Citrullination of LL-37 as a mechanism that selectively controls immunostimulatory potential of DNA

LL-37, jedyna ludzka katelicydyna, jest amfipatycznym, kationowym peptydem bakteriobójczym, który zawiera 37 reszt aminokwasowych. Dojrzały peptyd uwalniany jest z 18 kDa białka prekursorowego (katelicydyny) przez proteazę-3 w neutrofilach lub przez proteazy serynowe z rodziny kallikrein w keratynocytach. Oprócz zdolności bezpośredniego zabijania mikroorganizmów, LL-37 pełni funkcję immunomodulacyjną. Jedną z nich jest tworzenie kompleksów z ujemnie naładowanymi oligonukleotydami pochodzącymi zarówno z obumierających komórek gospodarza, jak i patogenów. Interakcja ta ułatwia rozpoznawanie DNA przez receptory wewnątrzkomórkowe, takie jak TLR-9 prowadząc do indukcji odpowiedzi immunologicznej. W ostatnim czasie udokumentowano, że zdolność peptydu LL-37 do pełnienia funkcji bakteriobójczych i immunomodulacyjnych zależy w dużym stopniu od jego modyfikacji potranslacyjnych, wśród których deiminacja odgrywa wyjątkową rolę. Struktura pierwszorzędowa LL-37 zawiera pięć reszt argininy (Arg), które są wydajnie cytrulinowane przez deiminazy peptydylo-argininowe (PADs), enzymy, które są zależnymi od wapnia hydrolazami katalizującymi konwersję dodatnio naładowanej argininy do neutralnej cytruliny. W związku z powyższym w niniejszej pracy skupiono się na roli deiminacji peptydu LL-37 w regulacji odpowiedzi zapalnej na kwasy nukleinowe. Uzyskane wyniki dowiodły, że cytrulinacja LL-37 katalizowana przez PAD2 ogranicza zależne od peptydu pochłanianie DNA przez fagocyty. Co ciekawe, karbamylacja peptydu (homocytrulinacja reszt lizyny) nie ma wpływu na jego interakcję z DNA, tak długo jak karbamylowany peptyd nie zostanie poddany działaniu PAD2. W badaniach zastosowano również syntetyczny peptyd LL-37 z resztami Arg zastąpionymi homoargininą (hArg-LL-37), która jest niewrażliwa na proces enzymatycznej deiminacji przez PADy. Dane te wykazały, że wiązanie peptydu LL-37 z DNA, a tym samym jego działanie immunoregulacyjne względem fagocytów nie zależy wyłącznie od ładunku dodatniego peptydu, ale od prawidłowego rozmieszczenia guanidynowych łańcuchów bocznych argininy w natywnej sekwencji peptydu. Aby zbadać, czy powyższe zjawisko odgrywa rolę w stanie zapalnym przeprowadzono badania z wykorzystaniem zewnątrzkomórkowych pułapek neutrofilowych (NETs, ang. neutrophil extracellular traps). Tworzenie tych struktur, zbudowanych z nici genomowego DNA udekorowanych białkami pochodzącymi z ziaren azurofilnych, w tym LL-37, zależy od aktywności PAD4. Szczegółowa analiza składu NETs i ich wpływu na profesjonalne fagocyty sugeruje, że katelicydyna/LL-37 jest cytrulinowana przez PAD4 podczas tworzenia pułapek neutrofilowych. Proces ten odgrywa istotną rolę w kontroli potencjału zapalnego NETs, zwłaszcza w regulacji odpowiedzi immunologicznej na uwolnione z obumierających komórek DNA, które jest rozpoznawane przez receptor TLR9. Przebieg procesu cytrulinacji w warunkach in vivo potwierdzają wyniki analizy serologicznej surowic pochodzących od pacjentów cierpiących na przewlekłe choroby zapalne, takie jak: paradontozę i reumatoidalne zapalenie stawów, w których wykazano podwyższony poziom przeciwciał rozpoznających cytrulinowaną formę peptydu LL-37. Ostatecznie, dowiedziono, że cytrulinowana forma peptydu LL-37 zwiększa powstawanie NETs, co sugeruje mechanizm pozytywnego sprzężenia zwrotnego. Podsumowując, cytrulinacja peptydu LL-37 odgrywa kluczową rolę w jego