This study explores understanding of primary and secondary prevention of anal cancer among human immunodeficiency virus (HIV)-infected foreign-born Latino gay and bisexual men (GBM). Between August 2015 and December 2016, researchers conducted 33 in-depth, semi-structured interviews with HIV-infected foreign-born Latino GBM. Interview questions sought to determine participants’ knowledge and perceived barriers and facilitators to primary and secondary prevention of anal cancer. Researchers analyzed interview transcripts using a qualitative content analysis approach. For primary prevention, men reported a lack of knowledge about the human papillomavirus (HPV) vaccine. However, for secondary prevention, roughly 60% of participants had previously screened for anal dysplasia via anal Papanicolaou (Pap) smear. However, participants reported willingness to screen, and provider recommendation was the most common screening facilitator. Men reported stigma related to their HIV status, sexual orientation, and anal Pap smear procedures as anal cancer screening barriers. Participants reported willingness to use a self-screening anal Pap smear test if it was commercially available. Health providers continue to be the leading source of health information. Therefore, provider recommendation for HPV vaccination and anal cancer screening among age-eligible foreign-born Latino HIV-infected GBM is critical. More work is needed to destigmatize HIV and sexual orientation to influence positive health behaviors among this population. Future intervention research could test the effects of provider-led interventions and also media campaigns aimed at influencing HPV vaccine uptake and anal cancer screening among this population. Most individuals exposed to HPV clear the infection without ever showing signs or symptoms. However, human immunodeficiency virus (HIV)-infected and other immunosuppressed populations experience an increased risk of persistent HPV infection.3 Over time, dysplasia can progress to high-grade squamous intraepithelial lesions (HSILs).4 Researchers and health-care practitioners believe that, similar to cervical HSIL and cervical cancer, untreated anal HSIL is the cause of anal cancer.5 The 2 greatest risk factors for developing anal cancer are the presence of anal HSIL6 and severe immunosuppression (CD4 count <200 cells/mm3).710 Other risk factors include age (being older than age 25),11 smoking (linked to prolonged infection with HPV oncogenic strains),12 infection with multiple high-risk HPV strains,13 increasing number of current sexual partners,14 history of developing anal warts,15 higher HIV viral loads,15 and reported regular receptive anal sex.15Rates of anal cancer, a relatively rare disease, which account for 0.5% of all new cases with cancer, have been steadily rising by 2.2% each year.17 To be most effective in preventing disease and saving money, all individuals should receive the vaccine prior to sexual debut.18 The vaccine is also safe and recommended for HIV-infected individuals between the ages of 9 and 26 years.19It is believed that HPV-related anal cancer can be prevented via primary (HPV vaccination) and secondary  prevention. For primary prevention, two commercially available HPV vaccines  offer the potential for immunity against HPV 16 and 18 (oncogenic strains) as well as HPV 6 and 11 .20 While high-resolution anoscopy (HRA) is a more sensitive and specific screening for detecting HSIL, numerous barriers  prohibit using HRA as the initial screening test.22 Although there are no national guidelines for anal dysplasia/anal cancer screening, researchers have found that screening HIV-infected populations for anal dysplasia every 2 to 3 years via anal Pap smear is a cost-effective, life-saving approach to detect  early signs of anal cancer.23For secondary anal cancer prevention, the anal Papanicolaou (Pap) smear is recommended as a first screening test to detect anal dysplasia among individuals asymptomatic for anal cancer and particularly for HIV-infected individuals.24 Past research identified barriers and facilitators to screening for anal cancer among HIV-infected gay and bisexual men (GBM) and MSM (predominantly non-Hispanic White) populations.26 Barriers included lack of awareness to screen, fear, and stigma related to receiving the anal Pap smear and cost of screening.HIV-infected men who have sex with men (MSM) are the population most at risk of anal cancer, given their immunosuppression, anal sex receptivity, and exposure to multiple high-risk HPV strains (including HPV 16).27 Among HIV-infected populations, foreign-born Latinos (including MSM)28 are the population least likely to be engaged and sustained in HIV primary care.29 Lack of sustained HIV primary care is associated with lower CD4 counts in HIV-infected patients’.30 Interruption of HIV primary care also places patients at a greater risk of other comorbidities , because patients are not being screened and treated for disease precursors.31 Although one literature review explored US Latino (including Puerto Rican) men’s HPV-related comorbidities,32 no studies, to our knowledge, report foreign-born Latino HIV-infected GBM’s perceptions about anal cancer prevention. Therefore, the objective of this study was to explore the perspectives, barriers, and facilitators related to both primary (HPV vaccine uptake) and secondary  prevention among foreign-born (not including Puerto Rican populations) Latino HIV-infected GBM. It is critical to understand the social determinants of these disparities among Latino immigrant populations in order to identify how to prevent and control anal cancer.Little is known about HIV-infected foreign-born Latino GBM’s risk of anal cancer. This population has previously reported inconsistent use of condoms during anal sex, which increases the risk of sexually transmitted infections, including HPV.33 and resides in Miami-Dade County), and a Google number that rang directly to the bilingual research assistant’s cellphone. After potential participants called the study number, the bilingual research assistant screened them for eligibility and asked them to recommend a date, time, and location for the interview.Upon receiving institutional review board approval from the University of Miami (IRB00000260) to conduct the study, the research team began recruiting a convenience sample of Latino HIV-infected GBM. They circulated Spanish-language study fliers to community-based organizations that provide outreach services to Latino GBM. Staff from the community-based organizations posted fliers at their organizations. The fliers provided information about the topic of interest (cancer prevention), inclusion criteria  that she and the bilingual research assistant expanded after conducting interactive rounds of coding together to achieve coder agreement. They conducted qualitative content analysis using an “intuitive” approach by reviewing all interview data and selecting the aspects most relevant to the research questions.Researchers interviewed 33 foreign-born Latino HIV-infected GBM. See Fifteen participants described that HPV is transmitted sexually. Five of these men had never heard of the HPV vaccine. Six participants who knew that HPV was sexually transmitted described the virus as affecting both men and women, whereas 4 believed it was a women’s health issue only see . Among tFifteen participants had heard of the HPV vaccine, one of whom (out of the 9 men who had been age-eligible to receive the vaccine) had received a single dose of the HPV vaccine. When asked where they first heard of the vaccine, 6 participants reported they received this information from their health-care providers, 4 from television public service announcements, and 2 from watching the news. The remaining participants reported hearing about the vaccine from reading a newspaper article, attending a health-related conference, and attending an HIV support group (single responses). The greatest barrier to receiving the vaccine was lack of knowledge about this resource (n = 10). (See Most participants had heard (n = 21) about anal cancer screening, 20 of whom reported having been screened for the cancer via anal Pap smear. These men described how provider recommendations influenced their decision to be screened. The 1 person who reported knowing about the anal Pap smear test but who had not actually been screened for anal dysplasia described how his health-care provider discouraged him from getting screened for anal dysplasia, telling him that he was too young to be screened.Psychosocial barriers to anal cancer screening included various forms of stigma, embarrassment, or discomfort related to anal dysplasia screening tests and fear of test results see . Less frMultiple forms of stigma prevented men from seeking HIV primary care and anal cancer screening. Specifically, participants reported stigma surrounding the following conditions as barriers to receiving sustained HIV primary care: (1) sexual orientation , (2) HIV infection, and (3) the combined stigma of being HIV-infected and identifying as gay/bisexual. Two men reported that self-perception or self-classification of being gay or bisexual influenced health-seeking behaviors, particularly if their providers told that being gay or bisexual increased their risk of negative health outcomes. They described how not all MSM regard themselves as gay or bisexual, and they also do not tell their providers that they are having sex with other men. In turn, providers may not promote the anal Pap smear to these patients. Four participants described the stigma against HIV as a reason why men did not seek HIV primary care. One participant described how the intersectionality of being a gay man who was HIV-positive increased his perceived stigma and fear of engaging in health-care practices, including screening for cancers.machismo. In addition, 4 men reported fear of receiving a cancer diagnosis as motivation to avoid anal cancer screening.Five participants described embarrassment-related screening procedures  as a reason why Latino men would choose not to test for precursors to anal cancer. These procedures both involve touching the anal canal. According to participants, this can embarrass men. This type of procedure may also violate cultural norms of masculinity, also known as Many participants (n = 20) were in favor of testing for anal cancer from home  if it was a viable, commercially- available testing option see . They noParticipants recommended various methods to disseminate health information about HPV, the HPV vaccine, and anal cancer. They most commonly recommended (n = 21) that health care providers inform patients about these health issues and provide the option to receive the vaccine and screen for anal cancer. Other common responses included using media such as Spanish-language online resources (n = 10), television public service announcements (n = 8), and Spanish-language radio shows (n = 2) to reach the local community with information about HPV. Eight participants also recommended various forms of print media  as health education resources.39 Similar to those studies, this research found high levels of HPV vaccine acceptability. The only causes for vaccine hesitancy among foreign-born Latino HIV-infected GBM were vaccine costs and fears of potential health complications due to their HIV status. No additional negative side effects from the vaccine (which does not consist of the live virus) have been reported among HIV-infected populations when compared to the general population.41 Human immunodeficiency virus-infected adults often test negative for oncogenic strains of HPV. Over time, however, they increase their risk of acquiring cancerous HPV strains if they increase their number of sexual partners.15 Further, past studies have reported that completing the HPV vaccine can reduce the risk of HSIL recurrence among MSM previously treated for these precancerous lesions.42 Given its safety for immunosuppressed populations, the HPV vaccine should be promoted, particularly by HIV primary care clinics, to all age eligible (up to age 26 years old). HIV-infected MSM (including those who identify as GBM). Completing this vaccine series will help reduce their risk of developing various HPV-related cancers and noncancerous genital warts. Social