Targeting TatD nuclease and the MAPK Pathway: Luteolin multifaceted approach against Mycoplasma gallisepticum infection.

Influenza A virus (IAV) noncanonical RNAs are bound by retinoic acid-inducible gene I (RIG-I). However, innate immune activation is infrequent and it is not understood why noncanonical IAV RNAs activate RIG-I in a sequence- or RNA structure-dependent manner. We hypothesized that multiple events need to occur before IAV RNA synthesis activates RIG-I and investigated whether RIG-I activation is stimulated by the noncanonical or aberrant transcription of mini viral RNAs (mvRNA), an RNA that is overexpressed in highly pathogenic IAV infections. We find that mvRNAs can cause noncanonical transcription termination through a truncated 5' polyadenylation signal or a 5' transient RNA structure that interrupts polyadenylation. The resulting capped complementary RNAs stimulate the release of an mvRNA and complement RIG-I activation in trans. Overall, our findings indicate that sequential rounds of noncanonical or aberrant viral replication and transcription are needed for innate immune signaling by IAV RNA synthesis.

Despite effective treatment, human immunodeficiency virus (HIV) persists in optimally treated people as a transcriptionally silent provirus. Latently infected cells evade the immune system and the harmful effects of the virus, thereby creating a long-lasting reservoir of HIV. To gain a deeper insight into the molecular mechanisms of HIV latency establishment, we constructed a series of HIV-1 fluorescent reporter viruses that distinguish active versus latent infection. We unexpectedly observed that the proportion of active to latent infection depended on a limiting viral factor, which created a bottleneck that could be overcome by superinfection of the cell, T cell activation, or overexpression of HIV-1 transactivator of transcription (Tat). In addition, we found that tat and regulator of expression of virion proteins (Rev) expression levels varied among HIV molecular clones and that tat levels were an important variable in latency establishment. Lower rev levels limited viral protein expression whereas lower Tat levels or mutation of the Tat binding element promoted latent infection that was resistant to reactivation even in fully activated primary T cells. Nevertheless, we found that combinations of latency reversal agents targeting both cellular activation and histone acetylation pathways overcame deficiencies in the Tat/TAR axis of transcription regulation. These results provide additional insight into the mechanisms of latency establishment and inform Tat-centered approaches to cure HIV.

Transgender, non-binary and gender-diverse patients utilizing feminizing gender-affirming hormone therapy (GAHT) may experience reduced fertility, which is currently addressed through fertility preservation and treatment cessation. Experimental research with animal models may identify mechanisms involved in testicular function impairment, their severity and duration, and may help to inform patients about reproductive health decisions. Feminizing GAHT may include combined estrogen, progesterone and/or antiandrogens (collectively referred to as E-GAHT) aiming to achieve individualized embodiment goals. This kind of treatment is highly desired, but it is expected to negatively impact the reproductive ability of transgender, non-binary and/or gender-diverse (TNG) individuals having testes, mainly by disrupting the biological pathways involved in spermatogenesis. There is little research on the impact of E-GAHT on the testis or on the potential recovery of spermatogenesis by stopping GAHT. Studies using animal models receiving GAHT are a novel promising method to help identify the reproductive pathways affected by GAHT and the degree of those effects. A literature search was performed to identify both clinical and experimental studies using rodent models applicable to the reproductive needs of E-GAHT users. We found large variability in clinical populations that has not been fully replicated in translational research. Furthermore, animal GAHT models have yet to be capitalized to examine fertility preservation. Some clinical studies indicated that sperm quality may be reduced in E-GAHT patients before initiating E-GAHT, which cannot be studied using GAHT-treated animals. As many TNG people of reproductive age may have the wish to create a future family, it is important to increase the knowledge that will inform and improve E-GAHT patients' fertility preservation outcomes and reproductive health.

