models are employed to compare PM effects on skin with a healthy and compromised barrier function. A disrupted barrier can be created by the conventional tape stripping method, which consists essentially of removing the SC of the animal skin model layer by layer –92. AnotAnimal models have various disadvantages, besides their different physiological structures. Animal studies are highly time and cost-ineffective; they are restricted to many regulations and countless ethical concerns . To effeHuman adult low calcium high temperature (HaCaT) cells are an immortalized keratinocyte cell line that is frequently used for 2D in vitro studies . HoweverThe two main types of 3D models used to mimic human skin are human epidermal equivalents (HEE) and human skin equivalents (HSE) , 107.Cultivation of HEE models starts with one cell type: either primary keratinocytes isolated from the human epidermis, or immortalized cell lines . These keratinocytes are grown in typical culture inserts on a supporting substrate, such as an inert membrane with a defined pore size or collagen matrix, using the air-liquid culture technique. This technique includes removing the cell culture medium from the upper side to expose the cells to air on one side, allowing the cells to ‘feed’ from the medium in the chamber underneath –110. UpoHSE models are comprised of both a dermal and an epidermal compartment consisting of fibroblasts embedded in a collagen matrix and keratinocytes, respectively. This two-compartmental model creates an interplay between the two skin layers, resulting in a better resemblance to human skin.Although 3D skin models like the HSE and HEE are practical models that well-resemble the human skin, their barrier properties are less defined, due to a different SC lipid composition and a higher barrier permeability compared to that of native human skin , 111–115There are four different barrier compartments in the epidermis: physical, microbial, chemical, and immunological compartments . In thisThe microbial barrier compartment consists of a diverse range of microorganisms, called the commensal skin microbiome that colonizes the skin. This includes colonization by millions of bacteria, fungi, and viruses, and the composition varies depending on the location of the skin . These cReported findings on the effects of PM on the skin’s barrier function in vivo and in vitro are summarized Table .Table 12.5 [The physical barrier compartment of the skin can be seen as a so-called “outside-in” and “inside-out” barrier. The “outside-in” barrier consists of the SC, which protects against the penetration of pathogens, allergens, and other exogenous substances such as PM , 147. Se2.5 . SPRR3 u2.5 , which i2.5 .The “inside-out” barrier, mainly provided by tight junctions, prevents excessive TEWL and, therefore, protects the skin against dehydration , 152. It2), resulting in decreased expression of structural barrier protein like keratins and filaggrin [In animal models, PM treatment mainly involved repetitive topical treatment for a longer time period (up to 2 weeks) of a relatively high dose  have beilaggrin , 158. HoIn 3D HSE models, a systemic treatment with PM (200 μg/mL) induced alterations in skin morphology and expression levels of structural proteins after a long time exposure  , 126. InPM was also reported to affect the skin’s cholesterol metabolism and increased cholesterol levels in 2D and 3D skin models . CholestKeratinocytes are crucial to the production of antimicrobial peptides (AMPs), serving as the chemical barrier compartment, preventing microbial invasion at sites of damaged epithelium by directly terminating the pathogen. These molecules can destroy bacteria either by creating holes in their cellular walls or by sequestering iron, a crucial nutrient for bacterial growth . ExampleOne of the primary mechanisms of the adverse effects of PM is through the generation of ROS. ROS are a group of highly reactive chemical species that are derived from the oxygen metabolism characterized by an unpaired electron  . When thPM can trigger the formation of exogenous ROS levels through the formation of free radicals as a result of the high particle surface reactivity , 189. Di2.5-treated keratinocytes and fibroblasts [2+ levels in vitro [Indirectly, PM or its adsorbed species  can increase endogenous ROS generation by inducing mitochondrial stress and increasing the activity of ROS producing enzymes. The mitochondria are commonly considered as the main source of ROS production in the cell. PM induces mitochondrial dysfunction, by inducing major structural damage , 195. Tyroblasts . This dyin vitro , 182. Asin vitro , 179–1862+, and, as a result, changes in the cellular Ca2+-balance activate ER stress [2+ [The endoplasmic reticulum (ER) is responsible for most of the intracellular CaR stress . PM was ress [2+ , 184–186ress [2+ , were acress [2+ , 175. Inress [2+ .CYP1A1, CYP1A2, and CYP1B1), the phase II metabolism  and a gene for the aryl-hydrocarbon receptor repressor (AhRR) [Sensing exogenous chemical substances is paramount to stimulating an immune response as the first line of defense against these compounds. An important sensor of environmental chemicals is the aryl hydrocarbon receptor (AhR), a transcription factor that is highly expressed in all skin cells . Besidesr (AhRR) . CYP enzr (AhRR) , 204–208r (AhRR) , 187.Other ROS producing enzymes are the members of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family . Once acThe skin is covered by a thin layer of sebum, that functions as a barrier against water evaporation  . Sebum lThe first line of defense against ROS is the cornified envelope, especially the SPRR family . It was Other defense mechanisms, to abrogate oxidative stress processes, are identified like the upregulation of antioxidant proteins and detoxifying enzymes that are activated by transcription factors via antioxidant response elements (ARE) . The expActivation of the AhR induces the nuclear translocation of Nrf2 by a mechanism that needs to be elucidated, but evidence suggests it involves protein kinases that induce transcription of detoxifying enzymes, leading to a decrease of ROS levels .2.5 [Oxidative stress can lead to the activation of programmed cell death, i.e., apoptosis . Protein2.5 , 175. Mi2.5 . Mitocho2.5 . It was 2.5 , 184, in2.5 , and act2.5 –184, 1862.5 , DNA fra2.5 , 182–184. Proteins involved in the initiation of autophagosome formation, such as light-chain 3B II (LC3B-II) and Beclin 1, were shown to be upregulated [ER stress is also related to autophagy, and PM exposure was shown to promote autophagy in vitroegulated , 224.2.5 induces senescence in human keratinocytes  [INK4A was found to be upregulated upon PM2.5 treatment, and its transcription was shown to be epigenetically regulated through promoter methylation [Another mechanism that can be activated upon ROS-induced DNA damage is cellular senescence. Senescent cells are in irreversible arrest, thereby limiting proliferation, that is implicated in skin aging . It was  HEK001) , 185. PM HEK001) . The senhylation . In micehylation .The skin plays a major role in protecting the body from harmful environmental factors. The keratinocytes and immune cells contribute to the first and second lines of defense of the skin’s immune system and are an indispensable part of the immunological barrier compartment. Additionally, the dermal fibroblasts play an important role in the sensing of pathogens, to protect the body further from