Preeclampsia is the most severe type of hypertensive disorder of pregnancy, affecting one in 10 pregnancies worldwide and increasing significantly maternal and neonatal morbidity and mortality. Women developing preeclampsia display an array of symptoms encompassing uncontrolled hypertension and proteinuria, with neurological symptoms including seizures at the end of pregnancy. The main causes of preeclampsia are still unknown. However, abnormal placentation and placenta vascularization seem to be common features in preeclampsia, also leading to fetal growth restriction mainly due to reduced placental blood flow and chronic hypoxia. An over activation of maternal immunity cells against the trophoblasts, the main cells forming the placenta, has been recently shown as an important mechanism triggering trophoblast apoptosis and death. This response will further disrupt the remodeling of maternal uterine arteries, in a first stage, and the formation of new placental vessels in a later stage. A consequent chronic hypoxia stress will further contribute to increase placental stress and exacerbate systemic circulatory changes in the mother. The molecular mechanisms driving these processes of apoptosis and anti-angiogenesis are also not well-understood. In this review, we group main evidences suggesting potential targets and molecules that should be better investigated in preeclampsia. This knowledge will contribute to improve therapies targeting a better placenta formation, having a positive impact on maternal disease prevention and on fetal development. Pregnancy is a major physiologic event in a woman’s life, leading to significant body and metabolic changes. During the implantation of the embryo, a new transient organ is formed connecting mother and fetus, the placenta . The strDuring the first three gestational weeks, the blastocyst implants in the decidual endometrium, developing the placenta. The implantation is mediated by the invasion of trophoblasts, the main cells forming the placenta . TrophobDuring the implantation process, endometrial cells, and uterine vessels, in addition to uterine immune cells, are modified to form the decidua. Trophoblasts will, in addition, invade the spiral arteries of the uterus. In order to reach this goal, trophoblasts differentiate into villous trophoblasts, ensuring the feto-maternal exchanges and the endocrine functions of the placenta, and in invasive extravillous trophoblasts, essential for the implantation and remodeling of the uterine vessels. The extravillous trophoblast proliferates and becomes invasive, migrating into the decidua and myometrium.The migration of the extravillous trophoblasts into the maternal spiral arteries also represents another key step of human placentation . There, However, some pregnancies are associated with complications. Indeed, several studies have demonstrated defects in the vascular remodeling process in more severe complications such as preeclampsia . In abnoIn this review, we will describe one of the most severe hypertensive disorders of pregnancy, preeclampsia. We will discuss how the molecular mechanisms linked to abnormal placentation involving maternal inflammation by activation of immune cells and apoptosis can alter placental vascularization and contribute to the unfavorable progression of preeclampsia.Preeclampsia is a disorder emerging around 20 gestational weeks; however, it is diagnosed through high blood pressure values (≥140/90 mmHg) and proteinuria of ≥ 0.3 g per day later during gestation. Other symptoms can be also associated such as edema, severe headaches and visual disturbance . ConsideIn addition, the fetus can also be affected by preeclampsia since it is one of the leading cause of premature birth and intrauterine growth restriction, mainly due to insufficient nutrient supply and chronic hypoxia exposure . In addiThe pathogenesis of preeclampsia is still not well-understood and seems to associate differential complex alterations in the placenta and maternal circulation, turning this disorder a major subject of a large amount of studies. Some, however, agree that the preeclampsia origin is an abnormal placentation, which can be defined by two distinct developmental stages: the pre-clinical and the clinical stages . While tThe pre-clinical stage corresponds to inefficient trophoblast invasion in the maternal decidua . If the Therefore, it is now well-understood that a healthy placentation depends on the essential steps of placental development and the ability to respond or adapt to different types of stress. The dysfunction of one of these processes can have harmful repercussions on maternal circulation and fetal development.In the pre-clinical and clinical stages, the maternal immune cells are also active. Lymphocyte T cells can significantly increase the production of inflammatory cytokines such as tumor necrosis factor alpha (TNFα) and interleukin-6 (IL6), as shown in preeclampsia. Pro-inflammatory responses are also associated with a reduction in anti-inflammatory cytokines . In mateInflammation can further activate and promote the programed death of trophoblasts, a process called apoptosis. In normal healthy pregnancies, the mechanisms of apoptosis can be of crucial importance to protect the trophoblasts from the attack of maternal immune cells and to promote the death of uterine arteries endothelial cells and their replacement by extravillous trophoblasts . HoweverIn addition, it has been described that abnormal placentation can be associated with an imbalance in the production of pro-angiogenic and antiangiogenic factors by the placenta. Increased secretion of antiangiogenic factors soluble fms-like tyrosine kinase 1 (sFlt1) and soluble endoglin (sEng), as well as reduced production of pro-angiogenic factors vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) during exposure to ischemia, likely contributes to the pathogenesis of preeclampsia . An incrIn the next sessions, we describe how these two interconnected mechanisms involving the maternal immune system and cell death act during placentation and placental vascularization in healthy pregnancies and in the pathology of preeclampsia.Several hypotheses have been proposed to explain an abnormal trophoblast invasion and the over activation of placental inflammation in early pregnancies with preeclampsia. Some support an impaired maternal immune response or a defective maternal immune-tolerance to the semi-allogeneic fetus . A succeIn normal pregnancies, the processes of trophoblast invasion and spiral artery remodeling are highly dependent on the maternal immune system to allow significant tissue changes . During However, an over activated immune system can play a significant negative role in placental development and the progression of preeclampsia. Epidemiological evidence supports this role, indicating an association between adverse changes in maternal immune responses and preeclampsia . In partOne hypothesis is that over activation of immune cells stimulates trophoblast apoptosis in preeclampsia . Indeed,In vitro, maternal macrophages could further induce apoptosis of periarterial extravillous trophoblasts  are major histocompatibility complex (MHC) molecules expressed on different cells of the placenta. Invasive extravillous trophoblasts express Class I HLA-C and the atypical class Ib antigens HLA-G, which promote immune-regulation between extravillous trophoblasts and maternal decidual NK cells. HLA-G is a ligand for the inhibitory receptor KIR2DL4 of NK cells . ConsequLower expression of other HLA genes can inversely protect the placenta. For example, syncytiotrophoblasts do not express HLA-A and HLA-B genes, which leads to not expressing MHC-I, a major binding site for lymphocyte T cells . TherefoAn important mechanisms involved in maternal-fetal immune tolerance is apoptosis, which is defined as a programed cell death that can be induced by two different pathways: the extrinsic and the intrinsic . The mecThe intrinsic pathway is usually activated during intracellular stress such as ischemia and DNA damage, or when cells receive signals from certain pro-apoptotic factors. During intracellular stress, P53 is also activated, a protein which main role is to stimulate cascades of pro-apoptotic factors mainly targeting the mitochondria . One of Evidence supports that in normal pregnancies, trophoblasts secrete FASL rather than expressing the FAS receptor , as showOn the other hand, changes in this process and on main players have a significant negative impact on placental development, contributing to the establishment of placental abnormalities. In hypertensive pregnancies, for example, increased trophoblast apoptosis and reduced cell invasion were described . More spConsistent with these findings, we and others have also shown that the expression of FAS and FASL in serum and in maternal lymphocytes also occurs in preeclampsia . We alsoIn addition, over activation of the intrinsic pathway may also occur due to reduced anti-apoptotic factors, being another potential origin of exacerbated apoptosis in preeclampsia  Hung et. Indeed,Syncytiotrophoblast apoptosis in preeclampsia has also been associated with larger numbers of syncytial knots and higher oxidative stress . RajakumThe increase of syncytial knots in trophoblasts has been observed and associated with down regulation of anti-apoptotic factors of the intrinsic pathway such as BCL2 and Mdm2 and with premature aging of the placenta in preeclampsia . In addiDespite the increasing number of studies reporting the involvement of the maternal immune system and apoptosis in the development of the placenta and preeclampsia, the main mechanisms promoting cell death and ineffective vascular remodeling in abnormal placentation are still unclear. It is well-known that close interaction between the maternal immune system and trophoblasts is essential during placentation. However, in the presence of an over activation of inflammation and apoptosis of the trophoblasts, it leads to abnormal placentation with impaired invasion, vascular remodeling and microcirculation in the placenta. Activation of different pathways of apoptosis also depends on pathological processes in the placenta. More specifically, activation of the FAS/FASL extrinsic pathway and of the intrinsic pathway mediated by P53, Bax, and BCL2 have been systematically reported in normal and complicated pregnancies such as preeclampsia although their mechanisms are not yet fully understood.Future studies are therefore necessary to better understand the mechanisms of activation of the extrinsic and intrinsic apoptosis pathways in hypertensive disorders of pregnancy and whether their activation may also depend on different pathological stimuli associated with preeclampsia. Current data support that activation of the extrinsic pathway is largely associated with systemic and local immune and inflammatory responses in the placenta. The intrinsic pathway is primarily described as a consequence of trophoblasts disruption under severe and chronic hypoxia, DNA damage and environmental stress conditions. Importantly, the close interaction of these two pathways can help promote abnormal placentation and vascularization, aggravating placental stress during preeclampsia. More studies evaluating these mechanisms are therefore needed to elucidate these interactions and to investigate new therapies capable of reducing the activation of apoptosis at different stages of the development of preeclampsia.NR wrote the manuscript and supervised figures edition. SM designed the figures and collaborated in writing. MB reviewed and edited the text and figures.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.