Transmasculine and gender diverse adolescents and young adults can often have nuanced considerations when utilizing contraception. This narrative review discusses contraception utilization patterns and specific considerations in this population and proposes a patient-oriented comparison of contraceptive options. Contraception care is an important aspect of comprehensive gender-affirming care in transgender and gender diverse (TGD) adolescents and young adults (AYAs). TGD AYAs seek contraception care for a variety of reasons, and several important considerations are unique to this population. In addition to pregnancy prevention, transmasculine and gender nonbinary persons may seek contraception to achieve amenorrhea and reduce gender dysphoria, or as part of their gender-affirming journey. While patients on testosterone therapy often experience amenorrhea, pregnancy has been reported and testosterone is not a form of contraception. Visits for contraceptive counseling provide an opportunity to discuss fertility goals and preservation planning as well as the possibility that some methods may provide endometrial protection. In this narrative review, we analyze the current literature and discuss specific considerations for contraceptive use in this population. We also propose a contraception overview for shared decision making for patients and their clinicians.

There are some unique aspects to providing trauma-informed reproductive care to transgender and nonbinary people, who are affected by minority stress, stigma, and particular forms of trauma; we review the evidence and suggest strategies for the provision of trauma-informed reproductive care to gender minorities. Stigma and minority stress affect the health of transgender and nonbinary (TGNB) people, leading to disparities across a range of outcomes. Barriers to accessing care, including reproductive care, further complicate these health disparities. Interpersonal stigma within the healthcare system, and high rates of physical and sexual violence survivorship, make TGNB people particularly vulnerable to healthcare trauma and poor care. This is particularly true among TGNB people with multiple intersecting marginalized identities. Trauma-informed care provides a framework for medical practitioners to provide safe, holistic, and sensitive care. Scant academic literature discusses trauma-informed reproductive care for TGNB individuals. We present a narrative review of the evidence for trauma-informed reproductive care for TGNB people and suggest potential application and implementation.

Many transgender and gender diverse (TGD) people want to have biologically related children. This review summarizes and discusses the options for fertility treatment and preservation in TGD adults and adolescents, with an emphasis on gender-affirming hormone therapy in the context of fertility treatment, clinical management strategies to minimize gender dysphoria during treatment and major factors in future use of cryopreserved gametes. Years of growing research demonstrate that TGD people desire fertility counseling and family building; however, social and medical factors can impact future fertility options. Fortunately, TGD individuals have many viable options for family building using their own gametes and/or reproductive organs. However, the nuanced ways in which different gender-affirming treatments affect reproduction, the interplay with nontreatment-related infertility factors and mitigation of likely dysphoria triggers are all critical to actual utilization. This review focuses on fertility treatment and preservation options for TGD patients and highlights these influential social and medical factors. Fertility treatments may be associated with worsening gender dysphoria in TGD people, and an affirming clinical environment and conscientious provider approach is paramount to treatment success. However, reducing gender dysphoria can also require specific changes to medically assisted reproduction and sperm collection protocols, some of which carry the potential for diminished outcomes or unknown effects. Adolescents undergoing fertility preservation treatments may need more support or additional protocol modifications, and outcomes may be poorer in this age group compared with adults. Testicular and ovarian tissue cryopreservation may present a fertility preservation option for prepubertal TGD children; however, in vitro gamete maturation remains experimental in this situation.

We describe a first-of-its-kind audit of LGBTQ+ inclusivity in fertility care providers across the United Kingdom. Despite efforts being made to improve LGBTQ+ inclusion in fertility care, our results paint a picture of widespread gaps in clinical and cultural expertise alongside significant barriers to LGBTQ+ inclusion. LGBTQ+ patients comprise one of the fastest-growing user demographics in fertility care, yet they remain under-represented in fertility research, practice, and discourse. Existing studies have revealed significant systemic barriers, including cisheteronormativity, discrimination, and gaps in clinical expertise. In this article, we present a checklist of measures that clinics can take to improve LGBTQ+ inclusion in fertility care, co-created with members of the LGBTQ+ community. This checklist focuses on three key areas: cultural competence, clinical considerations, and online presence. The cultural competence criteria encompass inclusive communication practices, a broad understanding of LGBTQ+ healthcare needs, and knowledge of treatment options suitable for LGBTQ+ individuals. Clinical considerations include awareness of alternative examination and gamete collection techniques for transgender and gender diverse patients, the existence of specific clinical pathways for LGBTQ+ patients, and sensitivity to the psychological aspects of fertility care unique to this demographic. The online presence criteria evaluate provider websites for the use of inclusive language and the availability of LGBTQ+-relevant information. The checklist was used as the foundation for an audit of fertility care providers across the UK in early 2024. Our audit identified a widespread lack of LGBTQ+ inclusion, particularly for transgender and gender diverse patients, highlighting deficiencies in clinical knowledge and cultural competence. Our work calls attention to the need for further efforts to understand the barriers to inclusive and competent LGBTQ+ fertility care from both healthcare provider and patient perspectives.