harmful intrusive species. The reported findings on the effects of PM on the activation of the inflammatory cascade in skin models have been summarized Table .Table 3Keratinocytes in the viable epidermis  and dermal fibroblasts defend the human body by sensing pathogens and mediating an immune response by differentiating between harmful and harmless pathogens . They doActivated TLRs can recruit and activate the adaptor molecule, myeloid differentiation primary response 88 (MyD88), to initiate signaling and activate the nuclear factor kappa B (NF-κB) pathway and members of the mitogen-activated protein kinase (MAPK) family , 240.TNF), interleukin 1 alpha (IL1A), interleukin 1 beta (IL1B) and chemokines such as C-X-C motif chemokine ligand 8 (CXCL8), have been shown to play a key role in the skin [ICAM1) and enzymes such as cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS) [2 (PGE2) derived from arachidonic acid from membrane phospholipids [NF-κB represents a group of five inducible transcription factors that, once activated, mediate transcription of target genes by binding to a specific DNA element. Its target genes include an enormous amount of chemokines and cytokines among which tumor necrosis factor (the skin . In turne (iNOS) . COX2 isholipids .2.5 was shown to be able to bind to TLR5 in keratinocytes [2 [PMinocytes . Additioinocytes . This, iinocytes . Indeed,inocytes , 233. Thinocytes , 243. PMcytes [2 , 232.NLRP3) gene, which is a crucial priming signal for the activation of the inflammasome [Activation of NF- κB also leads to the transcription of the NLR family pyrin domain containing 3 , can be recruited by TLRs  and, in addition to MyD88, can initiate signaling of both NF-κB and MAPK . This acA crosstalk between the previously described AhR and the MAPK pathway has been reported . The pro.MAPK activation and the resulting activation of AP-1 leads to transcriptional regulation of matrix metalloproteinases (MMPs). The major sources of MMPs in the skin are keratinocytes and fibroblasts , and theIt was shown in vitro and in vivo that PM-induced skin barrier dysfunction, oxidative stress, and inflammation are interconnected and exaggerate each other , was shown to suppress the expression of AhR and its nuclear translocation in human keratinocytes and attenuated the increase of intracellular ROS levels upon PM-treatment . In addipression . Camellicription . In addicription .The potent antioxidant vitamin E was shown to rescue PM-induced damage in keratinocytes by partially restoring the expression of proteins that are important for proliferation, differentiation, and desmosomal integrity, among others . ResveraSargassum horneri was shown to have metal ion chelating properties by decreasing the concentration of PM metal traces  in keratinocytes in a dose-dependent manner [Alginate extracted from t manner . This let manner .Cornus officinalis fruit (EECF) [Astragali Radix or its main compound formononetin resulted in the protection against PM-induced decrease in keratinocyte proliferation and differentiation markers and blocked PM-induced apoptosis in a 2D and 3D skin model [Ethanol extract of t (EECF) , a ROS st (EECF) . A pre-tin model . Green tin model . Additioin model . Moreovein model . In turnin model . A slighin model .10-induced pro-inflammatory response [Ecklonia cava extract and its compounds dieckol [2′-Fucosyllactose (2FL) was tested in HaCaT and shown to have protective effects against PMresponse . Eckol, response . Treatmeresponse . Additio dieckol , fucoste dieckol , diphlor dieckol , afzelin dieckol , and eup dieckol . Ethyl 2 dieckol .Niacinamide, a compound that is often used in various cosmetics, was shown to protect keratinocytes from PM-induced oxidative stress . Other rNanoparticle technologies, such as 734THI nanoparticle powder , eupafolAlthough most of the studies were carried out using an active ingredient only, protective effects against PM were demonstrated also from finished formulations. For example, a novel water-in-oil emulsion with a lecithin-modified bentonite, applied topically on a HEE 3D model, prevented skin damage from urban dust and cedar pollen, as indicated by the decrease in MMP-1 and IL-8 protein secretion .2D skin models lack the barrier properties that 3D skin models have, and PM treatment can result in an overestimation of the real effect. Since the greater part of PM research on skin has been done using 2D models, it is interesting though challenging to translate the results into 3D models and even in vivo outcomes. In a recent study, the irritation potency of several compounds, has been compared in both 2D and 3D models . By screWhen using 3D skin models, it is recommended to apply PM topically, that is a closer to real-life condition compared to systemic treatment. Additionally, the chemical interactions of PM with the skin and the PM properties upon interaction with the skin should be further investigated to perform exposure experiments that have a higher physiological relevance. It is important to study the correlation between PM source and composition on the effects that it has on the skin.Most of the PM is coming from reference standards that can be either outdated  or coming from a certain geographical region, leading to a wrong generalization of the current state of airborne PM. An example is testing of diesel particles: due to the continuously improving filtering systems of diesel cars, the emitted particles for the modern filter-equipped diesel cars are producing far less PM compared to gasoline cars . AlthougPM is only one of the major hazardous elements of air pollution, and other elements, such as ozone and UV radiation, are important to investigate as well. Combined exposures of skin models, including several pollutants, would allow for a more relevant exposure scenario and give more insight into the interactions between the pollutants and the skin; hence synergistic damage of these pollutants is suggested , 274.Taken together, these future directions could contribute to create recommendations for regulations to reduce airborne PM and for the dermato-cosmetic industry to design novel solutions for improved skin protection against air pollution.The effects of airborne PM on skin have been intensively studied, but the majority is done in 2D skin models, especially in HaCaT cells and primary keratinocytes. Those cells are exposed to dispersed PM in cell culture media and thus reflect the real-life situation to a lesser extent, where the outer skin layer is consisting of the SC and exposed to air.The results summarized do suggest not to limit our investigations to the assessment of oxidative stress and inflammation through ROS production and pro-inflammatory protein expression and secretion, respectively, but also to include implications in the complete mitochondrial respiratory chain, skin integrity, skin barrier function, epidermal differentiation and proliferation to the assessment.Airborne PM has shown to affect several distinct cellular processes to a high degree, making it a very intriguing subject to study. Investigating these effects on a model that better resembles the complexity of human skin, a HSE or HEE model, or more advanced 3D skin models, will further reveal the involved cellular processes and effects on the health of human skin. By targeting these processes in both healthy and diseased skin, skin damage could be prevented and possibly cured by providing new solutions for the development of skincare products.


