Based on self-report symptom checklists, 32% of the sample met diagnostic criteria for PTSD (n = 51), 24% for depression (n = 36), and 17% for both disorders (n = 25). Examining these data against the criteria for asylum in the U.S., we found that 70% of the overall sample (n = 159) met criteria for asylum, including 80% of those from El Salvador, 74% from Honduras, and 41% from Guatemala. These findings suggest that the majority of Central American migrants arriving at the U.S. border have significant mental health symptoms in response to violence and persecution, and warrant careful consideration for asylum status.In recent years, increasing numbers of families and individuals have arrived at the U.S. border from Central America, in particular, from Honduras, El Salvador, and Guatemala. This study sought to examine pre-migration trauma exposure and current mental health functioning of migrant families arriving at the U.S. border from the Northern Triangle region, with specific attention to the reasons offered for leaving their home country and the frequency with which migrant families appear to satisfy legal criteria for asylum We interviewed 234 adults in McAllen, Texas, using a structured interview and standardized questionnaires to assess exposure to trauma prior to migration, reasons for leaving their home country and symptoms of posttraumatic stress and depression. We found that 191 participants (83%) cited violence as a reason for fleeing their country, 119 individuals (69%) did not report the events to the police out of fear of gang-related retaliation or police corruption, and 90% ( In 2014, there was an unprecedented surge of migrants from the Northern Triangle countries  to the United States-Mexico border. Although public attention has focused primarily on unaccompanied minors, a comparable number of families arrived as well. In 2014 U.S. Customs and Border Patrol (CBP) apprehended roughly 50,000 unaccompanied minors in the Rio Grande Valley, versus about 20,000 in 2013, and roughly 50,000 family units, compared to only 7,000 in 2013. [Although the mass migration of Central American families has been attributed to gang violence and drug cartels, –5 littleIn response to the dramatic increase in displaced Central American families arriving at the U.S.-Mexico border, the U.S. sharply increased its capacity to detain families as part of its “aggressive deterrence strategy.”  Prior toThe growing number of families being detained and the lack of data on the reasons for increased rates of migration is disconcerting given that prolonged detention appears to contribute substantially to the risk of depression, posttraumatic stress disorder (PTSD) and other mental health disorders in refugees and asylum seekers. –12 For iChildren and families appear particularly vulnerable to the traumatizing, unpredictable environment of immigration detention. Mares et al. interviewed families held in two Australian immigration detention facilities, observing that a lack of opportunities for education and safe play, coupled with parental distress and an undermining of the parental role, places children at a high risk for developmental psychopathology.  Steel etThe present study sought to examine pre-migration trauma exposure and current mental health functioning of migrant families arriving at the U.S. border from the Northern Triangle region. Specifically, we examined the reasons offered for leaving their home country (with specific attention to past traumatic and persecutory experiences) and the nature and severity of psychological distress (particularly depression and PTSD symptoms). In light of public debate regarding the legitimacy of this population as asylum seekers, –14 this Data were collected from a quasi-consecutive sample of adults seeking assistance at the Church of the Sacred Heart in McAllen, Texas. The church, located near the U.S.-Mexico border, provides shelter, food, clothing, and medical care, primarily serving women and families with children that had been apprehended by CBP and released, pending a court hearing to determine whether they had a credible fear of returning to their native country.  All famin = 120, 51.3%) and described their economic status as “poor” . Most participants  had less than a high school education; 71 individuals reported having completed high school (31%), and 25 participants had completed a college education (10.9%).In total, 234 adults participated in the study; 114 from El Salvador (48.7%), 74 from Honduras (31.6%), and 46 from Guatemala (19.7%). The sample included 198 women (84.6%) and 36 men (15.4%), ranging in age from 18 to 62 years old . Most participants were married/partnered (n = 8) included medical doctors, psychologists, lawyers, and nuns, all of whom had previous experience working with an immigrant population. Interviewers were either bilingual (n = 7) or worked with the assistance of a Spanish-speaking interpreter (n = 1). Some participants were unable to complete the entire study, typically because they had scheduled transportation to meet family members living elsewhere in the U.S.Data were collected in private rooms at the church, using a structured interview developed by the study authors , and typA structured interview was developed to gather information about pre-migration exposure to traumatic events and acts of persecution, the extent to which the individual had access to legal recourse or governmental protection from violence and persecution, whether the individual was afraid to return to their home country. After pilot testing, the final version of the interview consisted of 41 questions, including demographic characteristics, reasons for leaving the home country, trauma and persecution events experienced in the home country, and experiences related to the journey to the U.S. and arrival at the border. All interviews were conducted in Spanish. Participants were also informed that their responses to the study interview and questionnaires would have no bearing on their legal status or services provided by the church.In order to ascertain whether migrants from the current sample described experiences that satisfied the criteria for consideration as an asylum-seeker, we developed an algorithm that mapped participant responses to the structured interview onto the core criteria for asylum see . This anTwo measures of psychological distress were administered during this study, the Harvard Trauma Questionnaire (HTQ)  and the Data analysis included descriptive statistics to characterize pre-migration trauma and rates of psychological symptoms, and frequency analyses to examine differences in rates of traumatic events across migrants from the three countries. Pearson product-moment correlation coefficients were used to quantify the association between psychological distress and length of time in ICE detention prior to release. This study was approved by the Institutional Review Board of the New York University Langone School of Medicine.n = 7) cited violence in their community as the primary reason for leaving their country. Overt threats and/or violence were significantly more common among migrants from El Salvador (92%) and Honduras (89%) compared to Guatemala (72%), Chi-Square = 12.55, df = 2, p = .002, phi = .23.p = .006, phi = .21; towards family: Chi-Square = 11.54, df = 2, p = .003, phi = .23. Physical violence towards a family member was also more common among those from El Salvador (45%) and Honduras (49%) compared to those from Guatemala (22%); Chi-Square = 8.78, df = 2, p = .012; phi = .20. On the other hand, rates of severe trauma were more comparable across countries, as the proportion reporting that a family member had been murdered was 34% among those from El Salvador, 37% from Honduras, and 20% from Guatemala; Chi-Square = 4.00, df = 2, p = .14, phi = .13. Likewise, rates of sexual assault were 2%, 4%, and 0% across the three countries ; Chi-Square = 2.36, df = 2, p = .31, phi = .10. Physical violence was also roughly comparable across groups, as 7% of participants from El Salvador reported experiencing physical violence themselves versus 8% of those from Honduras and 2% from Guatemala; Chi-Square = 1.84, df = 2, p = .40, phi = .09.Although the rates of reported trauma were extraordinarily high among the sample as a whole, some traumatic experiences were more common among migrants from El Salvador. For example, 55% of participants from El Salvador reported receiving death threats towards themselves and 58% reported death threats towards a family member. The corresponding rate of death threats was 40% (towards themselves) and 51% (towards another family member) for those from Honduras and 29% (towards themselves) and 30% (towards family) for those from Guatemala; towards self: Chi-Square = 10.20, df = 2, Participants were asked to describe the primary reason for fleeing their native country. The most common reason offered pertained to gang-related violence, reported by 138 individuals (60%). An additional 15 individuals cited domestic violence as their primary reason for fleeing (7%) and another 5 individuals cited other forms of violence (2%). However, when participants were asked about additional reasons for fleeing their country, another 33 individuals (14%) reported violence as the rationale. In total, 191 of 230 participants  indicated violence as a reason for fleeing their native country; of the 204 participants who reported experiencing threats or violence towards themselves or a family member, 182 (89%) cited violence as a reason for fleeing.n = 59; 31%), the police response was typically perceived as ineffective or even detrimental . Common reasons offered for not reporting violence to the police were fears of gang retaliation , concerns about police corruption , or belief that a police report would be ineffective or detrimental . The vast majority of participants (90%) reported being afraid to return to their native country. Based on the asylum algorithm described above (see n = 159) met criteria for asylum-seeking status, with significant differences in the rate across the three countries ; Chi-Square = 22.75, df = 2, p < .001; phi = .32.Most participants indicated that they did not report the problems they had experienced to the police, however of those who did  reporting symptoms indicative of PTSD and 24% (n = 36 of 148 that completed the PHQ-9) for major depressive disorder. Of the 144 individuals that completed both the HTQ and PHQ-9, 25 (17%) reported symptoms that satisfied the criteria for both PTSD and depression whereas 85 (59%) had neither disorder. Rates of PTSD and depression were roughly comparable across the three countries, with PTSD rates of 32%, 34%, and 30% among migrants from El Salvador, Honduras, and Guatemala respectively; Chi-Square = 0.15, df = 2, p = .93; phi = .03. Rates of depression were 27%, 29% and 8%, among individuals from El Salvador, Honduras and Guatemala; Chi-Square = 4.04, df = 2, p = .13, phi = .17. There was no association between length of time in ICE detention and severity of psychological distress .Rates of depression and PTSD were also high among migrants from all three countries, with 32% of participants  of participants had reported the threats or violence they experienced to the authorities and only six of these individuals described police intervention as effective (11% of those that reported the violence to the police). Rather, most participants indicated that they did not make a formal report, as they anticipated that this would either be ineffective or might even worsen the situation. It is not possible to determine whether the perceptions of study participants–that no effective recourse through existing legal channels was available to them–are accurate, but the low rate of effective intervention among the subset that did make reports to the authorities provides some support for this perception.There were a number of additional limitations to the study that should also be considered. First, the potential for distorted responding always exists, particularly given that all participants individuals were seeking entry into the U.S. Although these individuals were informed that their responses were confidential and would not impact their legal status, it is certainly possible that some participants deliberately fabricated or exaggerated the nature of their experiences. However, the relatively low frequency of many of the more “serious” traumatic events, such as physical or sexual assault, suggests that deliberate exaggeration or fabrication of trauma exposure was unlikely. The discrepancies observed across the three countries also support the credibility of these reports, as the three samples had significantly different rates of trauma exposure (presumably reflective of the relative frequency of violence across the three countries). Further, while rates of depression and PTSD were far higher than those found in typical community samples, they were roughly comparable to the rates observed in other studies of trauma-exposed refugees, including samples that had no discernible incentive to exaggerate psychological symptoms. In short, while the veracity of participant reports cannot be assured, these indicators support the credibility of these reports.per se. Finally, the extent to which this sample is representative of all individuals and families migrating to the U.S. from the Northern Triangle region is unknown. Although we are unaware of any systematic basis for transporting families through the study site, the potential for systematic bias in the sample exists.Another important limitation pertains to our analysis of the criteria for asylum. We based the algorithm used in this study on our review of the legal standards, but the extent to which this approach accurately captures these legal standards is largely unknown. Specifically, we did not have the opportunity to “validate” this algorithm by comparing the classification results to actual asylum determinations. Thus, these data are best characterized as an approximation of asylum criteria, rather than a legal standard These caveats notwithstanding, these data demonstrate high rates of trauma and psychological distress, and suggest that roughly two thirds of individuals from the Northern Triangle region of El Salvador, Honduras and Guatemala might meet the legal requirements for asylum in the U.S. Although some discrepancies in this rate were observed across the three countries, the vast majority of migrants from El Salvador and Honduras satisfied these requirements, as did nearly half of the migrants from Guatemala. Coupled with the high rates of severe psychological symptoms, there is little doubt that immigration policies such as the frequent use of detention need to be reconsidered.S1 Table(DOCX)Click here for additional data file.