It is essential to consider the deleterious secondary effects of coronavirus 2019 (COVID-19) disease and its consequences, especially in patients who develop the most severe forms. The survival of acute critical illness in the intensive care unit (ICU) may not reflect the patient’s quality of life after hospitalization.A study with survivors of acute respiratory distress syndrome (ARDS) found that 24 months after the disease, these patients had significantly lower exercise capacity and health status than healthy individuals. In addition, 29% of the survivors had not returned to work.,3 This syndrome is characterized by physical, functional, cognitive and psychiatric changes and by the development of posttraumatic stress disorder, which can lead to reduced quality of life.,5The interactions among the critical illness-related complications, comorbidities, life-support treatments, organizational aspects of intensive care and adaptation during the post-ICU period may contribute to the development of post-intensive care syndrome.In this context, it is suggested that the repercussions of COVID-19 resulting from treatment in the ICU include the development of ICU-acquired weakness (ICUAW) and that its consequences extend beyond the hospitalization period. In association with these factors, the duration of mechanical ventilation - another factor associated with ICUAW - for these patients is high (mean of 11.7 days), as are the lengths of stay in the ICU and hospital. Approximately 75-80% of patients hospitalized with COVID-19 have prolonged hospital stays of approximately 21 days.Many patients infected with COVID-19 require ICU admission due to severe acute respiratory failure and the development of ARDS, which is considered a risk factor for ICUAW. In addition, a systemic inflammatory process occurs via the release of proinflammatory cytokines, which contribute to the mechanism of muscle mass loss. Evidence has shown that organ dysfunction is strongly associated with muscle dysfunction. In addition, some of these patients have associated comorbidities, such as advanced age, renal dysfunction, hypertension, diabetes, heart disease, which may contribute to the incidence of ICUAW.,9 All these factors contribute to immobility, which, in turn, has deleterious effects on the cardiorespiratory, central nervous, musculoskeletal systems and metabolism.Thus, critically ill patients with COVID-19 may face a vicious cycle in which disease severity, the presence of comorbidities, prolonged invasive ventilatory support and the use of sedatives and neuromuscular blockers may contribute to the development of ICUAW and functional disorders in the short and long term (Patients with COVID-19 who are admitted to the ICU may have (multiple) organ failure, including ARDS, acute kidney injury, cardiac injury and liver dysfunction.ong term .Thus, these risk factors should be used as a means of evaluation and screening of these patients to promote early rehabilitation through early mobilization (EM) protocols in order to avoid and/or minimize complications and functional decline.,14EM helps to reduce the deleterious effects of the disease, especially on muscle and cardiopulmonary function, mobility and functionality. It is a safe and feasible practice that leads to improved muscle strength and functional recovery with improved quality of life.,15 EM also leads to better clinical outcomes, such as reduced mechanical ventilation durations and lengths of ICU and hospital stay.,15Thus, it helps to reduce hospital costs and may also prevent readmission to the ICU/hospital.Considering the clinical conditions caused by prolonged immobility and musculoskeletal deterioration, the implementation of systematized EM protocols is of fundamental relevance for patients with COVID-19, given the growing evidence of its benefit. It is known that in such settings, the priority may be the provision of advanced ventilatory care. However, rehabilitation should be incorporated into pandemic response plans at the onset of hospitalization and not only after patients experience consequences. In such cases, the multidisciplinary team plays a crucial role in the functional recovery and reintegration of these individuals into society.Currently, we are experiencing an overload of the health system, and ICUs are experiencing high occupancy rates or are at maximum capacity in Brazilian states; this leads to work overload for health professionals, given that ICUs are often not large enough to meet the demand. be initiated during hospitalization and continue in specialized rehabilitation programs after discharge in order to improve their functionality and quality of life and prevent rehospitalization.It is suggested that early rehabilitation interventions in patients with COVID-19, especially those who develop with severe muscle dysfunction, fatigue and dyspnea,-21Physical therapists have a role in providing interventions for mobilization, exercise and rehabilitation, especially for patients at risk of developing functional decline. In this context, strategies and recommendations were developed for workforce planning and preparation.-21Planning for the number of professionals needed to implement comprehensive patient care in the ICU and on the wards is recommended. The inclusion of professionals who have experience caring for critically ill patients in a hospital environment should be prioritized, i.e., physical therapists must have specialized knowledge, skills and decision-making capacity.-21Physical therapists with previous ICU experience should be identified and the return of these professionals to the ICU should be facilitated. It is also recommended that professionals who have no experience in the ICU be identified and used to support the care of COVID-19 patients in other areas of the hospital.-21Another important point is the implementation of training and learning resources, such as the development and management of critical care skills and training on the use of personal protective equipment. Thus, we suggest that future studies should develop similar approaches in Brazil to better elucidate the effects of COVID-19 on the functionality of affected individuals.Lastly, to minimize the impact of COVID-19 on patients undergoing home treatment postdischarge, televisits, telehealth consultations and telemonitoring services may be important treatment tools.


Collagen is the most abundant airway extracellular matrix component and is the primary determinant of mechanical airway properties. Abnormal airway collagen deposition is associated with the pathogenesis and progression of airway disease. Thus, understanding how collagen affects healthy airway tissue mechanics is essential. The impact of abnormal collagen deposition and tissue stiffness has been an area of interest in pulmonary diseases such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease. In this review, we discuss (1) the role of collagen in airway mechanics, (2) macro- and micro-scale approaches to quantify airway mechanics, and (3) pathologic changes associated with collagen deposition in airway diseases. These studies provide important insights into the role of collagen in airway mechanics. We summarize their achievements and seek to provide biomechanical clues for targeted therapies and regenerative medicine to treat airway pathology and address airway defects. The airway consists of both a conducting region  where air is humidified, warmed, and cleaned and a respiratory zone where gas exchange occurs. The airway is directly and continuously exposed to both macromechanical and micromechanical forces. Macromechanics is the study of organ-level mechanical and material properties. Intrathoracic respiratory forces, perfusion, and cough represent some of the dynamic macromechanical forces exerted on the respiratory system. As the airway is composed of heterogeneous components ), these constituents can be individually quantified using micromechanics. Micromechanical properties drive the mechanotransduction in the airway, driving cell–cell and cell–matrix interactions .The collagen family is the most abundant component of the airway ECM ,3,4,5, pIn this review, we appraise the role of collagen on normal airway mechanics. We then present studies of airway biomechanics, including the quantification of (1) airway micromechanics  and (2) micromechanical properties (cell- and matrix-level properties). In the last section, we review the role of pathologic collagen deposition and alteration in airway diseases and its resultant effect on airway mechanics. As collagen of airway ECM is becoming a primary biological source for airway tissue engineering, understanding its impact on airway mechanics will facilitate the development of biomaterials that can recapitulate the native airway.The primary role of collagen is to provide tensile strength to the ECM ,14, withAs the primary structural component of airway ECM, collagen provides biomechanical cues for cell adhesion and tissue growth . StudiesCollagens also play a vital role during airway regeneration and repair. Regenerative medicine has adopted the use of acellular airway constructs through decellularization in an effort to provide a biomimetic scaffold for tissue engineering. However, decellularization of a multi-lineage tissue  such as the trachea has resulted in ECM injury, loss of graft mechanical properties, and collapse in both pre-clinical and clinical applications ,28. New Airway mechanics has been quantified at macro and micro levels  to exploIn contrast, several modalities are used at the bedside to assess macromechanical properties of the airway in vivo. Functional assessment of the airway can be achieved quantitatively with the use of pulmonary function testing (PFT), for example with plethysmography to calculate lung volumes and spirometry to evaluate for the presence of intrathoracic or extrathoracic obstruction. Beyond pulmonary function tests, radiographic tests can be used to characterize changes in tissue thickness . Using aThe micromechanical properties of airway cells and extracellular compartment can define the mechanisms of repair, remodeling, and disease. This is typically quantified with the use of atomic force microscopy (AFM), a high-resolution imaging technique that permits not only nanometer-level resolution, but also the quantification of the stiffness (Young’s modulus) at the cellular level  41,42].,42.41,42Within the airway, bronchial and alveolar cells have also been studied extensively using AFM. In vitro studies of wound repair demonstrate localized changes in cellular stiffness during bronchial epithelial spreading and migration . Matrix In airway diseases, abnormal tissue remodeling is associated with the deposition of ECM components such as collagens, fibronectins, and proteoglycans, in and around the epithelium and surrounding vessels ,61,62,63Cystic fibrosis (CF) is an autosomal recessive disease that causes alterations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride ion channel, leading to thick mucus blocking the airway, causing infections and scarring of the lung . This reAsthma is chronic inflammation of the airway that leads to episodic narrowing of the airway, which is commonly exercise- or allergen-induced . Over tiThe correlation between collagen deposition and ECM stiffness has also been studied in vitro. Human bronchial fibroblasts (HBF) derived from asthmatic patients had a higher elastic modulus compared to non-asthmatic HBF . AsthmatIdiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial pneumonia of unknown cause . The incChronic obstructive pulmonary disease (COPD) is an inflammatory disease of the lungs, manifesting as incomplete airflow obstruction resulting in emphysema and chronic bronchitis . TypicalAs mentioned previously, emphysema is part of the pathophysiology of COPD . ASMC prAcute respiratory distress syndrome (ARDS) is a condition where the alveoli or alveolar vessels are injured, leading to inflammation and increased fluid in the alveoli. In patients with ARDS, mechanical ventilation can cause additional lung injury due to the barotrauma from high airway pressures ,99. In ALung function is known to deteriorate with age, resulting in poorer mucociliary clearance, loss of elastic recoil, and poorer lung function on PFT. One mechanism of lung aging is increased collagen and decreased elastin production by fibroblasts, thus increasing pulmonary stiffness and lowering compliance, increasing the elastic modulus ,101. In As the major component of extracellular matrix (ECM), collagen supports the macro- and micromechanical environment of the airway. Preservation of collagen type and quantity is necessary to maintain ECM stiffness for the native airway as well as for the development of tissue-engineered airway constructs. Macro and micro airway mechanics provide a fundamental knowledge of the contribution of collagen in the airway. Biomechanical properties can serve as a reference for biomaterial creation and scaffold fabrication, can provide a diagnostic tool for airway disease, and can also elucidate the mechanisms of disease. Future directions for collagen mechanics include their application as a biomarker for disease surveillance and the development of therapeutics.