Introduction. Central pontine myelinolysis is characterized by the occurrence of acute demyelinating lesions of cells in the pons secondary to abrupt oscillations of serum osmolarity. Its exact incidence is not well defined, but studies show a prevalence of 0.25 to 0.5% in the general population, 2.5% in the intensive care unit, and up to 10% in patients with risk factors, such as chronic liver disease and hepatic transplantation, alcoholism, malnutrition, diuretic therapy, electrolyte imbalance, hypoglycemia, and hyperglycemia. Case Report. A 70-year-old white female with extranodal diffuse large B-cell non-Hodgkin's lymphoma , stage IVA, developed pontine myelinolysis secondary to hypovolemic acute hypernatremia, which occurred due to diarrhea caused by chemotherapy . Discussion. Pontine myelinolysis occurs most often due to the rapid correction of chronic hyponatremia. But here, we describe a case of the disease secondary to the occurrence of hypovolemic acute hypernatremia in a patient with a hematological malignancy under treatment, who was on chronic treatment with thiazide diuretics and who presented with other electrolyte disturbances as risk factors for the development of pontine myelinolysis. Central pontine myelinolysis (CPM) was first described in 1959 by Adams et al. as a disease that affected alcoholic and malnourished patients and was characterized by acute demyelinating lesion of pontine cells secondary to abrupt oscillations of serum osmolarity , 3.The exact incidence of the disease is not well defined, but studies show a prevalence of 0.25 to 0.5% in the general population, 2.5% in the intensive care unit, and up to 10% in patients undergoing liver transplantation –7. Some The main etiopathogenic mechanism of ODS is the rapid correction of hyponatremia, especially in the scenario of hyponatremia with chronic evolution. A low serum sodium concentration leads to serum hyposmolarity and the consequent passage of free water into the intracellular environment of the cells of the central nervous system, which promotes cellular edema. In cases of chronic evolution, cells adapt and lose osmoles  and free water as a compensatory mechanism. In the scenario of rapid correction of hyponatremia, there is an acute increase in serum osmolarity and consequent dehydration of cells of the central nervous system, leading to noninflammatory demyelinating lesions and apoptosis , 16–20.This case report describes the occurrence of CPM related to hypovolemic acute hypernatremia secondary to diarrhea. It is a rare cause of ODS, which is often related to the correction of hyponatremia.A 70-year-old white female was diagnosed in June 2017 with extranodal diffuse large B-cell non-Hodgkin's lymphoma , stage IVA. Chemotherapy was initiated with the R–CHOP regimen  in the same month, performing the first two cycles using a Port-a-Cath catheter, without complications and with a substantial reduction of the left anterior thoracic mass. Comorbidities included hypertension for 3 years, hemorrhoidal disease (conservative treatment), bilateral knee arthrosis with total left knee arthroplasty 3 years earlier, and esophageal hiatus hernia (conservative treatment). Associated with this was a history of an imagining finding of an asymptomatic pulmonary thromboembolism during the staging of the hematological malignancy and history of poliomyelitis at 8 years of age with motor sequelae in the lower limbs . The patient had no history of smoking or drinking, and medications consisted in chronic use of chlortalidone 25 mg once daily, omeprazole 40 mg once daily, rivaroxaban 20 mg once daily, bromopride 10 mg once daily, and sulfamethoxazole/trimethoprim 800/160 mg 3x a week.3 (20% neutrophils), platelets 8000 cells/mm3, creatinine 2.6 mg/dL, urea 194 mg/dL, fasting glucose 82 mg/dL, sodium 164 mEq/L, magnesium 1.4 mEq/L, phosphorus 2.2 mg/dL, potassium 2.2 mEq/L, AST 23 IU/L, ALT 20 IU/L, albumin 2.1 g/dL, bilirubins normal, coagulation tests normal, arterial lactate 31 mg/L, pH 7.21, HCO3 16 mEq/L, chest X-ray normal, urinalysis normal, urine culture negative, and blood cultures negative.The patient underwent the third cycle of chemotherapy in September 2017, and 10 days later, she began to suffer from general malaise, hyporexia, prostration, lesions of the oral mucosa, acute voluminous diarrhea without pathological products, and fever (38.3°C). She was admitted to the intensive care unit of Hospital Renascentista with a diagnosis of high-risk febrile neutropenia with no defined infectious focus (MASCC score of 11 points), severe mucositis of the gastrointestinal tract with considerable oral lesions and diarrhea, prerenal acute renal failure, and severe pancytopenia after chemotherapy. Physical examination showed that the patient was dehydrated (3/4+), prostrate, less communicative and febrile (38.3°C), had poor peripheral perfusion with blood pressure 80/50 mmHg and heart rate 110 bpm, and had substantial oral mucositis, with no other changes in the general and neurological examination. Laboratory tests showed the following results: hemoglobin 6.7 g/dL, leukocytes 270 cells/mmImmediate empirical antibiotic treatment was initiated with piperacillin-tazobactam and vancomycin due to high-risk febrile neutropenia with multiple risk factors for Gram-positive infection , in addition to hemodynamic support of sepsis, volume resuscitation, transfusion support, and correction of water-electrolyte imbalance. She evolved with hemodynamic improvement, resolution of fever, improvement of renal function, and improvement of hypomagnesemia, hypokalemia, and hypophosphatemia, with progressive and slow sodium correction (<0.5 mEq/L/hour) until normalization after several days. However, considerable diarrhea and pancytopenia followed, which began to improve after two weeks of hospitalization. From a neurological point of view, despite clinical and laboratory improvement, she maintained prostration and developed a fluctuating lowered level of consciousness, grade I paresis of upper limbs and grade III paresis of lower limbs, mild dysarthria, upper and lower limb hyporeflexia, no signs of pyramidal release, and normal brain stem reflexes and ocular motricity. A complementary investigation was performed with cranial computed tomography, which showed only a previous small left lacunar stroke ; electroThe patient continued with clinical and laboratory improvement, with complete resolution of pancytopenia, water-electrolyte imbalance, renal dysfunction, and diarrhea. She had a slow neurological improvement and was discharged with normal level of consciousness and progressive recovery of motor deficits. After two years, her neurological status was the same as before the central pontine myelinolysis event, and her cranial computed tomography revealed a hypoattenuating focal area in the central region of the pons .The clinical picture of CPM includes a variety of neurological manifestations, which include flaccid quadriparesis with subsequent evolution to the spastic phase, dysarthria and dysphagia , oculomotor and pupillary changes, and lowered level of consciousness. In some situations, locked-in syndrome may occur. These neurological changes are due to the involvement of the pontine structures to different degrees, particularly the base of the pons  and tegmentum of the pons . The symThe diagnosis of CPM should be considered in cases of acute onset of neurological symptoms related to sodium imbalance, especially rapid correction of hyponatremia (>0.5 mEq/L/hour), where it is confirmed by imaging of the central nervous system, with prominence for magnetic resonance, whose findings are bilateral hypersignal in the central region of the pons in the T2 and FLAIR sequences and hyposignal in the T1 sequence . RadioloThe approach in CPM consists mainly in preventive measures based on the correct handling of the correction of sodium imbalance since there are no well-established treatments. Some approaches have been studied in recent years, with emphasis on immunoglobulin therapy or plasmapheresis, which possibly promote remyelinization and reduction of myelotoxicity. Rodriguez et al. described 3 cases of pontine myelinolysis treated with immunoglobulin and observed clinical improvement after treatment . Also, SPontine myelinolysis occurs most often due to rapid correction of chronic hyponatremia . HoweverWe describe here a case of CPM related to the occurrence of hypovolemic acute hypernatremia secondary to diarrhea in a patient with a hematological malignancy under treatment, presenting with the risk factors such as chronic use of a thiazide diuretic and the presence of other electrolyte disturbances .