marketing campaigns could conduct community-engaged formative work with HIV-positive Latino advocates (including foreign-born GBM) to determine the best methods for disseminating this health information in culturally and linguistically appropriate methods and language.35 Also, future studies should test the efficacy and cost-effectiveness of offering the HPV vaccine to all HIV-infected adults  to reduce HPV-related cancer risk and recurrence.To our knowledge, this is the first study to focus exclusively on foreign-born Latino HIV-infected GBM’s perceptions related to HPV, the HPV vaccine, and anal cancer screening. By extending cultural considerations for immigrant Latino GBM, our study’s findings add to the existing literature on various HIV-positive communities’ perceptions related to the prevention and control of anal cancer.33 Foreign-born Latino HIV-infected GBM noted that health-care provider recommendation alone was the driving factor for anal Pap smear completion. Therefore, it is critical that HIV primary care providers offer culturally sensitive anal dysplasia screening information for this population. It is important to note that, as described by research participants, not all foreign-born Latino MSM identify as being gay or bisexual. Past research studies have also confirmed that Latino men who have sex with other men do not necessarily self-identify as gay or bisexual.37 Therefore, social marketing campaigns that aim to link foreign-born Latino GBM to HIV-related health care and to anal Pap smears may be more effective if they market the information to MSM, without focusing on the language related to sexual orientation.36In this sample, more than half of the participants had previously been screened for anal dysplasia via anal Pap smear, a higher rate than what was previously reported in another study with English-speaking populations.39 Gwede and colleagues recommend promoting a home-based self-sampling cancer-screening tool as an early detection tool for colorectal cancer to overcome embarrassment related to screening procedures.39 Similarly, using an anal cytology self-sampling test that may be collected at home could potentially increase anal cancer screenings. When asked about this testing procedure’s acceptability, most participants believed that a home self-sampling test could reduce barriers to screening. Such tests are not currently commercially available. However, similar to colorectal cancer screening, home self-testing kits may increase anal dysplasia screenings if made available.Participants described how the anal cytology testing performed by a health-care provider could violate their sense of masculinity. Past studies have found similar barriers to colorectal cancer screening, predominantly among Latino and African American populations.41 Together, these various forms of stigma lead to social isolation, depression, and lack of retention in HIV primary care,42 and they are correlated with increased sexual risk behaviors.43 Culturally tailored community-engaged interventions aimed at reducing homophobia and these various forms of stigma may increase Latino HIV-infected GBM’s retention in HIV primary care and, in turn, increase cancer-screening behaviors.44Participants discussed the psychosocial barriers that could lead to a lack of screening and treatment for both HIV- and HPV-related comorbidities among Latino GBM. Although not specifically asked, men described various forms of stigma that are associated with sexual orientation, HIV status, and the intersection of sexuality and HIV status as barriers to care. Past research has also described the intersectionality of stigma related to HIV status, sexual orientation, and race as barriers to retention in HIV care among African American and Latino MSM.45 This would reduce their likelihood of learning about anal cancer and screening for anal cancer screening.There are potential limitations to this study. First, researchers asked foreign-born Latino GBM about HPV vaccine perspectives. At the time, only 9 participants had been age eligible (younger than age 26) to receive the vaccine since vaccine was first promoted for men (in October 2009). Therefore, it is likely that health-care providers did not offer this cancer prevention vaccine to the majority of study participants. Another threat may include interviewer variability. During this study, two bilingual graduate research assistants collected interview data. The graduate research assistants may have varied in the way that they interviewed participants, influencing participants’ responses. The first author tried to eliminate the threat of potential variability in interview conducted by training each graduate research assistant in interview techniques and practicing the interview with them. Finally, the research team did not ask about participants’ documentation status. Past research has shown that undocumented Latino populations have barriers to HIV primary care that impacts timeliness of HIV diagnosis and treatment as well as disease progression (when compared to documented populations).Our study’s findings highlight gaps in research. This study focused on patients’ barriers to anal cancer prevention. However, future research should explore HIV primary care providers’ practices and clinic-specific policies related to recommending the anal Pap smear among HIV-infected populations, including HIV-infected foreign-born Latino GBM. Researchers could work with health-care providers to craft linguistically and culturally appropriate messages and health education materials  to increase awareness about the need to screen for anal cancer, focusing on ways to destigmatize this screening. Provider-led and narrated media campaigns could also begin to increase communities’ understanding and intention to screen for anal dysplasia.Research that tests the feasibility, acceptability, and preliminary efficacy of using anal cytology self-sampling kits  to increase screening for this stigmatized cancer may provide further proof of the need for these self-testing kits. Other studies should focus on how the availability of these self-testing kits affects HIV primary care practice, particularly among clinics that do not currently have the resources to test for anal dysplasia. Findings from such studies could further inform the need to make these tests more widely available.HIV-infected men are at a higher risk of developing HPV-related anal cancer. This disease can be prevented and controlled by more focused efforts to ensure that (age-eligible) men complete the HPV vaccine series and screen for anal dysplasia. It is critical to take into consideration the cultural differences reported, such as the various forms of stigma against sexual orientation, HIV, and cancer screening procedures, in order for health-care practitioners to offer more acceptable and culturally competent care. Foreign-born Latino HIV-infected GBM need information from health-care providers and the media to increase their knowledge of the HPV vaccine and anal dysplasia screening. Given the lack of screening guidelines, more work is needed to inform clinical protocols for anal dysplasia screening among HIV-infected populations. Increased health outreach and increased provider recommendations to receive the vaccine and to screen for anal cancer will provide vulnerable populations such as foreign-born Latino HIV-infected GBM with the information they need to be empowered to prevent and control HPV-related anal cancer.


Euglycemic diabetic ketoacidosis (EDKA) is a rare variant of diabetic ketoacidosis which has been recently reported in association with sodium-glucose cotransporter 2 (SGLT-2) inhibitors. Empagliflozin, an agent belonging to this therapeutic class, was approved by the U.S. Food and Drug Administration (FDA) in 2014 for management of type 2 diabetes. Since then, sparse reports of its association with EDKA are emerging, similarly to its predecessors in the class. We report the case of a 58-year-old female who developed EDKA in the intensive care unit (ICU) 48 hours after her last intake of empagliflozin and a day after neurosurgery. Though expected to improve in the post-operative period, she developed a rapidly worsening and unexplained anion gap metabolic acidosis. She was eventually diagnosed with EDKA which was successfully treated with intravenous insulin infusion, dextrose-containing fluids and discontinuation of the offending drug. Metabolic abnormalities improved in less than 24 hours and patient recovered without complications. This report highlights the importance of recognizing EDKA as a complication of oral anti-diabetics and discontinuing SGLT-2 inhibitors days prior to surgery and ICU admission. Care should be applied to providing patient with low-dose ketogenesis-inhibiting basal insulin and close observation of laboratory values in order to minimize delays in diagnosis, prolonged hospital stays and complications of EDKA. Euglycemic diabetic ketoacidosis (EDKA) is an uncommon acute complication of diabetes mellitus first described by Munro et al. in 1973 . EDKA, uWe report the case of a 58-year-old female with history of type 2 diabetes mellitus who was admitted to the medical intensive care unit for altered mental status. Her past medical history was relevant for hydrocephalus requiring ventriculoperitoneal (VP) shunting 25 years ago, essential hypertension and obstructive sleep apnea. The patient was last seen at her baseline mental status three hours prior to presentation. Upon arrival, her primary survey was remarkable for a Glasgow Coma Scale score of 6. No focal neurologic deficits were appreciated. The patient was subsequently intubated for airway protection due to minimal responsiveness. Extensive laboratory workup including complete blood count (CBC), chemistries, urinalysis and illicit drug screen was unrevealing. Magnetic resonance imaging of the brain Figure  showed hCSF analysis was negative for infection. An electroencephalogram showed non-specific mild right temporal slowing and moderate generalized slowing. A VP shunt exchange was performed on day 2 of the hospitalization after obstruction was confirmed. Nevertheless, the patient’s clinical status worsened and severe metabolic acidosis was noted the following morning Table . Workup Collateral history obtained from the patient’s family revealed that her diabetes home regimen included insulin glargine 25 units subcutaneously daily, metformin 1000 mg twice daily, glipizide 10 mg daily and empagliflozin 25 mg daily. Her last confirmed intake of oral medications had been on the day prior to presentation. Moreover, it is worth mentioning that the patient was not receiving any enteral nutrition since admission and her diabetes was only being treated with correctional sliding scale insulin while in the intensive care unit. This constellation of information and laboratory findings raised concern for euglycemic diabetic ketoacidosis in the setting of SGLT-2 inhibitor use. The patient was therefore treated with an intravenous insulin infusion. Dextrose-containing maintenance fluids were added to avoid hypoglycemia. Ketoacidosis resolved over 48 hours (see evolution in Table This case illustrates the well-known role of surgery in triggering ketoacidosis. Counter-regulatory hormones, namely, cortisol, glucagon, growth hormone and catecholamines, are secreted at high levels in the post-operative setting . These hOur patient’s intake of empagliflozin one day before hospitalization, persistence of its effect manifesting as glucosuria for more than 48 hours post-interruption, concurrent increase in stress hormones after surgery and absence of adequate calorie intake all resulted in a decrease in the insulin:glucagon ratio and post-operative genesis of EDKA.With recent large studies confirming cardio and reno-protective properties of SGLT-2 inhibitors , 8, and SGLT-2 inhibitors like empagliflozin are known to increase the risk of euglycemic diabetic ketoacidosis, a condition some physicians may be unfamiliar with. This report adds to the growing body of literature demonstrating the risks encountered with surgery, prolonged starvation and insulin cessation in patients treated with these agents. Education of both physicians and patients is crucial to avert or decrease the negative impact of this complication.