Transgender and gender-diverse (TGD) people have similar desires for parenting as cisgender individuals but are likely to face greater barriers in accessing fertility treatment than their cisgender peers. Mental health professionals are well-positioned to advocate for and support TGD individuals seeking fertility care through pre-fertility treatment implications counseling regarding the psychosocial aspects of fertility treatment and family building. Transgender and gender-diverse (TGD) individuals experience significantly greater all-cause mortality and mental health disparities compared to their cisgender peers. Gender-affirming hormone therapy (GAHT) is a safe and effective treatment option for gender dysphoria that dramatically improves psychosocial health outcomes but may adversely impact fertility. Medical society guidelines recommend medical fertility preservation (FP) counseling and pre-fertility treatment psychoeducational implications consultation from qualified, reproductive mental health professionals (MHPs) for TGD individuals pursuing FP or third-party reproductive treatment. However, sparse literature exists specific to the structure of mental health psychoeducational consultation for TGD individuals pursuing FP. This narrative review highlights important areas for discussion in pre-fertility treatment mental health consultations. Results indicate that implications counseling should be conducted by an MHP with specialized training in reproductive mental health with TGD populations to reduce the risk of harm and promote successful emotional navigation of fertility treatment. Such counseling should be psychoeducational and not gatekeeping in nature and may include consideration of the psychosocial (e.g. emotional, relational, ethical, spiritual, social) risks and benefits of various family-building options. During these consultations, TGD individuals can explore their hopes and fears related to fertility and future family-building plans and discuss realistic treatment expectations, individual strengths, coping and communication strategies, and identify key support network members who may aid in navigating the fertility treatment process. MHPs can provide referrals to appropriate resources if necessary to help TGD individuals navigate treatment while coping with psychological symptoms and promoting behavior change.

Glucocorticoids (GCs) are widely used for treating hematological malignancies despite their multiple adverse effects. The biological response to GCs relies on glucocorticoid receptor (GR) transrepression (TR) that mediates the anticancer effects and transactivation (TA) associated with the side effects. Selective GR agonists (SEGRAs) preferentially activating GR TR could offer greater benefits in cancer treatment. One of the well-characterized SEGRAs, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride (CpdA), exhibited anticancer activity; however, its translational potential is limited due to chemical instability. To overcome this limitation, we obtained CpdA derivatives, CpdA-01-CpdA-08, employing two synthetic strategies and studied their anti-tumor activity: 4-(1-hydroxy-2-(piperidin-1-yl)ethyl)phenol or CpdA-03 demonstrated superior GR affinity and stability compared to CpdA. In lymphoma Granta and leukemia CEM cell lines, CpdA-03 ligand exhibited typical SEGRA properties, inducing GR TR without triggering GR TA. CpdA-03 effects on cell viability, growth, and apoptosis were similar to the reference GR ligand, dexamethasone (Dex), and the source compound CpdA. In vivo testing of CpdA-03 activity against lymphoma on the transplantable P388 murine lymphoma model showed that CpdA-03 reduced tumor volume threefold, outperforming Dex and CpdA. In conclusion, in this work, we introduce a novel SEGRA CpdA-03 as a promising agent for lymphoma treatment with fewer side effects.