We also evaluated Hg concentrations and maternal serum metallothionein (MT2) concentrations in relation to the number of dental amalgam fillings, and MT2A-5A/G (rs28366003) polymorphism. The levels of Hg and Se in maternal hair and blood/serum, placenta and cord blood/serum increased in relation to increasing fish consumption with the highest values in subjects from the coast. The concentrations of each element and between elements correlated across the matrices. Increasing amalgam number correlated linearly with increased Hg levels in maternal and cord serum and was not associated with serum MT2. No association of MT2A-5A/G polymorphism and Hg or Se levels were found. The results confirmed higher fish consumption in coastal vs. continental Croatia and increases of both Hg and Se related to fish consumption in all analyzed samples. Increased blood Hg reflected the predominant MeHg share from seafood, while increased serum Hg matched exposure from dental amalgams.The main source of mercury (Hg) exposure in the general population is fish. Another possible source is dental amalgam. Here, we compare the levels of Hg and selenium (Se) in samples of maternal and fetal origin collected shortly after childbirth of healthy postpartum women in the coastal ( Mercury (Hg) is a pervasive pollutant and toxicant of major concern for humans and wildlife. It is released in the environment via both natural and anthropogenic sources, and primary anthropogenic emissions (without re-emission processes) greatly exceed natural geogenic sources ,9,10,11.0) absorbed by inhalation after release from dental amalgam fillings, and exposure to ethylmercury through thiomersal-containing vaccines [Virtually everyone is exposed to Hg and its different chemical forms. Three main forms and sources of public concern are: exposure to MeHg by ingestion of fish, mercury vapor  Hg factors .A major factor underlying the biochemical properties of Hg and its compounds, and causing their biological effects and interactions in the body, is the great predilection of ionic species of Hg to bond to reduced sulfur atoms, especially on small endogenous molecules containing a thiol  group, such as glutathione, homocysteine, metallothionein (MT), and also albumin. Its binding affinity to thiol-containing pharmacological agents is used for chelation therapy to remove Hg from endogenous ligands by forming new –SH-Hg complexes. Different chemical forms of Hg also have different toxicokinetics in the body, all of which induce toxic effects, depending on the form of Hg present at the time of exposure, as well as the duration, level, and route of exposure. The primary target for toxicity of inorganic Hg is the kidney where it is taken up, accumulated, and its toxicity expressed. Although organic Hg compounds can also have nephrotoxic effects, they have more diffuse systemic distribution and affect other organs, including the nervous, hematopoietic, cardiovascular, immune, respiratory, gastrointestinal, and reproductive systems ,18,25,260 are predominantly transferred to the fetus who may already be affected in utero [During pregnancy, maternal Hg is partially accumulated in the placenta, and MeHg and Hgin utero ,31,32,33in utero ,35,36.MT2A-5A/G (rs28366003) polymorphism.More epidemiological studies are needed to assess special features of Hg exposure during pregnancy, especially by fish consumption, and to determine the potentially protective effects of nutrients closely interrelated with MeHg, such as Se, together with individual genetic susceptibility to Hg toxicity. The aim of this study, conducted as part of a broader cross-sectional population study in healthy postpartum women in Croatia, is to determine Hg and Se concentrations in samples of maternal and fetal origin  collected shortly after childbirth, and to compare these biomarkers between study participants from the coastal and continental areas with presumed different seafood/fish consumption and related Hg exposure. Irrespective of the geographic area of residence, we evaluated Hg concentrations in relation to the number of dental amalgam fillings as an additional source of Hg exposure, maternal serum metallothionein (MT2) concentrations  and n = 185 participants) and coastal—the city of Zadar and its surroundings in Zadar County located on the eastern Adriatic coast (n = 96 participants). Inclusion criteria for this study, as part of a wider study conducted within a national research project, were a term vaginal delivery (between 37 and 42 weeks of pregnancy) in the maternity ward and absence of any chronic illness, major complication during pregnancy or twin pregnancy. Study participants fulfilling these criteria were informed about the research scope, methods, and biological samples to be collected and included in the study after the endorsement of informed consent forms. The research protocol for studying exposure to the main toxic metals, cadmium (Cd), lead (Pb), and Hg, and their effects in pregnancy and postnatal period within the entire research project was reviewed and approved by the ethics committees of three collaborating institutions : Merkur University Hospital in Zagreb, Zadar County General Hospital, and the Institute for Medical Research and Occupational Health in Zagreb where the study was conducted. The ethical approvals and consent of study participants are still valid and encompass all future analyses. Accordingly, in 2015 an addendum to the previous ethical approval was obtained from the Institute for Medical Research and Occupational Health in Zagreb prior to starting additional analyses of the specific single nucleotide gene polymorphism and metallothionein (MT2) concentrations in previously collected biological samples of maternal origin within this research project.This study was carried out using personal data and selected biomarkers obtained in two cohorts of mother–infant pairs recruited from two geographic areas of residence in Croatia : continest and 5th min, a vitality index  that provides an accepted and convenient method for reporting the clinical status of the newborn infant immediately after birth based on a rating of 0, 1, or 2 for each of the five characteristics: A—appearance (skin color), P—pulse (heart rate), G—grimace (response to stimulation of the sole of the foot), A—activity (muscle tone), and R—respiration (respiratory effort), with 10 being a perfect score); maternal dietary history ; smoking habit ; and number of dental amalgam fillings . For the purpose of this study, we focused on the data on the self-reported frequency of seafood/fish consumption (expressed per week) and number of amalgam fillings as the main sources of Hg exposure, and the related parameters.Data on sociodemographic characteristics, medical history, mineral-vitamin supplement intake, number of amalgam fillings, and dietary habits of postpartum women focused on seafood consumption, were collected by a questionnaire as described elsewhere ,38 and fBased on self-reported data on seafood consumption, including fresh/frozen and tinned fish and shellfish, subjects were categorized into four subgroups: 0—no consumption; 1—consumption ≤1 per week; 2—consumption >1–2 per week; 3—consumption >2 per week. Based on the number of amalgam fillings, participants were categorized into four subgroups: 0—no amalgam fillings; 1–2—one or two amalgam fillings; 3–4—three or four amalgams fillings; ≥5—five or more amalgam fillings. According to MT2A-5A/G polymorphism, the participants were assigned into two subgroups: wild-type (AA genotype) and G allele carriers (AG/GG genotype).