Evidence is emerging for a role of opiates in various cancers. In this study, we aimed to investigate the association between regular opium use and cancer incidence.This study was done in a population-based cohort of 50 045 individuals aged 40–75 years from northeast Iran. Data on participant demographics, diet, lifestyle, opium use, and different exposures were collected upon enrolment using validated questionnaires. We used proportional hazards regression models to estimate hazard ratios (HRs) and corresponding 95% CIs for the association between opium use and different cancer types.trend<0·001). For site-specific cancers, use of opium was associated with an increased risk of developing oesophageal , gastric , lung , bladder , and laryngeal  cancers in a dose-dependent manner (ptrend<0·05). Only high-dose opium use was associated with pancreatic cancer . Ingestion of opium (but not smoking opium) was associated with brain  and liver  cancers in a dose-dependent manner (prend<0·01). We observed consistent associations among ever and never tobacco users, men and women, and individuals with lower and higher socioeconomic status.During a median 10 years of follow-up, 1833 participants were diagnosed with cancer. Use of opium was associated with an increased risk of developing all cancers combined , gastrointestinal cancers , and respiratory cancers  in a dose-dependent manner (pOpium users have a significantly higher risk of developing cancers in different organs of the respiratory, digestive, and urinary systems and the CNS. The results of this analysis show that regular use of opiates might increase the risk of a range of cancer types.World Cancer Research Fund International, Cancer Research UK, Tehran University of Medical Sciences, US National Cancer Institute, International Agency for Research on Cancer. The opiate crisis has resulted in thousands of deaths annually and billions in economic losses in many parts of the world.Previous studies have linked opium use with oesophageal,Because of the challenges inherent in studying opium, including gathering valid consumption data and the fear of stigma and prosecution, prospective studies of opium use with long-term follow-up are lacking. The Golestan Cohort Study (GCS) is the only population-based prospective study that includes a large group of regular opium users with validated opium use data.Evidence before this studyThe global crisis of opiate misuse and emerging evidence from experimental and case-control studies showing possible carcinogenic effects of some opium derivatives have raised substantial concern over the long-term effects of using opiates. We searched PubMed, Web of Science, and Scopus on Nov 1, 2019, for publications about use of opiates and cancer risk using the following Medical Subject Headings and relevant terms (opium OR opiate* OR opioid*) AND , with no language or date restrictions. Because of the challenges inherent in studying exposure, there is a paucity of prospective studies of opium use with long-term follow-up. Opium and its derivatives are widely used for cancer pain management and thus reverse causality is a major concern in the available case-control studies. Furthermore, most published studies identified by our search did not assess different types, routes, and doses of opium, which differ in their bioavailabilities, carcinogenic metabolites, and by-products. Additionally, some associations between opiates and specific cancer types have only been described in experimental studies and epidemiological investigations of these associations are lacking.Added value of this studyTo our knowledge, the Golestan Cohort Study (GCS) is the only population-based prospective study that includes a large group of regular opium users with validated opium use data. Furthermore, this report is the only prospective analysis from human studies on associations between opium use and overall and site-specific cancer incidence and is based on more than a decade of following up 50 000 participants of the GCS. This study documents the presence of dose-dependent associations between opium use and a broad array of cancer types, including oesophageal, gastric, laryngeal, lung, pancreatic, liver, bladder, and brain. We observed consistent associations between opium use and cancer among ever and never tobacco users, men and women, and individuals with lower and higher socioeconomic status. Furthermore, all routes and types of opium derivatives used by this population showed evidence of carcinogenic effects. Therefore, this study has important implications for public health and could aid the translation of knowledge and implementation of evidence into practice and policy decision making.Implications of all the available evidenceThe overall evidence indicates that the carcinogenic effects of regular use of opium derivatives might be greater than expected and extend beyond the upper aerodigestive tract to include several organs of the respiratory, digestive, and urinary systems and the CNS. Given the huge increase in use of opiates in the past few years, further global initiatives to reduce opiate misuse and implement preventive strategies to mitigate their hazardous long-term effects are needed.The design and objectives of the GCS have been published previously.The GCS was approved by the institutional review boards of the Digestive Disease Research Institute of Tehran University of Medical Sciences, the International Agency for Research on Cancer, and the US National Cancer Institute.Upon enrolment, two validated questionnaires were completed for the participants: a detailed general questionnaire Opiates are a subgroup of opioids that contain various products derived from the opium poppy plant, including opium, morphine, and heroin.Literature from the 1980s suggested opium as a potential risk factor for oesophageal cancer.The current analysis is based on 531 789 person-years of follow-up. Participants were followed up from enrolment by annual telephone surveys and home visits. If participants or their families reported incident cancers or deaths, a staff member was sent to the home of the patient or the deceased individual to collect detailed information and a team was sent to the corresponding medical centres to gather copies of relevant medical reports. Collected documents were reviewed separately by two expert physicians to verify the diagnosis of cancer. In cases of disagreement, a third expert physician finalised the diagnosis. The final diagnosis of cancer was recorded based on the Ninth Revision of the International Statistical Classification of Diseases and Related Health Problems. For this study, we matched the recorded cancer cases to the Golestan Population-based Cancer Registry database to minimise any possible misclassifications. 1833 (92%) of 1991 self-reported cancer cases were confirmed using the explained quality control steps and were included in this analysis. We used first primary cancer cases for our site-specific analysis. For gastric cancer, we did a subgroup analysis of cardia and non-cardia subtypes. Most oesophageal cancer cases were squamous cell carcinoma so we did not do a subgroup analysis by histology.We used Cox proportional hazards regression models to estimate hazard ratios (HRs) and corresponding 95% CIs for the association between opium use and risk of cancer. We set age as the timescale and defined the entry time as the age at enrolment in the GCS. The exit time was defined as the age at first cancer diagnosis for the cancer cases, the age at death for deaths from other causes, and the age at last follow-up for other participants, until Jan 1, 2019 (exit date was censored at this point).vs female), ethnicity (Turkman vs non-Turkman), residence , wealth score (quartiles), smoking cigarettes (ever vs never), cumulative pack-years of smoked cigarettes (continuous), and regular alcohol drinking (never vs ever). The second model (model 2) additionally included chewing nass (never vs two quantiles of the cumulative nass-years), regular consumption of hookah (never vs ever), predominant household fuel , and diet (tertiles of the HEI score). The results of the two models were very similar  included sex  in the main model, we stratified the analyses by ever versus never use of tobacco and further assessed the interaction between opium and tobacco use because of concerns about residual confounding from tobacco smoking. The questionnaire responses on tobacco use were validated previously through reinterviewing a subgroup of participants and comparing questionnaire responses with the presence of cotinine in their urine samples.The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.50 045 participants were enrolled in the GCS. We excluded 11 individuals who had been diagnosed with cancer before enrolment, leaving 50 034 participants for this analysis. During follow-up, 1833 (3·7%) participants were diagnosed with various cancers—1464 (79·9%) of these had histological confirmation and 369 (20·1%) were identified through verbal autopsy and other available medical records. Participants who developed cancer tended to be older, male, Turkman, and have a lower wealth score . FurtherDetails of opium use and the demographics of opium users are shown in Ever-use of opium and the duration of opium use, regardless of the type and route of use, showed a significant association with the risk of developing all cancers combined, gastrointestinal cancers, and respiratory cancers in the entire cohort, and within never and ever tobacco users  in a dosWhen we assessed site-specific cancers, use of opium was associated with an increased risk of developing oesophageal , gastric , lung , bladder , and laryngeal  cancers  in a dosWhen we compared the route of opium use, we observed similar associations between both ingesting and smoking opium with all cancers combined , gastrointestinal cancers combined , respiratory cancers combined , lung cancer , bladder cancer , and laryngeal cancer . By contrast, associations were stronger for opium smoking with oesophageal cancer  and gastric cancer , whereas associations for opium ingestion were stronger with brain cancer  and liver cancer  of 8486 opium users in the cohort used raw opium and very few users used other types, such as heroin or opium dross .Stratification of the analyses by tobacco use ; table 4In the GCS, opium use was associated with a higher risk of multiple cancers occurring in the respiratory, digestive, and urinary tract and CNS. Both ingesting and smoking opium were associated with cancer. Consistent associations were observed among ever and never tobacco users, men and women, and individuals with lower and higher socioeconomic status.Within the gastrointestinal system, opium was associated with a dose-dependent increase in the risk of developing oesophageal, gastric, pancreatic, and liver cancers. Our results are supported by previous, albeit scarce, studies. The relationship between opium use and oesophageal cancer was first suggested in the 1970s,We also observed associations between opium use and non-gastrointestinal cancers, including lung, laryngeal, bladder, and brain cancers. The relationship between opium use and bladder cancer was suggested in the 1970s, and since then has been shown in several case-control studies.17Three mechanisms have been proposed for the causal association between opium use and cancer. The first potential mechanism is the genotoxic or mutagenic effect of opium smoke and pyrolysates, and some opium alkaloids.The current study shows that both opium smoking and opium ingestion can increase cancer risk in different organs. Furthermore, we previously showed that both routes of opium use can increase the risk of deaths due to circulatory, respiratory, digestive, and infectious diseases.The strengths of this study are the large sample size, long follow-up period with less than 1% loss to follow-up, little missing data, presence of a uniquely large group of regular opium users, validation of self-reported opium consumption using urinary biomarkers, stringent sensitivity analyses (including among never tobacco users), and relatively low prevalence of important confounders such as alcohol and tobacco .This study also has some limitations. Like any observational study, we cannot rule out potential errors in exposure and outcome measurements. However, because of the prospective design of this study, any errors in measuring the exposure are likely to be non-differential differences in potential exposure errors between those who developed the studied outcomes and those who did not develop any of the studied outcomes. Furthermore, to minimise the possibility of outcome measurement errors we followed a strict approach towards case verification using several independent sources and did a sensitivity analysis restricted to histologically confirmed cases. In this study, we did not test the contents of the opium used, and therefore might have missed the presence of contaminants (including lead), which could have contributed to carcinogenicity. However, observing the relationship between opium use and cancers in different populations, documentation of different carcinogenic mechanisms for opiates in experimental studies, and observing the increased cancer risk with all routes and types of used opium make it unlikely that the effects were due to contaminations. Although our study shows higher cancer risk among opium smokers and those who ingest opium compared with those who have never used opium, caution is required in interpreting these results, as low statistical power for some cancer types prevents discrimination between the risks of the two routes of administration. Finally, despite adjustments for potential confounders and different sensitivity analyses, we cannot exclude the possibility of residual confounding, particularly for some cancer types that showed a modest risk increase associated with opium use. Also, the small number of cases in some cancer types and analytic strata might have resulted in spurious associations or unstable results and therefore requires investigation in further studies.In conclusion, regular use of opium might be associated with increased cancer risk in multiple sites of the respiratory, digestive, urinary, and central nervous systems, among ever and never tobacco users, men and women, and individuals with lower and higher socioeconomic status. Given the recent increase in using opium derivatives, further global initiatives to reduce the misuse and implement preventive strategies to mitigate hazardous long-term effects are needed.