Inclusion of TAT and NLS sequences in lipopeptide molecules generates homogenous nanoparticles for gene delivery applications

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Approximately 890,000 new cases are diagnosed each year, while incidence continues to rise and is anticipated to increase by 30% by 2030. HNSCC occurs most often in men in their 50-60s, although the incidence among younger individuals is increasing. Current standard of care practices include surgical resection, followed by adjuvant radiation or chemoradiation. HNSCC tumors exhibit frequent mutation of CDKN2A (22% of tumors) and TP53 (72% of tumors). Additionally, two members of the TP53 gene family, TP63 and TP73, are frequently altered in HNSCC. TP63 encodes two major isoforms, ΔNp63 which lacks the main transcription activation domain and acts as a dominant-negative inhibitor of transactivation (TA), and TAp63 (contains a p53-like TA domain). ΔNp63 is overexpressed in a majority of HNSCC tumors. Our lab has reported ΔNp63α interacts with activated c-Rel (a nuclear factor-KB family member) in HNSCC, thereby promoting uncontrolled proliferation, a key alteration in the pathogenesis of cancers. Consistent with a role in growth regulation, ΔNp63α:c-Rel complexes bind a promoter motif and repress the cyclin-dependent kinase inhibitor p21WAF1 in both human HNSCCs and normal keratinocytes overexpressing ΔNp63α. The direct relationship between ΔNp63α and activated c-Rel is observed by strong nuclear co-localization in the proliferating compartment of primary head and neck SCC. Stimulation of HNSCC cells with TNFα results in the induction of the c-Rel oncoprotein that binds to ΔNp63, displacing and inactivating the tumor suppressor TAp73 from ΔNp63-TAp73 complexes. Using western blot analysis in human HNSCC cell lines, we confirmed that stimulation with TNFα enhances nuclear c-Rel localization. We then set out to design a high throughput screening bioassay for the purpose of evaluating compounds that can inhibit c-Rel accumulation in the nucleus under TNFα stimulation. By preventing activated c-Rel from interacting with ΔNp63α in the nucleus we aim to decrease uncontrolled proliferation and restore the tumor-suppressive functionality of the ΔNp63-TAp73 complex. A 384-well immunocytochemistry assay was developed using a confocal microscopy readout and an automated image analysis algorithm to quantify nuclear translocation. The optimized assay will be used to screen the NCATS collection of approved drugs and investigational oncology agents. Top candidates will undergo further investigation in vitro, ex vivo, and in vivo to explore the possibility of repurposing the small molecules for HNSCC therapeutics. Citation Format: Kristin Ann Altwegg, Kathryn E. King, Dvir Blivis, Ty C. Voss, Natalia J. Martinez, Wendy C. Weinberg, Mark J. Henderson. Developing a drug repurposing screen for head and neck squamous cell carcinoma using a novel c-Rel bioassay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3101.