® Trace Element, Becton Dickinson, New Jersey, NJ, USA) with K2-EDTA anticoagulant (4 mL) and without anticoagulant (6 mL). Whole placentas were placed in marked zip-lock polyethylene bags. Hair samples were collected by cutting with clean scissors from the nape of the neck as close as possible to the root. Blood serum was separated by centrifugation at +4 °C and 3000 rpm for 20 min  and decanted into clean polyethylene tubes. All collected samples were transported in cooler bags to the laboratories of the Institute of Medical Research and Occupational Health in Zagreb where placentas were sampled, as described earlier [Biological samples collected in the period between 2008 and 2010 in maternity wards included: maternal blood and hair, placenta, and umbilical cord blood. Immediately after childbirth, maternal and cord blood were sampled in two vacutainer tubes  TX-100 [Blood and serum samples were diluted before analysis with a solution containing 0.7 mM NH) TX-100 . Dilution = 71) than originally collected from the participants of the continental area because part of the samples was used for nicotine analysis related to maternal cigarette smoking and its association with Cd exposure biomarkers [The number of hair samples available for element analysis was lower  system combined with high efficiency particulate air (HEPA) filters.Concentrations of Hg and Se in maternal hair, blood and serum, placenta and cord blood, and serum were determined by inductively coupled plasma–mass spectrometer (ICP-MS) Agilent 7500cx . The ICP-MS optimized working conditions are shown in ® Whole Blood Controls  and Serum Controls (Level I and II) , SeronormTM Trace Elements Whole Blood (Level I and II) and Serum (Level I and II) , bovine liver BCR 185R, pig kidney BCR 186R, mussel tissue BCR 278R , human hair IAEA-086 , and human hair CRM 13  , according to the method provided by the manufacturer, and stored at −20 °C until used. BioSpec-nano  was used to check the concentration and purity of the isolated DNA. To amplify the signal of the fragment 241-bp polymerase chain reaction (PCR), GeneAmp PCR System 2700  was used with the following primers:MT2A-5A/G was genotyped by the method of restriction fragment length polymorphism (RFLP) by incubation of the PCR products at 37 °C for 1 h and 45 min with BsgI restriction enzyme  as described in our previously published paper [Single nucleotide polymorphism (SNP) of ed paper . Digesten = 94) using enzyme-linked immunosorbent assay (ELISA) kit SEB868Hu and the double-antibody sandwich method as recommended by the manufacturer . TECAN Infinite F50  was used to measure MT2 absorbance at 450 nm.The levels of metallothionein (MT2) in serum were determined in a part of the study participants . The Kolmogorov–Smirnov test was used to test the normality of variables and logarithmic (ln) transformation was used to normalize variables before statistical analysis. Differences between the groups were tested by Mann–Whitney U test or by one-way ANOVA with post hoc Bonferroni test in the case of three or more subgroups . The relationships between Hg and Se in all measured samples, maternal seafood consumption, amalgam fillings, and maternal serum MT2 were analyzed by Spearman’s rank correlation. Forward stepwise multiple linear regression analysis was used to evaluate the effects of maternal seafood consumption and amalgam fillings on Hg levels in maternal blood, serum and hair, placenta and cord blood, and serum. The independent variables included in the regression models were residence area, seafood consumption per week, maternal amalgam fillings , maternal age, education, parity, and maternal weight gain during pregnancy. For the final model, only variables at p < 0.05 were included. The software package TIBCO Statistica™, version 13.5.0.17  was used for statistical analysis and GraphPad Prism 7  for graphic illustration of results.Data were expressed as the median (25–75% interquartile range) and/or mean with standard deviation (SD). The values of elements’ concentration below the limit of detection (LOD) were substituted with the LOD/2 value. Statistical significance was set at 5% (n = 185) and coastal area (n = 96), with the available data on SNP MT2A-5A/G frequency (n = 268) and the number of dental amalgams (n = 208) in participants from both study areas. Average maternal age was 29 years. The study participants matched in parity, body weight and height before pregnancy, average weight gained during pregnancy, gestation week at delivery, birth weight and length, median APGAR vitality scores in the 1st and 5th min, and trimmed fresh placental weight. Regarding seafood consumption, most self-declared data actually referred to fresh and frozen marine fish, and only partially to tinned fish. There were marked differences between the studied cohorts; in the continental area 54% of subjects self-reported that they ate fish less than once a week and 11% ate no fish at all, whereas among subjects from the coastal area more than 50% of participants reported fish consumption at least once or more than twice a week. With regard to determined genotype frequencies of MT2A-5A/G polymorphism, among 268 participants, 94.4% subjects were typical homozygote (AA) and 5.6% subjects were G-allele carriers. Among the latter, 5.2% subjects were heterozygote (AG) and 0.4% atypical homozygote (GG). The allele frequencies were in accordance with the Hardy–Weinberg equilibrium . Regarding the presence of dental amalgam fillings, 18% of participants had no dental amalgams and 82% of participants had at least one amalgam filling; specifically, 30% had one to two, 22% had three to four, and 30% had five or more dental amalgams.Significantly higher concentrations of both Hg and Se were found in all measured samples of the maternal-placental-fetal unit in the coastal vs. continental areas. These increases in Hg concentrations were 2.3 to 3.5-fold, while the ratio between Se concentrations in the two study groups was up to 1.2 .The concentrations of Hg in maternal hair and blood, placenta, and cord blood in the study participants from the continental and coastal area with regard to the frequency of seafood (mainly fish) consumption per week based on self-reporting are presented in p < 0.0001) with Hg levels that were higher 3–7-fold in maternal hair, 7–8-fold in maternal blood, 4–6-fold in the placenta, and >10-fold in cord blood.In both geographic areas of residence, all Hg biomarkers increased with increasing maternal seafood consumption. In participants who consumed seafood at least once per week compared to those who ate no seafood, Hg levels were 1.5-fold higher in maternal hair, 2-fold in maternal blood, 1.5-fold in the placenta, and 2–3-fold in cord blood. In subjects who consumed seafood twice per week vs. subjects who ate no seafood, these differences were highly significant  consumption per week based on self-reporting are presented in In all study participants from both geographic areas, no differences were found in the concentrations of MT2 in maternal serum between the subgroups in relation to the frequency of seafood (fish) consumption . No diffIn subjects with dental amalgams (one or more) compared to subjects without dental amalgams , Hg concWe found no difference between the subgroups of study participants related to the number of dental amalgams in either concentrations of Hg in maternal and cord serum  or MT2 cs) between maternal age, seafood (fish) consumption, number of amalgam fillings, maternal serum MT2, and the concentrations of Hg and Se in the measured samples are presented in s = 0.62–0.86) were found between Hg levels in all measured samples of maternal and fetal origin and for Se in maternal blood and serum (ρs = 0.8), whereas correlations between Se levels in other measured samples were weak (ρs = 0.18–0.55). In all analyzed samples, the levels of Hg and Se were significantly positively correlated (ρs = 0.19–0.52). MT2 concentration was inversely correlated with Se in maternal blood (ρs = −0.28), while maternal amalgam number (as represented in s = 0.16). Maternal age correlated with seafood intake, Hg in maternal and umbilical cord blood and serum, Hg in placenta, and Se in maternal blood and serum (ρs = 0.12–0.2). These results, together with the significant correlations between Hg biomarkers and residence area, education, parity, and weight gain (results not shown), suggest a complex association among the variables. Each of these studied variables was