Cold-adapted microorganisms represent a large fraction of biomass on Earth because of the dominance of low-temperature environments. Extreme cold environments are mainly dependent on microbial activities because this climate restricts higher plants and animals. Himalaya is one of the most important cold environments on Earth as it shares climatic similarities with the polar regions. It includes a wide range of ecosystems, from temperate to extreme cold, distributed along the higher altitudes. These regions are characterized as stressful environments because of the heavy exposure to harmful rays, scarcity of nutrition, and freezing conditions. The microorganisms that colonize these regions are recognized as cold-tolerant (psychrotolerants) or/and cold-loving (psychrophiles) microorganisms. These microorganisms possess several structural and functional adaptations in order to perform normal life processes under the stressful low-temperature environments. Their biological activities maintain the nutrient flux in the environment and contribute to the global biogeochemical cycles. Limited culture-dependent and culture-independent studies have revealed their diversity in community structure and functional potential. Apart from the ecological importance, these microorganisms have been recognized as source of cold-active enzymes and novel bioactive compounds of industrial and biotechnological importance. Being an important part of the cryosphere, Himalaya needs to be explored at different dimensions related to the life of the inhabiting extremophiles. The present review discusses the distinct facts associated with microbial ecology from the Himalayan cryosphere perspective. Our planet is a cold planet because the majority experiences temperatures below 15 °C throughout the year. Himalaya is one of the geographic locations that is recognized as a unique low-temperature environment since it holds the highest glacial coverage after the polar regions . FurtherBesides the polar region, Himalaya possesses the highest altitude approx. 8000 m asl) and represents similar environmental conditions to the other extreme colder regions on Earth. Its widely known low-temperature regions restrict the growth of higher plants and animals. The normal life in such locations is challenging; therefore, very few animals or plants can be seen surviving in these regions . The ext000 m aslcsp, ctr, rbps, etc.) has been studied to support growth under extreme low temperature [Cold-adapted microorganisms have evolved metabolic processes to cope with the adverse effects of low temperatures . Freezinperature ,16. Thesperature . Antifreperature ,19. Exceperature . Apart fperature . Recentlperature .Investigations on the cold-adapted microorganisms have been focused on the most extreme low-temperature environments only. However, the climatic conditions in the Himalaya are somewhat different. Because of the relatively broad spectrum of environmental conditions that prevail, the Himalayan region favors colonization of the “tolerants” in comparison to “philes”. While growth of the latter is restricted to specific climatic conditions, the tolerants, being more diverse, can survive and colonize under a range of external factors. Investigations on the cellular and metabolic processes of these cold-tolerant microorganisms can help in revealing the connection between obligates (philes) and generalists (tolerants). The life processes associated with tolerants are more complicated and important in environmental and industrial perspectives. Himalaya is one of the regions representing low-temperature environments  on Earth . EarlierThe existing studies from different regions of Himalaya show the complicated composition of microbial communities ,30,31,32Towards bacterial communities, Gangwar et al.  reportedThe abundance of Actinobacteria, Proteobacteria, and Firmicutes in the Himalayan region was similar to the bacterial genera found in the other colder regions (such as Antarctic and Arctic) ,55. BecaThe soils at high altitude at the retreating of glaciers have become an important ecosystem in the climate change perspective as these barren soils are transforming due to colonization of various new microbial communities. These communities consist of diverse microorganisms, heterotrophs to chemotrophs, that contribute significantly to the biogeochemical cycles in the developing stage of soils. The abundance of cyanobacteria indicates their key role in increasing soil carbon and nitrogen content through other environmental processes . SimilarThe Tibetan Plateau represents the world’s highest plateau that contains glaciers with extremely cold and unique habitats for colonization of microorganisms in the ice sheets . ProbablBesides glaciers, snow, lakes, or sediments from the barren lands, unique sites with high temperatures  have also been located in the Himalayas . Even inApart from the extensive molecular studies of microbial communities, there are some studies available that provide the status of the microbial biomass in the soil affected by the climatic conditions/anthropogenic activities ,77. ForeAlthough a plethora of information on the microbial communities associated with the colder environments is available, understanding the ecological processes and the interactions among the microbial communities is still in its infancy. As per the records, bacterial groups are frequently targeted, whereas information of the fungal component remains scarce. Further, apart from high-throughput methods used in ecological studies, environmental factors still restrict the understanding on microbial interactions effectively. More investigations are required targeting functional attributes that can help us to understand the complex network of the energy flow in such microbial communities. It is widely known that the microbial communities of the low-temperature environments are highly sensitive to temperature changes; therefore, they can serve as indicators of climate change. Out of all these important issues, the cold-adapted microbial communities require more investigation regarding their isolation, characterization, preservation, and careful utilization for various industries. The cold-adaptive microorganisms are recognized as potential source of cold-active enzymes and bioactive compounds . In the The microorganisms colonizing the extreme colder regions are adapted to cope with various stress conditions  and produce novel metabolites that are absent in the mesophiles. These metabolites have significant importance in various industries such as food, textiles, therapeutics, and so forth ,85. BioaThe low-temperature environments of Himalaya are seen as a promising source for novel products, bioactive compounds, and other industrially relevant substances/compounds ,27,87. MPseudomonas, Acinetobacter, Rhodococcus, Bacillus, and Sphingobium have been found to contribute significantly to the degradation of complex hydrocarbons (such as petroleum products) under low-temperature environments [Not only the biotechnological benefits but also the inhabiting cold loving microorganisms contribute to several ecological processes in different ecosystems located in Himalaya . In the ronments ,103,104.ronments .The biodegradation process in low-temperature environments is very slow and restricted. Organic carbon is sequestered and accumulates in these environments . These cold-adapted bacteria were also found to possess different hydrolytic enzymatic activities indicating their contribution in the cycling of nutrients including carbon ,40,106. Rhodococcus, Pseudomonas, Bacillus, Serratia, and Ascomycetous fungi  have been recognized as promising candidates in agriculture and forestry [Stenotrophomonas with multiple plant growth promoting activities and possibilities to utilize as biofertilizer has been reported [Apart from degradation, the inhabiting microbial communities have shown their immense contribution to improve the agriculture in low to extreme low temperature environments of Himalaya. These psychrotrophic/psychrophilic microorganisms have been reported for their potential in plant growth promotion activities. They are found to possess activities, such as phosphate solubilization, indole acetic acid production, and siderophore secretion, that are useful in agriculture under low-temperature environments ,108. In forestry ,111,112,reported . In lastreported ,115; excreported . Towardsreported . By consAdditional information on the taxonomic and functional aspects of microbial communities of Himalayan regions is provided in So far, we have discussed the importance of Himalaya as a fraction of the cryosphere, which contributes significantly to the global ecology. Although the microbial diversity of Himalaya has been explored by various research groups, still the information related to the microbial ecology of this unique region is scarce. Most researchers have focused on the taxonomic aspects, while the microbial interactions and functional ecology remain neglected. Further, the diversity or the composition of the most important micro-eukaryotes in the Himalayan region is not targeted well, neither with culture-dependent nor with culture-independent methods. Since Himalaya shares climatic similarities with the polar regions, information on the microbial composition and the metabolic activities needs to be investigated so that it can be compared to the extreme colder regions to identify microbial resources . The gene pool of several unique sites of Himalaya  is still untouched. The gene pool is precious because of its wide tolerance against a range of environmental conditions, such as high-temperature fluctuation and precipitation in lush green forests to barren cold deserts.Majority of these studies report high abundance of extremotolerant species that might be due to a broad fluctuation in temperature  of various regions of Himalaya. This broad range of temperature change allows the mesophiles and the tolerants to grow actively in the environment. This could be one the reasons for the distinct metabolic ability of Himalayan microorganisms from the microbial communities of the Antarctic region .Besides, in the present scenario of global warming, these cold environments are being recognized as highly affected regions because of their sensitivity towards temperature change. While most of the studies provide information on the shifts in microbial communities in various environments of the polar regions, the ecology of the Himalayas still needs focused attention. Very rare or negligible information is on record about the transformation of the microbial activities under the changing environmental conditions. The change in green cover and glacier retreat are documented for Arctic and Antarctic, but no such authentic information is provided in the context of Himalaya.It will be important to explore the extreme cold regions of Himalaya to enhance our understanding on the metabolic activities in the low-temperature environments. This would help us to identify ecological problems associated with the mountain system, leading to the efficient use of natural resources along with the preservation of the unique biodiversity, the microbial diversity in particular, of Himalaya.