immunomodulacyjnym działaniu przeciwdziałając utracie tolerancji gospodarza na własne cząsteczki, w tym DNA.LL-37, the only human cathelicidin, is an amphipathic, cationic antimicrobial peptide that contains 37 amino acids residues. The mature peptide is released from an 18-kDa precursor protein (cathelicidin) by protease-3 in neutrophils or by serine proteases of the kallikrein family in keratinocytes. Besides playing antimicrobial role, LL-37 exerts several immunomodulatory functions that regulate host responses to pathogens. Among them is formation of complexes with negatively charged oligonucleotides derived from both dying host cells and pathogens. Such interaction facilitates the recognition of DNA by intracellular receptors such as TLR9, thereby stimulating the immune response. It was recently documented that the ability of peptide to execute its bactericidal and immunomodulatory functions strongly depends on posttranslational modifications, among which enzymatic deimination (citrullination) of Arg residues plays a unique role. The primary structure of LL-37 contains five arginine (Arg) residues that are effectively citrullinated by peptidyl-arginine deiminases (PADs), enzymes that are calcium-dependent hydrolases catalyzing the conversion of positively charged arginine to neutral citrulline. Therefore, presented study focused on the role of LL-37 deimination in the regulation of the host immune response to nucleic acids. Presented data revealed that citrullination of LL-37 by PADs hindered peptide-dependent DNA uptake and sensing by pDCs. In contrast, carbamylation of the peptide (homocitrullination of lysine residues) had no effect on its interaction with DNA. This activity was abolished by citrullination of Arg residues in the peptide by PAD2. We implemented in the study the synthetic peptide LL-37 with Arg residues substituted by homoarginine (hArg-LL-37), which is insensitive to enzymatic deimination. Using such peptide, we observed that regardless of PAD2 treatment hArg-LL-37 preserved full biological activity. These data have showed that peptide binding to DNA and its immunoregulatory effects on myeloid cells do not depend entirely on the positive charge of LL-37 but on the proper distribution of guanidium side chains of Arg (or homo-Arg) in the native peptide sequence. To explore if above phenomenon plays a role in inflammatory conditions, we introduced in the study neutrophil extracellular traps (NETs). Formation of those structures, composed of genomic DNA and decorated with azurophilic proteins including LL-37, depends on PAD4 activity. Detailed analysis of NETs composition and their influence on myeloid cells suggest that cathelicidin/LL-37 is citrullinated by PADs during NETs formation. Such process makes a marked contribution to the inflammatory potential of NET structures especially in regulating the immunostimulatory response of cell-free DNA, which is mediated by TLR9. Such conclusion was additionally supported by an elevated antibody response against citrullinated LL-37 in blood serum obtained from patients suffering from chronic inflammatory diseases (periodontitis, rheumatoid arthritis). Finally, obtained data revealed that citrullinated LL-37 itself enhances the generation of NETs formation suggesting the mechanism of positive feedback. Taken together, by preventing the breakdown of tolerance to self-molecules, citrullination of LL-37 plays a critical role in immunomodulation of the host response