therefore considered as potentially explanatory/confounding in their influence on Hg levels.Spearman’s correlation coefficients , it has a very diverse topography; from the Mediterranean to Central European, mountainous to flat, coastal to continental. It ranks as one of the top five European countries with regard to biodiversity [This paper presents data on Hg and Se levels measured in samples collected shortly after childbirth that represent concentrations in three compartments of the maternal-placental-fetal unit, comprising maternal hair, blood and serum, placental tissue, and umbilical cord blood and serum. The results were partly already published  and hereiversity . There iiversity .During its formation, hair sequesters MeHg by binding to –SH groups in keratin, and after exposure to MeHg, hair Hg consists of just over 80% MeHg. In group studies, hair specimens are regarded as accurate measures for the evaluation of exposure to Hg, especially in the assessment of MeHg exposure by fish consumption ,16,47,48Our results confirm an association of seafood/fish consumption and Hg concentration in maternal hair ,42,50,51In comparison with population groups in North America having traditionally high seafood/fish intake, participants from the coastal area in this study who consumed seafood more than twice per week had three-fold lower Hg levels in hair compared to Hg levels in the maternal hair of, for example, Northern Québec Inuit postpartum women in the third trimester . HoweverAs in hair, MeHg in blood is also the main contributor to total Hg levels and the concentrations of Hg in blood show a direct relationship with hair Hg levels. The typical hair-to-blood ratio is estimated at 250, corresponding to the overall average of values for individuals reported in published studies from 140–370, although large variations exist, especially in persons with infrequent fish consumption ,12,16,28p < 0.02) in the ratios of Hg concentrations in cord blood to maternal blood that were 1.6 in coastal and 1.2 in continental mother–infant pairs. Based on the meta-analysis, the ratios between cord and maternal blood concentrations of MeHg and inorganic Hg are estimated at 1.89 and 1.01 [Exposure to MeHg during prenatal life, especially during critical periods of central nervous system development, may cause serious and irreversible impairments . Organicand 1.01 ; hence cand 1.01 ,28,58, wand 1.01 ,69; howeand 1.01 ). Studieand 1.01 ,71. In oThe strong positive correlation (ρ > 0.8) between Hg levels in maternal blood and cord blood is in line with previous findings ,63,72. FThe literature data on Hg concentrations in the placenta are scarcer than on the concentrations of two other main toxic metals, Cd and Pb. The reported Hg levels in the placenta under conditions of low to moderate environmental exposure vary from 2 to 13 μg/kg with an average of 8 μg Hg/kg, based on wet weight ,31. In t0 released into the mouth and saliva during chewing and placement and/or removal. The estimated daily absorbance of Hg from amalgam fillings ranges from 3–17 µg [0 depends on the number, surface areas, and chemical characteristics of the amalgam filling(s), personal eating habits, food texture, and personal physiological factors. To this end, amalgam fillings have been the subject of an ongoing dispute for centuries, known as “amalgam wars”, due to concern of health effects of released Hg [Amalgam fillings have been used for dental restoration for over 150 years and they are still used in many countries, including Croatia, due to their advantages, such as simple preparation, hardness, effectiveness, duration, and low cost. Apart from an unattractive appearance, the major disadvantage of dental amalgam is the potential risk of exposure to Hg 3–17 µg , whereaseased Hg ,80,81,82eased Hg ,84,85,86eased Hg ,78,87. Aeased Hg . Althougeased Hg , cord bleased Hg ,42, and eased Hg , or no aeased Hg . Our reseased Hg ,47,86,89External factors that influence vulnerability to the toxic effects of Hg are gender, age, physiological status, nutritional status, and the intake of other foods or nutrients that might influence the absorption, uptake, distribution, and metabolism of MeHg ,90. GeneWe are well aware that both our research approach  and the study sample size (less than 300 participants) are far too small to draw any firm conclusions on the influence of this particular MT2 polymorphism on Hg levels in the measured biological samples of maternal and fetal origins related to the main sources of Hg exposure, seafood, and dental amalgams. This, together with the broader assessment of MT2 levels in the measured matrices, is recognized as the major weakness of this study.Women of child-bearing age should consume fish since a fish-rich diet is beneficial for the health of both mother and developing offspring, and supplies nutrients including essential elements and omega-3 fatty acids. For example, docosahexaenoic acid (DHA) levels were found to decline during pregnancy and lactation, but the higher the intake of seafood and eicosapentaenoic acid (EPA)/DHA-supplements, the higher the DHA status in the maternal circulation . HoweverThe results reported here corroborate previous studies that showed no association between birth weight or birth length and maternal seafood consumption ,101,102 Concentrations of Hg in commercial fish species, including European pilchard and hake, as commonly consumed species by the Croatian population, are reported to range between 0.017–0.130 mg/kg wet weight of muscle fish tissue ,107. Accth week of pregnancy who consumed fish one to two times per week [A number of studies have reported that maternal serum concentrations of Se decrease with the progress of pregnancy due to increased Se transfer to the developing fetus, especially during the third trimester when Se is retained in the fetal liver ,115,116.per week . These lper week . In our per week ,118,119 per week ,121, whiper week , Canada per week , and theper week . Differeper week ,124. We per week . As fishper week ,83,125, per week . In contper week . The complex relationship between Se intake, biomarkers of its status in the body, and risk of disease has not yet been fully elucidated. Appropriate biomarkers, together with the impact of gender and genotype, especially polymorphisms, should be studied further and considered when updating the current dietary recommendations to aid preventive public health measures and therapeutic clinical practice . There iThe study of mother–infant pair cohorts in continental and coastal areas of Croatia , that differ in traditional dietary habits of fish consumption, shows that maternal seafood/fish consumption is associated with increased levels of Hg and Se in maternal hair and blood, umbilical cord blood, and also in placenta, as a tissue sample generally underrepresented as a matrix with determined Hg concentrations. Coastal participants with a cultural tradition of a fish-rich diet were observed to have higher levels of both trace elements, toxic Hg and essential Se. New evidence is related to the lack of association between either maternal serum MT2 and Hg exposure from dental amalgams or MT2A-5A/G (rs28366003) polymorphism and the levels of Hg and Se in the measured matrices. These results confirm that Hg levels in maternal hair and blood are associated with seafood consumption and mainly reflect MeHg exposure, and this is additional evidence of the association of Hg levels in serum and the number of dental amalgams, reflecting inorganic Hg exposure. The results of this study provide new and original data on Hg exposure in the vulnerable population groups of mother–infant pairs in this part of the world. Future studies of Hg and Se effects and their interactions related to fish consumption should include a detailed seafood consumption questionnaire and collection of detailed data on intake frequency, serving sizes, and consumed fish species, together with a larger number of study participants  to assess the impact of the related gene polymorphism.