Diagnostic Uncertainty has been developed in the Wolfram Language. The program provides six modules with nine submodules for calculating and plotting the standard combined, measurement and sampling uncertainty and the resultant confidence intervals of various diagnostic accuracy measures of screening or diagnostic tests, which measure a normally distributed measurand, applied at a single point in time to samples of non-diseased and diseased populations. This is done for differing sample sizes, mean and standard deviation of the measurand, diagnostic threshold and standard measurement uncertainty of the test. The application of the program is demonstrated with an illustrative example of glucose measurements in samples of diabetic and non-diabetic populations, that shows the calculation of the uncertainty of diagnostic accuracy measures. The presented interactive program is user-friendly and can be used as a flexible educational and research tool in medical decision-making, to calculate and explore the uncertainty of diagnostic accuracy measures.Screening and diagnostic tests are applied for the classification of people into diseased and non-diseased populations. Although diagnostic accuracy measures are used to evaluate the correctness of classification in clinical research and practice, there has been limited research on their uncertainty. The objective for this work was to develop a tool for calculating the uncertainty of diagnostic accuracy measures, as diagnostic accuracy is fundamental to clinical decision-making. For this reason, the freely available interactive program  The terThere is a persistent need of estimating the uncertainty of diagnostic accuracy measures, especially regarding screening and diagnostic tests of life-threatening diseases. The current pandemic of novel corona virus disease 2019 (COVID-19) has exposed this unequivocally ,4,5,6,7.Diagnostic Uncertainty has been developed to explore the combined, measurement and sampling uncertainty of diagnostic accuracy measures as:Diagnostic accuracy is fundamental to clinical decision-making ,Defining the permissible measurement uncertainty is critical to quality and risk management in laboratory medicine .Sampling uncertainty is decisive for clinical study design to evaluate a screening or diagnostic test .The program Se), specificity (Sp), overall diagnostic accuracy (ODA), Youden’s index (J), Euclidean distance (ED) and concordance probability (CZ).Error-based measures, estimating misclassification rates. These include sensitivity (PPV), negative predictive value (NPV), likelihood ratio for positive result (LR+) and likelihood ratio for negative result (LR−).Information-based measures, assisting the interpretation of each single test result. These include positive predictive value (DOR).Association-based measures, estimating the strength of the association between the test results and the reference diagnostic method. These include diagnostic odds ratio (There are diagnostic accuracy measures (DAM) used for evaluating the discriminative ability of a screening or diagnostic test in clinical research and practice . These aThe true disease condition status: sensitivity, specificity, overall diagnostic accuracy, diagnostic odds ratio, likelihood ratio for positive result, likelihood ratio for negative result, Youden’s index, Euclidean distance and concordance probability.The test outcome: positive predictive value and negative predictive value.Defined conditionally onInvariant: sensitivity, specificity, diagnostic odds ratio, likelihood ratio for positive result, likelihood ratio for negative result, Youden’s index, Euclidean distance and concordance probability.Dependent: positive predictive value, negative predictive value and overall diagnostic accuracy.As prevalenceThey can be further classified as following:The natural frequency and the probability definitions of the above diagnostic accuracy measures are presented in Uncertainty is an expression of imperfect or deficient information. When quantifiable it can be represented with probability . The folAs measurements are inherently variable, measurement uncertainty is defined as a “parameter, associated with the result of a measurement, that characterizes the dispersion of the values that could reasonably be attributed to the measurand” . MeasureDiagnostic accuracy measures are estimated by applying a screening or diagnostic test to samples of populations. Sampling heterogeneity contributes to the combined uncertainty of the diagnostic accuracy measures . Even whprobable .There is a reference (“gold standard”) diagnostic method classifying correctly a subject as diseased or non-diseased .Either the values of the measurand or their transforms ,24 are nMeasurement uncertainty is normally distributed and homoscedastic in the diagnostic threshold’s range.The sampling is simple random.If the measurement is above the threshold the patient is classified as test-positive, otherwise as test-negative.For the calculation of the uncertainty of the diagnostic accuracy measures of a screening or diagnostic test based on a measurand, it is assumed that:The calculation of the diagnostic accuracy measures is based on their probability definitions . The senThe uncertainty of an input parameter or a diagnostic accuracy measure bility p .The standard measurement uncertainty edicine” . Bias maedicine” .If If If interval .The standard combined uncertainty sure see , accordiWhen there are The effective degrees of freedom  formula ,32:(7)veIf t-distribution with If The resultant confidence interval (CI) of Diagnostic Uncertainty was developed in the Wolfram Language [® Ver. 12.2, Wolfram Research, Inc., Champaign, IL, USA [To calculate the uncertainty of the diagnostic accuracy measures, the interactive program Language , using W IL, USA . The pro® or Wolfram Mathematica® (see The program is freely available as a Wolfram Mathematica Notebook (.nb) . It can ica® see .The flowchart of the program is presented in The modules and submodules of the program include panels with controls which allow the interactive manipulation of various parameters, as described in detail in The values of the standard combined, measurement and sampling uncertainties of diagnostic accuracy measures of a screening or diagnostic test are plotted versus the diagnostic threshold of the test .The values of the relative standard combined, measurement and sampling uncertainties of diagnostic accuracy measures of a screening or diagnostic test are plotted versus the diagnostic threshold of the test .The values of the lower and upper bounds of the confidence intervals of diagnostic accuracy measure of a screening or diagnostic test, at a selected confidence level, are plotted versus the diagnostic threshold of the test .The values of the standard combined, measurement and sampling uncertainties of diagnostic accuracy measures of a screening or diagnostic test are plotted versus the measurement uncertainty of the test .The values of the relative standard combined, measurement and sampling uncertainties of diagnostic accuracy measures of a screening or diagnostic test are plotted versus the measurement uncertainty of the test .The values of the lower and upper bounds of the confidence intervals of diagnostic accuracy measures of a screening or diagnostic test, at a selected confidence level, are plotted versus the measurement uncertainty of the test .The values of the standard combined, measurement and sampling uncertainties of diagnostic accuracy measures of a screening or diagnostic test are plotted versus the total population sample size .The values of the relative standard combined, measurement and sampling uncertainties of diagnostic accuracy measures of a screening or diagnostic test are plotted versus the total population sample size .The values of the lower and upper bounds of the confidence intervals of diagnostic accuracy measures of a screening or diagnostic test, at a selected confidence level, are plotted versus the total population sample size .The values of the standard combined, measurement and sampling uncertainties of diagnostic accuracy measures of a screening or diagnostic test, at a selected diagnostic threshold, are calculated and presented in a table .The values of the relative standard combined, measurement and sampling uncertainties of diagnostic accuracy measures of a screening or diagnostic test, at a selected diagnostic threshold, are calculated and presented in a table .The point estimations and the lower and upper bounds of the confidence intervals of diagnostic accuracy measures of a screening or diagnostic, at a selected confidence level and diagnostic threshold, are calculated and presented in a table .The program was applied to a bimodal distribution of log-transformed blood glucose measurements in samples of non-diabetic and diabetic populations. The data were derived from a national health survey conducted in Malaysia in 1996 . A glucoIn the plots of In the chart of Figure 22In the tables of The results of the illustrative example are presented:The parameter settings of The combined uncertainty and the resultant confidence intervals increase with measurement uncertainty  and decrcu(x)has has see :Little eIn addition, measurement uncertainty is the main component of the combined uncertainty of specificity, overall diagnostic accuracy, positive predictive value, diagnostic odds ratio and likelihood ratio for a positive result.Diagnostic Uncertainty explores the combined, measurement and sampling uncertainty of diagnostic accuracy measures of a screening or diagnostic test The existence of a “gold standard” diagnostic method. If a “gold standard” does not exist, there are alternative approaches for the estimation of diagnostic accuracy measures .The normality of either the measurements or their applicable transforms ,24,40,41The bimodality of the measurands, that is generally accepted, although unimodal distributions could be considered ,48.The simple random sampling.The measurement uncertainty homoscedasticity in the diagnostic thresholds range. Nevertheless, if measurement uncertainty is heteroscedastic, thus skewing the measurements distribution, appropriate transformations may restore homoscedasticity .The assumptions used for the calculations:Limitations of this program, that could be improved by further research, are the following:(2)The first order Taylor series approximations for the uncertainty propagation calculations ,30. High(3)The uncertainty of prevalence rate approximation by the Agresti–Coull adjusted Waldo interval , althougIf the above assumptions are not valid, there are other components of uncertainty which are not calculated by this program.However, addressing these limitations, would increase exponentially the computational complexity.®, NCSS®, R, SAS®, SPSS®, Stata®, MedCalc®, NIST Uncertainty Machine, UQLab, metRology), provides this range of plots and tables without advanced programming.The program presented in this work complements our previously published software , which eDiagnostic Uncertainty calculates the combined, measurement and sampling uncertainty of diagnostic accuracy measures and the resultant confidence intervals and can be used as a flexible, user-friendly, interactive educational or research tool in medical decision-making.The presented program


