Modeling diverse genetic subtypes of lung adenocarcinoma with a next-generation alveolar type 2 organoid platform.

Lung cancer is the leading cause of cancer-related death worldwide. Lung adenocarcinoma (LUAD), the most common histological subtype, accounts for 40% of all cases. While existing genetically engineered mouse models (GEMMs) recapitulate the histological progression and transcriptional evolution of human LUAD, they are time-consuming and technically demanding. In contrast, cell line transplant models are fast and flexible, but these models fail to capture the full spectrum of disease progression. Organoid technologies provide a means to create next-generation cancer models that integrate the most advantageous features of autochthonous and transplant-based systems. However, robust and faithful LUAD organoid platforms are currently lacking. Here, we describe optimized conditions to continuously expand murine alveolar type 2 (AT2) cells, a prominent cell of origin for LUAD, in organoid culture. These organoids display canonical features of AT2 cells, including marker gene expression, the presence of lamellar bodies, and an ability to differentiate into the AT1 lineage. We used this system to develop flexible and versatile immunocompetent organoid-based models of KRAS, BRAF, and ALK mutant LUAD. Notably, organoid-based tumors display extensive burden and complete penetrance and are histopathologically indistinguishable from their autochthonous counterparts. Altogether, this organoid platform is a powerful, versatile new model system to study LUAD.

Protamines and the sperm nuclear basic proteins Pandora's Box of insects.

Insects are the largest group of animals when it comes to the number and diversity of species. Yet, with the exception of Drosophila, no information is currently available on the primary structure of their sperm nuclear basic proteins (SNBPs). This paper represents the first attempt in this regard and provides information about six species of Neoptera: Poecillimon thessalicus, Graptosaltria nigrofuscata, Apis mellifera, Nasonia vitripennis, Parachauliodes continentalis, and Tribolium castaneum. The SNBPs of these species were characterized by acetic acid urea gel electrophoresis (AU-PAGE) and high-performance liquid chromatography fractionated. Protein sequencing was obtained using a combination of mass spectrometry sequencing, Edman N-terminal degradation sequencing and genome mining. While the SNBPs of several of these species exhibit a canonical arginine-rich protamine nature, a few of them exhibit a protamine-like composition. They appear to be the products of extensive cleavage processing from a precursor protein which are sometimes further processed by other post-translational modifications that are likely involved in the chromatin transitions observed during spermiogenesis in these organisms.

Rapid interrogation of cancer cell of origin through CRISPR editing.

The increasing complexity of different cell types revealed by single-cell analysis of tissues presents challenges in efficiently elucidating their functions. Here we show, using prostate as a model tissue, that primary organoids and freshly isolated epithelial cells can be CRISPR edited ex vivo using Cas9-sgRNA (guide RNA) ribotnucleoprotein complex technology, then orthotopically transferred in vivo into immunocompetent or immunodeficient mice to generate cancer models with phenotypes resembling those seen in traditional genetically engineered mouse models. Large intrachromosomal (∼2 Mb) or multigenic deletions can be engineered efficiently without the need for selection, including in isolated subpopulations to address cell-of-origin questions.

Intersectional stigma and the arc of intranational migration: experiences of transgender adolescents and women who migrate within Peru.