Species-specific observed relationships are established between cumulative weather severity in autumn-winter and duck population rate of change. Dynamically downscaled projections of weather severity are developed using a high-resolution regional climate model, interactively coupled to a one-dimensional lake model to represent the Great Lakes and associated lake-effect snowfall. Based on the observed relationships and downscaled climate projections of rising air temperatures and reduced snow cover, delayed autumn-winter migration is expected for all species, with the least delays for the Northern Pintail and the greatest delays for the Mallard. Indeed, the Mallard, the most common and widespread duck in North America, may overwinter in the Great Lakes region by the late 21st century. This highlights the importance of protecting and restoring wetlands across the mid-latitudes of North America, including the Great Lakes Basin, because dabbling ducks are likely to spend more time there, which would impact existing wetlands through increased foraging pressure. Furthermore, inconsistency in the timing and intensity of the traditional autumn-winter migration of dabbling ducks in the Mississippi and Atlantic Flyways could have social and economic consequences to communities to the south, where hunting and birdwatching would be affected.Projected changes in the relative abundance and timing of autumn-winter migration are assessed for seven dabbling duck species across the Mississippi and Atlantic Flyways for the mid- and late 21 Waterfowl and their habitats are ecologically, economically, and socially valuable, providing food, income, recreation, and ecosystem stability . It has Because waterfowl are relatively large-bodied migratory birds that number in the millions, they have considerable energy needs during migration and winter . RedistrAnas platyrhynchos) [The autumn migration of dabbling ducks is thought to be controlled by changes in photoperiod; habitat suitability and management; food accessibility; weather severity, including effects from both temperature (thermoregulatory cost) and snow cover; feeding ecology and competition ,12,17–32hynchos) . Waterfohynchos) –45, withhynchos) ,46–48 orhynchos) ,49–50.Anas rubripes), a short-distance migrant, has shown signs of delayed migration and wintering at more northern areas [Anthropogenic climate change, particularly rising air temperatures, longer growing seasons, and diminished snow accumulation at many locations , has alrrn areas ,67,71–74rn areas ,75–78.Anas discors), which is a long-distance migrant that largely responds to photoperiod. In general, the primary migration cue for long-distance migrants  wintering in stable habitats is photoperiod, with decreasing day length synchronizing their circadian and circannual rhythms, while weather cues have a greater impact on short-distance migrants that winter in unstable habitats [Recent studies by ,12,21,23habitats –80. Mosthabitats .st century were based on statistically downscaled climate projections from nine global climate models (GCMs) within the Coupled Model Intercomparison Project Phase Three (CMIP3) and the application of an operational snow accumulation and ablation model. December-January WSI was projected to decline substantially during this century, leading to increased likelihood of delays in the timing and intensity of autumn-winter waterfowl migrations. Besides being restricted to only one dabbling duck species, the previous study was limited because the statistical downscaling approach used to derive snowfall projections did not consider projected changes in water temperature, ice cover, and evaporation for the Great Lakes and thus the earlier projections poorly represented future lake-effect snowfall dynamics [ investigdynamics . This isdynamics  and incrdynamics  and lakedynamics ,85 that dynamics ,87 produst century on the relative abundance and autumn-winter migratory behavior of seven common dabbling duck species across the Mississippi and Atlantic Flyways of North America. While the current study focuses on dabbling ducks as an example of the application of downscaled weather severity projections, the general approach can be more broadly applied to other wildlife investigations.The objective of the current study is to investigate the likely impacts of dynamically downscaled weather severity projections for the mid- and late 21The following summarizes the statistical models developed by  for the Anas americana), the index, WSIMEAN, is introduced and defined as:Anas strepera, RateG), Green-Winged Teal , and Northern Shoveler (Anas clypeata) are as follows:Anas acuta), the WSI index is computed as follows,For the American Black Duck, principal component (PC) analysis of the individual elements of weather severity yields the following index for PC1:rrelated ,90. The Given the quadratic fit of most of the aforementioned population rate formulas, biologically unrealistic values can be generated for PC1 and WSIMEAN by the aforementioned models when WSI is calculated for extremely mild or severe values not within the range of the collected data sample. As a result, positive population rates are assumed in the study region for the American Black Duck when PC1 falls below the threshold of -19 to -15 (depending on latitude) or for the Mallard when PC1 falls below the threshold of -23 to -11, indicative of mild, snow free conditions. Furthermore, negative population rates are assumed for the American Wigeon and Gadwall when WSIMEAN exceeds the thresholds of 85 to 150 and 80 to 185, respectively, indicative of cold, snow-covered conditions.th, mid-21st, and late 21st centuries are limited here to 1980–1999, 2040–2059, and 2080–2099, according to the representative concentration pathway 8.5 (RCP8.5) [The output from six CMIP5 GCMs is dynamically downscaled using the Abdus Salam International Centre for Theoretical Physics (ICTP) Regional Climate Model version four (RegCM4) , interac(RCP8.5) , which i(RCP8.5) –87,96–99Prior to calculating species-specific weather severity indices, RegCM4-simulated output of daily 2-meter air temperature and snow depth is debiased against observational data, both in terms of the daily mean and interannual standard deviation for each day. The source of observed daily mean air temperature for 1984–2013 is the 1-km, gridded Daily Surface Weather and Climatological Summaries (Daymet) product –101. Forst century, ranging from +1.9°C for GFDL to +3.1°C for MIROC5, compared to the late 20th century  by 2.4°C by the mid-21 century . The mear MIROC5 . The uncst century, based on an analysis of the frequency of days within specific mean air temperature bins, between -35°C to -30°C and 20°C to 25°C  during autumn-winter (September through February) is expected to decrease substantially during the 21 to 25°C . During st century, especially in February-March, with declines during the two-month period ranging from -31% in GFDL to -50% in MIROC5 by the late 21st century (not shown). Declines of this magnitude would mean that the Great Lakes are projected to become mostly open water during winter by the late 21st century, with a dramatically shortened ice season. As described by [st century and by -17% in GFDL to -45% in ACCESS by the late 21st century.In response to the aforementioned warming in autumn-winter, ice cover on the Great Lakes is projected to experience pronounced reductions during the 21ribed by , future ribed by ,96 introth century) are simulated by the mid-21st century, ranging from -4.3 days in GFDL to -10.9 days in ACCESS, and of -15.0 days (-48%) are simulated by the late 21st century, ranging from -10.4 days in GFDL to -17.8 days in MIROC5. Within the study region, the zone of most pronounced reductions in the duration of snowpack is identified around 44–45°N, across the Great Lakes region, partly associated with the projected decline in lake-effect snowfall.Due to reduced snowfall and accelerated snowmelt, the mean number of days with at least 2.54 cm (1 inch) of snow on the ground is projected to decline across the Mississippi and Atlantic Flyways . Reductith century  during migration are projected to shift for each of the dabbling duck species. Projected changes in the probability (among 20 years) of negative population rates across the Mississippi and Atlantic Flyways, and thus the tendency for autumn-winter migration, are presented in Figs st century, when averaged across the six models  projected delays in migration are expected for modern-day late (early) migrant species. The Northern Pintail is projected to experience the least delay in migration out of the Great Lakes region 40–50°N), on the order of +12 days and +23 days by the mid- and late 21x models , S5 Fig. century , Fig 5. 0°N, on tMallards . The modst century across the Mississippi and Atlantic Flyways. Based on aerial and ground-based survey data of waterfowl abundance, species-specific empirical relationships are established, specific to the Mississippi and Atlantic Flyways, between the rate of change in abundance of dabbling ducks and cumulative weather severity indices, based on daily mean air temperature, the number of consecutive days with mean air temperature below freezing, daily snow depth, and the number of consecutive days with at least 2.54 cm of snow on the ground. These weather severity indices reflect both energy conservation theory and energy acquisition theory by capturing the impacts of cold atmospheric conditions on the energy expenditure of ducks and of snow cover, lake ice cover, and wetland icing on food availability to ducks [Projected changes in the relative abundance and timing of autumn-winter migration for seven dabbling duck species are investigated for the mid- and late 21to ducks . Of the to ducks ,104–106.th, mid-21st, and late 21st centuries using a high-resolution regional climate model, RegCM4, interactively coupled to a one-dimensional lake model to represent the Great Lakes. This effort expands upon the initial pool of downscaled models presented by [Statistical downscaling can be a powerful and efficient way to translate GCM output to spatial scales that are more relevant for planners and resource managers. However, a key assumption of statistical downscaling is that the drivers of local spatial variation in climate remain unchanged over time. This stationarity assumption has not held true for the Great Lakes Basin, where lake ice cover has rapidly declined in recent decades, leading to enhanced evaporation and lake-effect snowfall –85. Pastented by ,87.st century across the Mississippi and Atlantic Flyways, including substantial mean warming during autumn-winter, fewer days below freezing, and diminished lake ice cover, lake-effect snowfall, and snow depth. The most pronounced reductions in the number of days with snow on the ground is identified around 44–45°N, across the Great Lakes region, consistent with the findings of [th century climatological near-surface air temperature for December-March, such that as the isotherm shifts northward in the future, projected trends in declining snowfall accelerate in this zone. Further to the north, mean temperatures remain low enough, even later this century, for much of the wintertime precipitation to still occur as snow and for the snowpack to persist. From a climatological perspective, we recommend that future work expand the number of applied RCMs, to better capture the spread of climate change uncertainty for the study region, and couple the RCM to a three-dimensional lake model, in order to represent the circulation of deep lakes and reduce biases in lake temperatures, timing of stratification, and ice cover.Dramatic reductions in weather severity are projected for the mid- and late 21dings of . As discdings of , this zost century, overwintering in the Great Lakes region may become increasingly likely for the American Black Duck and Mallard. This appears to be supported by the study of [The projected reduction in weather severity should lead to delayed autumn-winter migration