Purpose: To analyze human and bacteria proteomic profiles in bile, exposed to a tumor vs. non-tumor microenvironment, in order to identify differences between these conditions, which may contribute to a better understanding of pancreatic carcinogenesis.Patients and Methods: Using liquid chromatography and mass spectrometry, human and bacterial proteomic profiles of a total of 20 bile samples  patients, and 13 from pancreatic head ductal adenocarcinoma (PDAC) patients) that were collected during surgery and taken directly from the gallbladder, were compared. g:Profiler and KEGG (Kyoto Encyclopedia of Genes and Genomes) Mapper Reconstruct Pathway were used as the main comparative platform focusing on over-represented biological pathways among human proteins and interaction pathways among bacterial proteins.Results: Three bacterial infection pathways were over-represented in the human PDAC group of proteins. IL-8 is the only human protein that coincides in the three pathways and this protein is only present in the PDAC group. Quantitative and qualitative differences in bacterial proteins suggest a dysbiotic microenvironment in the PDAC group, supported by significant participation of antibiotic biosynthesis enzymes. Prokaryotes interaction signaling pathways highlight the presence of zeatin in the GS group and surfactin in the PDAC group, the former in the metabolism of terpenoids and polyketides, and the latter in both metabolisms of terpenoids, polyketides and quorum sensing. Based on our findings, we propose a bacterial-induced carcinogenesis model for the biliary tract.Conclusion: To the best of our knowledge this is the first study with the aim of comparing human and bacterial bile proteins in a tumor vs. non-tumor microenvironment. We proposed a new carcinogenesis model for the biliary tract based on bile metaproteomic findings. Our results suggest that bacteria may be key players in biliary tract carcinogenesis, in a long-lasting dysbiotic and epithelially harmful microenvironment, in which specific bacterial species' biofilm formation is of utmost importance. Our finding should be further explored in future using in vitro and in vivo investigations. Pancreatic ductal adenocarcinoma (PDAC) is a malignant and highly lethal neoplasm of unknown etiology and is usually diagnosed at advanced stages . The curBacteria have been associated with benign and malignant disease, and bacterial carcinogenesis is a process still being characterized in detail. The knowledge from such study may be the starting point to drive clinical interventions focused on cancer prevention. The carcinogenesis associated with viruses is based on the integration of the viral genome into the host DNA  and has been extensively studied and characterized . ConversThe biliary tract including intra-pancreatic bile ducts, is a semi-closed duct system possessing its own microbiota –15, lineBile is stored and concentrated in the gallbladder, which is a clean reservoir where this biological fluid can be extracted for protein analysis . In reseWe analyzed samples of human bile taken directly from the gallbladder, and not by ERCP, exposed to a pancreatic tumor vs. non-tumor microenvironment. The aim of the study, once samples were analyzed by mass spectrometry, was to find meaningful biological information through pathway inference analysis of the identified human and bacterial proteins. Biological pathway analysis was initially performed using the g:Profiler platform to compare and generate a complete panorama of the gene-protein sets being analyzed, including over-represented KEGG biological pathways . Then, wThe Institutional Human Ethics Committee at CES University and Clinic approved this study, and patients must give informed consent. Samples were de-identified before performing proteomic analysis. A surgical pathologist collected a total of 20 gallbladder bile samples; seven from patients with gallstones (GS), and 13 from patients with PDAC arising from the head of the pancreas. All patients were Colombians, and residents of Medellín (Colombia). For GS patients, bile was obtained in the operating room immediately after laparoscopic extraction of the gallbladder, puncturing the gallbladder fundus with a syringe, and aspirating at least 5 mL of bile. For PDAC patients, bile was similarly collected, by aspirating bile with a syringe from the gallbladder pancreatoduodenectomy specimens were sent to the pathology lab for a cryosection margin report. Immediately after collection, bile samples were transported on ice, aliquoted, and stored at −80°C until further analysis. Patients with a clinical history of previous malignant neoplasms, chemotherapy, prior biliary tract surgery or biliary stent placement, HIV, pregnancy, chronic pancreatitis, choledocholithiasis, cystic fibrosis, hepatolithiasis, primary biliary cholangitis, liver cirrhosis, primary sclerosing cholangitis, or acute cholecystitis were excluded from this study.Bile samples were thawed at room temperature and processed as previously described with slight modifications . BrieflyProteomic analysis was performed by Creative Proteomics , briefly, the techniques used are described as follows:Total proteins were precipitated from the protein solution using methanol and chloroform. Approximately 10 μg of total protein was dissolved in 6 M urea aqueous solution and was denatured with 10 mM DL-dithiothreitol, incubated at 56°C for 1 h, followed by alkylation with 50 mM iodoacetamide, and incubated for 60 min at room temperature, protected from light. Next, 500 mM ammonium bicarbonate (ABC) was added to the solution to make a final concentration of 50 mM ABC with a pH of 7.8. Promega Trypsin was added to the protein solution for digestion at 37°C for 15 h. The generated peptides were further purified with the C18 SPE column (Thermo Scientific) to remove salt. Samples were dried in a vacufuge and stored at −20°C until use.An Easy-nLC1000  coupled to a 100 μm × 10 cm in-house made column packed with a reversed-phase ReproSil-Pur C18-AQ resin  was used. A sample volume of 5 μL was loaded, with a total flow rate of 600 nL/min, and a mobile phase of A: 0.1% formic acid in water; and B: 0.1% formic acid in acetonitrile. The analytical separation was run using a gradient: from 6 to 9% B for 15 min, from 9 to 14% B for 20 min, from 14 to 30% B for 60 min, from 30 to 40% B for 15 min and from 40 to 95% B for 3 min, eluting with 95% B for 7 min.An Orbitrap Q Exactive™ mass spectrometer  set on a spray voltage of 2.2 kV and a capillary temperature of 270°C was used. Mass spectrometry resolution was set to 70,000 at 400 m/z and precursor m/z range: between 300.0 and 1800.0. The production scan range starts from m/z 100, activated by collision-induced dissociation (CID), and an isolation width of 3.00. The raw files were analyzed and searched against the human protein database from Uniprot using Maxquant (1.5.6.5). The parameters were set as follows: the protein modifications were carbamidomethylation (C) (fixed), oxidation (M) (variable); the enzyme specificity was set to trypsin; the maximum missed cleavages was set to 2; the precursor ion mass tolerance was set to 10 ppm, and MS/MS tolerance was 0.6 Da.Peptide-protein analysis was performed at ICMT-CES University. Contaminants, albumin, hemoglobin related peptides, and peptides with zero intensity were eliminated from the full human and bacteria list of peptides-proteins. The identifiers of protein were standardized, missing gene names were manually completed, and protein taxonomy was verified.t-test, considering a logarithmic change of 2.5 and adjusting the false discovery rate to 0.42% . The biological validity of imputing non-existent values for non-observed proteins, in order to compare the exclusive groups of proteins, was explored. However we chose to perform the analysis based only on observed values in the two groups, GS and PDAC (shared proteins).Then, the full list of shared proteins was adapted to meet the requirements of the Prostar platform online version 1.18.1 , seekinghttp://www.uniprot.org/, UniProt release 2019_10). Then, in order to provide mechanistic insights into the biologically integrated function, Uniprot-standardized human protein lists of entries for each group were analyzed in the g:Profiler web page (https://biit.cs.ut.ee/gprofiler/gost) , were used for further analysis in the KEGG Mapper Reconstruct Pathway. KEGG identifiers were obtained from