Intra- and interindividual variation in drug responses is one major reason for the failure of drug therapy, drug toxicity, and even the death of patients. Precision medicine, or personalized medicine, is a field of medicine that customizes an individual's medical diagnosis and treatment based on his/her genes, microbiomes, environments, etc. Over the past decade, a large number of studies have demonstrated that gut microbiota can modify the efficacy and toxicity of drugs, and the extent of the modification varies greatly from person to person because of the variability of the gut microbiota. Personalized manipulation of gut microbiota is an important approach to rectify the abnormal drug response. In this review, we aim to improve drug efficacy and reduce drug toxicity by combining precision medicine and gut microbiota. After describing the interactions between gut microbiota and xenobiotics, we discuss (1) the effects of gut microbiota on drug efficacy and toxicity and the corresponding mechanisms, (2) the variability of gut microbiota, which leads to variation in drug responses, (3) the biomarkers used for the patient stratification and treatment decisions before the use of drugs, and (4) the methods used for the personalized manipulation of gut microbiota to improve drug outcomes. Overall, we hope to improve the drug response by incorporating the knowledge of gut microbiota into clinical practice. Drug responses vary among patients in terms of efficacy and toxicity, and this variation presents great challenges in optimizing drug therapy in the clinic 15 microbial cells and 5 × 106 unique microbial genes, both of which outnumber human cells and genes With the rapid progress in omics such as genomics and metabolomics, gut microbiota has become a focus in understanding the responses of human bodies to drugs. Gut microbiota is a complex group of microorganisms that dwell in the gastrointestinal tract in a mutualistic fashion with hosts. The intraindividual composition of gut microbiota is extremely dynamic, and the interindividual composition of gut microbiota varies greatly as well The term precision medicine is often used interchangeably with the terms personalized medicine, stratified medicine and person-centered medicine When orally administered, the xenobiotics that have passed through the small intestine will ineluctably contact and thus chemically interact with gut microbiota. Gut microbiota synthesizes a battery of enzymes that are capable of transforming xenobiotics. These reactions include but are not limited to, rearrangement, oxidation, isomerization, esterification, condensation, reduction, and hydrolysis Salmonella typhimurium and Salmonella enteritidis at suitable concentrations and under the appropriate pH conditions Codonopsis pilosula can increase the antimicrobial peptide levels in the Drosophila gut after sodium dodecyl sulfate-induced damage Direct contact between xenobiotics and gut microbiota can lead to compositional changes in gut microbiota Figure A. XenobiN-oxide (TMAO). Lipases in the small intestine and microbial glycyl radical enzymes in the colon sequentially metabolize dietary phosphatidylcholine and produce trimethylamine (TMA). Absorbed TMA can be further oxidized by flavin monooxygenases (FMO1 and FMO3) in the liver to produce TMAO, a compound that is responsible for an increased risk of cardiovascular diseases Direct contact between xenobiotics and gut microbiota can also result in the transformation of xenobiotics and changes in gut microbiota metabolites Figure B. EquippIn vitro incubation of L-dopa with Helicobacter pylori showed a conspicuous decrease of L-dopa, and H. pylori pre-incubated with L-dopa exhibited remarkably decreased adhesion to gastric epithelial cells L-dopa can bind to the outer membrane proteins of H. pylori, and as a result the concentration of L-dopa in bacterial suspension was significantly decreased. The gastrointestinal epithelium has evolved to confine the gut microbiota and maintain the symbiotic relationship between the host and gut microbiota. Disruption of this architecture allows the infiltration of gut bacteria and the excess influx of materials such as lipopolysaccharides. The infiltration of gut bacteria and materials activates the immune systems of hosts, and finally leads to several diseases such as depressive disorder, cirrhosis and inflammatory bowel disease Xenobiotics can directly and indirectly interact with gut microbiota by direct binding and modulation of the host immune system Figure B. In vitThe impact of the direct and indirect interactions between gut microbiota and xenobiotics on the pharmacological and toxic effects of drugs is so profound that these interactions can cause the failure of drugs and even the death of patients. Therefore, to achieve precision medicine, it is necessary to understand the effects of gut microbiota on the efficacy and toxicity of drugs. Here, we discuss the effects of gut microbiota on drug efficacy and toxicity and the corresponding mechanisms. Because direct binding is the simplest type of interaction Figure A, we focβ-glucosidase and endo-β-glucosidase in vitro antiplatelet activities of these flavonoid aglycones such as quercetin, hesperetin and narigenin are stronger than those of their parental compounds Gut microbiota can inactivate, activate and change the potency and bioavailability of drugs via direct enzymatic metabolism Figure B. DigoxiHirsutella sinensis mycelium exhibited anti-obesity effects on mice via selective promotion of the growth of Parabacteroides goldsteinii, and no alteration of SCFAs was observed Escherichia, Intestinibacter, Lactobacillus, Bacteroides fragilis, Akkermansia muciniphila, etc., and the ensuing effects of increased production of beneficial SCFAs, the alteration of the bile acid pool, the enhancement of the release of gut hormones glucagon-like peptide 1 and peptide YY, and secretion of peptide Amuc_1100, which can enhance barrier function and decrease endotoxemia The normal composition, metabolism and spatial distribution of gut microbiota play indispensable roles in the homeostasis of hosts. Drugs, especially ethnomedicines, can change the composition, metabolism and spatial distribution of gut microbiota to (partly) achieve therapeutic effects especially those drugs that cannot be absorbed or exhibit poor bioavailability Figure C. A typeβ-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors that are used for the treatment of coronary artery disease. It has been reported that some patients are statin-resistant while others are statin-intolerant Competition and modulation of host genes and enzymes that are involved in transportation and metabolism of xenobiotics can also impact the efficacy of drugs Figure E. Statinβ-glucuronidases. SN-38 in the intestine is toxic toward epithelial cells and is the cause of diarrhea in patients with colon cancer. β-glucuronidases also play a similar role in the toxic effects of NSAIDs β-glucuronidases release the aglycones. The gut epithelial cells then absorb and metabolize the aglycones into reactive metabolites that are toxic to the intestinal wall.Gut microbiota can modulate the toxicity of drugs via direct enzymatic metabolism Figure B. In thiRaoultella planticola, and the products of deglycosylation exhibited reduced toxicity on model species Caenorhabditis elegansBifidobacterium longum HY8001 and B. fragilis, can transform the herbal compound geniposide into genipin, a compound that shows increased toxicity toward HepG2 cells In vitro and in vivo studies have demonstrated that gut microbiota can modulate the toxicity of arsenic via direct metabolism of arsenic (including oxidative methylation and glutathione conjugation), and this process also increases the bioavailability of arsenic Direct enzymatic metabolism can also influence toxicity by changing the toxicity and bioavailability of drugs. Doxorubicin is a widely used drug for carcinomas. However, its clinical use is limited due to its cumulative toxicity in normal tissues that can manifest as diarrhea, vomiting, hair loss and life-threatening cardiotoxicity B. fragilis and an increase in macrophage density B. fragilis significantly decreased inflammatory reactions induced by methotrexate and restored the polarization of macrophages, suggesting the involvement of gut microbiota and host immune system in methotrexate-induced intestinal mucositis. In addition to direct metabolism induced by gut microbiota, heavy metals can cause dysbiosis and disruption of the gut barrier, which may further activate the host immune system et al. showed that ingestion of 10 ppm arsenic for four weeks significantly perturbed the composition of mouse gut microbiota and its metabolism various substances, such as bile acids and amino acids Drugs can change the composition, metabolism and spatial distribution of gut microbiota, which in turn affect the toxicity of drugs Figure C. MethotN-acetyl-p-benzoquinone imine by CYP450 enzymes CYP2E1 and CYP3A4 p-Cresol is a microbial product produced during the metabolism of tyrosine and phenylalanine by gut microbiota. In the liver, p-cresol is metabolized to p-cresol sulfate, a substrate of the human cytosolic sulfotransferase 1A1 (SULT1A1), which is shared by acetaminophen p-cresol sulfate exhibit reduced acetaminophen sulfate to acetaminophen glucuronide ratios in urine p-cresol and acetaminophen hampers the capability of hosts to reduce the toxicity of acetaminophen by increasing the possibility of producing N-acetyl-p-benzoquinone imine.Competition with gut microbiota metabolites for host proteins and enzymes that participate in transportation and metabolism of xenobiotics can also impact the toxicity of drugs Figure D. A typiE)-5-(2-bromovinyl) uracil (BVU). BVU is an inhibitor of liver dihydropyrimidine dehydrogenase, a catabolic enzyme that is responsible for detoxification of 5-FU. Thus, combined administration of sorivudine and 5-FU, increases the circulating 