for all seven dabbling duck species, with results suggesting that the delays will be least for the Northern Pintail and greatest for the Mallard . By the late 21study of , which nstudy of , which lstudy of . Projectstudy of .Large economic losses might be expected for the southern flyway states due to diminished hunter and bird watching opportunity, especially because the most common species of duck in North America is projected to overwinter in some years in the Great Lakes Basin. This could reduce the number of migrating waterfowl in more southerly states significantly. As emphasized by , in lighS1 Figst and late 21st century, compared to the late 20th century (fourth and fifth columns). Results are shown for CNRM, MIROC5, IPSL, MRI, ACCESS, and GFDL, from top to bottom row.The probabilities are shown for each day from 1 September through 31 March, with the x-axis labeled as days since 1 September. Projected changes in these probabilities are shown for the mid-21(EPS)Click here for additional data file.S2 Figst and late 21st century, compared to the late 20th century (fourth and fifth columns). Results are shown for CNRM, MIROC5, IPSL, MRI, ACCESS, and GFDL, from top to bottom row.The probabilities are shown for each day from 1 September through 31 March, with the x-axis labeled as days since 1 September. Projected changes in these probabilities are shown for the mid-21(EPS)Click here for additional data file.S3 Figst and late 21st century, compared to the late 20th century (fourth and fifth columns). Results are shown for CNRM, MIROC5, IPSL, MRI, ACCESS, and GFDL, from top to bottom row.The probabilities are shown for each day from 1 September through 31 March, with the x-axis labeled as days since 1 September. Projected changes in these probabilities are shown for the mid-21(EPS)Click here for additional data file.S4 Figst and late 21st century, compared to the late 20th century (fourth and fifth columns). Results are shown for CNRM, MIROC5, IPSL, MRI, ACCESS, and GFDL, from top to bottom row.The probabilities are shown for each day from 1 September through 31 March, with the x-axis labeled as days since 1 September. Projected changes in these probabilities are shown for the mid-21(EPS)Click here for additional data file.S5 Figst and late 21st century, compared to the late 20th century (fourth and fifth columns). Results are shown for CNRM, MIROC5, IPSL, MRI, ACCESS, and GFDL, from top to bottom row.The probabilities are shown for each day from 1 September through 31 March, with the x-axis labeled as days since 1 September. Projected changes in these probabilities are shown for the mid-21(EPS)Click here for additional data file.S1 File(NC)Click here for additional data file.S2 File(NC)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S4 File(NC)Click here for additional data file.S5 File(NC)Click here for additional data file.S6 File(NC)Click here for additional data file.S7 File(NC)Click here for additional data file.S1 TableHere, the data includes the day since 1 September, daily mean air temperature (°C), daily snow depth (cm), four components of weather severity , the PC1 index, and computed population rate of change for Mallards. Grey shading indicates conditions favorable for declining populations related to migration. It might be expected that Mallards would migrate south from this location around early December that year.(DOCX)Click here for additional data file.


Nonalcoholic fatty liver diseases (NAFLD) is the major cause of hepatocellular carcinoma and increases the risk of mortality. Understanding the trends of its clinical and biochemical changes is essential to identify patients with NAFLD that are at the greatest risk of nonalcoholic steatohepatitis (NASH) and cirrhosis in Iran. Patients with NAFLD confirmed by ultrasonography were enrolled into the current study. They had negative serologic markers of viral or autoimmune hepatitis, no findings in favor of metabolic liver disease, and had not received medications that affect liver, such as silymarin and Ursobil. Biochemical and clinical symptoms and histological variables were evaluated for each patient. Descriptive statistics were used to compute all variables.A total of 206 patients, including 109 male and 97 female, with the mean age of 41.2 years were enrolled. The number of patients without obesity and diabetes were 34 (16.4%) and 48 (23.1%), respectively. Sleep disorder, delayed sleep, daytime sleepiness, and late dinner were noticeably common in patients with NAFLD. Furthermore, anxiety, thirst sensation, bloating, warming sensation, defecation disturbances, and upper abdominal pain were common among patients with NAFLD. NAFLD is a heterogeneous disorder with vast clinical presentations. It seems that anxiety and gastrointestinal problem are common among such patients. Moreover, inadvertent sleep could have a considerable effect on developing NAFLD. Patients with diabetes have more severe NAFLD, based on clinical and histological findings. NASH) is part of a broad range of common, chronic, and progressive nonalcoholic fatty liver disease (NAFLD). NAFLD is a clinical term indicating the deposition and accumulation of fat in liver. More than 5% of liver weight has hepatotoxic effects and could cause liver damage. The term NASH was introduced for the first time by Ludwig in 1980 for the females with obesity, without history of high intake of alcoholic beverages  was calculated according to weight (Kg) divided by the square of height (meters). Waist circumflex (WC) was measured at midway between the uppermost border of the iliac crest and the lower border of the costal margin (rib cage). The quality and quantity of sleep were also asked from the patients. The quality of sleep was evaluated and scored between 0 and 10, in which <3= bad, 3 to 5= not good, 6 to 8= good, and 9to 10= very good. In this context, the falling asleep time less than 15 minutes, staying asleep without medication no obvious cause of sleep interruptions such as sleepwalking, apnea or night terrors, no feels of light sleep, fragmented or unrefreshing sleep, and sleeping during 10:00 PM to 4:00 AM were considered. The quantity of sleep was defined as the hours of sleeping during the night. The intensity of gastrointestinal symptoms, including abdominal distention and bloating, were evaluated and scored from 0 to 4, in which 0= absent, 1= mild, 2= moderate, 3= severe, and 4= intolerable.Liver function tests including, alkaline phosphatase (ALP), serum albumin, aspartate aminotransferase (AST), bilirubin, alanine aminotransferase (ALT), platelet count, international normalized ratio (INR), total cholesterol (Chol), triglycerides (TGs), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and hemoglobin A1c (HbA1c) were assayed by central laboratory of Firouzgar Hospital. The presence of diabetes mellitus (DM) and hypertension were defined based on American Diabetes Association (ADA) (11) and the Joint National Committee (JNC) criteria (12). Blood pressure was measured in 3 positions . Insulin resistance was assessed with homeostatic model assessment—insulin resistance (HOMA-IR) based on the following formula: [fasting glucose (mg/dL) × insulin (μU/mL)] divided by 405.Statistical analysisThe data were analyzed by SPSS software for windows . Descriptive statistics was employed to measure all variables; data were reported as means ±standard deviation (SD) for continuous variables or frequencies, and percentages for categorical variables. Ethics ApprovalThis study protocol was approved by the Ethics Committee of Iran University of Medical Sciences, Tehran, Iran, with ID number: 93038626182 in accordance with the declaration of Helsinki. The procedure was explained to all the participants and a signed written informed consent forms was obtain. During the intervention, 206 patients were enrolled into the study. The mean±SD age of the cases was 41.2± 8.3 years, and 109 (52.7%) cases were male. The mean BMI was 31±1.5; also, 34 patients (16.4%) had not obesity and 48 (23.1 %) had diabetes mellitus. The basic characteristics of the patients are summarized in NAFLD, nonalcoholic fatty liver disease; FBS, fasting blood sugar; ALT, alanine aminotransferase; AST, aspartateaminotransferase; HOMA-IR, homeostatic model assessment—insulin resistanceRegarding the clinical presentations, the most common symptoms were upper abdominal pain, fatigue, thirst, and anxiety. The common clinical manifestations are illustrated in NAFLD is defined as a condition where ≥5% to 10% of hepatocytes exhibit macroscopic steatosis by light microscopy in the absence of other etiologies of liver disease. NAFLD is one of the most common causes of chronic liver disease. The results of the current study showed how the clinical symptoms of NAFLD differ among patients. The severity of clinical and biochemical NAFLD presentations is under investigation , 14. BasRegarding the symptoms, although the patients are usually nonspecific, fatigue is the common symptom. However in clinical practice, the study faced many patients with vast clinical complaints. On the other hand if human is considered as a whole; hence, manifestation of liver disease may present differently. Therefore, concerning the fact the patients were examined, which resulted in interesting clinical findings. In the present study, anxiety, thirst, feeling of changing temperature, and bloating were the most common complaints. Disguise and upper abdominal pain, particularly in the left upper quadrant, were also a common complaint. The upper abdominal pain may have sharp or dull quality. The complaint of frequent bloating, disguise, thirst, and headaches were almost present recently, before medical consultation and were usually treated as dyspepsia and reflux disease. Sleep disorder and late onset of sleeping were noticeable. Recently, many attempts are made to evaluate the effects of sleep quality and quantity in the development, and progression of inflammation and chronic diseases .This is almost a new issue in NAFLD, but it has a long history in the Iranian traditional medicine; as it is indicated in major reference books. Based on the Iranian traditional medicine, the best time to sleep is 10:00 PM to 4:00 AM, which is almost comparable with solar circadian. In fact, many of the patients in the current study stayed up late at night and woke up late in the morning. Brensmeier et al., reported a case–control study in which short sleep duration, delayed onset, and poor quality of sleep were common among patients with NAFLD. They also confirmed the association of NAFLD with late dinner. The association between sleep disturbances and dinner time needs further studies , 17. PreIt is noteworthy that, in the current study the majority of subjects were male. Nevertheless, in many studies females were predominant; while, recent studies revealed the antecedence of male in this condition and the current results were comparable with those of them. This could be related to life style and dietary changes and the fact that males are more susceptible to the same risk factors. In addition, some studies illustrated that stress may be involved in NAFLD progress and males are prone to daily stress , 20, 21.Blood pressure is considered as a manifestation of metabolic syndrome and NASH. In the current study, the mean blood pressure was about normal 125/83 mmHg). An exact cannot be offered, but it could be due to the limited number of participants. In former studies, the presence of DM along with other factors, such as higher BMI, hypertension and older age, were associated with severity of NAFLD/NASH . In the  mmHg. AnIn conclusion, NASH could be presented with different clinical manifestation as long time before the diagnosis .Although fatigue is a common complaint of patients, the consultant must consider other related symptoms. Furthermore, it seems that NASH is not actually a female prominent condition. Moreover, poor quality of sleep and sleep disorder could be considered as the risk factors that should be investigated.