the Uniprot FASTA file of the total, exclusive, and differentially abundant protein list, through BlastKoala  (https://www.genome.jp/kegg/tool/map_pathway.html)   . g:Profitkoala/) . KEGG Maay.html) . On the lt.org/) , by perflt.org/)  with BonThe biological context was analyzed as a whole for the total protein groups by correlating findings with the specific proteins identified for each condition. Many biological pathways were enriched over the Bonferroni p-adjusted value threshold in the total protein groups, but just three of them were related to bacterial infection .For bacterial proteins, the same protocol for contaminant elimination, quality control, and differential abundance analysis was performed as for human proteins. We did not use g:Profiler for bacterial protein analysis because this platform is not conceived for multi-species analysis. The bacterial protein lists of total and exclusive proteins from GS and PDAC patients were also included in the retrieve ID/mapping module of the Universal Protein consortium resource. KEGG identifiers were obtained using BlastKoala from the Uniprot FASTA files, and we focused our attention on prokaryote interaction signaling pathways during the analysis in KEGG Mapper Reconstruct Pathway .A total of 20 bile samples extracted from gallbladders were analyzed, seven of which were taken from patients with GS (mean age of 48 years) , and 13 After excluding peptides that were unassociated with any known proteins, a total of 10,834 human peptides were identified with a mean of 542 peptides per sample, 8,877 peptides in the GS group and 7,413 in the PDAC group. Peptides were associated with a total of 2,198 human proteins, 1,837 proteins in the GS group, and 1,932 proteins in the PDAC group. Upon comparison, a total of 1,571 proteins were common to both groups, while 266 proteins were exclusively found in the GS group, and 361 proteins in the PDAC group . For bacThe g:Profiler platform was used for the over-representation analysis in KEGG signaling pathways. Analyzing the total list of proteins, the platform identified from the 1,837 proteins in the GS group 1,832 (99.7%) and from 1,932 in the PDAC group 1,929 (99.8%). Regarding exclusive and differentially abundant proteins, the platform identified 100% of proteins in the GS and PDAC groups. In the total protein lists, we found five over-represented pathways in the GS group and seven in the PDAC group , and in The over-represented pathways were analyzed in KEGG Mapper Reconstruct Pathway focusing our attention in the three g:Profiler over-represented pathways in the PDAC total protein group related to the bacterial infections Shigellosis, Pertussis, and Legionellosis. Analyzing the list of proteins in these three pathways , it is nN = 3), bile from the gallbladders of PDAC patients (N = 11) and common biliary brush over the tumor from PDAC patients (N = 11) as samples. The results of the analysis show two statistically significant over-represented pathways, pyrimidine deoxyribonucleotide biosynthesis and isoprene biosynthesis (unpublished results).Proportional participation in some taxonomic levels of the imputed protein species is summarized in Upon comparison of the GS and PDAC total protein groups, we found qualitative and quantitative differences regarding quorum sensing, biofilm formation, antibiotic synthesis (biosynthesis of other secondary metabolites) and metabolism of terpenoids and polyketides . RegardiTo the best of our knowledge, this is the first study to compare human and bacterial proteins, using a metaproteomic approach, bile samples exposed to a tumor vs. non-tumor environment in human PDAC and GS patients, respectively. The characterization of a single species protein profile is known as proteomics, while the characterization of a multi-species protein profile is known as metaproteomics . The metFinding meaningful biological information from omics' science data sets has been one of the major challenges of science in recent years . The relIL-8 was identified as a common protein in the three g:Profiler over-represented signaling pathways in PDAC human total proteins, associated with bacterial infections. IL-8 is a human chemotactic interleukin of the C-X-C family also known as CXCL8, originally discovered in macrophages , but alsHigh levels of IL-8 are described as poor outcome predictors in many malignant neoplasms, including PDAC. The cellular endpoint effects induced by the IL-8 CXCR1/CXCR2 axis, in normal epithelial cells, tumor cells or other cells in the tumor microenvironment, promote cellular survival, proliferation, angiogenesis, and a stem cell phenotype , 44. ConF. nucleatum and A. naeslundii, and not the planktonic form, stimulates the synthesis of IL-8 by human squamous epithelial cells  . BacteriThe change in bacteria association, from free-living or planktonic to biofilm formation, relies upon many genetic factors and local conditions . BiofilmWe hypothesized that there is no such thing as a dysbiotic microbiota, regarding the presence or absence of certain bacterial species. A dysbiotic microbiota is a haphazard composition of bacterial species with products harmful to bacteria and epithelial cells, specific to a particular individual vis-à-vis microbiota-modifying factors. Based on our metaproteomic findings regarding bacterial and human proteins, and its associations, we proposed a biliary tract carcinogenesis model. We are aware of that our proteomic analysis is a snapshot of established PDAC cases, and the propose bacteria-induced carcinogenesis model for the biliary tract is still speculative, not validated, and therefore must be interpreted with caution . The modin vitro and in vivo investigations. Microbiota analysis and dysbiosis alone have not answered the question of the bacteria-induced pathology model. Future research may concentrate on improving the throughput of protein identification from complex biological fluids like bile and consider a combined microbiota and metaproteomic approach to analyze bacterial communities and bacterial and human proteins. It is necessary to start thinking of a change in the dysbiosis paradigm, as we hypothesized dysbiosis is not a specific bacterial composition but rather a harmful protein microenvironment that can be created by several “dysbiotic” microbiotas.There are no clear indications for sample size calculations in proteomics research, and the results from a specific protein extraction method, imputation pipeline, and bioinformatic analysis must be validated in further The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020151.The studies involving human participants were reviewed and approved by Ethical Committee—Universidad CES. The patients/participants provided their written informed consent to participate in this study.AA and NC-C design the study. DD, OP, and AA participated in sample acquisition. MS-J, NC-C, and AA participated in the laboratory procedures, protein analysis, and biological pathways interpretation. AA and NC-C wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Schistonchus caprifici was recovered in Iran during the present study. The molecular characters of this population were studied using partial sequences of large subunit ribosomal DNA (LSU rDNA D2-D3) for the first time, and its phylogenetic relationships with other isolates of the species were discussed. Compared to previous available data, no remarkable genetic distance was observed between the presently studied isolate and other isolates.A population of  Aphelenchoides , Aprutides , Basilaphelenchus , Cryptaphelenchus , Devibursaphelenchus , Ektaphelenchus , Ektaphelenchoides , Laimaphelenchus , Robustodorus , Seinura , and Sheraphelenchus  are known that occur in the country  Cobb, 1927, being reported in three conference abstracts  during the present study, the morphological and morphometric characters of this population were found to be in accordance with the data presented by Hunt (1993), Vovlas and Larizza (1996) and S. caprifici calculated using MEGA6  have been reported from Iran, and currently the genera try e.g. . Schistobstracts  with no ng MEGA6  ranged fS. caprifici. This is the first molecular phylogenetic study of the species from Iran, showing D2-D3 sequences of the studied Iranian population is identical to those of the majority of previously sequenced populations.In the Bayesian phylogenetic tree inference under the GTR + I + G evolutionary model , the new