N = 602; Mage = 13.50; 46.42% female at baseline), who were mostly between12 and 15 years old. Additionally, using multi-group models, and a stringent p-value of p < 0.01, we explored whether gender and adolescent phase  further moderated these links. The majority of the analyses, resulted in non-significant findings. Specifically, in our non-multi group model, we found no significant peer, and family effects for the entire sample. However, for our multi-group models, we found that higher levels of negative mother-adolescent relationship quality exacerbated the link between peer pressure and subsequent early adolescent boys' delinquency 1 year later, while low levels of mother-adolescent negative relationship quality reversed the association. That is, low levels of mother-adolescent negative relationship quality attenuated the link from higher levels of peer pressure to higher levels of delinquency, but only in early adolescent boys. These findings existed above and beyond significant links from prior adolescent delinquency (T1) to future adolescent delinquency (T2). To conclude, although this was not the case for most adolescents, for early adolescent boys fewer negative interactions between mother and adolescents at an earlier time point (in advance) could potentially curtail the negative effects that delinquent peer pressure has on delinquency in the future. Implications for theory and practice are discussed.Social learning theories assume that delinquent peer norms and/or peer pressure are the components of delinquent peer socialization that lead to subsequent adolescent delinquency. However, these specific peer influences are rarely investigated. Moreover, social learning theories such as coercion theory posit that parenting behaviors also play an important role in the development or prevention of delinquency. However, surprisingly, little research has investigated whether parent behaviors could moderate the link between the above-described peer influences and adolescent delinquency. Hence, using structural equation modeling, the current 1-year longitudinal study investigated these questions among ethnically-diverse Dutch adolescents ( It is often assumed that modeling is facilitated via conformity to perceived delinquent peer norms  or via direct/overt peer pressure ] ssue see ]. Furthesocial neurodevelopmental imbalance models suggest that peers (particularly the presence of peers) are socially rewarding during adolescence. Peer presence is theorized to trigger the reward-related brain regions, leading adolescents to place higher value on arousing motivational or rewarding stimuli  at time point one, investigated parent and peer predictors of minor delinquency  1 year later at time point 2. Specifically, extrapolating from social learning theories, such as Coercion Theory, the current study was designed to test whether higher levels of negative mother-adolescent relationship quality moderates the hypothesized links between delinquent peer norms and peer pressure to engage in delinquency and subsequent adolescent delinquency 1 year later, while controlling for delinquency levels in the previous year. Additionally, we explore gender and adolescent phase (early vs. middle adolescence) moderation effects in all of these independent and interaction links.Taken together, the current 1-year longitudinal study including two age groups of early and mid-adolescents between the ages 12–15  and 582  adolescents respectively. At baseline (year 1) adolescents were 13.50 years (SD = 1.23) and were in their 1st  or 3rd year  of high school. At baseline, most adolescents in their 1st year of high-school were between the ages 12–13 and most adolescents in their 3rd year of high school were between the ages 14–15. These age periods  were used to categorize the adolescents into “early adolescence phase” and “middle adolescence phase,” respectively.Adolescents in the current study were from the first two waves of a larger longitudinal study in the Netherlands on adolescent risk-taking . Data-cThe data-collections took place at schools throughout The Netherlands during regular school hours, and were led by trained research assistants, who were all bachelor and master psychology students. Parents received information letters about the research project as well as dissent letters that could be returned to the schools if they wished to not allow their adolescents to participate. Participants could choose to receive a chocolate candy worth 2 euros as a participation prize, or have their name entered in a raffle for a chance to win a 50 euro gift voucher.Delinquency was measured with 7 items, that tapped vandalism  and property crime (4 items that related to theft) subscales of the International Self-Reported Delinquency questionnaire  that coNegative mother-adolescent relationship quality was measured with the Negative Interaction scale of the Network of Relationships Inventory , and on delinquent peer norms . However, there were no significant gender differences in delinquency at year 1 , for perceived peer pressure  and mother-adolescent negative relationship quality . As for adolescent phase, no significant differences existed in the mean scores of early vs. middle adolescents at a p < 0.01 level (Table T-tests information: delinquency T1 ; delinquency T2 ; peer norms ; perceived peer pressure ; mother-adolescent negative relationship quality . All predictor variables were significantly correlated with delinquency in year 1. Furthermore, peer norms and mother-adolescent negative relationship quality were significantly correlated with delinquency in year 2. All correlations were in the expected directions.In Table el Table . Boys sc on negative mother-adolescent relationship quality, peer pressure to engage in delinquency positively predicts delinquency levels 1 year later. However, adolescents who score 0.35 or below average (p = 0.048) on negative mother-adolescent relationship quality, peer pressure negatively predicts delinquency levels 1 year later. Thus, on the one hand there is an exacerbating effect of high mother-adolescent negative relationship quality, while on the other hand there is a buffering/protective effect of mother-adolescent negative relationship quality on how peer pressure predicts delinquency in early adolescent boys. Follow-up comparisons of the significant interaction in the early adolescent boys subgroup vs. the same interaction in the other three subgroups, showed an overall significant moderation effect of adolescent phase and gender .The model without the multi-group comparisons  moderate these linkages. Our main analyses without the adolescent phase by gender multi-group models only yielded significant results for prior adolescent delinquency. That is, higher levels of delinquency in 12–15 year old boys and girls predicted higher levels of delinquency 1 year later, however there were no significant effects for delinquent peer norms, perceived peer pressure, negative mother-adolescent relationship quality, or for the interaction between these peer and parent factors.When looking specifically at early adolescents, results showed that for early adolescent boys, lower than average mother-son negative relationship quality  attenuated the relationship between delinquent peer pressure and adolescent delinquency. Conversely, higher than average mother-son relationship quality exacerbated the link between of delinquent peer pressure and delinquency. In other words, the lesser the negative mother-adolescent relationship quality, the less delinquent behavior adolescents will portray when confronted with increased delinquent peer pressure. However, the higher negative mother-adolescent relationship quality, the more delinquency early adolescent boys will portray when faced with increased delinquent peer pressure. Thus for early adolescent boys, low mother-adolescent negative relationship quality acts as a protective factor against the effect of peer pressure on adolescent delinquency, whereas high mother-adolescent negative relationship quality acts as a risk factor. Finally, we did not find any significant interaction effects for middle-adolescents, or early adolescent girls, however.boys compared to early adolescent girls. This finding of gender differences in Mason et al.  are more vulnerable to delinquent peers, perhaps because early adolescents have not yet fully developed the capacity to resist peer influence . It shoOn a side note, it is relevant to mention that although coercion theory is an influential theory on externalizing problem behavior development, it has also been criticized. Namely, coercion theory claims that it is parental behaviors that causes their offsprings to act in similar ways to their parents. In other words, adolescents learn behaviors from their parents. However, parents and their offsprings might act in similar ways because of genetic confounding [for a discussion see ], an impThe current study showed that when investigating adolescent delinquency, interactions between peer and parent predictors as well as moderation by adolescent phase and gender are important to consider. Namely, particularly for early adolescent boys, we found that influences of delinquent peers and parents do not operate independently, as it is the interplay between peer pressure to engage in delinquency and high negative mother-adolescent relationship quality that predicted higher levels of delinquency. However, importantly, the reverse is also true. That is, low levels of negative relationship quality between mothers and their early adolescent sons, can minimize the amplifying effect that deviant peers have on adolescent delinquency. Thus the current findings have highlighted potentially amendable characteristics of parent-adolescent relationship quality that could possibly make early adolescent boys less vulnerable to delinquent peer pressure, and thus this could be valuable findings for interventions. Accordingly, the findings could perhaps be valuable for interventions if considered within the broader context of what is known on relative parent and peer influences on adolescent delinquency. For example, the results suggest that delinquent peers increase adolescent delinquency, but only under certain conditions, such as when there are higher levels of negative mother-adolescent relationship quality in combination with higher levels of peer pressure to engage in delinquency, and this is only the case for early adolescent boys. Having a healthy relationship with mothers could then possibly break the vicious cycle of the effect of delinquent peer affiliation on delinquency. Namely, adolescents who engage in delinquency typically have delinquent friends who might pressure them to (further) engage in delinquent behaviors. However, it is likely not feasible to include adolescent's entire delinquent peer group in therapy sessions. Hence in such a case, interventions could focus on improving the relationship quality between mothers and their adolescent offsprings, and this could minimize the amplifying effect that delinquent peer pressure has on adolescent delinquency. One of such notable interventions that has proven to be effective for Dutch families  is the prevention efforts in addition to the above-described intervention efforts.Considered together, the longitudinal nature of our results suggest that ensuring fewer negative interactions between mothers and adolescents at an earlier time point (in advance) could potentially curtail the adverse effects delinquent peer pressure has on early adolescent boys' delinquency in the future. Thus, the current findings could also provide useful implications for This study was carried out in accordance with the ethical standards that were in place at the Faculty of Social and Behavioral Sciences, Utrecht University at the time the study began. Standardized questionnaires and computer tasks were used that were not deemed likely to yield physical or emotional risks for the participants and therefore review by the ethical committee was not deemed necessary and therefore no formal approval was given. All subjects' parents gave passive informed consent for their adolescent's participation in the study and participants could withdraw their participation at any time.ID developed the study concept and design, and JD and MvA gave advice and feedback. ID oversaw the data-collection. ID performed the data-analysis and interpretation. ID drafted the manuscript, and JD and MvA provided critical revisions.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.