5-FU levels, leading to 5-FU-associated death Lactobacillus spp. and Bacteroides spp. In addition to the combinational use of ethnomedicines, combinational use of chemical drugs and ethnomedicines is very common in China. Some studies have preliminarily demonstrated that gut microbiota is responsible for the efficacy-enhancing and toxicity-reducing effects of the combinational use of chemical drugs and ethnomedicines Gut microbiota can also responsible for drug-drug interactions. The increasing antimicrobial drug resistance has become a serious global health issue, leading to an increase in infection-associated death. The use of drug combinations is encouraged to treat multidrug-resistant bacterial infections in the clinic; however, their effects on microbiota remain unexplored. Profiling of approximately 3,000 combinations of antibiotics, human-targeted drugs and food additives showed that 70% of drug-drug interactions are species-specific and 20% showed strain specificity, and antagonism is more common than synergism A. muciniphila are responsible for the variation in the therapeutic effects of PD-1 blockade Proteobacteria became predominant by the 12th week in non-responders  Ruminococcaceae in responding patients Although the progress in understanding the roles of gut microbiota in the modulation of drug efficacy and toxicity has been striking, the extent of modulation still varies greatly person-to-person, leading to the unpredictable reactions to drugs, as evidenced by typical examples of the response of patients to PD-1/PD-L1 blockers Bacillus megaterium ATCC 14581 is strongly inducible in the presence of phenobarbital The functions of gut microbiota include but are not limited to the metabolism of xenobiotics, biosynthesis of vitamins, steroid hormones, and neurotransmitters, modulation of host immune maturation, cell proliferation, vascularization and neurologic signaling The factors that shape the composition of gut microbiota can also impact the function of gut microbiota Figure . For exaCurrently, determination of the causation or association between bacterial species and diseases such as obesity and ulcerative colitis has allowed the development of gut microbiota-based biomarkers for precision disease diagnosis and risk stratification Berberis vulgaris and Coptis chinensis. In China, berberine is extensively applied to treat bacteria-associated diarrhea and cardiovascular diseases such as hypercholesterolemia Eggerthella lenta in the gut is responsible for the conversion of active digoxin to cardio-inactive dihydrodigoxin E. lenta cannot accurately predict the metabolism of digoxin in the gut, as E. lenta-colonized patients still show remarkable variation in dihydrodigoxin production cgr) operon is specific to drug-metabolizing strains of E. lentaGut microbiota and gut microbiota-associated components including genes, enzymes, bacterial strains or species, and metabolites can be developed as biomarkers for the personalized use of drugs Figure A. PerhapFaecalibacterium prausnitzii was positively correlated with the increase in tacrolimus dosing in recipients F. prausnitzii in metabolizing tacrolimus into two different metabolites, and the major metabolite showed much less bioactivity in vitroF. prausnitzii may act as a biomarker for an increase in the dosage of tacrolimus  and colonized with cgr-expressing E. lenta showed significantly higher concentrations of serum and urinary digoxin than the control mice. In contrast, the serum and urinary digoxin-elevating effects were not observed in mice colonized with E. lenta that lacked cgr operon. In another study, 3,4-dihydroxy- phenylacetic acid, a compound produced by gut microbiota metabolism of quercetin, attenuated acetaminophen-induced liver injury in mice Dietary intervention represents a good alternative to manipulate the composition and function of gut microbiota because of the convenience of this approach. It has been shown that the diet can rapidly and profoundly change gut microbiota and can also influence the interindividual variation of host microbial communities Lactobacillus and Bifidobacterium while inhibiting Odoribacter growth and increasing the fecal level of SCFAs in mice with constipation Bacillus, Escherichia, and Propionibacterium can produce helpful effects on hosts by inhibiting the growth of harmful bacteria, producing beneficial metabolites, competing for nutrients, and facilitating the host immune response Lactobacillus rhamnosus synthesizes various molecules such as adhesive pili and lipoteichoic acid, and thus exerts immunomodulatory, probiotic, and antimicrobial effects Lactobacillus casei variety rhamnosus mitigated FOLFOX-induced diarrhea and intestinal mucositis in a mouse model of colorectal cancer β blockade and Escherichia coli Nissle 1917, a probiotic strain, significantly improved the antitumor and immune suppression effects of TGF-β blockade in mice Prebiotics and probiotics have been extensively studied in the clinic for their beneficial effects on gut microbiota. Dietary supplementation with prebiotics promotes the growth of beneficial bacteria and the production of beneficial metabolites. For example, dietary supplementation with oligosaccharides promotes the growth of Although conventional approaches for the modification of gut microbiota have shown great potential for precision medicine, these approaches usually lack specificity as they may lead to extensive modification of untargeted gut bacteria. For example, the antibiotics prescribed for the elimination of specific bacteria can produce extensive and long-lasting effects on other gut bacteria and even influence the metabolism of hosts et al. In their study, they engineered gut bacteria to have the capability to sense and report the levels of two compounds in mice with dextran sodium sulfate-induced colitis Salmonella typhimurium and Shewanella baltica. The two sensors were reprogrammed to drive the expression of green fluorescent protein and were ported to the gut‐adapted probiotic E. coli Nissle 1917. After the gavage of engineered strains into mice, flow cytometry was used to detect the engineered bacteria in the colon and feces and to analyze the sensor outputs. The results showed that the engineered thiosulfate sensor was activated by colon inflammation, suggesting the success of the biosensor in detecting colon inflammation and the thiosulfate content. In addition to tetrathionate and thiosulfate, gut bacteria have been engineered to sense and report signals that include but are not limited to cytokines such as TNF, IFN-γ and IL-1β, hormones such as adrenaline, metabolites such as γ-aminobutyric acid, physiological stimuli such as temperature, and quorum sensing signals   Figure A.ex vivo clinical samples Because the signals sensed and reported by engineered bacteria are so comprehensive that they almost encompass all the signals from surrounding bacteria, an emerging aim of bacteria engineering is the development of gut bacteria as diagnostic tools C. difficile infection  et al. showed that reprogramming of Cas9 to target virulent genes can efficiently kill the virulent Staphylococcus aureus, but not the avirulent strain in mice For some drugs, interindividual differences in efficacy and toxicity can be ascribed only to certain gut bacterial species; hence, what we desire is the precise depletion of the species without significant modification of the levels of other bacteria. One attractive approach for achieving this goal is to utilize phages to selectively delete those culprits in the gut. Phages are naturally present in gut microbiota and are important for maintaining the balance of gut microbiota  et al. conferred E. coli Nissle with the ability to express genes encoding phenylalanine-metabolizing enzymes  E. coli, into phenylketonuria model mice reduced the blood concentration of phenylalanine by 38% in comparison with that in the control mice, and the reduction was independent of dietary protein intake. Another advantage of this study was that, hippurate, the final product of phenylalanine excreted in the urine, can act as a predictive biomarker for strain activity. In addition to phenylketonuria, bacteria have been engineered for treatment of cholera, colitis, type 1 diabetes, Pseudomonas aeruginosa infections, obesity, etc., by conferring bacteria with the capability to produce proteins, polypeptides and metabolites exhibiting immunoregulation and blood glucose regulation abilities such as IL-10, CAI-1, bacteriocin and linoleic acid isomerase In contrast to the precise removal of gut bacteria, introducing engineered bacteria into gut, a method similar to the use of probiotics, can also confer beneficial effects to hosts Figure D. UnlikethyA dapA gene, encoding 4-hydroxytetrahydropicolinate synthase, enables biocontainment by rendering the cell wall biosynthesis and growth of the engineered strain SYNB1618 dependent on exogenous diaminopimelate To survive and thrive, the introduced strains have to compete with native gut bacteria that share similar niches. Once the goal of introducing engineered strains into the gut is achieved, the scenario we don't want to see is that the introduced strains profoundly perturbed the original gut microbiota composition, or overexpression of targeted genes. Hence, the ideal introduced strains are those that carry a “switch” that can be used to precisely control the proliferation of strains. Or in an alternative way, their expression of targeted genes can be regulated. To control the proliferation of specific strains, the most commonly used strategy is to engineer the strains to have auxotrophy Figure E. An exaCurrently, microbiome engineering is an exciting and promising approach that can be used for diagnostic and therapeutic applications Clostridium difficile infection; however, single FMT is still not effective in about 10-20% of patients. Treatment with antibiotics can increase the colonization of implanted bacteria, and thus enhance the efficacy of FMT in rats In clinical practice, attention should be paid to the monitoring of biomarkers and the methods of manipulating gut microbiota. The function of gut microbiota and other factors that can shape drug outcomes should be monitored not only before the usage of drugs but also during the drug treatment. This is because the composition and function of gut microbiota are rather dynamic and are highly influenced by many factors such as emotional stress, diet, and exercise Figure . In addiAlthough great progress in the standardization of donor screening, sample preparation, banking and delivery have been made concerning conventional methods such as FMT etc. In contrast to drugs mined outside of the gut that can undergo metabolism by gut microbiota, drugs mined from the gut may bypass the metabolic effects of gut microbiota and thus eliminate the variability of the drug response induced by gut microbiota.Direct mining of drugs from the gut is a promising approach that can minimize the variability of drug responses induced by gut microbiota. The gut microbiota can synthesize numerous natural products that enable microbe-microbe communication and microbial modulation of the host immune, nervous and metabolomic systems. However, the identities and functions of these molecules, for the most part, remain unknown. Currently, a burgeoning field pertaining to gut microbiota is to mine molecules with powerful bioactivities, and this field has generated plenty exciting achievements de novo by gut microbiota and molecules that are transformed from orally administered drugs. In addition to avoiding metabolism by gut microbiota, the drugs mined by this approach possess several other advantages. For example, traditional antibiotics often cause long-term and widespread destruction of the gut microbiota composition and even lead to antibiotic resistance, which may further promote the development and aggravation of diseases There are two types of drugs that can be mined in the gut: molecules that are synthesized