BACKGROUND: Migration is recognized as a key determinant of health. Yet, limited research addresses the arc of intranational migration and, even less, the experiences of transgender (trans) adolescents and women migrants and the associated health vulnerabilities. Using intersectional stigma as a theoretical frame, this study seeks to better understand the sexual health vulnerabilities and needs of trans women migrants in Peru. METHODS: Between October and November 2016, in-depth interviews (n = 14) and two focus groups (n = 20) were conducted in Spanish with trans women in three Peruvian cities. To explore pre- and during migration experiences, focus groups were conducted in Pucallpa and Iquitos, key cities in the Amazon where trajectories often originate. To assess during migration and post-migration experiences, we conducted interviews in Pucallpa, Iquitos, and Lima to better understand processes of relocation. Audio files were transcribed verbatim and analysed via an immersion crystallization approach, an inductive and iterative process, using Dedoose (v.6.1.18). RESULTS: Participants described migration as an arc and, thus, results are presented in three phases: pre-migration; during migration; and post-migration. Intersectional stigma was identified as a transversal theme throughout the three stages of migration. The pre-migration stage was characterized by poverty, transphobia, and violence frequently motivating the decision to migrate to a larger city. Exploitation was also described as pervasive during migration and in relocation. Many participants spoke of their introduction to sex work during migration, as key to economic earning and associated violence (police, clients). CONCLUSION: Findings advance understandings of intranational migration and forced displacement as key determinants of trans women's health. Dimensions of violence at the intersection of classism and cisgenderism render trans women highly vulnerable at every step of their migratory journeys. Experiences of intranational mobility and relocation were described as uniquely tied to age, intersectional transphobic stigma, engagement in sex work, and multiple forms of violence, which impact and can magnify sexual health vulnerabilities for transgender women in Peru who migrated intranationally.

Mental health and substance use risks and resiliencies in a U.S. sample of transgender and gender diverse adults.

PURPOSE: Victimization contributes to mental and behavioral health inequities among transgender and gender diverse (TGD) people, but few studies have simultaneously examined health-promoting resiliencies. We sought to identify classes of risk and resilience among TGD adults, assess characteristics associated with these classes, and examine their relationship with mental health and substance use outcomes. METHODS: Cross-sectional data were from the 2015 US Transgender Survey, a non-probability study including 26,957 TGD adults. Using latent class analysis, we classified patterns of vulnerability and resilience based on risk (past-year denial of equal treatment, verbal harassment, physical attack, bathroom-related discrimination; lifetime sexual assault, intimate partner violence) and protective (activism; family, work, classmate support) factors. Regression models were fit to (1) determine the association between sociodemographic and gender affirmation characteristics and latent classes; (2) model associations between latent classes and mental health (current serious psychological distress, past-year and lifetime suicidal thoughts and attempts, and lifetime gender identity/transition-related counseling) and substance use (current binge alcohol use, smoking, illicit drug use; past-year drug/alcohol treatment) outcomes. RESULTS: Three latent classes were identified: high risks, with activism involvement ("risk-activism," 35%); low risks, with not being out about one's TGD identity ("not-out," 25%); and low risks, with high family support ("family-support," 40%). Gender affirmation and sociodemographic characteristics, such as race/ethnicity and sexual orientation, were associated with latent classes. Risk-activism class membership was associated with higher odds of negative mental health and substance use outcomes, while the family-support class had lower odds of these outcomes. CONCLUSIONS: Interventions leveraging family support, and policy protections from discrimination and victimization, may promote TGD mental and behavioral health.

Covid-19 en la población LGBTIQ+ en Ecuador: Perspectivas de una junta asesora comunitaria.

No disponible.

Sexual Behaviors and HIV/STI Prevention Strategies Among Sexual Minority Men in Ecuador Who Use Geosocial Networking Apps.

Around the world, geosocial networking apps have become widely popular among sexual minority men (SMM). This research analyzed the sexual behaviors and HIV and STI prevention strategies (HIV/STI testing, HIV/STI inquiry, and HIV/STI disclosure) of an online-recruited sample of 284 SMM living in Ecuador. Sexting and oral sex were the most common sexual behaviors among SMM in the sample. Most participants had low perceptions of HIV and STI risk; 85% reported being tested for HIV and 70% for STIs. Being older predicted higher odds of being tested for either HIV or STIs at least once. Being single also predicted HIV testing. Future interventions in the country should explore apps' utility as intervention tools to spread information about sexual health and HIV prevention strategies, such as condom use and event-driven PrEP. Apps can also facilitate connections to sexual health services, including programs for PrEP initiation and linkage to HIV treatment. They should also focus on promoting sexual harm reduction conversations among potential app-met sexual partners.

Perceptions of power and sexual pleasure associated with sexual behaviour profiles among Latino sexual minority men.