Altered expression of many genes and proteins is essential for cancer development and progression. Recently, the affected expression of metadherin (MTDH), also known as AEG-1 (Astrocyte Elevated Gene 1) and Lyric, has been implicated in various aspects of cancer progression and metastasis. Elevated expression of MTDH/AEG-1 has been reported in many cancers including breast, prostate, liver, and esophageal cancers, whereas its expression is low or absent in non-malignant tissues. These expression studies suggest that MTDH may represent a potential tumor associated antigen. MTDH also regulates multiple signaling pathways including PI3K/Akt, NF-κB, Wnt/β-catenin, and MAPK which cooperate to promote the tumorigenic and metastatic potential of transformed cells. Several microRNA have also been found to be associated with the increased MTDH expression in different cancers. Increased MTDH levels were linked to the tumor chemoresistance making it an attractive novel therapeutic target. In this review, we summarize data on MTDH function in various cancers. Chen etin situ (DCIS) of the breast  leads to increased MTDH and acts as a prognostic marker for breast cancer outcome  play important, if subtle, roles in the regulation of gene expression and translation in cellular differentiation and proliferation. Unsurprisingly, aberrant expression of oncogenic miRs (onco-miRs) and tumor suppressor miRs is implicated in cancer pathogenesis , 50. MTDmiR-21 . In contt cancer   tumor suppressor via NF-κB mediated pathways and contributes to the HER2-targeting therapy resistance . Thus, M+ mediated immune response inhibited cancer cell proliferation and lung metastases in a mouse breast cancer xenograft model (Immunotherapy targeting the tumor antigens has been an area of intense research. Expression of MTDH is increased in many different cancer types. Importantly, auto-antibodies against the MTDH protein have been detected in cancer patients confirming its immunogenicity and supports MDTH as a potential immunotherapy target . We receft model . Given tMTDH is pro-oncogenic factor playing multifaceted and diverse roles in cancer progression. Its association and central role in regulating signaling pathways such a MAPK, wnt/β-catenin, PI3K/AkT, NF-κβ pathways in various cancers shows that it plays a vital role in metastasis. MTDH contribution to chemo and radiotherapy resistance provides a new direction for the development of anticancer therapeutics. Multiple mechanisms converge to promote expression of MTDH in cancers. Further studies are therefore warranted to determine whether the elevated MTDH expression has prognostic value for development of the malignancy. Given its increased expression in many cancer types it would be important to determine whether MTDH represents a feasible target for cancer therapy, including immune-therapy. For these reasons there is now an urgent need to determine the clinical and therapeutic significance of increased MTDH expression in cancer.GD originated the idea for writing the review and wrote about MTDH association in different cancers. NS and MG wrote the following sections: micro RNAs regulation of MTDH expression and MTDH target for cancer therapy. ER, JL-R, and NM were involved in writing the MTDH regulation and molecular functions and preparing the original figures. RM and SK contributed to the clinical significance of MTDH expression in cancer. AR managed the different collaborations during the writing of the review and contributed to editing the manuscript. MB was overall responsible for coordinating and managing multisite collaboration, and writing 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.


To report our findings in a young patient with unilateral retinitis pigmentosa (RP)-like appearance who developed pigmentary changes in his left retina after an episode of bilateral pars planitis.A 17-year-old man presented with 6 months of blurry vision in both eyes. He was diagnosed with bilateral pars planitis. Progressive, intraretinal bone crepuscule pigmentation developed in his left retina during the following three months. An electroretinogram showed subnormal response only in the left eye, suggesting the diagnosis of unilateral pseudoRP.An inflammatory disease like pars planitis can accelerate the pigmentation of the retina and mimic a RP in young patients. Causes of pseudoRP may be considered, especially in those rare cases with unilateral affection. Retinitis pigmentosa (RP) refers to a group of inherited diseases causing retinal degeneration. It is a major cause of visual disturbance and blindness in all age groups. It usually comes with a bilateral, symmetric impairment of visual functions along with night blindness and gradually concentric loss of peripheral vision . Howeversine pigmento has been used [Retinal pigmentary changes as coarse clumps in a “bone spicule” configuration are typically observed in RP, but fundus appearance depends on the stage of retinal degeneration. In the earliest stages, especially in young patients without symptoms, the fundus usually appears normal and the diagnosis may be delayed. When no pigmentation is observed in the fundus despite documented abnormalities of retinal cell function, the term RP een used .Some disorders as melanoma of the choroid can lead to retinal changes with pigment irregularities, mimicking RP . Few infWe present a case of a young patient who quickly developed pigmentary changes in his left retina mimicking a unilateral RP after an episode of bilateral pars planitis.A 17-year-old man with no known past medical history, presented with 6 months of blurry vision in both eyes. He had no other ocular, medical, or surgical history.Baseline visual acuity (VA) was 20/20 in the right eye (RE) and 20/63 in the left eye (LE). His intraocular pressure was normal (17/16 mmHg). Slit lamp examination revealed normal anterior structures and no anterior segment inflammation. A subclinical keratoconus was described in the topography study. 1+ cells in the vitreous , snowballs and snow banking in the inferior pars plana as well as peripheral vasculitis were observed in both eyes (BE) on dilated fundus examination.Toxoplasma gondii, Borrelia burgdorferi or Treponema pallidum were negative, as well as for antinuclear (ANA) and antineutrophil cytoplasmatic (ANCA) antibodies.Serological tests for HIV, He was diagnosed with bilateral pars planitis. Optical coherence tomography (OCT) showed cystoid macular edema in the LE, so that sub-tenon injection of triamcinolone was performed in order to control inflammation, which was slowly reduced.Subsequent follow-up visits showed improvement in vision to 20/25 in the LE, resolving progressively the inflammation of the posterior pole although 0.5+ cells were observed in the vitreous cavity. Then, no additional therapy was needed. Nonetheless, the patient complained of loss of visual field in his LE.In-depth fundus examination showed a mild, diffuse, granular appearance of the retinal pigment epithelium (RPE) throughout the LE. Progressive, intraretinal bone crepuscule pigmentation developed during the following three months Fig. . The RE An electroretinogram (ERG) showed subnormal response only in the LE. The diagnosis of unilateral RP-like appearance was made.We report a rare case of pars planitis with progressive unilateral pigmentation of the retina. Unilateral RP  is a rare and controversial condition that has been described in the literature, but there are still doubts about its existence as an isolated clinical entity and its relation to RP. Some mechanisms purposed are the occurrence of genetic mosaics (mutations that affect only some of the cells) or somatic mutations (instead of germline mutations) , 8. SincThe differential diagnosis for unilateral retinal pigment epithelium changes and peripheral field loss starts with asking for prior blunt trauma, retinal detachment or retained metallic intraocular foreign body. Treatment with antipsychotics from the group of phenothiazines can induce a peripheral pigmentary retinopathy . Since oSome patients with cancer can develop antibodies against retinal antigens (antirecoverin antibodies), leading to a paraneoplastic syndrome called carcinoma-associated retinopathy or autoimmune retinopathy . Since tRetinal pigmentary changes can also occur in the context of past ocular infection or inflammatory eye disease. Syphilis, toxoplasmosis, cytomegalovirus, rubella, measles or diffuse unilateral subacute neuroretinitis (DUSN) must be ruled out . They caThe patient was first diagnosed of pars planitis. Few publications of uveitis coexisting with RP have been reported, specially a form of non-granulomatous uveitis called Fuchs’ heterochromic iridocyclitis –14. DustThe typical bone-spicule pigmentation represents migration of pigment into the interstitial spaces of the retina from disintegration of RPE cells. This pigment accumulation is greater in the surrounding spaces of the retinal vessels, producing perivascular pigmentary cuffing and spicule-shaped deposits. Almost all forms of RP go through a stage where no pigmentary changes exist in the retina. This stage may exist for decades before typical RP signs appear.Pigmentation of the retina developed in only three months in our patient. Probably some grade of epithelitis developed in the left eye of our patient. We hypothesize that inflammation may accelerate the pass to the pigmentary stage. It is well known that inflammatory eye diseases like rubella, syphilis or DUSN present with pigmentary abnormalities within the retina . InflammIn summary, an inflammatory process like pars planitis can accelerate the pigmentation of the retina. Asymmetrical RP must be ruled out in these unilateral RP-like appearance post-inflammatory changes.