The pH of the tumor microenvironment drives the metastatic phenotype and chemotherapeutic resistance of tumors. Understanding the mechanisms underlying this pH-dependent phenomenon will lead to improved drug delivery and allow the identification of new therapeutic targets. This includes an understanding of the role pH plays in primary tumor cells, and the regulatory factors that permit cancer cells to thrive. Over the last decade, carbonic anhydrases (CAs) have been shown to be important mediators of tumor cell pH by modulating the bicarbonate and proton concentrations for cell survival and proliferation. This has prompted an effort to inhibit specific CA isoforms, as an anti-cancer therapeutic strategy. Of the 12 active CA isoforms, two, CA IX and XII, have been considered anti-cancer targets. However, other CA isoforms also show similar activity and tissue distribution in cancers and have not been considered as therapeutic targets for cancer treatment. In this review, we consider all the CA isoforms and their possible role in tumors and their potential as targets for cancer therapy. Since their first discovery in 1933, CAs have been at the forefront of scientific discovery; from basic enzymology, to the application of structural biology and in silico approaches to study protein dynamics. In addition, CAs have been shown to be important in drug discovery and clinical medicine. CAs comprise a family of metalloenzymes that catalyze the reversible hydration of COfixation . The moslciparum . 2 reactions in many physiological processes. In humans, 15 CA isoforms are encoded. Among these, only 12 coordinate a zinc in the active site making them catalytically active . Isoforms CA VIII, X, and XI are termed CA-related proteins (CA-RPs) as they lack the required metal ion within the active site [The human CAs (α-class) share the same 3D tertiary structure, but differ in sequence . Furtherive site ,7. Whileive site ,9. Aminoive site ,7,10,11.ive site ,14. CA Iive site ,17,18, wive site , the memive site ,21, CA Xive site , and CA ive site ,24, and ive site ,26,27 weive site , whereasive site .+ retention, calcification, bone resorption, signal transduction, electrolyte secretion, gluconeogenesis, ureagenesis, and lipogenesis. Due to their broad roles in metabolism, CAs have served as therapeutic targets for several diseases, including glaucoma and epileptic seizures, altitude sickness, and more recently in the treatment of obesity and pain [2 gas exchange in artificial lungs [The α-CAs are involved in many physiological processes including respiration, pH regulation, Naand pain ,31,32. Iand pain ,39,40,41al lungs ,45,46,47al lungs . The kinOf the 15 CA isoforms expressed in humans, only CA IX and CA XII have been implicated in cancer. These enzymes are transmembrane proteins in which their extracellular domain contains the catalytic activity, positioning them in the regulation of the tumor microenvironment. CA IX is of particular interest because of its high expression in solid tumors while exhibiting low expression in normal tissues ,48,49,50Because of the established roles that both CA IX and CA XII play in the process of tumorigenesis, cancer cell signaling, tumor progression, acidification, and metastasis, many classes of CA IX/CA XII-targeted inhibitors and biologics have been studied in the preclinical setting. These studies have yielded promising results showing that inhibition of CA IX/CA XII catalytic activity decreased the growth, proliferation, and metastatic potential of several aggressive cancers both in vitro and in vivo ,53,54,55In this review, the current understanding of CAs role in tumor physiology and pH regulation is discussed. The expression and function of each CA isoform in both normal and tumor cells/tissues, as well as some of the commonly found mutations in tumor specimens, will be discussed. The goal of this review is to provide a comprehensive overview of the CA family and their combined role in cancer and current anti-cancer therapies.e and pHi, respectively). In part, this is postulated to be due to the over production of lactate because of high glycolytic rates and inhibition of pyruvate decarboxylation in the mitochondria. Export of metabolic acids ultimately lowers pHe [Primary tumors are often described as heterogeneous, in that cells of different types, metabolic states, and stages within the cell cycle can exist at any given time-point ,66. Thiswers pHe . i and pHe such that the extracellular space maintains a slightly more basic environment (pHe ≥ 7.3) relative to the intracellular environment (pHi = 7.2) [e drops to values ranging from 6.5–7.1 with only a marginal decrease in pHi (≥ 7.2) [e becomes favorable for extracellular matrix (ECM) remodeling, limits HCO3− dependent dynamic buffering, and induces acid activation and expression of proteases, resulting in the facilitation of tumor cell dissemination and invasion. Additionally, the slight decrease in pHe favors tumor cell proliferation, metabolic adaptation, migration pathways, and results in evasion of apoptosis. This ultimately sets up conditions that benefit tumor cell survival and proliferation, resulting in an unfavorable prognosis for cancer patients [i, when compared to the more acidic pHe, favors flux through glycolysis and inhibition of gluconeogenesis (mostly in the liver and pancreas) [i ≥ 7.2 stimulates lactate dehydrogenase (LDH) activity, which has an in vitro pH optimum of ~pH 7.5. This enzyme mediates the conversion of pyruvate to lactate and regenerates NAD+, which is required for continued glycolytic activity [i increases expression of several glycolytic enzymes, thus contributing to the high rate of observed glycolytic activity within the tumor cell. Alternatively, a lower pHi (<7.2) will reverse these conditions and decrease expression of glycolytic enzymes such as LDH, and transporters like GLUT1 [In most normal cells, a pH differential is maintained between pHi = 7.2) ,78,79. T (≥ 7.2) ,78,80. Tpatients ,76,78,81ancreas) ,83. Specactivity ,85. Furtke GLUT1 ,87. e is caused by the rapid extrusion of lactic acid and free protons from tumor cells, resulting from the upregulation of glycolysis in the cytosolic compartment. It has been postulated that glycolytic enzymes cluster at the inner surface of cell membranes and interact with ion and proton transporters at the cell surface. This allows for a rapid transport of protons both in and out of the cell depending on the shift in metabolic equilibrium achieved within the cellular microenvironment [e establishes a favorable environment for cell metastasis and invasion [e enhances expression and activities of ECM reorganizational proteases, such as matrix metalloproteinases (MMPs) and cathepsin B [i ≥ 7.2 creates an environment that favors de novo actin filament formation through the expression and activation of actin-binding proteins such as cofillin, villin, profilin, twinfilin, and talin [The decrease in pHironment ,89. In ainvasion . Specifihepsin B ,92. In cnd talin ,96,97,98nd talin ,96,97,98i ≥ 7.2, there is an increase in the activity of CDKs, specifically CDK1, which increases the efficiency of MAPK pathways [i suppresses DNA damage checkpoints that would typically slow the progression of a cell through the G2/M phase and restrict proliferation [i ≥ 7.2 favors a suppression of apoptotic pathways [i = 7.2), a reduction in pHi to < 7.2 would result in a conformational change in the pro-apoptotic factor, BAX, causing its activation and interaction with the mitochondrial membrane [i becomes slightly more alkaline, these pathways are suppressed, allowing the tumor cells to resist apoptosis [i ≥ 7.2, which is the case in most tumor cells, there is a high probability that this anti-apoptotic pH level will be maintained, even in cases where there may exist a small influx of protons. Taken together with pH-induced ECM and metabolic transitions, it is clear that the unique pH differential across membranes drives tumor cell proliferation and survival. This unique pH profile in tumor cells also permits cancer cell proliferation through bypassing cell cycle check points and evasion of apoptotic pathways ,100. Whepathways . This stpathways . In addiferation . In combpathways . In normmembrane . This inIf we can understand the complex factors that establish the unique pH environment of tumor cells, it may be possible to develop therapeutics strategies that can reduce or inhibit these factors to re-normalize pH. In recent years, the carbonic anhydrase family has been shown to create and maintain the pH differential in tumor cells . EvidencIt is widely accepted that CAs are the driving force in pH regulation in primary tumor cells as they are for normal tissue. These enzymes function, through their hydratase/CA activity, to regulate the production of bicarbonate, the universal physiological buffer. Along with this, CAs produce or sequester protons. In addition to their hydratase activity, CAs also have a slower esterase activity, which is mediated by the same catalytic pocket with a mechanism similar to that of the hydratase/CA activity . Thus, mHypothesis 1 .2 present in the extracellular space, and convert this to HCO3− and protons. Adjacent ion transporters, such as anion exchangers (AE1–AE3), can then traffic HCO3− [e. Inside the cell, the newly imported HCO3− can be converted back to CO2, which can be used in metabolic pathways or diffuse back outside of the cell carrying that sequester proton. This restores pHi to more normal levels (e decreases (<6.5). Originally, it was proposed that CA IX, one of the most prevalent extracellular CAs in hypoxic tumors, retained its activity in acidic and basic environments through the use of an ‘internal buffer’, which exists as an extended N-terminal domain, called the PG domain [. The PG domain of CA IX has a large quantity of aspartate residues [This hypothesis suggests cooperative activity between cytosolic (such as CAs I and II) and extracellular CAs  that ‘cycle’ substrates for pH