Phylogenetic and comparative genomic studies have revealed complex patterns of alx gene duplications during deuterostome evolution. Remarkably, alx genes have been implicated in skeletogenesis in both echinoderms and vertebrates. In this review, we provide an overview of current knowledge concerning alx genes in deuterostomes. We highlight their evolutionarily conserved role in skeletogenesis and draw parallels and distinctions between the skeletogenic gene regulatory circuitries of diverse groups within the superphylum.Members of the  Biomineralization, the formation of mineral by living organisms, is an exceptionally widespread phenomenon and is thought to have evolved independently and rapidly in many different metazoan phyla through the deployment of a wide range of biomineralization mechanisms and chemistries. Depending on the type and extent of the mineral components, biomineralized tissues are used for structural support, resource acquisition, and protection. There are three predominant classes of biogenic mineral in metazoans: calcium carbonates, calcium phosphates, and silica. The carbonate and phosphate salts of calcium are widely used as skeletal material by vertebrates and invertebrates, while silica biomineralization is prevalent in sponges . The emeWithin the deuterostome superphylum, only vertebrates and echinoderms produce extensive biomineralized skeletal structures. The vertebrate endoskeleton consists primarily of the skull, vertebrae, ribs, and limb bones all of which are composed of matrix proteins  and calcium phosphate crystals. Vertebrate biomineralization is predominantly orchestrated by chondrogenic cells (chondrocytes) and osteogenic cells (osteoblasts and osteoclasts). The vertebrate skeleton is formed during early development by cartilage and/or connective tissue membranes, which are subsequently replaced by bony tissues through the process of ossification. There are two forms of ossification, endochondral and intramembranous ossification. Endochondral ossification is associated with the formation of long bones and requires the presence of a hyaline cartilage template formed by chondrocytes . During via metamorphosis, and these two life history stages bear little morphological resemblance to one another. In some echinoderm clades, specifically echinoids (sea urchins) and ophiuroids (brittle stars), the feeding larva also possesses an intricate and extensive calcitic endoskeleton, which is first laid down during embryonic development and further elaborated after feeding begins. The founder cells of the embryonic skeletogenic lineage, the large micromeres, arise early in development and are specified by a combination of localized maternal factors and unequal cell division. At the mesenchyme blastula stage, the large micromere descendants undergo an epithelial-to-mesenchyme transition (EMT) and ingress into the blastocoel as primary mesenchyme cells, or PMCs  consisting of signaling and gene regulatory pathways that was independently co-opted and fine-tuned for biomineralization in diverse animal taxa. One common regulator of deuterostome skeletogenesis is the Alx transcription factor family, which has been shown to have an ancient, conserved role in this process in both vertebrates and echinoderms. In this review, we examine the current state of knowledge concerning deuterostome alx gene family encodes Paired-class homeodomain transcription factors that contain a highly conserved DNA-binding homeodomain and a C-terminal Otp, Aristaless, and Rax (OAR) domain, features that are shared by many Paired-class homeodomain proteins. Phylogenetic and comparative genomic studies have revealed considerable variability in the number of alx genes in different deuterostomes, pointing to a complex evolutionary pattern of lineage-specific gene duplication and loss  that arose through two duplication events. Through the course of evolution, one of the paralogues, alx3, was lost from amphibian and reptile lineages  are located close to each other in the genome and have very similar intron-exon organizations. Molecular phylogenetic analysis of Alx proteins indicate that Bf-Alx1 and Bf-Alx2 form a monophyletic group, providing further support for the view that they arose from a lineage-specific gene duplication event , a group that includes most of the vertebrate species used for developmental studies. These genes are expressed most prominently in distinct but partially overlapping patterns in neural crest-derived craniofacial mesenchyme and in mesenchyme of the limb bud, both of which are sources of cartilage and bone  and have shown that alx1 expression in this region is under the direct control of the transcription factors Emx2 and Pbx1  in Gallow cattle . Members of the alx gene family may also have other, less well-characterized, developmental functions, although some of the effects of mutations in these genes on non-skeletal tissues are likely to be indirect. In the cranial region, it is well-established that alx-family genes are expressed robustly and selectively by neural crest cells  vertebrates and cephalochordates (amphioxus), animals that possess only cartilaginous skeletons, al crest . Cephaloal crest . Amphioxl stages . At presalx1 is one of the earliest regulatory genes expressed during development, and it plays a pivotal role in specifying the fate of PMCs, the embryonic skeletogenic cells , and expression remains restricted to this cell lineage throughout embryogenesis  cells, which ultimately reform a larval skeleton  and holothuroids (sea cucumbers), and is required and for skeletogenesis in these species  receptor, vegfr-Ig-10, one of the two vegfr genes in sea urchins  and VEGF receptors (VEGFR2 and VEGFR3) are expressed by chondrocytes and chondrogenic cells, and autocrine signaling through this pathway regulates morphogenesis and differentiation  is expressed specifically in chondrocytes  and tissue inhibitors of metalloproteinases (TIMPs). MMPs constitute a class of enzymes that function in the degradation of extracellular matrix (ECM) proteins  family of membrane transport proteins are differentially expressed in the PMCs . In addilc5a11/2 . While t family  family . In vert) family . Fam20C ) family . Mutatioclerosis . In vitral cells , and famnd teeth .otop2L, the single sea urchin ortholog of the vertebrate otopetrin genes , it will be important to learn more about the single alx gene of hemichordates, including its pattern of expression, gene targets, and role in the formation of the small, calcareous skeletal elements of adult hemichordates  and other sox genes in the chondrogenic lineage, and runx2 and osterix in the osteoblast lineage  development, alx1 is co-regulated by Emx2 and Pbx1, but the orthologous echinoderm genes have not been studied in detail.In vertebrates, as noted above, the embryonic lineages of cells in the limbs and limb girdles that express  lineage . Becausealx-related genes in biomineralization is the cranial neural crest. There is agreement that a definitive neural crest is found only in vertebrates, but the evolutionary history of this cell population, particularly the origins of the skeletogenic  compartment, remains a subject of much debate  has been identified  in the anterior, pharyngeal mesoderm of ancient chordates, in cells that produced pharyngeal cartilage (As noted above, in jawless vertebrates and cephalochordates, the expression patterns of volution . With thartilage .alx1; i.e., the acquisition of a new role in robustly mediating biomineralization, as reflected by the direct transcriptional inputs this transcription factor provides into a large fraction of biomineralization effector genes (alx-family genes have not been characterized, and therefore, it is not known whether they include effectors of biomineralization. It should be noted that possible signals of evolutionary conservation between echinoderms and vertebrates in this context would likely be obscured by the well-documented, rapid evolution of many biomineralization-related proteins (alx-family genes in echinoderms and vertebrates initially involved the sharing and/or duplication of cis-regulatory elements among paralogs, as indicated by the overlapping patterns of expression of paralogous alx-family genes in both taxa. The recruitment of duplicated, alx-related genes to a biomineralization-related function would likely have been facilitated if the ancestral gene was already expressed in an embryonic tissue that produced an extensive extracellular matrix, a prerequisite for the assembly and growth of biomineral (alx-family genes and to better reconstruct the evolutionary relationships among those cell lineages.A hypothesis that emerges from these comparative studies is that a rudimentary, ancestral program of chondrogenesis, perhaps deployed in mesenchyme cells derived from embryonic mesoderm, was present in the ancestral deuterostome and provided a suitable gene regulatory system onto which biomineralization-promoting circuitry could be layered. We propose that in echinoderms, gene duplication was followed by the neo-functionalization of or genes . A similproteins . Presumaomineral . In thisJK and CE contributed to the conception of the review and co-wrote the paper. Both the 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.