Transthoracicechocardiography, magnetic resonance angiography, and computed tomography (CT)angiography are first line noninvasive tests available while invasive tests such ascoronary angiography and intravascular ultrasound are second line diagnosticalternatives. Treatment approaches are still controversial and election of theoptimal surgical procedure, whenever applicable, must be an individualized andpatient-centered decision.We present the case of a young patient with an anomalous left coronary artery arisingat the right sinus of Valsalva with severe stenosis and hypoplasia throughout theinterarterial segment, which hampered corrective surgery. The timely detection andadequate treatment of this specific anomaly gains relevance because of itsassociation with increased risk of sudden cardiac death. Although the trueprevalence of interarterial anomalous left coronary artery is unknown, owing to thelack of population-wide screening studies and at times asymptomatic course, itsfrequency has been reported at 0.03%.A fourteen-year-old otherwise healthy boy with no family history of disease presentedwith severe chest pain while he had been jogging for 5 minutes. The pain lasted for2 hours and was followed by generalized weakness, dyspnea and confusional state. Hewas initially treated on a secondary care local clinic in which a baselineelectrocardiogram reported ST segment depression in all precordial leads and serumTroponin I taken within 24 hours of symptom onset reached > 30 ng/mL (referencelevel of fluorescence immunoassay 0-0.4 ng/mL). The patient developed pulmonaryedema and spent 7 days in the intensive care unit. After stabilization, he wasreferred to our tertiary care hospital. On hospital admission, he washemodynamically stable, cardiac and pulmonary examination were normal. Plain chestx-ray was normal and the electrocardiogram showed sinus rhythm with ST segmentdepression and repolarization abnormalities in precordial leads V1 to V3. Completeblood count reported leukocytosis with neutrophilia; lipid profile and thetoxicologic screening, including cocaine, came back normal. A transthoracicechocardiogram was performed which revealed a hypokinetic anteroseptal wall withnormal systolic and diastolic function; no report of coronary anomalies wasdocumented in the first place. Polymerase chain reaction tests for various viruses on peripheral blood samples werenegative. He was pharmacologically managed with aspirin, atenolol and ivabradine. Arest perfusion magnetic resonance imaging detected an anterior, anteroseptal andlateral nontransmural myocardial infarction with systolic left ventriculardysfunction (ejection fraction of 45%) alongside an anomalous origin of the leftcoronary artery arising at the right sinus of Valsalva with an interarterialstenotic tract. A CT angiography demonstrated a left coronary artery arising at theright sinus of Valsalva from a separate ostium with an acute take-off angle andproximal oval-like narrowing with an extension of 11 mm running throughout theinterarterial segment  and 2. CWe deem important the presentation and discussion of this case considering its uniquehigh-risk features, particular evolution and nonstandard surgical approach with goodclinical outcome.4,5 Clinicians must bear in mind that anomalous origin of the leftcoronary artery is a differential diagnosis in every previously healthy youngpatient with acute onset chest pain and evidence of acute myocardial ischemia.Transthoracic echocardiography can be the ideal diagnostic test in low-incomesettings; however, it must be noted that its accuracy is limited6 and a specific evaluation may obtainbetter results in the diagnosis. The detailed description of the anomaly shouldalways be sought considering that there are three anatomical features that have beenlinked to a worse prognosis: intramural course, slit-like coronary ostium, and acutetake-off angle of the anomalous coronary.7 In this case, apart from an acute take-off angle, a stenoticand hypoplastic course added to the disease burden. Given the aggravating coronaryfeatures encountered in this patient, aborted cardiac arrest or even sudden cardiacdeath could have been an expected outcome. Furthermore, the surgical approach couldnot tackle the coronary anomaly. Regardless of these apparently adverse factors, thepatient fully recovered and reports asymptomatic with no evidence of cardiac lesionat more than one year follow up, which gives light to the fact that there must beother factors, such as vasoreactive ability and early collateral circulation, thatcan influence the course of this disease.Structural heart diseases are among the causes of sudden cardiac death in youngpatients.7 Although the safety of correctivesurgery has been demonstrated,8,9its efficacy in the prevention of sudden cardiac death in the long term remains tobe proven with further prospective studies. Besides, whenever we encounteraggravating features that make corrective surgery a difficult approach, coronaryartery bypass grafting poses an alternative without undermining patient’s short- andlong-term prognosis. Comparing these surgical approaches in cohort studies should beadvocated.Corrective surgery, such as coronary translocation, must be offered to symptomaticpatients with this coronary anomaly and high-risk features.The poorly understood physiopathology and natural history of this coronary anomalyhinder the development of risk stratification strategies and causes controversies inmanagement algorithms. The presence of knowledge gaps regarding true worldwideprevalence, specific mechanisms of myocardial ischemia and optimal surgical optionscall for ongoing research to improve evidence-based decision making.


We read with great interest the recent systematic review and meta-analysis of high-flow nasal cannula (HFNC) oxygen therapy versus conventional oxygen therapy (COT) in patients after planned extubation . We greaFirst, randomized controlled trials (RCTs) and non-RCTs may be inappropriately combined together in the meta-analysis, which goes against the principle of pooling studies with the similar design , 3. Thus2 and respiratory rates may be improper. The standardized mean difference is utilized as the summary statistic in the meta-analysis when the trials all assess the same outcome, but measure it in various ways [Second, using the standardized mean difference as the summary statistic for the meta-analyses of PaOous ways . Moreoveous ways . TherefoZ-curve neither crossed the line of the trial sequential monitoring boundary for benefit nor the required information size boundary, which established inconclusive evidence [Z-curve obviously crossed the line of the trial sequential monitoring boundary for benefit, which may mislead the interpretation because of the inaccurate trial sequential analysis. Therefore, the figure of trial sequential analysis may better be not drawn based on a rough estimated RRR.Third, trial sequential analysis for comparison of postextubation respiratory failure between two groups may better be drawn based on the accurate relative risk reduction (RRR) of 37.17%  and cross-over studies were not pooled together for primary outcome and main secondary outcomes in our article, only pooled together for secondary outcomes of respiratory rate, PaOentified . Further2. However, as the comfort score was measured with different scales among the included studies, the standardized mean difference (SMD) should be used.Second, we thank Dr. Luo and agree that the weighted mean difference (WMD) should be used when outcome measurements in all trials are made on the same scale. WMD would be better for analyzing respiratory rate and PaOχ2 = 7.82, df = 4, P = 0.10, I2 = 49%) among the included studies, which might have been due to the heterogeneous population of patients and various treatment measures after extubation, the accurate RRR of 37.17% could not reflect the realistic RRR. Hence, basing on the RRR of 37.17% to draw the trial sequential analysis for comparison of postextubation respiratory failure between two groups is improper and may also mislead the results. Furthermore, we are cautious to our conclusion and underlined in our article that “a decisive conclusion should be made cautiously. Further large-scale, multicenter studies are needed to confirm our results.”Third, according to the user manual for trial sequential analysis (TSA), the estimation of the control group event proportion and an anticipated intervention effect are important determinants of the calculated required information size when doing TSA . The ant


Fluidity in cell fate or heterogeneity in cell identity is an interesting cell biological phenomenon, which at the same time poses a significant obstacle for cancer therapy. The mammary gland seems a relatively straightforward organ with stromal cells and basal- and luminal- epithelial cell types. In reality, the epithelial cell fates are much more complex and heterogeneous, which is the topic of this review. Part of the complexity comes from the dynamic nature of this organ: the primitive epithelial tree undergoes extensively remodeling and expansion during puberty, pregnancy, and lactation and, unlike most other organs, the bulk of mammary gland development occurs late, during puberty. An active cell biological debate has focused on lineage commitment to basal- and luminal- epithelial cell fates by epithelial progenitor and stem cells; processes that are also relevant to cancer biology. In this review, we discuss the current understanding of heterogeneity in mammary gland and recent insights obtained through lineage tracing, signaling assays, and organoid cultures. Lastly, we relate these insights to cancer and ongoing efforts to resolve heterogeneity in breast cancer with single-cell RNAseq approaches. The mammary gland is comprised of many different interacting cell types. Epithelial cells form a primitive structure early during embryonic development, which later form the nipple and a ductal network that expands into the fat pad. These epithelial ducts are surrounded by fat cells, and become innervated with a variety of stromal cells including endothelial cells, immune cells, and fibroblasts . MaturatMammary epithelial cells (MECs) typically form a bilayered epithelium and can be broadly separated into two distinct compartments: an inner layer of luminal and outer layer of basal MECs. However, additional heterogeneity exists within both luminal and basal MECs. Luminal populations are often sub classified based on hormone and growth factor receptor status. The basal epithelium contains a subset of so-called myoepithelial