A high-throughput screen identifies that CDK7 activates glucose consumption in lung cancer cells.

Elevated glucose consumption is fundamental to cancer, but selectively targeting this pathway is challenging. We develop a high-throughput assay for measuring glucose consumption and use it to screen non-small-cell lung cancer cell lines against bioactive small molecules. We identify Milciclib that blocks glucose consumption in H460 and H1975, but not in HCC827 or A549 cells, by decreasing SLC2A1 (GLUT1) mRNA and protein levels and by inhibiting glucose transport. Milciclib blocks glucose consumption by targeting cyclin-dependent kinase 7 (CDK7) similar to other CDK7 inhibitors including THZ1 and LDC4297. Enhanced PIK3CA signaling leads to CDK7 phosphorylation, which promotes RNA Polymerase II phosphorylation and transcription. Milciclib, THZ1, and LDC4297 lead to a reduction in RNA Polymerase II phosphorylation on the SLC2A1 promoter. These data indicate that our high-throughput assay can identify compounds that regulate glucose consumption and that CDK7 is a key regulator of glucose consumption in cells with an activated PI3K pathway

Generating health technology assessment evidence for rare diseases

Objectives: Rare diseases are often heterogeneous in their progression and response to treatment, with only a small population for study. This provides challenges for evidence generation to support HTA, so novel research methods are required. Methods: Discussion with an expert panel was augmented with references and case studies to explore robust approaches for HTA evidence generation for rare disease treatments. Results: Traditional RCTs can be modified using sequential, three-stage or adaptive designs to gain more power from a small patient population or to focus trial design. However, such designs need to maintain important design aspects such as randomization and blinding and be analyzed to take account of the multiple analyses performed. N-of-1 trials use within-patient randomization to test repeat periods of treatment and control until a response is clear. Such trials could be particularly valuable for rare diseases and when prospectively planned across several patients and analyzed using Bayesian techniques, a population effect can be estimated that might be of value to HTA. When the optimal outcome is unclear in a rare disease, disease specific patient reported outcomes can elucidate impacts on patients’ functioning and wellbeing. Likewise, qualitative research can be used to elicit patients’ perspectives, with just a small number of patients. Conclusions: International consensus is needed on ways to improve evidence collection and assessment of technologies for rare diseases, which recognize the value of novel study designs and analyses in a setting where the outcomes and effects of importance are yet to be agreed.</p

Experience of BRCA1/2 mutation-negative young women from families with hereditary breast and ovarian cancer: a qualitative study

BACKGROUND: Little is known about the experience of young women who become aware of their parent’s BRCA1 or BRCA2 (BRCA) mutation status as adolescents or young adults. There is also currently a gap in the literature pertaining to those who are found to be negative for their familial mutation. We aimed to investigate the experience of these mutation-negative young women from hereditary breast and ovarian cancer (HBOC) families. METHODS: Using a semi-structured questionnaire we interviewed 8 women. All of the women were non-carriers of their familial mutation and had learned of the mutation in their family as adolescents or young adults at least 6 months prior to undergoing genetic testing. All interviews were audio recorded, transcribed, and independently analyzed by the investigators. This was followed by an in-depth cross-case analysis, enabling the formulation of emergent themes. RESULTS: The women’s age ranged from 22 to 37 years old and all were of Ashkenazi Jewish descent. Prominent emergent themes from the interviews included the impact of how and when the familial mutation status was disclosed, the factors influencing when a young woman chooses to undergo predictive genetic testing, the predictors of post-test adjustment and risk perception, as well as the impact of familial cancer experience versus the familial mutation. CONCLUSIONS: By eliciting detailed patient narratives we have begun to show that this generation of BRCA mutation-negative young women is likely still affected by the degree of cancer history in their family, even with their understanding of the genetic contribution to disease. Larger studies with tightened participant characteristics, as well as studies involving women from different cultural backgrounds, are needed to further define the experience and needs of true negative young women from HBOC families