In a body of research typically focused on risk reduction and disease prevention, other factors motivating the sexual behaviours of Latino sexual minority men, such as resource-based power and sexual pleasure, are less well understood. To this end, Latino immigrant sexual minority men living in New York City were surveyed about their sexual behaviours, perceived power differentials, and pleasure from insertive and receptive anal intercourse. Power and pleasure were examined as associations with behavioural profiles identified through latent class analysis, adjusting for age and partner type. Four latent classes of Latino sexual minority men were identified based on behaviours reported during the most recent sexual event: behaviourally insertive (14.2%), behaviourally versatile (25.9%), behaviourally receptive (29.2%), and limited penetrative behaviour (30.7%). Participants who derived pleasure from insertive and receptive anal intercourse had higher odds of belonging in the behaviourally insertive and behaviourally receptive class, respectively. Perceptions of resource-based power were not associated with class membership. Findings highlight the importance of sexual pleasure as a driver of sexual behaviour, irrespective of power dynamics. Sexual health curricula and interventions for sexual minority men should consider sexual pleasure and sex-affirmative frameworks when providing sexuality education and promoting sexual wellbeing.

[Dermatological manifestations of monoclonal gammopathies]. / Patologías cutáneas asociadas a gammapatías monoclonales: ¿qué sabemos hasta ahora?

Monoclonal gammopathies of uncertain significance (MGUS) correspond to pre-malignant hematological disorders characterized by the production of a monoclonal protein and infiltration of less than 10% of the bone marrow by plasma cells. Its importance lies in the risk of progression to malignant disorders and in the association with different renal, neurological and skin manifestations. There are pathophysiological mechanisms that support a causal relationship between monoclonal gammopathies (MGs) and different skin diseases, such as type I cryoglobulinemia (CG), primary systemic amyloidosis (PSA) or necrobiotic xanthogranuloma (NXG). However, there is a group of skin diseases associated with MGs whose pathogenesis has not been elucidated. In this context, the role of the dermatologist is crucial in the suspicion of different haematological disorders based on skin manifestations and in the multidisciplinary treatment of these patients. In this article, we carry out an exhaustive review of the literature published in this area and propose a screening algorithm for MGs in patients with specific skin diseases.

Rapid modelling of cooperating genetic events in cancer through somatic genome editing.

Cancer is a multistep process that involves mutations and other alterations in oncogenes and tumour suppressor genes. Genome sequencing studies have identified a large collection of genetic alterations that occur in human cancers. However, the determination of which mutations are causally related to tumorigenesis remains a major challenge. Here we describe a novel CRISPR/Cas9-based approach for rapid functional investigation of candidate genes in well-established autochthonous mouse models of cancer. Using a Kras(G12D)-driven lung cancer model, we performed functional characterization of a panel of tumour suppressor genes with known loss-of-function alterations in human lung cancer. Cre-dependent somatic activation of oncogenic Kras(G12D) combined with CRISPR/Cas9-mediated genome editing of tumour suppressor genes resulted in lung adenocarcinomas with distinct histopathological and molecular features. This rapid somatic genome engineering approach enables functional characterization of putative cancer genes in the lung and other tissues using autochthonous mouse models. We anticipate that this approach can be used to systematically dissect the complex catalogue of mutations identified in cancer genome sequencing studies.

Functional epigenetics approach identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers.