cells that lie along the outside of the ductal epithelial tree and assist in the motility of milk protein along the lumen. The MEC system also contains subsets of basal and luminal stem and progenitor cells, which will be covered in greater detail later. For example, within the luminal compartment a population of luminal progenitor cells can go on to form ductal or alveolar cells. In addition, basal cells contain a subset of mammary stem cells (MaSCs) forming a small, heterogeneous, and to date poorly defined population that drive development, repair and organ reconstitution when transplanted into epithelium-cleared fat pads of recipient mice .Puberty is the most dynamic and striking period during the development of the mammary gland. The rudimentary duct undergoes significant expansion, resulting in the formation of bulbous multilayered structures called terminal end buds (TEBs)  Figure . TEBs arThe luminal compartment contains different mature, differentiated cell populations with specified functions as well as progenitor-cell populations that are each distinguished by specific gene and extracellular ligand profiles.loCD29loCD24+CD14−EpCAMhic-kit−Sca1+CD61−CD49b− [loCD29loCD24+CD14−EpCAMhiSca1loCD61− expression pattern. Alveolar cells can be distinguished by their secretory morphology and histologically by the accumulation of milk proteins within.Several classifications of luminal cells exist within the mammary gland. Luminal cells are segregated based on functional, morphological, and expression profiling evidence. Histologically, luminal cells can be broadly separated into two types: ductal and alveolar. Mature ductal cells can be either estrogen receptor (ER) positive or negative, and feature the following marker expression profile: CD49f1−CD49b− , in whicTranscription factors have been demonstrated to drive luminal cell fate commitment throughout development. The Elf5 (E74-like factor 5) transcription factor is one of the major drivers of luminal cell specification and differentiation. Elf5 is able to directly repress Slug (Zinc Finger Protein SNAI2) transcription, thereby blocking basal cell determination and promoting luminal cell fate ,9. Proge+, CD49b+, CD14+, and c-kit+ whereas their mature counterparts are negative for the surface expression of these proteins [Luminal progenitor populations can be distinguished using many of the above extracellular markers. For example, ductal progenitors are CD61proteins ,14,15. Sproteins . The fathiCD49fhiCD24+EpCAMlo/med [hiCD29hiCD24hiEpCAMlo/medSca1−. Markers that are specific for a restricted basal cell progenitor have not been identified and neither have specific markers for a restricted myoepithelial cell progenitors. Luminal progenitors are thought to arise from an early basal cell population.Unlike luminal cells, the factors that control and label different basal cell populations are less clear. Both stem cells and bipotent progenitor cells have been identified within the broader basal cell lineage. The use of extracellular markers to distinguish these discrete populations, however, has not yet yielded clear profiles. Mature myoepithelial basal cells are typically CD29AMlo/med . The undSeveral epithelial-mesenchymal transition (EMT) factors have been identified as molecular regulators of the basal/MaSC population. The transcription factor Slug is a key for the basal/MaSC lineage as it suppresses luminal cell fate. Slug-deficient mice have delayed ductal morphogenesis and aberrantly express luminal signature genes in basal cell populations ,18. The Lineage tracing is a powerful tool to observe and track the function of adult-, stem- and progenitor-cells during normal homeostasis, and various studies have utilized this approach to shed light on the lineage hierarchies of the MECs. K8-creER targeted cells exclusively differentiate into luminal cells when observed in vitro . MECs maLineage tracing studies were performed in order to hone-in on the exact nature of mammary progenitor populations. In 2011, van Keymeulen et al. established that all mammary epithelial lineages are derived from cytokeratin-14 (K14) expressing embryonic progenitors . FollowiIn the late 1980s and early ′90s, three-dimensional (3D) cultures were instrumental in the discovery of the cell biology of mammary gland development and breast cancer. We term these classical three-dimensional spheroid cultures in our review, as these did not yet focus so much on the function and preservation of stem cells through Wnt signaling. We will cover more on Wnt signals and organoids later. For an excellent overview with historic perspectives on the development of these 3D culture platforms we refer you to a review by Drs Simian and Bissell . EssentiClassical 3D spheroid cultures also aided identification of restricted luminal- or basal-cells and bipotent progenitors. In vitro experimentation confirmed that, the restricted differentiation potential of both luminal cells and basal, myoepithelial cells can be maintained, given specific culture conditions . In mammThe EGFR family consists of EGFR (ErbB1/Her1), ErbB2/Her2, ErbB3/Her3, and ErbB4/Her4. ErbB receptors can both homo- and heterodimerize, with 10 possible combinations-six heterodimers and four homodimers. There is a high level of homology between kinase domains of the four EGFRs (~60–80%) with divergence occurring predominantly in the C terminus (shared identity is only ~10–25%). ErbB proteins have tissue specific expression, though they are commonly expressed together. EGFRs are found in several epithelial cell types such as the lung, intestine, and the breast. ErbB2 has no known ligands , but freUp to 13 different ligands have been found to bind EGFR family proteins: EGF, HB-EGF, transforming growth factor (TGF), amphiregulin (AREG), epiregulin (EREG), epigen (EPG), betacellulin (BTC), and neuregulins 1–6 (NRG) ,35,36. LEgfr-deficient mice perish just after birth, which complicated deciphering the exact nature of EGFR during mammary organogenesis [Waved-2 mice to circumvent this problem. The Waved-2 allele has a point mutation near EGFR’s cytoplasmic kinase domain that reduces activity; it is hypomorphic [Waved-2 mice have defective mammary development with diminished branching and a reduction in ductal invasion [Egfr’s importance in the stroma was confirmed via the generation of mixed tissue recombinants from transplanting neonatal epithelial cells from wild type or egfr-deficient mice [The vast majority of what is understood about the EGFR family’s role in mammary development comes through the use of genetic mouse models. ogenesis ,40. Luetomorphic . Waved-2invasion ,43. Use invasion . Egfr’s ent mice ,46.ErbB2–4 results in impaired ductal outgrowth during puberty. Deletion of ErbB2 shunts ductal outgrowth [Perturbations in other EGFR family genes also result in dramatic mammary developmental phenotypes. Specifically, deficiency in the utgrowth ,48. ErbBMammary epithelial cells are organized into a developmental hierarchy based on extracellular receptor and gene expression patterns. The exact nature of these populations, and the factors that balance their proliferation with differentiation, are not well understood. Recent evidence has emerged, however, that EGFR signaling in MECs may be a key player in better defining this hierarchy as depicted in In 2011, a report by Pasic et al. began to decipher EGFR’s potential role in controlling MEC fate decisions during development. An ex vivo organoid model was utilized using cells taken from normal human breast tissue. They observed that different EGFR ligands could elicit discrete cell fate decisions. EGF stimulation of human breast organoids initiated a significant expansion of the basal  population. Conversely, AREG stimulation drove organoids towards a luminal  cell fate. Interrogation of the downstream effector revealed that this deviance in cell fate decisions was due in part to the strength of downstream MEK-ERK signals, in which stronger activation EGFR-Ras-MEK-ERK selectively expanded the basal cell population and weaker activation drives luminal expansion .loEpCAMhi and EpCAMlo cells [Mukhopadhyay et al. expanded our insights into this initial model in 2013 . Using alo cells . Taken tSince many of the signaling effectors triggered by the EGFR lay downstream of Ras, it is of interest to consider the strength and duration of Ras activation as the cell fate determination factor. A historic study reported that nuances in receptor-Ras signaling can affect cell fate in a PC-12 cell line system. Stimulation of rat adrenal carcinoma cells (PC-12) with different EGFR ligands produced altered cell fate. In the PC-12 system, EGF is a weaker agonist compared to the strong nerve growth factor (NGF). EGF stimulation led to a short pulse of Ras-MEK-ERK activation and cell proliferation, while NGF stimulation elicited prolonged Ras-MEK-ERK signals, exit from the cell cycle and differentiation . Since tDuctal morphogenesis is the process in which the mammary epithelium invades the fat pad during puberty to form a fully branched ductal epithelial tree. It is known to occur in a somewhat stochastic process, regulated by the combinatorial input of diverse signals. The stochastic aspect is perhaps best exemplified by the fact that there are substantial differences between mammary glands of mouse littermates. This has led the field to conclude that predetermined genetic control of pubertal development is not a possibility, unlike in the development of other epithelial tissues . DevelopThe pubertal developmental process is initiated in large part by the expression of ovarian and pituitary hormones . These sEstrogen is not the only steroid hormone important for pubertal mammary gland development, however. In fact, estrogen alone is insufficient to restore ductal morphogenesis when other input (like from the pituitary gland) is missing. Exogenous administration of growth hormone, normally produced by the pituitary gland, can restore the impaired branching phenotype of pituitary gland-deficient mice . Growth fgfr2 disrupts ectodermal and placode formation during embryonic mammary organogenesis [fgfr2 have a proliferative disadvantage when compared to their wild-type and fgfr2-heterozygous counterparts, and are depleted within TEBs [fgfr2−/− reveal a similar phenotype; proliferation is significantly attenuated and TEBs are completely absent from the glands [fgfr2 is a key regulator of luminal epithelial cells and it plays a specific role in the TEBs of elongating ducts.Several growth factor receptors and receptor tyrosine kinases (RTKs) are involved in the integrated signaling