Abstract The docking protein GAB2 binds to growth factor, cytokine and antigen receptors via the adaptor GRB2. Thereby, GAB2 amplifies the signaling output of the SHP2/Ras/ERK, PI3K/AKT and STAT5 pathways, leading to survival, proliferation and migration. Beside these physiological functions, GAB2 is often overexpressed or hyperphosphorylated in different types of cancer, in particular melanoma, breast cancer and various leukemia entities. Previous studies demonstrated that mice with constitutive Gab2 deficiency are protected against breast cancer, acute and chronic myeloid leukemia driven by HER2, BCR::ABL1 and FLT3-ITD, respectively. While these studies support a critical role in tumor initiation, nothing is known about the relevance of GAB2 for tumor maintenance and progression. To this end, we have developed two novel conditional loss- and gain-of-function mouse models to further analyze the role of GAB2 in cancer. In the first model, the transgenic GAB2 mouse, GAB2 is expressed under the control of the TET-O promotor, allowing the tetracycline dependent expression in tissues expressing a tetracycline trans activator (tTA). We have already started by crossing in the SCLtTA mouse, in which tTA is expressed under the control of the murine stem cell leukemia 3’ enhancer. Here we show that the expression of transgenic GAB2 is tightly regulated by tetracycline and restricted to hematopoietic cells. This system is now perfectly suited to analyze the role of GAB2 in hematologic malignancies. By expressing tTA under different promotors, we propose that GAB2 overexpression can be directed to other organs or cell types. Combining this mouse line with cancer mouse models will allow to study the role of GAB2 in different cancer entities. With the second mouse model, the conditional Gab2 KO mouse, it is possible to study the therapeutic potential of GAB2 impairment in vivo. In this mouse, exon three of the Gab2 gene is flanked by LoxP sites. This allows for its CRE recombinase mediated deletion, leading to GAB2 deficiency. The combination of this mouse model with inducible and tissue specific CRE strains will allow to analyze the role of GAB2 in various cancer mouse models. To demonstrate the efficient conditional deletion of exon 3, we first crossed in the tamoxifen inducible ROSA26 CreERT2 mouse line. So far, we were able to show the functionality of this system in vitro in mouse embryonic fibroblasts (MEFs) generated from the conditional Gab2 KO mouse. In a next step we will verify the results in vivo and will cross this mouse line with a mouse model for Chronic Myeloid Leukemia (CML) to investigate the therapeutic potential of GAB2 in CML. In summary, we are convinced that these two novel mouse models will help to identify GAB2 as a potential target or biomarker in various cancer entities. In particular, the conditional approach will allow to alter GAB2 expression in already established tumors, thus mimicking pharmacological targeting. Citation Format: Moritz Angel, Vanessa Klappstein, Jule Schrimpf, Melanie Langhammer, Khalid Shoumariyeh, Cornelius Miething, Ulrich Kloz, Brittney Armstrong, Franciscus van der Hoeven, Tilman Brummer, Sebastian Halbach. Novel mouse models to study the role of the docking protein GAB2 in general and in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5374.

Transcriptional control of CCAAT/enhancer binding protein zeta gene in chicken adipose tissue

The prognosis for patients with metastatic melanoma is poor and treatment options are limited. Genetically-engineered T cell therapy targeting chondroitin sulfate proteoglycan 4 (CSPG4), however, represents a promising treatment option, especially as both primary melanoma cells as well as metastases uniformly express CSPG4. Aiming to prevent off-tumor toxicity while maintaining a high cytolytic potential, we combined a chimeric co-stimulatory receptor (CCR) and a CSPG4-directed second-generation chimeric antigen receptor (CAR) with moderate potency. CCRs are artificial receptors similar to CARs, but lacking the CD3ζ activation element. Thus, T cells expressing solely a CCR, do not induce any cytolytic activity upon target cell binding, but are capable of boosting the CAR T cell response when both CAR and CCR engage their target antigens simultaneously. Here we demonstrate that co-expression of a CCR can significantly enhance the anti-tumor response of CSPG4-CAR T cells in vitro as well as in vivo. Importantly, this boosting effect was not dependent on co-expression of both CCR- and CAR-target on the very same tumor cell, but was also achieved upon trans activation. Finally, our data support the idea of using a CCR as a powerful tool to enhance the cytolytic potential of CAR T cells, which might open a novel therapeutic window for the treatment of metastatic melanoma.

O2-generating multifunctional polymeric micelles for highly efficient and selective photodynamic-photothermal therapy in melanoma

Salt stress severely damages the growth and yield of crops. Recently, long noncoding RNAs (lncRNAs) were demonstrated to regulate various biological processes and responses to environmental stresses. However, the regulatory mechanisms of lncRNAs in cotton (Gossypium hirsutum) response to salt stress are still poorly understood. Here, we observed that a lncRNA, trans acting of BGLU24 by lncRNA (TRABA), was highly expressed while GhBGLU24-A was weakly expressed in a salt-tolerant cotton accession (DM37) compared to a salt-sensitive accession (TM-1). Using TRABA as an effector and proGhBGLU24-A-driven GUS as a reporter, we showed that TRABA suppressed GhBGLU24-A promoter activity in double transgenic Arabidopsis (Arabidopsis thaliana), which explained why GhBGLU24-A was weakly expressed in the salt-tolerant accession compared to the salt-sensitive accession. GhBGLU24-A encodes an endoplasmic reticulum (ER)-localized β-glucosidase that responds to salt stress. Further investigation revealed that GhBGLU24-A interacted with RING-type E3 ubiquitin ligase (GhRUBL). Virus-induced gene silencing (VIGS) and transgenic Arabidopsis studies revealed that both GhBGLU24-A and GhRUBL diminish plant tolerance to salt stress and ER stress. Based on its substantial effect on ER-related degradation (ERAD)-associated gene expression, GhBGLU24-A mediates ER stress likely through the ERAD pathway. These findings provide insights into the regulatory role of the lncRNA TRABA in modulating salt and ER stresses in cotton and have potential implications for developing more resilient crops.