regulation between the extracellular and intracellular tumor environment. Specifically, extracellular CAs take advantage of the large quantity of COic HCO3−  into thel levels A [30,57,G domain . The PG residues . It is tresidues ,112. Recresidues .3− can be readily transported into the cell rather than diffuse away. This is supported by recent evidence showing that glycolytic enzymes, in association with CA, cluster near transporters at the cellular membrane [The above hypothesis also relies on the ability of CA IX (or other membrane-associated CA) to cluster close enough to bicarbonate transporters, such that newly synthesized HCOmembrane ,114,115.i > 150 mM) representing an evolutionary adaption of the enzyme to harsh conditions such as high lactate levels, increased acidity and hypoxia [i of 1.12 mM) more potent than HCO3− (Ki of 13 mM) where as its reduced form, under hypoxic conditions, is not [2 hydration activity in tumors exposed to more harsh conditions than in the physiological setting. Another factor that must be taken into account is extracellular lactate concentration and its effect on CA IX activity. In 2001, Innocenti et al. showed that CA IX has low sensitivity to inhibition by lactate .e and produce substrate (HCO3−) for cytosolic CAs, but instead buffer pHe to levels (between 6.5–7.1) that promote tumor cell survival and avoid tumor necrosis. In this case, the tumor cell has begun to transition to a hypoxic state and its metabolic shift toward glycolysis has taken place. This causes a rapid extrusion of lactic acid and a drastic reduction in pHe, which has been recorded at levels as low as 5.5. At this pH, the cell would soon undergo necrosis resulting in its death. But we know that cancer cells continue to survive and proliferate within the acidic microenvironment. It is postulated that the extracellular CAs act to sequester protons in the form of CO2 to raise and maintain the pHe to a level that favors tumor cell growth, proliferation, and survival  and preserve the core, folded structure (as low as pH = 2.0). Both of these attributes are necessary for the enzyme to stabilize pHe in an acidic environment. Sweitach et al. has also suggested that the net effect of CA IX on pHe will depend on the emission of CO2 relative to lactic acid [i (~6.3) and pHe was measured at pH ~ 6.9 at the core [i (~6.6) was observed and extracellular acidity was increased (pHe ~ 6.6) [e but the direction of the reaction is ultimately dependent on substrate availability, i.e., lactate levels and the pH of the tumor microenvironment.This hypothesis is similar to Hypothesis 1, in that both extracellular transmembrane CAs and cytosolic CAs play a role in tumor cell pH regulation. However, in this alternative hypothesis, it is proposed that extracellular CAs do not necessarily lower pHsurvival B. This hn et al.  These sttic acid . In theitic acid . When CAthe core . Howevere ~ 6.6) . While te in tumors. Furthermore, this result suggests that the presence of the PG domain of CA IX has limited involvement in pH regulatory processes, in contrast to previous thought. If it is true that extracellular CA IX acts to regulate pH through sequestering a single proton via a His residue then several questions arise: (1) what is the maximum pHe regulating potential of all extracellular CAs on the tumor cell surface? And (2) is this significant enough to contribute to the pH differential observed in tumor cells, which would validate this hypothesis? To answer these questions, the pH regulating potential, or change in pHe (∆pHe), induced by an extracellular CA acting as a proton-sequestering agent can be estimated. Consider that the concentrations of CA IX and/or XII dimer (common extracellular CAs in tumor cells) is ~19 nM for a single hypoxic tumor cell with a diameter = 20 µm and an initial pHe of 6.5. In addition, assume that a monomer of extracellular CA can sequester protons in a ~1:1 ratio via a single active site histidine. With these assumptions, the following expression can be used to establish the relationship between pH and proton concentration in a solution: Recent evidence found by Jamali et al.  has sugge represents the initial pH of the extracellular environment (in this case is 6.5). Considering only these parameters, the total quantity of extracellular CA in a single tumor cell contributes an overall Here, unctions , this co+ + H2CO3− → CO2 + H2O) over hydration (CO2 + H2O → H+ + H2CO3−), while the opposite is expected to happen at much higher pH [2O ~ 6.3), work not only to prevent the unfavorable acidification of the tumor microenvironment, as a result of the metabolic switch, but to “adjust” the pHe to favor tumor growth, progression, and eventually metastasis. The role of cytosolic CAs in both hypotheses remains similar. Cytosolic CAs will utilize available substrates in the form of CO2, water, HCO3−, and protons, allowing them to buffer the intracellular environment to maintain the slightly alkaline pH that is observed. It is also important to note that CAs catalyzes a reversible reaction and this reaction depends on substrate/product availability. Hypothesis 2 takes this bi-directionality into account, hence the buffering capacity of extracellular CAs. For instance, at pH values lower than ~6.8, CA IX has been shown to favor its dehydration reaction  isoforms include CA I, II, III, VII, VIII, X, XI, and XIII ,48. Becatabolons ,134,135.High levels of cyt-CAs I and II expression are found in red blood cells and are necessary for maintaining physiological pH of the blood. It is suggested that CA II activity is the more dominant because of its greater expression and increased catalytic efficiency (in vitro) compared to CA I, although the intracellular environment might dictate how these enzymes behave in vivo . In addiThe potential of CA I as a tumor-associated isoform has not been extensively studied. However, according to multiple genomic databases including the cBioPortal of the cancer genome atlas (TCGA) and the human protein atlas, medium to high levels of CA1 mRNA was detected in acute myeloid leukemia, colorectal cancer, and renal carcinoma patients, based on RNA sequencing ,141,142.CA I has also been shown to be upregulated in human pancreatic cancer (PDAC) where its expression correlated with tumor de-differentiation. Biopsies of patients with PSA positive gray-zone levels (serum prostate-specific antigen levels ranging from 4 to 10 ng/mL is considered a diagnostic gray zone for detecting prostate cancer) also tested positive for plasma CA I . This suUnlike CA1, detection of CA2 mRNA expression using RNA sequencing showed a more widespread upregulation in cancers. These include but are not limited to prostate, melanomas, bladder, thyroid, breast, lung, liver, pancreas, gliomas (with the highest expression observed in glioblastomas), renal cell carcinomas, and head and neck cancers ,142. LikIn normal cells, CA III expression has been detected in both skeletal muscles and adipose tissues (both white and brown fat), with medium expression also observed in the breast ,137,152.There are few published studies that have focused on the expression and function of CA III and CA VII in tumors. However, according to the RNA sequencing and IHC data deposited to the TCGA and human protein atlas respectively, CA3 mRNA transcripts were observed in multiple cancers, with the highest median expression in glioblastomas and thyroid cancers ,141,142.The median expression for CA7 mRNA transcript in tumors is much lower than the previously mentioned isoforms, with the highest expression observed in thyroid carcinoma, colorectal adenocarcinoma, and lower-grade brain gliomas ,142. In A study published by Kuo et al. discovered reduced levels of CA I, II and III in human hepatocellular carcinoma (HCC) compared to adjacent normal tissue. In 2008, a study showed that CA III expression promotes the transformation and invasive capacity of hepatoma cells through the focal adhesion kinase (FAK) signaling pathway . In this5) comparable to CA I  in response to cyclic adenosine monophosphate (cAMP) activates CA IX activity, under hypoxic conditions . EpidermLike CA IX, CA XII expression has also been shown to be upregulated in many tumor types. However, its expression is also abundant in normal tissues. The most abundant cancer-related CA12 expression, according to RNA sequencing datasets deposited in the TCGA database, was observed in renal cell carcinomas, colorectal, breast, bladder glioblastomas, and head and neck cancers ,142. TheOne current research interest in the field of CA drug discovery is enzymes’ role in cancer, either as a prognostic marker or as a potential drug target. To date, experimental evidence has indicated an important relationship between pH regulation and tumor cell proliferation and survival. Many of these studies show direct involvement of CA in these processes, marking them as important avenues for pH-disruptive anti-cancer treatments. This is particular true in tumors that have undergone hypoxic transition, where CAs establish pH gradients that favor cancer cell growth at the expense of adjacent normal cell (and patient) survival. Of the 15 human CAs, however, attention has only been directed toward two isoforms, CA IX and XII. Of course, this has been fruitful as inhibitors are now in clinical trials that specifically target each isoform. Despite this achievement, the roles of the remaining CAs have only recently been considered in cancers. This area of research has strong potential as many of the human isoforms, beyond CA IX and XII, have shown an association with tumorigenesis in primary cancers. With much still to understand in terms of the “combined role” of CAs in different cancer types, it will be important moving forward for researchers to consider the effects beside those of CA IX and XII. As the search for viable and novel treatments against the most aggressive and detrimental cancers continues, priority should be placed on deciphering the roles of cancer-related CAs.


Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum constitutes a broad range of organisms with an intriguing array of ultrastructural morphologies, including intracellular membranes and compartments and their corresponding complex genomes encoding these forms. The phylum Chlamydiae are all obligate intracellular bacteria and, although much is already known of their genomes from various families and how these regulate the various morphological forms, we know remarkably little about what is likely the deepest rooting clade of this phylum, which has only been found to contain pathogens of marine and fresh water vertebrates. The disease they are associated with is called epitheliocystis; however, analyses of the causative agents is hindered by an inability to cultivate them for refined in vitro experimentation. For this reason, we have developed tools to analyse both the genomes and the ultrastructures of bacteria causing this disease, directly from infected tissues. Here we present structural data for a member of the family Ca. Similichlamydiaceae from this deep-rooted clade, which we have identified using molecular tools, in epitheliocystis lesions of gilthead seabream (Sparus aurata) in Greece. We present evidence that the chlamydial inclusions appear to develop in a perinuclear location, similar to other members of the phylum and that a chlamydial developmental cycle is present, with chlamydial forms similar to reticular bodies (RBs) and elementary bodies (EBs) detected. Division of the RBs appeared to follow a budding process, and larger RBs with multiple condensed nucleoids were detected using both transmission electron microscopy (TEM) and by focused-ion beam, scanning electron microscopy (FIB-SEM). As model hosts, fish offer many advantages for investigation, and we hope by these efforts to encourage others to explore the biology of fish pathogens from the PVC.The  Lepomis machrochirus)   and barramundi   in a Ugandan lake system  used as cleaner fish in salmon farms off the Norwegian coast    larvae , we identified two species of a novel beta-proteobacterial genus, Candidatus Ichthyocystis, as the main pathogens  was carried out in Selonda SA farms in Saronikos  and Argolida , as previously described  and sterile sea water and sent chilled by overnight courier to Switzerland. All samples were kept chilled and further processed immediately upon arrival.Sampling of juvenile gilthead seabream  staining.For electron microscopy, formalin-fixed gill tissues were post-fixed in a mixed solution of 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.5 at 4°C overnight. Samples were prepared for TEM by embedding into epoxy resin according to standard procedures  using the FIBICS Nanopatterning engine . The gallium-ion beam for milling was used at 30 kV, 600 pA current and the images were acquired at an acceleration voltage of 1.5 kV using an in-lens energy selective backscattered electron detector (ESB) with a grid voltage of 1.3 kV. The resolution was set to 5 nm in the XY axes and 5–10 nm in the Z axis. The image stacks were aligned with TrackEM2  for 4 min. Following addition of 50 ng probe per slide in a hybridization buffer comprising 6 × SSC, 5 × Denhardt's solution, and 12% dextran sulfate, samples were denatured at 90°C for 4 min and hybridized at 48°C overnight. A wash with 2 × SSC at 48°C was followed by manual post staining with 4′-6-diamin-2-phenylindole (DAPI) at 10 μgml−1 to visualize bacterial and host DNA for 10–30 min.FISH was performed on the Ventana Discovery XT automated platform using the probe shown in Table FISH and HE sections were scanned on a Hamamatsu Nanozoomer 2.0 HT scanner for an overview of the section, and high resolution imaging was performed on the Leica SP5 resonant confocal laser scanning microscope. Deconvolution was performed using Huygens  and 16 samples from Argolida (2013) were screened for the presence of chlamydial sequences. All samples had visible epitheliocystis lesions under histopathological examination, although infection intensities were higher in Argolida fish. The majority of the cysts were attributed to Chlamydiae-specific primers  over 1,075 bp from Norwegian ballan wrasse , indicating that they are likely to be distantly related members of this genus  multiply to fill the inclusion before condensing to form EBs, which are released from the cell to initiate a fresh infectious cycle. Both the Chlamydiae and members of the Planctomycetes lack the tubulin homolog FtsZ which is involved in forming the septum regulating the binary fision of most bacterial cells, raising the question as to how this process is regulated. In FtsZ-less members of the Planctomycetes this occurs by a polar budding of bodies from a larger intermediate form , all members of which have only been found as intracellular bacterial pathogens of marine or fresh water vertebrates, indicative of their early evolutionary origins and specialization for an aqueous environment  densely packed particles Figure , resemblChlamydiaceae RBs undergo binary fission, as most other bacteria, prior to transformation into the infectious particles or EBs. This may not be the case. As we were preparing this manuscript, an elegant study was forthcoming, indicating that at least with Chlamydia trachomatis, EBs are generated by polar budding from RBs, possibly also containing multiple chromosomes (Abdelrahman et al., Ca. Similichlamydia (Figure It was long thought that the a Figure . This stChlamydia and how the members of this phylum share their origins within the PVC superphylum. Detailed morphological studies are critical to these efforts (Draghi et al., Indeed, a concerted effort to analyse the genomes of this deep-rooted chlamydial clade would provide an invaluable insight into the essence of what makes a Conceived and designed the experiments: AF, HSS, JM, MR, ND, PK, and LV. Performed the experiments: AF, HSS, JM, LN, MS, MR, ND, PK, and LV. Analyzed the data: AF, HSS, JM, LN, MR, MS, PK, and LV. Contributed reagents/materials/analysis tools: HSS, JM, MS, ND, and PK. Contributed to writing the manuscript: HSS, JM, MR, PK, and LV.This work was supported by the European Union through Marie Curie Intra-European Fellowship grant number 332058 to HSS and an FP7 Aquaexcel-TNA project 01-05-15-0004-B to LV and PK. AF was partly supported by SNF project 310030_138533 to LV. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.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.


Herbal supplements are a significant source of drug-drug interactions (DDIs), herb-drug interactions, and hepatotoxicity. Cytochrome P450 (CYP450) enzymes metabolize a large number of FDA-approved pharmaceuticals and herbal supplements. This metabolism of pharmaceuticals and supplements can be augmented by concomitant use of either pharmaceuticals or supplements. The xenobiotic receptors constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) can respond to xenobiotics by increasing the expression of a large number of genes that are involved in the metabolism of xenobiotics, including CYP450s. Conversely, but not exclusively, many xenobiotics can inhibit the activity of CYP450s. Induction of the expression or inhibition of the activity of CYP450s can result in DDIs and toxicity. Currently, the United States (US) Food and Drug Administration does not require the investigation of the interactions of herbal supplements and CYP450s. This review provides a summary of herbal supplements that inhibit CYP450s, induce the expression of CYP450s, and/or whose toxicity is mediated by CYP450s. The aim of this review is to highlight the effects of herbal supplement use on xenobiotic metabolism and the development of hepatotoxicity or modulation of therapeutic efficacy, either alone or with concurrent use of other herbal supplements and Food and Drug Administration (FDA)-approved pharmaceuticals. Cytochrome P450 (CYP450) enzymes metabolize a large number of xenobiotics, which may lead to the formation of hepatotoxic compounds. Fortunately, pharmaceuticals are evaluated for the potential to inhibit or induce CYP450 enzymes before being marketed in the United States (US) . HoweverThe expression of CYP450 enzymes is induced by nuclear receptors that respond to xenobiotics. Many CYP450 enzymes are transcriptionally regulated by the xenobiotic receptors pregnane X receptor  and constitutive androstane receptor  ,7,8,9. WConcurrent use of pharmaceuticals with herbal supplements, as well as the use of multiple herbal supplements, presents additional problems. Many patients do not report herbal supplement use to their physicians ,16,17,18To develop this review concerning the association of herbal supplement use with P450 modulation and hepatotoxicity, we used the search terms “herbal supplement” or “herb” combined with “hepatotoxicity” or “liver injury” to search the PubMed database. From this initial search, we selected compounds associated with hepatotoxicity in an in vivo model. We then searched for each of these supplements with “P450”, “CYP450”, or “cytochrome P450”. Finally, we searched for “herbal supplement” or “herb” combined with “P450”, “CYP450”, or “cytochrome P450”. Care is taken to include reports where the effect (P450 modulation or hepatotoxicity) is attributable to a single causative compound or when an extract of defined herbal ingredients is provided.The most commonly used herbal supplements in the United States are echinacea, garlic, ginko biloba, saw palmetto, ginseng, grape seed extract, green tea, and St. John’s wort . This reGardenia, fucomarins in grapefruit juice, and Echinacea extract may inhibit the activity or decrease the expression of cytochrome P450 enyzmes -Epight loss . Toxicitght loss . Green tght loss . Green tght loss .Actae/Cimicfuga racemose) is native to the eastern region of North America. The roots and rhizomes of black cohosh are used as a hormone replacement and as an anti-inflammatory [Black cohosh  is native to North America and is consumed to treat wounds, prevent urinary tract infections, and to treat diabetes [Cranberry  is the result of an accidental cross between the pomelo (C. grandis) and the sweet orange (C. sinesis) that is believed to have originated in Barbados [The grapefruit  [w/v), caused increased aspartate transaminase (AST) and alanine aminotransferase (ALT) in rats and histologic changes in rat liver [Fructus gardeniae and geniposide all cause hepatoxicity in rats [Echinacea, and Gardenia alone may not be associated with toxicity in humans, but their effects upon the P450 system warrant concern.Plants of the pressive ,89,90. Rtathione . Genipin1 (P-gp) . Genipos1 (P-gp) . The adm1 (P-gp) . Adminisat liver . The aqu in rats ,93. CranGingko biloba, piperine in black and white pepper, diallyl sulfide in garlic, and grapeseed extract increase p450 expression (The hydroxylation of xenobiotics and endobiotics by P450 enzymes can produce daughter compounds that are more toxic than the parent compound . This ispression ..Activation of PXR by ligands such as rifampicin and many other compounds results in the transcription of genes that are involved in the transport and metabolism of xenobiotics. PXR is a nuclear hormone receptor (NHR), a class of proteins that are characterized by a DNA-binding domain, as well as a ligand-binding domain (LBD). The LBD of human PXR (hPXR) binds to many ligands with a wide range of different structures ,109. NHRO-oxime binds to and activates human CAR, but not mouse CAR [CAR also controls the expression of xenobiotic metabolizing enzymes and transporters. Unlike PXR, CAR is constitutively active in the absence of ligand . Agonistouse CAR ,123. TheHypericum perforatum) is used to treat anxiety and depression. St. John’s wort is native to Europe, but is now also cultivated in the US, Canada, and Australia [Hypericum perforatum induced liver injury that is associated with the use of copaiba, a herbal supplement that is used as an anti-inflammatory [St. John’s wort  and Cyp2b10, Cyp1a1, and Cyp3a11 mRNA in WT mice, but not in Car−/− mice [Ginkgo biloba consumption. Ginkgo biloba extract activates the transcription of a CYP3A4 promoter when an expression construct is overexpressed with either mouse or human PXR in HepG2 cells [Ginkgo biloba extract also increases the expression of CYP3A4, CYP3A5, and ABCB1 (MDR1) mRNA in LS180 colorectal cancer cells [Gingko biloba extract demonstrated that ginkgolide A and ginkgolide B induce the expression of CYP2B6, CYP3A4, UGT1A1, MDR1, and MRP2 mRNA in primary human hepatocytes [Ginkgo biloba is associated with hepatotoxicity in humans [Ginkgo biloba, as well as the isolated compounds gingkolide A and B themselves activate hPXR, thereby increasing the expression of CYP3A4, CYP3A5, and CYP2B6 and other genes controlled by hPXR. Whether the toxicity reported by humans taking Ginkgo biloba is a result of hPXR activation or of induction of the P450 system is unknown.tinnitus ,128,129.