environment that directs mammary morphogenesis. Fibroblast growth factors (FGF) and their receptors (FGFRs) are critically important for growth and branching. FGF2 and FGFR2 in particular shape the profile of ductal outgrowth and MEC proliferation. Genetic deletion of ogenesis . Mammaryhin TEBs . Mice wie glands . In summErbB2 shunts ductal outgrowth [ErbB2 also controls TEB formation through its regulation of cellular compartmentalization. ErbB3 deficiency results in small TEBs and increased branch density with decreased TEB size as a result in an increase in apoptosis, controlled via observed changes in the PI3K (Phosphoinositide 3-kinase) signaling pathway. Anti-apoptotic transcription factor Bcl-2 is reduced in −/−ErbB3 mice [ErbB4 have defect occurring later in breast development, specifically during the formation of milk-producing luminal cells [The EGFR family of signaling proteins plays a pivotal role in directing pubertal mammary gland development in conjunction with FGF and FGF2. In brief, perturbations to each of the four EGFR family members result in developmental defects. In ex vivo culture, FGF2 addition can rescue growth and branching in EGFR-null 3D spheroids . Mice wiutgrowth ,48. ErbB−/− mice . Mice deal cells .Dr Schlessinger put forth an elegant and simple model for EGFR activation with EGF as an external signal leading to the conversion of a monomeric receptor to a ligand-induced dimer , which sWhereas it is clear that the EGFR family plays a role in mammary gland development and maturation as well as breast cancer, a lot less is known about the specific effector kinase pathways that lie downstream of the receptor families such as the EGFR. Elegant imaging studies revealed that levels of phosphorylated ERK kinases (a type of MAPK) are highest in MECs near the front of elongating ducts whereas levels of phosphorylated Akt kinase were equal throughout and Huebner et al. proposed a model of receptor-induced proliferation leading to highly motile cells with high phospho-ERK . Two thoWhile there is a general consensus about some of the factors that mark and or regulate the MaSC population, the field has been left with more questions than answers when trying to specifically define MaSCs . For exa+ cells have increased repopulating activity and whether they trend towards quiescence or self-renewal has not been clearly delineated. The exact nature and potential of Lgr5+ and Lgr5-negative MaSC populations is an ongoing field of research [Some progress has been made towards separating MaSCs from mature myoepithelial and basal progenitors by using transgenic mouse models. MaSCs residing in the terminal end bud (TEB) cap cells exclusively express the phosphatase s-Ship , but MaSresearch ,86,87. Tresearch . Furtherresearch . The hetresearch ,91. Receresearch . In sum,In summary, in vitro and in vivo findings suggest that breast epithelial cells can be arranged into a developmental hierarchy , but it Organoids are miniature, three-dimensional, in vitro tissue cultures that retain stem cell function, and are generated from pieces of tissue . OrganoiFurthermore, organoids can be cultured indefinitely and can be cryopreserved, whereas classical mammosphere cultures generally survive for shorter time periods . Indeed,Lgr5 [A crucial part of successfully culturing organoids  is to prLgr5 . R-spondLgr5 . StructuLgr5 .In addition to surrogate Wnt and R-spondin ligands, EGF and Noggin are required to indefinitely expand organoids . FurtherAdvances in next generation sequencing and microfluidic-based handling of cells and reagents now enable us to explore cellular heterogeneity in an unbiased manner using various single-cell genomics modalities to profile genomic features in individual cells . The curAmong the genomics modalities available to date, single-cell RNA sequencing (scRNAseq) is the most advanced and most widely accessible to the research community, and has recently been applied to both human and mouse mammary epithelial samples . A recenElf5) and mature, hormone-responsive luminal cells . In addition to the main cell types, intermediate states were identified marked by expression of both luminal and basal genes, which could be indicative of transitional cell states between luminal and basal cells and thus support the notion of one continuous lineage trajectory that maintains all MEC cell types. Another similar study focused on the differentiation dynamics of the mouse mammary epithelium during various developmental stages of adult virgin, pregnant, lactating and involuting mammary gland using scRNAseq, which defined the lineage hierarchies as a differentiation continuum rather that discrete differentiation stages [The mouse mammary epithelium at post-pubertal stage generally revealed a comparable cell type and state composition with some differences in terms of marker genes expressed in each cell type. Pal et al.  used scRn stages . The mosn stages ,120. It n stages , or highn stages ,123, incn stages ,125, andn stages . More isIn addition to gene expression programs, the epigenetic makeup of the cell is a critical determinant of cellular identity that is not detectable in scRNAseq data. Recent technological advances now allow for profiling chromatin accessibility using the Assay for Transposase-Accessible Chromatin using sequencing (ATACseq) to reconstruct cis/trans regulatory elements associated with cellular identity . FurtherIn summary, cartoons such as the one depicted in


Klebsiella gavage on the microbiome. Deficient Paneth cell function induced significant compositional changes in the cecal microbiome with a significant increase in Enterobacteriacae species. Further, the bloom of Enterobacteriaceae species that occurs is phenotypically similar to what is seen in human NEC. This further strengthens our understanding of the importance of Paneth cells to intestinal homeostasis in the immature intestine.Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal morbidity and mortality in premature infants. Human and animal studies suggest a role for Paneth cells in NEC pathogenesis. Paneth cells play critical roles in host-microbial interactions and epithelial homeostasis. The ramifications of eliminating Paneth cell function on the immature host-microbial axis remains incomplete. Paneth cell function was depleted in the immature murine intestine using chemical and genetic models, which resulted in intestinal injury consistent with NEC. Paneth cell depletion was confirmed using histology, electron microscopy, flow cytometry, and real time RT-PCR. Cecal samples were analyzed at various time points to determine the effects of Paneth cell depletion with and without  A key regulator of small bowel homeostasis and the intestinal microbiome is the Paneth cell . Paneth Enterobacteriaceae prior to NEC development ) overnight at 4°C. Following fixation, samples were post-fixed with 1% osmium tetroxide for 1.5 h and then dehydration with ethanol and embedded in Epon 12 . Ultrathin sections (70 nm) were post-stained with uranyl acetate and lead citrate and viewed with a JEOL 1230 transmission electron microscope (TEM) .Samples were deparaffinized and rehydrated. To unmask antigens, citrate buffer (pH 6.0) was used in a Biocare Company Decloaking Unit at 110 degrees for 15 minutes followed by TBST washing (5 minutes x 2) and blocking in 5% normal goat serum . Rabbit-Anti HA , chicken-anti GFP , anti-lysozyme  were used as primary antibodies at manufacturers recommended concentrations. D1C2 antibody was developed through the Intestinal Stem Cell Consortium  and Firmicutes (p < 0.0001) , compared to control mice of the same age , the mice were euthanized and their cecal samples were collected and examined as above. Microbiome alterations following dithizone-induced Paneth cell depletion, although not statistically significant in the short term, exhibit significant changes at three weeks. PC-DTR) which has a human diphtheria toxin receptor bound to the Cryptdin-2 promotor on the Paneth cell membrane [PC-DTR mice to evaluate for strain differences. The predominant organisms in the PC-DTR mice were statistically equivalent to those in C57Bl6 mice and included Firmicutes, Bacteroidetes, and Proteobacteria. To determine the effects of Paneth cell depletion on the microbial community of PC-DTR mice, cecal samples were collected 1, 24 and 72 hours after DTX exposure. Paneth cell depletion from DTX exposure in PC-DTR mice resulted in significant acute alterations to the microbial composition, which trend back towards normal over time. The most significant change was seen in the 24 hour post-treatment time point although minor differences were also seen at 1 hr and 72 hrs post treatment. Importantly, the two models of Paneth cell depletion, dithizone and DTX, induce distinct changes to the composition of the cecal microbiome . Using this reagent, we examined the effect on crypts of animals treated with dithizone or DTX compared to sham controls , we lastly wanted to determine if the bloom of Enterobacteriaceae seen in our model was simply a result of the gavaged bacteria in our NEC model. Timed samples from Rosa mice gavaged with GFP-labeled Klebsiella were harvested and evaluated. Tagged Klebsiella were present in the ileum as early as 30 minutes after gavage and absent from the small intestine by 5 hours (Klebsiella gavage, and then gradually returned to baseline levels within 4 hours after bacteria administration. The bloom of Enterobacteriaceae present following induction of clinical NEC via Paneth cell depletion was seen more than 8 hours after Klebsiella gavage, well after the administered Klebsiella was shown to have passed through the small intestine.Since  5 hours . Additio 5 hours . OverallKlebsiella not only induces intestinal injury that resembles human NEC [Enterobacteriaceae family [The host-bacterial axis that exists between humans and their intestinal microbes is critical to maintenance of health. Neonates are exposed to an environment teeming with bacteria at birth leading to sequential colonization by waves of bacteria in predictable patterns , 43. Thiuman NEC , but alse family . This fuPaneth cells represent an important component of our intestinal innate immunity, preventing translocation and overgrowth of potential pathogenic bacteria . ConstitBacteroidetes and diminishes the relative amount of Firmicutes, treatment with DTX induces an expansion of Firmicutes and a decrease in Proteobacteria. In considering why these two treatments induce different compositional changes in the biome, we first considered their mechanisms of action. Dithizone is a heavy metal chelator and acts on Paneth cells by binding to their abundant zinc stores, which potentially deprives bacteria of this essential micronutrient [The findings from the above studies support the idea that Paneth cell physiology is important to the host/microbiome axis in the immature intestine. Our data show that depletion of Paneth cells causes alteration in the microbiome composition acutely in the 72 hours following exposure and continues through the next several weeks of life despite no new interventions. It is also interesting to note that our two complimentary models of Paneth cell depletion using dithizone or DTX, induce significant but different changes in the microbiome Figs . While inutrient , 46. Hownutrient . Althougnutrient , 46. Ournutrient , 42. WhiEnterobacteriaceae family, part of the Proteobacteria phylum regardless of the fact that in both scenarios the proportion of Proteobacteria decreased. This bloom was not seen in any of the control mice or in the dithizone-only or DTX-only cohorts. Multiple human NEC studies have demonstrated increased numbers of Enterobacteriaceae prior to development of NEC [Importantly, our data show that both models of Paneth cell depletion induced NEC show significant blooms of the t of NEC –15. Thust of NEC , but alsKlebsiella, a genus in the Enterobacteriaceae family, to the treatment mice could potentially confound our results, we performed additional studies to determine if the Enterobacteriaceae bloom included the gavaged Klebsiella. Overall, bacterial load, determined by spectrophotometry, showed that small intestine bacteria levels rose dramatically between 1 and 1.5 hours following Klebsiella gavage. Bacterial levels returned to baseline within 4 hours after gavage. Ileal samples from Rosa mice gavaged with GFP-labeled Klebsiella demonstrated that GFP-labeled Klebsiella was no longer present in the small intestine by 5 hours post-gavage. These results confirm that the administered Klebsiella pneumonia exited the small intestine before the bloom of Enterobacteriaceae was seen in the dithizone and DTX NEC models.Recognizing that administration of Klebsiella-induced dysbiosis in mice induces a similar phenotype to what is seen in the microbiota of human infants who develop NEC. These results provide support to the idea that Paneth cell depletion or dysfunction may play a key role in development of intestinal disease, especially NEC. However, further investigation is needed to determine if dysbiosis is a causative factor or a result of progression to NEC-like injury.In summary, our data clearly show that Paneth cell depletion in the immature small intestine induces significant alterations of the composition of the intestinal microbiota. Our data also show that Paneth cell depletion followed by


Our results indicate that ATPCL has a dispensable role in histone acetylation and prevents massive chromosome fragmentation when citrate efflux is altered.The Citrate Lyase (ACL) is the main cytosolic enzyme that converts the citrate exported from mitochondria by the SLC25A1 carrier in Acetyl Coenzyme A (acetyl-CoA) and oxaloacetate. Acetyl-CoA is a high-energy intermediate common to a large number of metabolic processes including protein acetylation reactions. This renders ACL a key regulator of histone acetylation levels and gene expression in diverse organisms including humans. We have found that depletion of ATPCL, the Drosophila ortholog of human ACL, reduced levels of Acetyl CoA but, unlike its human counterpart, does not affect global histone acetylation and gene expression. Nevertheless, reduced ATPCL levels caused evident, although moderate, mitotic chromosome breakage suggesting that this enzyme plays a partial role in chromosome stability. These defects did not increase upon X-ray irradiation, indicating that they are not dependent on an impairment of DNA repair. Interestingly, depletion of ATPCL drastically increased the frequency of chromosome breaks (CBs) associated to mutations in  Acetyl coenzyme A (acetyl-CoA) is a high-energy intermediate common to a large number of metabolic processes, including lipogenesis and cholesterogenesis that take place in different intracellular compartments. Acetyl-CoA is also required for protein acetylation reactions, which are important in post-translation modifications of proteins such as histones.Acetyl coenzyme A may be synthesized in mitochondria and exported to the cytosol. This transport is strictly dependent on the citrate-malate-pyruvate shuttle. For this process, mitochondrial acetyl-CoA is first condensed with oxaloacetate by citrate synthase thus producing citrate and free CoA. Citrate is then exported to the cytosol through the mitochondrial citrate carrier SLC25A1. Here, acetyl-CoA generation is mediated by the activity of the ATP-cytrate lyase (ACL), which catalyzes the ATP-dependent cleavage of mitochondrial-derived citrate into oxaloacetate and acetyl-CoA . FinallyAlthough ACL resides mainly in the endoplasmic reticulum in mammalian cells , it can de novo fatty acid synthesis. Glycolysis generates citrate by tricarboxylic acid (TCA) cycle, the citrate is preferentially exported from mitochondria to cytosol and then cleaved by ACL to produce cytosolic acetyl-CoA, the building block for de novo lipid synthesis. Thus, by coupling energy metabolism with fatty acids synthesis, ACL plays a critical role in supporting cancer cell growth. It is not therefore unexpected that inhibition of ACL dramatically suppresses tumor cell proliferation  indicating that this enzyme is partially required for chromosome stability in mitotic cells. Interestingly, the number of CBs is substantially enhanced when the export of citrate from mitochondria is inhibited suggesting that the ATPCL role on chromosome integrity is essential when cytosolic citrate is limited. We also show that Drosophila, the citrate lyase-encoding gene  maps on 52D9-11 chromosome 2 region and consists of 10 exons (including the 5′ and 3′ UTR sequences) that, according to the FlyBase annotation, specifies four potential transcripts  that retain all exons albeit with few differences Exel7138ATPCL hemizygotes (Df(2R)Exel7138 is a deficiency in the 52D1-52D12 polytene region that removes ATPCL) were late lethal while either DG23042ATPCL homozygotes and hemizygotes displayed semi-lethality. By mobilizing the DG23042ATPCL P-element, we generated an additional lethal ATPCL mutant allele (Δ7), which potentially bears partial deletions of the gene and severely affected ATPCL expression (see below). This suggests that loss of function of ATPCL might prevent development as it occurs also for ACL knock-out mice  confirming that both phenotypes are due to lesions in the ATPCL gene.We obtained from the Bloomington stock center two putative P-element induced ectively . ATPCL01out mice . FinallyATPCL mutants (01466/ATPCLATPCLΔ7trans heterozygotes yielded to the strongest reduction (∼80%) of expression of the ATPCL protein indicating that this mutant allele combination represents the most severe condition  analysis on larval brain extracts detected a band of expected size (∼130 KDa), which decreased in  mutants . ATPCL01ondition . Immunofondition . Howeverondition . Thus, londition , ATPCL lATPCL mutants with respect to wild-type (1.30 pm/μl) indicating that, similarly to its mammalian counterpart, ATPCL is required for Acetyl-CoA synthesis  are required for chromosome integrity in mammalian and Drosophila cells, respectively  could genetically interact. The cytological analysis of mitotic metaphases from late lethal ATPCL sea double mutants revealed that CBs frequency is synergistically increased  with respect to the sum of break frequencies of both ATPCL (4.5%) and sea ∼25%; . As SLC25A1 and Scheggia . Surprisingly, only ∼5% (772) of the genes significantly changed their transcriptional profile indicating that loss of ATPCL only marginally affected global gene transcription. Of these, approximately 23% (182) were > 2-fold either down- or up-regulated . However, it cannot be ruled out the possibility that ATPCL could be required for the acetylation of either nuclear or cytoplasmic factors, instead of histones, that in turn are required for chromosome integrity. Alternatively, as ATPCL plays a direct role in the homeostasis of lipids, whose modifications  are also required to modulate DNA repair (ATPCL mutants defects on chromosome integrity could arise as a consequence of protein acetylation-independent activity. Proteomic studies will be fundamental to verify these hypotheses.Here we have molecularly characterized for the first time A repair , it is pWe have previously demonstrated that mutations in the mitochondrial citrate carrier Sea/SLC25A1, led to frequent CBs in Drosophila mitotic cells . The finsea or ATPCL, which are almost genetically null and exhibited a strong reduction of corresponding transcripts. It is conceivable that citrate deprivation in sea mutants causes much greater effects than reduction of Acetyl-CoA in ATPCL mutants. Indeed, citrate may play different tissue-specific metabolic roles and function as a general chelator of physiologically important cations. Thus its deprivation in the cytosol may compromise several intracellular pathways. Citrate reduction as consequence of inhibition of Sea/SLC25A1 transport activity might also inhibit ATPCL activity and/or expression thus exacerbating the ATPCL mutant phenotype. It has been recently demonstrated that in Drosophila citrate may indeed regulate a feedback mechanism that coordinates intracellular metabolism with glycolysis and TCA cycle (ATPCL depletion in sea mutants yields to a synergic CBs phenotype suggest that ATPCL prevents chromosome fragmentation when citrate metabolism is impaired.The different effect of loss of Sea and ATPCL on Drosophila chromosome integrity is intriguing. This is not due to the molecular nature of mutant alleles of either CA cycle . It coulde novo lipogenesis. Whether a similar histone-acetylation independent effect takes place also in Drosophila will be an interesting issue to address in the future.The mild phenotype associated with loss of ATPCL in Drosophila does not preclude the possibility that also the overexpression of ATPCL may impact Drosophila somatic cell proliferation. Overexpression of the human ortholog ACL supports cancer growth by fueling the glucose-dependent PM, MDG, and AP performed gene characterization and mutants analysis. VL and RN carried out the microarray analysis. YR supervised gene characterization and discussed the data. GC supervised the experiments and wrote 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.