Alteración en los Hábitos del Consumidor Saltillense a Consecuencia del COVID-19

El estudio pretende indagar sobre la modificación en el comportamiento del consumidor saltillense debido a la contingencia por la epidemia del nuevo coronavirus COVID-19. El trabajo incluyó a 354 participantes adultos residentes en la Ciudad de Saltillo, Coahuila, México. La muestra está conformada por 195 mujeres, es decir, un 55.1% y 159 hombres lo que constituye un 44.9%. Se realiza un trabajo de tipo Cuantitativo, exploratorio, descriptivo y transversal no experimental. Las preguntas son categóricas y la escala de respuesta es tipo Likert del 1 al 7, validando un instrumento de 25 elementos obteniendo un Alfa de Cronbach .786. Con base en las variables analizadas se infiere que los saltillenses se han vuelto consumidores más racionales, prefiriendo alimentos caseros, cuidando sus gastos, preocupándose por el futuro y extrañando la experiencia de comprar en tiendas físicas favoreciendo las ventas por internet y el servicio a domicilio. Y concluye que existe un nuevo perfil de consumidor no solo en Saltillo, sino a nivel mundial, que procura el bienestar en su salud, modificando hábitos alimenticios y conductuales adaptándose a esta nueva normalidad. &nbsp; The study aims to investigate the modification in the behavior of the Saltillo consumer due to the contingency caused by the epidemic of the new coronavirus COVID-19. The work included 354 adult participants residing in the City of Saltillo, Coahuila, Mexico. The sample is made up of 195 women, that is, 55.1% and 159 men, which constitutes 44.9%. A quantitative, exploratory, descriptive and non-experimental cross-sectional work is carried out. The questions are categorical and the response scale is Likert type from 1 to 7, validating an instrument of 25 elements obtaining a Cronbach's Alpha .786. Based on the variables analyzed, it can be inferred that the people of Saltillo have become more rational consumers, preferring homemade foods, taking care of their expenses, worrying about the future and missing the experience of buying in physical stores, favoring internet sales and home delivery. And it concludes that there is a new consumer profile not only in Saltillo, but also worldwide, which seeks the well-being in their health, modifying eating and behavioral habits, adapting to this new normal

Loss of Gata6 causes dilation of the hair follicle canal and sebaceous duct

The uppermost aspect of the hair follicle, known as the infundibulum or hair canal, provides a passageway for hair shaft egress and sebum secretion. Recent studies have indicated that the infundibulum and sebaceous ducts are lined by molecularly distinct differentiated cells expressing markers including Keratin 79 and Gata6. Here, we ablated Gata6 from the skin and observed dilation of both the hair canal and sebaceous ducts, independent of gender and hair cycle stage. Constitutive loss of Gata6 yielded only a mild delay in depilation‐induced entry into anagen, while unperturbed mutant mice possessed overtly normal skin and hair. Furthermore, we noted that Keratin 79 and Gata6 expression and localization did not depend upon each other. Our findings implicate Gata6 in maintaining the upper hair follicle and suggest that regulation of this transcription factor may be compromised in pathologies such as acne or infundibular cystic diseases that are characterized by abnormal expansion of this follicular domain.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149235/1/exd13757_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149235/2/exd13757-sup-0001-FigS1-S9.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149235/3/exd13757.pd

Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression.

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease

Recruiting Volunteers for New Horizons\u27 Apprenticeship Program for Homeless Young Adults

Introduction New Horizons is a Seattle-based organization focused on meeting the basic needs of homeless young adults ages 18-25 through food, shelter, clothing, health clinics, and social work appointments. To help their clients achieve a sustainable lifestyle, New Horizons addresses the most challenging obstacles including job and house hunting through an innovative apprenticeship program through their café, Street Bean Coffee Roasters. Clients can learn skills transferable to other careers, including barista basics, work experience, and networking. Our project is to help New Horizons find volunteers or employees for their apprenticeship program. In finding volunteers or employees for the café, we are providing professional training and networking opportunities for the apprentices so when the apprenticeship program is completed, the participants have job experience for their resume and connections to assist in their job search. In addition to recruiting staff for the café, we are also building a connection between New Horizons and Seattle Pacific University’s (SPU) volunteer groups in hopes of providing more volunteer opportunities and creating a quarterly volunteer event. Background Homelessness is a local and national crisis, disproportionately affecting Black, Native American, Hispanic, and LGBTQ populations (Huffman et al., 2021). According to the National Conference of State Legislators (NSCL, 2022), an estimated 4.2 million youth and young adults experience homelessness each year. Of these, about 700,000 are unaccompanied minors, meaning they are not part of a family or accompanied by a parent or guardian. In Seattle, the homeless population has risen exponentially. According to New Horizons (2018), “The number of unstably housed persons in King County has steadily risen by roughly 20% each year for the past three years”; over 1,500 youth in King County are affected by homelessness. The barriers the homeless youth encounter can be divided into four groups: domestic, health, bureaucratic, and social support (Huffman et al., 2021). Issues identified within these categories include maintaining personal hygiene, inability to find and receive proper medical treatment, education and employment gaps, criminal records, and limited support from potential employers. These issues are multifactorial, overlapping and intersecting on multiple planes, “mutually reinforcing the structures undergirding work and home” (Huffman et al., 2021), creating the ‘scaffolded city’ phenomenon the homeless population lives within. A survey conducted to analyze the needs insecurity among Florida college students identified extensive support programs as essential to the inclusion of students traditionally excluded by higher education via remediation, transfer, vocational training, and contract education (Nix et al., 2021). New Horizons confronts most of these challenges by providing an abundance of holistic resources for the homeless youths of Seattle. Although there is a lot known about the social inequities that the homeless youth population face, there is not much information regarding sustainable resources to transition off the streets and become included in the socioeconomic world. A quasi-experimental longitudinal study aimed to assess the feasibility of improving socioeconomic inclusion outcomes by supporting identity capital in youths who struggled to shake the identity of homelessness (Thulien et al., 2021). Identity capital includes fostering hope, focusing on personal strengths, and improving self-esteem. Most notably, many participants expressed gratitude for the normalization of strategies and skills they learned, framing them as something one needs (Thulien et al., 2021). These findings suggest that targeting identity capital is feasible and may be a promising approach to incorporate into a more complex intervention that includes housing, education, and employment resources to help youth transition out of homelessness. New Horizons offers an apprenticeship program through Street Bean Coffee that gives a chance to gain work experience and skills, but also provides networking opportunities and higher chances of long-term employment. Activities with Rationale To support the community of New Horizons our group has developed flyers with QR codes and other deliverables to help find adequate staffing, volunteers, and providing supplies. The QR codes itself has direct links to their amazon wish list, donation needs, volunteer page, street bean coffee roaster barista position. With these codes people can access their website to not just look for a job position but it provides awareness to a community in need of support. We also were able to connect New Horizons with SPU’s volunteer programs Latreia an Urban Involvement. Our group provided resources and points of contact to the coordinator of these SPU volunteer programs in hopes the relationship between New Horizons and SPU would strengthen. This connection allows for there to be volunteer events to better assist and help serve the community. Outcomes We had short term goals of providing for the population of New Horizons through creation of resources so that they can utilize them to cater to their needs and New Horizons will receive student volunteers from Latreia before March 9th 2022. Our long-term goals were to have the homeless youth population located around New Horizons gain more knowledge regarding resources that are available to them, such as job opportunities via Street Bean Coffee Roasters within 3 months and have New Horizons obtain a consistent flow of volunteers and resources via collaboration with SPU\u27s Urban Involvement, quarterly. These short term goals were both met while the long term goals are still in progress. Evaluation The utilization of mixed methods, both qualitative and quantitative data, proves to help evaluate the success of our interventions more efficiently (Abilgaard et al., 2016). The qualitative data analyzed was whether the barista position was filled and whether SPU’s volunteer programs formed a connection with New Horizons. After the initiation of our interventions, there was the removal of the job application on New Horizons website, the barista position filled, and Latreia partnering with New Horizons where a group of SPU volunteers painting their garage to be used as a blank canvas for young adults to do graffiti artwork. These outcomes display evidence of an effective relationship between SPU’s volunteering programs and New Horizons and our goals being met. The quantitative data analyzed was the number of people that attended the Latreia event and how many people scanned the flyer QR codes. Ten volunteers attended the event compared to their average range of 10-15 people that regularly attend their events. Data collected from the QR code tracking website suggested that 34 people scanned the QR code for the barista position, 28 people scanned the QR code for New Horizon’s Amazon wish list, and 27 people scanned the QR code for the volunteering page. These evaluations proved that the SPU community interacted with the flyers and were successful in bringing attention to New Horizons’ needs. Conclusion To help New Horizons and the community they serve we were able to help them form long term relationships that will continue on when we leave. By creating this relationship with SPU volunteer programs and access to a new barista, New Horizons can better serve the population of homeless youth through job training and acts of service. With the aid of our interventions bringing awareness to New Horizons by various means, we were able to engage with their culture to help change their world. Limitations of Research There are limitations to our project and research. Due to the COVID-19 pandemic, there is a limited amount of recent national and local research and statistics available to analyze and collect around homelessness (NSCL, 2022). Due to social distancing and isolation precautions, surveys and data collection cannot be completed or are very limited (NSCL, 2022). The small sample size or cross-sectional instead of longitudinal methods can limit a study. Diversity in age groups and selected sample size may not represent the total population (Thulien et al., 2021). Different geographic locations or urban vs rural areas may affect the availability of resources, programs, or tactics utilized based on the prevalence of the homeless population. In Nix et al. (2021), the inadequacy of faculty and staff training was evident in varied faculty and staff awareness of basic need opportunities. For future interview-based studies such as Huffman et al. (2021), training young adults who have experience being homeless to conduct the interviews may mediate socioeconomic differences between interviewers and interviewees, allowing for more fully open reflection, trust, and nuanced interpretation. Shared experiences between the interviewers and interviewees may also demonstrate effectiveness of apprenticeship programs for homeless youths while providing a supportive role model who has transitioned from their homeless identity into a successfully employed individual with professional networking connections. References Abildgaard, J., Saksvik, P., & Nielsen, K. (2016). How to measure the intervention process: An assessment of qualitative and quantitative approaches to data collection in the process evaluation of organizational interventions. Frontiers in Psychology, 7, Article 1380. https://doi.org/10.3389/fpsyg.2016.01380 Huffman, T., Leier, C., Generous. M., Hinrichs, M., & Brenneman, L. (2021). Climbing the ‘scaffolded city’: Tactics used by homeless young adults to navigate employment barriers. Journal of Applied Communication Research, 49(2), 148-167. https://doi.org/10.1080/00909882.2020.1839119 National Conference of State Legislatures. (2022). Youth homelessness overview. National Conference of State Legislatures. https://www.ncsl.org/research/human-services/homeless-and-runaway-youth.aspx New Horizons. (2018). About: Youth homelessness. New Horizons. https://nhmin.org/youth-homelessness-seattle/ Nix, A., Bertrand Jones, T., Daniels, H., Hu, P., & Hu, S. (2021). “There’s so much that we’re doing”: How Florida college system institutions address basic needs insecurity among students. Community College Review, 1, 1-20. https://doi.org/10.1177/00915521211047674 Thulien, N., Wang, A., Mathewson, C., Wang, R., & Hwang, S. (2021). Tackling exclusion: A pilot mixed method quasi-experimental identity capital intervention for young people exiting homelessness. PLoS ONE, 16(8), 1-19. https://doi.org/10.1371/journal.pone.025628

Serum Apolipoprotein AI and B Are Stronger Biomarkers of Diabetic Retinopathy Than Traditional Lipids

10.2337/dc10-0793Diabetes Care342474-479DICA