−/− mice . This diG2 cells . Ginkgo er cells . Furtheratocytes . Ectopicatocytes . Ginkgo n humans . Whole ePanax quinquefolius) and in eastern Asia (Panax ginseng). The roots and leaves are available as pills and in energy drinks, teas, and coffee beverages. Panax quinquefolius is used to reduce the incidence and severity of colds, whereas Panax ginseng is believed to enhance cognitive ability and to lower blood sugar levels; both contain ginsenosides and gintonin [Panax ginseng extract inhibits the induction of Cyp1a1 mRNA and protein expression induced by benzo-pyrene. Cyp1a1 metabolizes benzypyrene, and this activation is required for toxicity [Panax ginseng as a constituent of an energy drink for three months, there may have been an interaction between imatinib and the ginseng that resulted in liver injury [S)-protopanaxadiol, a ginseng sapogenin, in human liver and intestinal microsomes [Ginseng is found in North America . White and black pepper are taken for GI distress, bronchitis, malaria, cholera, and cancer [P. nigrum are thought to cause ROS production and to cause oxidative damage in cancer cells [4, piperine increased the amounts of plasma liver enzymes, hepatic lipid peroxidation, and NADPH-cytochrome c reductase activity [4 may be due to P450-mediated ROS production [4 administration. Further studies of hPXR transgenic mice may fully elucidate the role of piperine in CCl4-mediated HILI.Piperine is an alkaloid that is responsible for the flavor and smell of black and white pepper  is used to treat a variety of medical conditions. Consumption of large amounts of garlic is believed to reduce the number of bites by insects, such as ticks [−/− mice [−/− mice [Garlic  represents the clinical presentation of toxicity and the pathological effects of the injury and is a necessary step to assigning causation to a particular agent . CholestCharacterization of liver injury by pathogenesis considers intrinsic versus idiosyncratic types of injury . IntrinsPredicting the risk of liver injury due to herbal supplements requires both a standardized causality assessment method and a repository of this information reported in the clinic. The Council for International Organizations of Medical Sciences (CIOMS), Working Group IX, developed the Roussel Uclaf Causality Assessment Method (RUCAM), a list of tools and guidelines for researchers and clinicians to evaluate and manage the risk of medicinal products  ,194,195.Mentha pulegium) is native to Europe, North Africa, and the Middle East. Another species (Hedeoma pulegioides) is native to North America. Pennyroyal is traditionally used in cooking and as an abortifacient. It is also used for colds, pneumonia, and dyspepsia [Menthol is prepared from corn mint or peppermint, or is made synthetically. Menthol is administered topically to treat minor muscle and joint pain by causing the skin to feel cool and then warm, distracting patients from feeling deeper pain . Pennyroyspepsia ,159,199.yspepsia ,167. Penyspepsia ,161,162.yspepsia . Pugelonyspepsia . Purifieyspepsia .Menthofuran is a monoterpene that is found in several species of mint plants and is oxidized by cytochrome P450 enzymes to hepatotoxic metabolites. The metabolism of menthofuran requires CYP1A2, CYP2E1, and CYP2C19 . These cS,3S,4S)- and (−)--trans-p-methane-3,8-diol derivatives [R)-(+)-pulegone causes increases in the marker of liver injury, glutamate pyruvate transaminase, in mice treated with pulegone that are mitigated by pretreatment with disulfiram and cimetidine, inhibitors of CYP2E1 and CYP1A2, respectively [CYP2A6 is the major cytochrome P450, which is involved in the oxidative metabolism of menthol in human liver microsomes to form (+)-(1ivatives . (−)-Menivatives ,33. Addiectively . Additioectively . Peppermectively . In addiCinnamomum camphora), which is native to Asia. Camphor is now predominantly produced from turpentine and is used in such products as Vicks VapoRub™. Camphor products are usually applied topically to reduce dermatologic complaints and the symptoms of respiratory tract disease [Camphor was traditionally prepared from the wood of the camphor laurel  disease . Camphor disease . In fact disease ; howeverTeucrium chamaedrys and other species of the genus Teucrium) is used to treat gallbladder disease, fever, diarrhea, gout, and for weight loss [Germander  and their N-oxides are found in approximately 3% of flowering plants. These plants are predominantly members of the Asteraceae, Boraginaceae, Compositae, and Fabaceae families, and can also be  species ,215. PAs species ,217. (±) species . Senecio species . 9-Gluta species . Monocro species . Corresp species ,169. Ret species . The hepHerbal supplement use can lead to HILI due to interactions with the P450 system. These interactions may not be predicted until a series of reports of HILI associated with the use of a particular herbal supplement leads to an investigation of potential interactions. Unlike prescription drugs regulated by the FDA , the potential of herbal supplements to induce or inhibit P450 enzymes is not investigated before the supplements are made available to the general population. This lack of information before a supplement is marketed is compounded by the difficulties in event-reporting and history-taking concerning herbal supplement use. Difficulty in assigning an etiologic designation to a particular xenobiotic in patients with positive histories of multi-pharmaceutical, multi-supplement, or mixed pharmaceutical and supplement regimens complicates what “post-marketing” reporting is available. Many networks for reporting exist, but are weakened by difficulties of self-reporting of herbal supplement use by patients and by poor history-taking. However, careful history-taking by physicians may prevent an interaction from taking place and identify the likely offending agent.Previous articles have discussed herbal supplements and HILI, but this review has focused on herbal supplements that may interact with the P450 system to exacerbate the toxicity of other drugs or to cause toxicity themselves ,223,224.Careful analysis of existing literature combined with extensive history-taking concerning the use of herbal supplements can help to prevent herb-drug interactions and HILI. The recognition of and reporting of HILI events by physicians can lead to greater knowledge of potential interactions and hepatotoxicity, enabling them to better educate patients of the dangers of some combinations.


The tumor suppressor p53 as the “guardian of the genome” plays an essential role in numerous signaling pathways that control the cell cycle, cell death and in maintaining the integrity of the human genome. p53, depending on the intracellular localization, contributes to the regulation of various cell death pathways, including apoptosis, autophagy and necroptosis. Accumulated evidence suggests that this function of p53 is closely involved in the process of cancer development. Here, present knowledge concerning a p53-autophagy-metastasis link, as well as therapeutic approaches that influence this link, are discussed. Being one of the major suppressors of tumorigenesis, p53 is involved in regulation of the cell cycle, cell death, maintenance of genome integrity and DNA repair. Therefore, it is traditionally considered a “guardian of the genome.” In recent years, however, p53 has also been reported to possess some novel “non-canonical” functions, including the regulation of metabolism and autophagy.p53 gene is modified by a number of transcription activators  [The structure of p53 reflects the complexity of its functions. Based on gel electrophoresis analysis, it is a nuclear protein with a molecular mass of 53 kD; however, the real mass of this protein is 43.7 kDa. It contains 393 amino acids and acts mainly as a transcription factor for multiple genes ,2,3. It  RBP-Jκ)  and repr RBP-Jκ)  an antis RBP-Jκ) ,16. It s RBP-Jκ)  acetylat RBP-Jκ)  and conf RBP-Jκ) .Structural peculiarities of p53 give a clue toward understanding the diversity of its functions. First classified as an oncogene, p53 exhibits various antiproliferative functions, and suppresses growth and tumorigenesis in cell culture. The best-understood, canonical functions of p53 include regulation of the cell cycle and cell death. It may induce cell cycle arrest at G1/S and G2/M checkpoints in response to various disturbances through transactivation of p21 and 14-3-3σ, respectively ,21. Undep63 and p73 are close homologues of p53 and they are all members of the p53 family of transcription factors. They share a similar domain structure, but p53 lacks a specific C-terminal region, which is alternatively spliced in p63 and p73. Thus, the latter exist as different isoforms, which exhibit different, even opposite functions. Full-length isoforms (TAp63 and TAp73) act synergistically with p53. They can bind to classical DNA p53-binding sites and consequently induce cell cycle arrest, apoptosis and senescence in the same manner as p53 . Amino-tMutations of p53 lead to increased susceptibility to tumor transformation and are found in over 50% of cancers. They are considered to be necessary for the development of most tumors, especially ovarian cell carcinoma, squamous lung cancer and triple-negative breast cancer. Their presence often contributes to rapid disease progression and resistance to conventional therapeutics and is associated with poor prognosis . These mMacroautophagy (hereafter referred to as autophagy) is a highly conserved catabolic process that captures and degrades misfolded proteins and cellular organelles and sustains cell survival during different stress processes ,44. AutoAMPK, LKB1, PTEN and TSC1/2 induce autophagy while, conversely, oncogenes that promote mTOR signaling, such as class I PI3K, AKT, Ras and RHEB inhibit autophagy [Beclin 1 has been detected in 40% to 75% of human ovarian, prostate, and breast cancers. Beclin 1-deficient mice were shown to be tumor prone [Beclin 1 may act as a tumor suppressor gene involved in the pathogenesis of human cancers and that autophagy may prevent the development of these tumors [Unlike their normal counterparts, cancer cells may have high levels of basal autophagy and can be dependent on autophagy for survival. It is now well known that autophagy may play a tumor-suppressing or tumor-promoting roles depending on the context ,47,48. Tutophagy ,51. In lor prone . In contor prone ,45. These tumors ,54.Heterozygous deletion of several other core autophagy genes is reported to promote a tumor-suppressing role of autophagy in cancer . Thus, dATG5 or ATG7 deletion, leads to chronic oxidative stress, accumulation of damaged mitochondria, tissue damage and inflammation which all favor tumor initiation [As well as mitochondria being considered to be the main source of ROS (Reactive Oxygen Species), autophagy can carry out its tumor suppressor function by enabling the elimination of damaged mitochondria, thereby preventing ROS accumulation . This tyitiation ,70.-/-SQSTM1/p62 mice were protected from Ras-induced lung carcinomas compared with wt animals [Defects in autophagy are associated with the accumulation of aggregated proteins and the autophagy substrate SQSTM1/p62, a ubiquitin-binding protein that is a target of cargo-selective autophagy. Such events are linked to the increased production of ROS, ER (endoplasmic reticulum) stress and activation of the DNA damage response . Knockdo animals .Finally, autophagy also represents the mechanism that may limit tumorigenesis by restraining tumor necrosis and chronic inflammation, which are mediated by the release of proinflammatory high-mobility group box protein 1 (HMGB1) . This prWhile autophagy has a tumor-suppressing role in the early stage of carcinogenesis, in advanced cancers it often acts as a tumor survival or even tumor promoter mechanism . This isATG7 deletion in a K-ras-driven non-small cell lung cancer (NSCLC) significantly reduces tumor growth. Moreover, the ablation of ATG7 leads to the progression of adenomas and adenocarcinomas harboring K-ras mutation to benign oncocytomas, tumors characterized by the accumulation of dysfunctional mitochondria. ATG7 deficiency activates p53, which in turn contributes to tumor inhibition, as co-deletion of p53 in part rescue impairment of tumor growth [A high level of autophagy is detected in cancer cells bearing K-ras mutation promoting their proliferation and survival, whereas the deletion of core autophagy genes in tumor cells has been shown to stimulate the induction of cell death ,74. Thusr growth . Likewisr growth .The role of autophagy in cancer is highly dependent on the type of tumor and its developmental stage. Activation or inactivation of autophagy can contribute differently to tumorigenesis. Reduced autophagy can contribute to tumor progression, whereas increased autophagy may be a mechanism for tumor survival under hypoxic, metabolic or therapeutic stress conditions. Thus, the modulation of the autophagy process is a promising, but complex, therapeutic strategy for the enhancement of anticancer treatments.Recent evidence suggests that p53 may also contribute to the relationship between autophagy and malignancy ,78. As mIn the nucleus, p53 induces autophagy through regulation of the mTOR pathway in a transcription-dependent manner. Many p53 target genes, including PTEN, TSC2 and AMPKβ, are known to negatively regulate mTOR . The cruCDKN2A gene, is considered to be a tumor suppressor, and has been shown to regulate p53 positively through inhibition of MDM2-induced degradation [CDKN2A exon2, suppresses the ability of ARF to induce autophagy [Another link between p53and autophagy could be provided by p14ARF signaling. The ARF protein, encoded by radation ,83. At tutophagy .p53 has also been shown to induce the autophagy pathway through DRAM, which is a lysosomal protein that modulates autophagy in response to nutrient starvation ,86,87. IMeanwhile, inactivation of cytoplasmic p53 might trigger autophagy, suggesting that the nonnuclear p53 pool is a potent autophagy repressor ,92. IndeOne of the p53 target genes the TP53-induced glycolysis and apoptosis regulator (TIGAR) has been shown to fulfill an anti-autophagic functions . OriginaBeclin 1, limits mammary tumorigenesis driven by Palb2 deletion in a wt Trp53, but not in p53 null tumors [The mechanism by which cytoplasmic p53 inhibits autophagy is not completely understood . Howeverl tumors . Thus, tAs mentioned above, autophagy can reduce oxidative stress through the removal of organelles generating ROS, such as mitochondria and peroxisomes. On the other hand, oxidative stress leads to p53 activation, and thus, mitigating oxidative stress autophagy may suppress p53’s activity . AutophaMetastasis is a multi-step process that promotes cancer cell migration to distant organ sites ,103. TheThe complex interplay between autophagy and p53 during tumorigenesis has been discussed above. However, it remains unclear whether they interact during the metastatic process. At the same time, multiple studies have shown that p53 and autophagy regulate the same processes at different stages of metastasis development ,109.In line with its dual role in cancer, the role of autophagy in the regulation of metastasis appears to be controversial and highly dependent on the cell type and/or tumor stage .At the early stages of the metastatic process, autophagy may function as a suppressor of metastasis by reducing tumor necrosis, and inflammatory cell infiltration, and by mitigating oncogene-induced senescence . At the Specifically, autophagy may reduce inflammation, which is required for initiation of metastasis. Indeed, in promoting cell survival during various stress conditions, such as hypoxia, autophagy suppresses necrosis and further macrophage infiltration of the tumor site . AutophaAutophagy has been shown to control EMT providing degradation of specific EMT regulators, such as Twist1, Snail and Slug, which are the transcription factors involved in the regulation of EMT ,117,118.In turn, p53 may also act as a suppressor of metastasis, for example negatively regulating factors involved in the execution of the EMT program ,122. OveATG7 knockdown reduced the expression of the mesenchymal markers vimentin and N-cadherin, thereby blocking EMT and subsequently migration of NSCLC cells [As previously mentioned, autophagy may also promote metastasis regulating different stages of the metastatic cascade. Induction of autophagy sustains cancer cell survival under stress, which subsequently may promote tumor progression and metastasis ,127. ConLC cells .Interestingly, autophagy also promotes resistance to anoikis, another form of programmed cell death occurring when cells detach from the ECM . AutophaAutophagy may promote metastasis regulating the Rho family of small GTPases. Members of this family, including Rac, cdc42 and Rho, are small proteins that play a crucial role in the reorganization of actin cytoskeleton, and regulate the cell cycle progression, motility and invasion ,136,137.A recent study suggests that Rho A level is controlled by autophagy . In contAdditionally, it was demonstrated that the loss of p53 is associated with high level of GTP-bound Rho A, Rock1 and cdc42 . In ordeAutophagy is also essential for RAS-driven oncogenic transformation and migration. Epithelial cells bearing RAS mutation display highly invasive phenotype associated with an EMT ,145. It Taken together, these observations provide evidence that p53 and autophagy are involved in the regulation of different processes during metastatic progression including EMT and resistance to anoikis. However, despite close interaction during tumorigenesis the data about their direct interplay during metastasization are not clear enough. Better understanding of p53 perturbations in human cancer and their relationship with autophagy may be essential in order to provide more precise prognoses and improve therapeutic strategies.Mutations of p53 are found in more than 50% of cancers. They are considered to be necessary for the development of most tumors. Their presence often contributes to rapid disease progression and resistance to conventional chemotherapy and is associated with poor prognosis, especially when the tumor bears gain-of-function mutant p53 ,150. ResThe interplay between autophagy and mutant p53 is multifaceted. On the one hand, p53 has been demonstrated to inhibit autophagy, but on the other, chaperone-mediated autophagy triggers mutant p53 degradation and, thus, may regulate the progression of the tumor and its response to chemotherapeutic agents . One canAnother approach to altering mutant p53 for therapy was suggested to target downstream pathways through which p53 acts. Despite the diversity of various p53 mutants, many of them share a common feature—they interact and inhibit p73, another potent tumor suppressor, which structurally and functionally resembles p53. A molecule called RETRA was shown to inhibit p73 and p53 interaction, thereby releasing p73 and activating transcription of its target genes, which suppress tumor growth . One mayMany types of cancer have been reported to evade cell death via inhibiting p53-dependent autophagy. It was found, for instance, that in renal cell carcinoma, transglutaminase 2 (TG2) crosslinks p53 in autophagosomes, thereby decreasing the p53 level in the cell and evading apoptosis . Hence, It is now clear that modulation of autophagy is promising therapeutic approach for certain cancer types ,165. HowAlthough in recent years there has been extensive discussion about whether inhibition of autophagy enhances or reduces cancer therapy, nowadays consensus has been reached that both may take place . RosenfeLikewise, it was reported that mutant p53 becomes more stable when the cells are treated with the antimalarial drug hydroxychlorochine . AnotherTP53 gene.To sum up, the role of autophagy and its interplay with p53 depends on different factors and the clinical approach depends on the type of the tumor, p53 status and many other aspects. Future studies are required to confirm the efficacy of autophagy inducers in animal models and in cancer patients carrying the mutant There is ample evidence to indicate that a functional interplay between the tumor suppressor p53, its mutants and autophagy during tumorigenesis exists. An increasing number of studies also highlight the emerging roles of autophagy and p53 in the regulation of different metastatic cascade steps. Both p53 and autophagy were shown to contribute to anoikis resistance, EMT activation/suppression, ECM interactions, cell migration regulation, Ras-driven invasion, and several more. Importantly, at present, many issues remain unanswered. For example, how do p53 and autophagy interact biochemically during the metastatic process? How might the type of p53 mutation influence the relationship between autophagy and metastasis?It is clear that the contribution of each of the different p53 target genes might vary according to the tumor type, tissue specificity, molecular context, stress signal and other circumstances. The contribution of autophagy in carcinogenesis is also context-dependent and today the consequences of its modulation are difficult to predict. Further studies are required in order to improve our understanding of the p53-autophagy-metastasis link and provide a new promising therapeutic approach to combat cancer.