Defects in epigenetic regulation play a fundamental role in the development of cancer, and epigenetic regulators have recently emerged as promising therapeutic candidates. We therefore set out to systematically interrogate epigenetic cancer dependencies by screening an epigenome-focused deep-coverage design shRNA (DECODER) library across 58 cancer cell lines. This screen identified BRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex, as being essential for the growth of tumor cells that harbor loss of function mutations in BRG1/SMARCA4. Depletion of BRM in BRG1-deficient cancer cells leads to a cell cycle arrest, induction of senescence, and increased levels of global H3K9me3. We further demonstrate the selective dependency of BRG1-mutant tumors on BRM in vivo. Genetic alterations of the mSWI/SNF chromatin remodeling complexes are the most frequent among chromatin regulators in cancers, with BRG1/SMARCA4 mutations occurring in ∼10-15% of lung adenocarcinomas. Our findings position BRM as an attractive therapeutic target for BRG1 mutated cancers. Because BRG1 and BRM function as mutually exclusive catalytic subunits of the mSWI/SNF complex, we propose that such synthetic lethality may be explained by paralog insufficiency, in which loss of one family member unveils critical dependence on paralogous subunits. This concept of "cancer-selective paralog dependency" may provide a more general strategy for targeting other tumor suppressor lesions/complexes with paralogous subunits.

Evolution of high mobility group nucleosome-binding proteins and its implications for vertebrate chromatin specialization.

High mobility group (HMG)-N proteins are a family of small nonhistone proteins that bind to nucleosomes (N). Despite the amount of information available on their structure and function, there is an almost complete lack of information on the molecular evolutionary mechanisms leading to their exclusive differentiation. In the present work, we provide evidence suggesting that HMGN lineages constitute independent monophyletic groups derived from a common ancestor prior to the diversification of vertebrates. Based on observations of the functional diversification across vertebrate HMGN proteins and on the extensive silent nucleotide divergence, our results suggest that the long-term evolution of HMGNs occurs under strong purifying selection, resulting from the lineage-specific functional constraints of their different protein domains. Selection analyses on independent lineages suggest that their functional specialization was mediated by bursts of adaptive selection at specific evolutionary times, in a small subset of codons with functional relevance-most notably in HMGN1, and in the rapidly evolving HMGN5. This work provides useful information to our understanding of the specialization imparted on chromatin metabolism by HMGNs, especially on the evolutionary mechanisms underlying their functional differentiation in vertebrates.

Characterization of mussel H2A.Z.2: a new H2A.Z variant preferentially expressed in germinal tissues from Mytilus.

Histones are the fundamental constituents of the eukaryotic chromatin, facilitating the physical organization of DNA in chromosomes and participating in the regulation of its metabolism. The H2A family displays the largest number of variants among core histones, including the renowned H2A.X, macroH2A, H2A.B (Bbd), and H2A.Z. This latter variant is especially interesting because of its regulatory role and its differentiation into 2 functionally divergent variants (H2A.Z.1 and H2A.Z.2), further specializing the structure and function of vertebrate chromatin. In the present work we describe, for the first time, the presence of a second H2A.Z variant (H2A.Z.2) in the genome of a non-vertebrate animal, the mussel Mytilus. The molecular and evolutionary characterization of mussel H2A.Z.1 and H2A.Z.2 histones is consistent with their functional specialization, supported on sequence divergence at promoter and coding regions as well as on varying gene expression patterns. More precisely, the expression of H2A.Z.2 transcripts in gonadal tissue and its potential upregulation in response to genotoxic stress might be mirroring the specialization of this variant in DNA repair. Overall, the findings presented in this work complement recent reports describing the widespread presence of other histone variants across eukaryotes, supporting an ancestral origin and conserved role for histone variants in chromatin.

Bordetella pertussis filamentous hemagglutinin itself does not trigger anti-inflammatory interleukin-10 production by human dendritic cells.

Filamentous hemagglutinin (FHA) is an important adhesin of the whooping cough agent Bordetella pertussis and is contained in most acellular pertussis vaccines. Recently, FHA was proposed to exert an immunomodulatory activity through induction of tolerogenic IL-10 secretion from dendritic cells. We have re-evaluated the cytokine-inducing activity of FHA, placing specific emphasis on the role of the residual endotoxin contamination of FHA preparations. We show that endotoxin depletion did not affect the capacity of FHA to bind primary human monocyte-derived dendritic cells, while it abrogated the capacity of FHA to elicit TNF-α and IL-10 secretion and strongly reduced its capacity to trigger IL-6 production. The levels of cytokines induced by the different FHA preparations correlated with their residual contents of B. pertussis endotoxin. Moreover, FHA failed to trigger cytokine secretion in the presence of antibodies that block TLR2 and/or TLR4 signaling. The TLR2 signaling capacity appeared to be linked to the presence of endotoxin-associated components in FHA preparations and not to the FHA protein itself. These results show that the endotoxin-depleted FHA protein does not induce cytokine release from human dendritic cells.