Endocrine therapy is the frontline treatment for estrogen receptor (ER) positive breast cancer patients. However, the primary and acquired resistance to endocrine therapy drugs remain as a major challenge in the clinic. Here, this work identifies an estrogen-induced lncRNA, LINC02568, which is highly expressed in ER-positive breast cancer and functional important in cell growth in vitro and tumorigenesis in vivo as well as endocrine therapy drug resistance. Mechanically, this work demonstrates that LINC02568 regulates estrogen/ERα-induced gene transcriptional activation in trans by stabilizing ESR1 mRNA through sponging miR-1233-5p in the cytoplasm. Meanwhile, LINC02568 contributes to tumor-specific pH homeostasis by regulating carbonic anhydrase CA12 in cis in the nucleus. The dual functions of LINC02568 together contribute to breast cancer cell growth and tumorigenesis as well as endocrine therapy drug resistance. Antisense oligonucleotides (ASO) targeting LINC02568 significantly inhibits ER-positive breast cancer cell growth in vitro and tumorigenesis in vivo. Furthermore, combination treatment with ASO targeting LINC02568 and endocrine therapy drugs or CA12 inhibitor U-104 exhibits synergistic effects on tumor growth. Taken together, the findings reveal the dual mechanisms of LINC02568 in regulating ERα signaling and pH homeostasis in ER-positive breast cancer, and indicated that targeting LINC02568 might represent a potential therapeutic avenue in the clinic.

Some viruses are known to be associated with the onset of specific cancers. These microorganisms, oncogenic viruses or oncoviruses, can convert normal cells into cancer cells by modulating the central metabolic pathways or hampering genomic integrity mechanisms, consequently inhibiting the apoptotic machinery and/or enhancing cell proliferation. Seven oncogenic viruses are known to promote tumorigenesis in humans: human papillomavirus (HPV), hepatitis B and C viruses (HBV, HCV), Epstein-Barr virus (EBV), human T-cell leukemia virus 1 (HTLV-1), Kaposi sarcoma-associated herpesvirus (KSHV), and Merkel cell polyomavirus (MCPyV). Recent research indicates that SARS-CoV-2 infection and COVID-19 progression may predispose recovered patients to cancer onset and accelerate cancer development. This hypothesis is based on the growing evidence regarding the ability of SARS-CoV-2 to modulate oncogenic pathways, promoting chronic low-grade inflammation and causing tissue damage. Herein, we summarize the main relationships known to date between virus infection and cancer, providing a summary of the proposed biochemical mechanisms behind the cellular transformation. Mechanistically, DNA viruses (such as HPV, HBV, EBV, and MCPyV) encode their virus oncogenes. In contrast, RNA viruses (like HCV, HTLV-1) may encode oncogenes or trigger host oncogenes through cis-/-trans activation leading to different types of cancer. As for SARS-CoV-2, its role as an oncogenic virus seems to occur through the inhibition of oncosuppressors or controlling the metabolic and autophagy pathways in the infected cells. However, these effects could be significant in particular scenarios like those linked to severe COVID-19 or long COVID. On the other hand, looking at the SARS-CoV-2─cancer relationship from an opposite perspective, oncolytic effects and anti-tumor immune response were triggered by SARS-CoV-2 infection in some cases. In summary, our work aims to recall comprehensive attention from the scientific community to elucidate the effects of SARS-CoV-2 and, more in general, β-coronavirus infection on cancer susceptibility for cancer prevention or supporting therapeutic approaches.

A heterozygous missense variant in DLX3 leads to uterine leiomyomas and pregnancy losses in a consanguineous Iranian family