Comparative structure of vertebrate sperm chromatin.

A consistent feature of sperm nuclei is its exceptionally compact state in comparison with somatic nuclei. Here, we have examined the structural organization of sperm chromatin from representatives of three vertebrate lineages, bony fish (Danio rerio), birds (Gallus gallus domesticus) and mammals (Mus musculus) using light and transmission electron microscopy (TEM). Although the three sperm nuclei are all highly compact, they differ in morphology and in the complement of compaction-inducing proteins. Whereas zebrafish sperm retain somatic histones and a nucleosomal organization, in the rooster and mouse, histones are largely replaced by small, arginine-rich protamines. In contrast to the mouse, the rooster protamine contains no cysteine residues and lacks the potential stabilizing effects of S-S bonds. Protamine driven chromatin compaction results in a stable, highly condensed chromatin, markedly different from the somatic nucleosome-based beads-on-a-string architecture, but its structure remains poorly understood. When prepared gently for whole mount TEM, the rooster and mouse sperm chromatin reveal striking rod-like units 40-50 nm in width. Also present in the mouse, which has very flattened sperm nuclei, but not rooster, where nuclei take the form of elongated cylinders, are toroidal shaped structures, with an external diameter of about 90 nm. In contrast, similarly prepared zebrafish sperm exhibit nucleosomal chromatin. We also examined the early stages in the binding of salmine (the salmon protamine) to defined sequence DNA. These images suggest an initial side-by-side binding of linear DNA-protamine complexes leading to the nucleation of thin, flexible rods with the potential to bend, allowing the ends to come into contact and fuse to form toroidal structures. We discuss the relationship between these in vitro observations and the rods and toroids seen in nuclei, and suggest an explanation for the apparent absence of these structures in TEM images of fully condensed sperm nuclei.

The neuroendocrine transition in prostate cancer is dynamic and dependent on ASCL1.

Lineage plasticity is a recognized hallmark of cancer progression that can shape therapy outcomes. The underlying cellular and molecular mechanisms mediating lineage plasticity remain poorly understood. Here, we describe a versatile in vivo platform to identify and interrogate the molecular determinants of neuroendocrine lineage transformation at different stages of prostate cancer progression. Adenocarcinomas reliably develop following orthotopic transplantation of primary mouse prostate organoids acutely engineered with human-relevant driver alterations (e.g., Rb1-/-; Trp53-/-; cMyc+ or Pten-/-; Trp53-/-; cMyc+), but only those with Rb1 deletion progress to ASCL1+ neuroendocrine prostate cancer (NEPC), a highly aggressive, androgen receptor signaling inhibitor (ARSI)-resistant tumor. Importantly, we show this lineage transition requires a native in vivo microenvironment not replicated by conventional organoid culture. By integrating multiplexed immunofluorescence, spatial transcriptomics and PrismSpot to identify cell type-specific spatial gene modules, we reveal that ASCL1+ cells arise from KRT8+ luminal epithelial cells that progressively acquire transcriptional heterogeneity, producing large ASCL1+;KRT8- NEPC clusters. Ascl1 loss in established NEPC results in transient tumor regression followed by recurrence; however, Ascl1 deletion prior to transplantation completely abrogates lineage plasticity, yielding adenocarcinomas with elevated AR expression and marked sensitivity to castration. The dynamic feature of this model reveals the importance of timing of therapies focused on lineage plasticity and offers a platform for identification of additional lineage plasticity drivers.

The Bordetella pertussis type III secretion system tip complex protein Bsp22 is not a protective antigen and fails to elicit serum antibody responses during infection of humans and mice.

The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice.