Near chromosome-level and highly repetitive genome assembly of the snake pipefish Entelurus aequoreus (Syngnathiformes: Syngnathidae).

The snake pipefish, Entelurus aequoreus (Linnaeus, 1758), is a northern Atlantic fish inhabiting open seagrass environments that recently expanded its distribution range. Here, we present a highly contiguous, near chromosome-scale genome of E. aequoreus. The final assembly spans 1.6 Gbp in 7,391 scaffolds, with a scaffold N50 of 62.3 Mbp and L50 of 12. The 28 largest scaffolds (>21 Mbp) span 89.7% of the assembly length. A BUSCO completeness score of 94.1% and a mapping rate above 98% suggest a high assembly completeness. Repetitive elements cover 74.93% of the genome, one of the highest proportions identified in vertebrates. Our demographic modeling identified a peak in population size during the last interglacial period, suggesting the species might benefit from warmer water conditions. Our updated snake pipefish assembly is essential for future analyses of the morphological and molecular changes unique to the Syngnathidae.

Efficacy of a therapist-guided smartphone-based intervention to support recovery from bulimia nervosa: Study protocol of a randomized controlled multi-centre trial.

OBJECTIVE: Although inpatient treatment is highly effective for patients with bulimia nervosa (BN), some patients show a resurgence of symptoms and relapse after discharge. Therefore, the aim of this study is to evaluate the efficacy of a guided smartphone-based aftercare intervention following inpatient treatment to support recovery. METHOD: 172 female patients with BN (DSM-5: 307.51) will be randomized to receive a 16-week smartphone-based aftercare intervention (German version of 'Recovery Record') with therapist feedback as an add-on element to treatment as usual (TAU) or TAU alone. Assessments will take place at baseline (discharge, T0), during the intervention (after 4 weeks, T1), post-intervention (after 16 weeks, T2) and at 6-month follow-up (T3). Primary outcome will be remission at T2. Moderator and mediator analyses will investigate for whom the aftercare intervention suits best and how it works. CONCLUSIONS: This is the first randomized controlled trial to examine a guided smartphone-based aftercare intervention following inpatient treatment of patients with BN. We expect that this innovative aftercare intervention is highly accepted by the patients and that it has the potential to support recovery after inpatient treatment and thereby could contribute to improving aftercare for patients with BN.

A systems biology approach to define mechanisms, phenotypes, and drivers in PanNETs with a personalized perspective.

Pancreatic neuroendocrine tumors (PanNETs) are a rare tumor entity with largely unpredictable progression and increasing incidence in developed countries. Molecular pathways involved in PanNETs development are still not elucidated, and specific biomarkers are missing. Moreover, the heterogeneity of PanNETs makes their treatment challenging and most approved targeted therapeutic options for PanNETs lack objective responses. Here, we applied a systems biology approach integrating dynamic modeling strategies, foreign classifier tailored approaches, and patient expression profiles to predict PanNETs progression as well as resistance mechanisms to clinically approved treatments such as the mammalian target of rapamycin complex 1 (mTORC1) inhibitors. We set up a model able to represent frequently reported PanNETs drivers in patient cohorts, such as Menin-1 (MEN1), Death domain associated protein (DAXX), Tuberous Sclerosis (TSC), as well as wild-type tumors. Model-based simulations suggested drivers of cancer progression as both first and second hits after MEN1 loss. In addition, we could predict the benefit of mTORC1 inhibitors on differentially mutated cohorts and hypothesize resistance mechanisms. Our approach sheds light on a more personalized prediction and treatment of PanNET mutant phenotypes.

Pain Catastrophizing and Impact on Pelvic Floor Surgery Experience.

IMPORTANCE: Understanding patients' perceptions of symptoms and outcomes of urogynecologic surgery is essential for providing high-quality care. OBJECTIVE: The aim of the study was to assess association of pain catastrophizing with pelvic floor symptom distress and impact, postoperative pain, and voiding trial in patients undergoing urogynecologic surgery. STUDY DESIGN: Individuals whose self-identified gender was female and were undergoing surgery March 2020-December 2021 were included. Participants completed the Pain Catastrophizing Scale (range 0-52), Pelvic Floor Distress Inventory, and Pelvic Floor Impact Questionnaire preoperatively. Pain catastrophizing was score ≥30 and describes the tendency to magnify the overall threat of pain. Voiding trial failure was inability to void ≥2/3 of instilled volume (≤300 mL). The association between pain catastrophizing and symptom distress and impact was assessed with linear regression. A P < 0.05 is significant. RESULTS: Three hundred twenty patients were included (mean age, 60 years, 87% White). Forty-six of 320 participants (14%) had a pain catastrophizing score ≥30. The pain catastrophizing group had higher body mass index (33 ± 12 vs 29 ± 5), more benzodiazepine use (26% vs 12%), greater symptom distress (154 ± 58 vs 108 ± 60), and greater urogenital (59 ± 29 vs 47 ± 28), colorectal (42 ± 24 vs 26 ± 23), and prolapse (54 ± 24 vs 36 ± 24) subscale scores, all P ≤ 0.02. The pain catastrophizing group had greater impact (153 ± 72 vs 72 ± 64, P < 0.01) and urogenital (60 ± 29 vs 34 ± 28), colorectal (36 ± 33 vs 16 ± 26), and prolapse (57 ± 32 vs 22 ± 27) subscale scores, P < 0.01. Associations remained controlling for confounders ( P < 0.01). The pain catastrophizing group had higher 10-point pain scores (8 vs 6, P < 0.01) and was more likely to report pain at 2 weeks (59% vs 20%, P < 0.01) and 3 months (25% vs 6%, P = 0.01). Voiding trial failure did not differ (26% vs 28%, P = 0.98). CONCLUSIONS: Pain catastrophizing is associated with greater pelvic floor symptom distress and impact and postoperative pain but not voiding trial failure.

Systematic Review of Lymphangioleiomyomatosis Outcomes in Pregnancy and a Proposed Management Guideline.

OBJECTIVE: Lymphangioleiomyomatosis (LAM) is a rare, multisystem disease that primarily affects women of reproductive age. Disease progression has been linked to estrogen exposure, and as such many patients are advised to avoid pregnancy. Data are limited regarding the interaction between LAM and pregnancy, and as such we performed a systematic review to summarize available literature reporting outcomes of pregnancies complicated by maternal LAM. STUDY DESIGN: This was a systematic review including randomized controlled trials, observational studies, systematic reviews, case reports, clinical practice guidelines, and quality improvement studies with full-text manuscripts or abstracts in the English language with primary data on pregnant or postpartum patients with LAM. The primary outcome was maternal outcomes during pregnancy as well as pregnancy outcomes. Secondary outcomes were neonatal outcomes and long-term maternal outcomes. This search occurred in July 2020 and included MEDLINE, Scopus, clinicaltrials.gov, Embase, and Cochrane Central. Risk of bias was ascertained using the Newcastle-Ottawa Scale. Our systematic review was registered with PROSPERO as protocol number CRD 42020191402. RESULTS: A total of 175 publications were identified in our initial search; ultimately 31 studies were included. Six (19%) studies were retrospective cohort studies and 25 (81%) studies were case reports. Patients diagnosed during pregnancy had worse pregnancy outcomes compared to those diagnosed with LAM prior to pregnancy. Multiple studies reported a significant risk of pneumothoraces during pregnancy. Other significant risks included preterm delivery, chylothoraces, and pulmonary function deterioration. A proposed strategy for preconception counseling and antenatal management is provided. CONCLUSION: Patients diagnosed with LAM during pregnancy generally experience worse outcomes including recurrent pneumothoraces and preterm delivery as compared to patients with a LAM diagnosis prior to pregnancy. Given that there are limited studies available, and that the majority are low-quality evidence and subject to bias, further investigation of the interaction between LAM and pregnancy is warranted to guide patient care and counseling. KEY POINTS: · Data are limited on the effects of lymphangioleiomyomatosis on pregnancy outcomes.. · We performed a systematic review to summarize pregnancy outcomes complicated by LAM.. · Patients diagnosed with LAM during pregnancy experience worse outcomes..

Single-cell RNA-based phenotyping reveals a pivotal role of thyroid hormone receptor alpha for hypothalamic development.

Thyroid hormone and its receptor TRα1 play an important role in brain development. Several animal models have been used to investigate this function, including mice heterozygous for the TRα1R384C mutation, which confers receptor-mediated hypothyroidism. These mice display abnormalities in several autonomic functions, which was partially attributed to a developmental defect in hypothalamic parvalbumin neurons. However, whether other cell types in the hypothalamus are similarly affected remains unknown. Here, we used single-nucleus RNA sequencing to obtain an unbiased view on the importance of TRα1 for hypothalamic development and cellular diversity. Our data show that defective TRα1 signaling has surprisingly little effect on the development of hypothalamic neuronal populations, but it heavily affects hypothalamic oligodendrocytes. Using selective reactivation of the mutant TRα1 during specific developmental periods, we find that early postnatal thyroid hormone action seems to be crucial for proper hypothalamic oligodendrocyte maturation. Taken together, our findings underline the well-known importance of postnatal thyroid health for brain development and provide an unbiased roadmap for the identification of cellular targets of TRα1 action in mouse hypothalamic development.

Increased disparities associated with black women and abnormal cervical cancer screening follow-up.

Background: To determine whether race and ethnicity impacts patient adherence to follow-up for colposcopy after abnormal cervical cancer screening. Methods: This retrospective chart review included women that were randomly selected from patients presenting to our colposcopy clinic from 1/2019 to 12/2019. Inclusion criteria were females age ≥21 years-old and appropriate referral for colposcopy. Patients were grouped into three categories: (1) ADHERENT to follow-up if they came to their first scheduled appointment; (2) DELAYED if they presented more than three months from their original referral (usually missing 1-3 appointments); and (3) NOT ADHERENT if they did not show for their appointment after referral. Analysis was performed using SPSS v.26. Results: 284 women met inclusion criteria for the study. The majority of women were Black (65.2 %) followed by non-Hispanic Whites (20.0 %) and Latinx (14.8 %). Overall, 39.1 % were ADHERENT, 18.6 % were DELAYED, and 42.3 % were NOT ADHERENT. When compared with non-Hispanic White women, there was a significant difference between race/ethnicity and timing of follow-up (p = 0.03). Blacks were more likely to be NOT ADHERENT (45.9 %; p = 0.03), and Latinx and Blacks were the most likely to be DELAYED (35.7 % and 21.1 %; p = 0.03). Private insurance patients were more likely to be ADHERENT for care compared with un-/underinsured patients (78.9 vs 27.8 %, p = 0.0001). Conclusion: There is inadequate follow-up after abnormal cervical cancer screening across all races/ethnicities; however, lack of adherence is higher in Black patients. Moreover, 25% of Hispanic and Black women present in a delayed fashion. Culturally relevant assessments and interventions are needed to understand and address these gaps.

CARD9 Forms an Alternative CBM Complex in Richter Syndrome.

Richter syndrome (RS) is defined as the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, mostly diffuse large B-cell lymphoma (DLBCL). Despite intensive therapy, patients with RS have an unfavorable clinical outcome. The detailed pathobiology of Richter transformation still needs to be elucidated. Here, we report high mRNA and protein levels of CARD9 in the RS cell line U-RT1. Co-immunoprecipitation revealed the assembly of a CBM complex using CARD9 instead of CARD11. CARD9 is known to be an activator of NF-кB signaling in myeloid cells. U-RT1 Western blot analyses showed phosphorylation of IκB as well as IKK, indicating a constitutively active canonical NF-кB pathway. This was further supported by the significant reduction in cell viability and CYLD cleavage products after CARD9 siRNA knockdown. We also showed immunostaining for CARD9 in 53% of cases analyzed in a series of RS tissue specimens, whereas other lymphomas rarely show CARD9 expression. This is the first report on ectopic expression and function of CARD9 in an aggressive B-cell lymphoma. Our findings suggest that CARD9 may contribute to the pathogenesis of RS.

The effects of offering immediate postpartum placement of IUDs and implants to pregnant patients with heart disease.

OBJECTIVE: To evaluate the effects of offering immediate postpartum long-acting reversible contraception to pregnant patients with heart disease. STUDY DESIGN: Retrospective cohort of pregnant patients with cardiac disease managed by a Comprehensive Pregnancy & Heart Program. Patients were divided into 2 cohorts: pre-immediate postpartum LARC Program implementation (March 2015 to January 2017) and post-implementation (February 2017 to June 2019). The primary outcome was LARC (intrauterine device [IUD] or etonogestrel implant) use postpartum, defined as LARC either immediately postpartum or at the postpartum visit. Secondary outcomes included contraception intent at delivery and IUD expulsion rate of IUDs placed immediately postpartum. RESULTS: Of 159 included patients, 96 (60%) delivered during the post-implementation period. LARC use tripled after program implementation, 11% vs 35%, p < 0.01. Specifically, immediate postpartum IUD use increased from 1 (1.6%) to 10 (10.4%), p = 0.05, and use of immediate postpartum implant increased from 0 to 14 (14.6%), p = 0.002. Rates of women without contraception plans at delivery decreased from 32% to 14%, p < 0.01, as did the number of women using medroxyprogesterone acetate: 16% vs 4%, p = 0.01. Tubal ligation rates were not different before and after program implementation: 24% and 29%, p = 0.46. Postpartum visit rates were similar between Pre and Post groups: 70% and 72%, p = 0.78, respectively. One immediate postpartum IUD expulsion occurred. CONCLUSION: LARC use tripled in pregnant patients in an obstetric heart disease program after implementation of an immediate postpartum LARC Program. Access to immediate postpartum IUDs and implants should be a public health priority for women with heart disease to reduce their disproportionate burden of maternal morbidity and mortality. IMPLICATIONS: Access to immediate postpartum IUDs and implants should be a public health priority for women with heart disease - as well as all people with high-risk health conditions - to reduce their disproportionate burden of maternal morbidity and mortality.

Racial disparities in reliable contraceptive use in women with heart disease.

OBJECTIVE: The aim of the study was to investigate the differences in reliable contraceptive use between black women and white women with maternal cardiac disease. METHODS: The study comprised a retrospective cohort of women with maternal cardiac disease managed by the University of Alabama at Birmingham (UAB) Comprehensive Pregnancy and Heart Program (CPHP). Women were included if they had attended one or more prenatal visits at the UAB CPHP and delivered at the UAB hospital between March 2015 and June 2019. The primary outcome was reliable contraceptive use within 2 months postpartum, defined by receipt of long-acting reversible contraception (i.e., an intrauterine contraceptive device or an etonogestrel implant) or female sterilisation. All outcomes were compared based on self-reported race. RESULTS: One hundred and forty-nine women met the inclusion criteria. Black women (n = 63) were more likely than white women (n = 86) to use reliable contraception (65% vs 43%; p = 0.008). Black women were less likely than white women to be undecided or decline contraception at the time of admission (13% vs 27%; p = 0.037). There was no difference in reliable contraceptive use between black women (n = 20, 63%) and white women (n = 23, 72%) with modified World Health Organisation (WHO) class III/IV lesions (p = 0.42). CONCLUSION: Black women with maternal cardiac disease were more likely than white women to receive reliable contraception. Interventions to prevent unintended pregnancy in women with maternal cardiac disease should focus on improving reliable contraceptive use, especially for women with modified WHO class III/IV lesions.

CD47-targeted cancer immunogene therapy: Secreted SIRPα-Fc fusion protein eradicates tumors by macrophage and NK cell activation.

CD47 protects healthy cells from macrophage attack by binding to signal regulatory protein α (SIRPα), while its upregulation in cancer prevents immune clearance. Systemic treatment with CD47 antibodies requires a weakened Fc-mediated effector function or lower CD47-binding affinity to prevent side effects. Our approach combines "the best of both worlds," i.e., maximized CD47 binding and full Fc-mediated immune activity, by exploiting gene therapy for paracrine release. We developed a plasmid vector encoding for the secreted fusion protein sCV1-hIgG1, comprising highly efficient CD47-blocking moiety CV1 and Fc domain of human immunoglobulin G1 (IgG1) with maximized immune activation. sCV1-hIgG1 exhibited a potent bystander effect, blocking CD47 on all cells via fusion protein secreted from only a fraction of cells or when transferring transfection supernatant to untransfected cells. The CpG-free plasmid ensured sustained secretion of sCV1-hIgG1. In orthotopic human triple-negative breast cancer in CB17-severe combined immunodeficiency (SCID) mice, ex vivo transfection significantly delayed tumor growth and eradicated one-third of tumors. In intratumoral transfection experiments, CD47 blockage and increased migration of macrophages into the tumor were observed within 17 h of a single injection. Natural killer (NK) cell-mediated lysis of sCV1-hIgG1-expressing cells was demonstrated in vitro. Taken together, this approach also opens the opportunity to block, in principle, any immune checkpoints.

U-RT1 - A new model for Richter transformation.

The advent of highly effective treatments targeting the disease biology of chronic lymphocytic leukemia (CLL) has transformed the therapeutic field tremendously. However, transformation into an aggressive B-cell lymphoma, called Richter syndrome (RS), remains highly challenging since the treatment options for this condition are still insufficient. Exploratory drug testing and experimental studies are restricted by the lack of satisfactory models. We have established U-RT1, a cell line derived from a highly proliferating RS clonally related to the patient's underlying CLL. The cell line shows morphological features and an immunophenotype of RS-DLBCL (non-GCB). Molecular analysis revealed a complex karyotype with driver aberrations characteristic for RS such as loss of TP53 and CDKN2A. Furthermore, U-RT1 displays a chromosomal gain of the NOTCH1 gene locus and strong immunoreactivity for BCL-2. These features suggest that U-RT1 is the first eligible model system for investigations on the pathogenesis of RS and novel treatment options.

Blocking Y-Box Binding Protein-1 through Simultaneous Targeting of PI3K and MAPK in Triple Negative Breast Cancers.

The multifunctional protein Y-box binding protein-1 (YB-1) regulates all the so far described cancer hallmarks including cell proliferation and survival. The MAPK/ERK and PI3K/Akt pathways are also the major pathways involved in cell growth, proliferation, and survival, and are the frequently hyperactivated pathways in human cancers. A gain of function mutation in KRAS mainly leads to the constitutive activation of the MAPK pathway, while the activation of the PI3K/Akt pathway occurs either through the loss of PTEN or a gain of function mutation of the catalytic subunit alpha of PI3K (PIK3CA). In this study, we investigated the underlying signaling pathway involved in YB-1 phosphorylation at serine 102 (S102) in KRAS(G13D)-mutated triple-negative breast cancer (TNBC) MDA-MB-231 cells versus PIK3CA(H1047R)/PTEN(E307K) mutated TNBC MDA-MB-453 cells. Our data demonstrate that S102 phosphorylation of YB-1 in KRAS-mutated cells is mainly dependent on the MAPK/ERK pathway, while in PIK3CA/PTEN-mutated cells, YB-1 S102 phosphorylation is entirely dependent on the PI3K/Akt pathway. Independent of the individual dominant pathway regulating YB-1 phosphorylation, dual targeting of MEK and PI3K efficiently inhibited YB-1 phosphorylation and blocked cell proliferation. This represents functional crosstalk between the two pathways. Our data obtained from the experiments, applying pharmacological inhibitors and genetic approaches, shows that YB-1 is a key player in cell proliferation, clonogenic activity, and tumor growth of TNBC cells through the MAPK and PI3K pathways. Therefore, dual inhibition of these two pathways or single targeting of YB-1 may be an effective strategy to treat TNBC.

Eating disorders in times of the COVID-19 pandemic-Results from an online survey of patients with anorexia nervosa.

OBJECTIVE: The COVID-19 pandemic and the resulting public restrictions pose a psychological burden for humans worldwide and may be particularly detrimental for individuals with mental disorders. Therefore, the current study explored effects of the COVID-19 pandemic on eating disorder (ED) symptoms and other psychological aspects in former inpatients with anorexia nervosa (AN). METHOD: One-hundred and fifty-nine patients with AN-discharged from inpatient treatment in 2019-completed an online survey on contact history with COVID-19, changes in ED symptoms and other psychological aspects, health care utilization, and strategies patients employed to cope during the pandemic. RESULTS: Approximately 70% of patients reported that eating, shape and weight concerns, drive for physical activity, loneliness, sadness, and inner restlessness increased during the pandemic. Access to in-person psychotherapies and visits at the general practitioner (including weight checks) decreased by 37% and 46%, respectively. Videoconference therapy was used by 26% and telephone contacts by 35% of patients. Patients experienced daily routines, day planning and enjoyable activities as the most helpful among the most used coping strategies. DISCUSSION: The COVID-19 pandemic poses great challenges to patients with AN. ED-related thoughts and behaviors may be used as dysfunctional coping mechanisms to regain control over the current circumstances. E-mental health interventions appear to be promising for supporting AN patients during these hard times. Furthermore, interventions addressing symptoms of depression and anxiety, as well as intolerance of uncertainty might help them manage their ED symptoms.

Combined Chemisorption and Complexation Generate siRNA Nanocarriers with Biophysics Optimized for Efficient Gene Knockdown and Air-Blood Barrier Crossing.

Current nucleic acid (NA) nanotherapeutic approaches face challenges because of shortcomings such as limited control on loading efficiency, complex formulation procedure involving purification steps, low load of NA cargo per nanoparticle, endosomal trapping, and hampered release inside the cell. When combined, these factors significantly limit the amount of biologically active NA delivered per cell in vitro, delivered dosages in vivo for a prolonged biological effect, and the upscalability potential, thereby warranting early consideration in the design and developmental phase. Here, we report a versatile nanotherapeutic platform, termed auropolyplexes, for improved and efficient delivery of small interfering RNA (siRNA). Semitelechelic, thiolated linear polyethylenimine (PEI) was chemisorbed onto gold nanoparticles to endow them with positive charge. A simple two-step complexation method offers tunable loading of siRNA at concentrations relevant for in vivo studies and the flexibility for inclusion of multiple functionalities without any purification steps. SiRNA was electrostatically complexed with these cationic gold nanoparticles and further condensed with polycation or polyethyleneglycol-polycation conjugates. The resulting auropolyplexes ensured complete complexation of siRNA into nanoparticles with a high load of ∼15,500 siRNA molecules/nanoparticle. After efficient internalization into the tumor cell, an 80% knockdown of the luciferase reporter gene was achieved. Auropolyplexes were applied intratracheally in Balb/c mice for pulmonary delivery, and their biodistribution were studied spatio-temporally and quantitatively by optical tomography. Auropolyplexes were well tolerated with ∼25% of the siRNA dose remaining in the lungs after 24 h. Importantly, siRNA was released from auropolyplexes in vivo and a fraction also crossed the air-blood barrier, which was then excreted via kidneys, whereas >97% of gold nanoparticles were retained in the lung. Linear PEI-based auropolyplexes offer a combination of successful endosomal escape and better biocompatibility profile in vivo. Taken together, combined chemisorption and complexation endow auropolyplexes with crucial biophysical attributes, enabling a versatile and upscalable nanogold-based platform for siRNA delivery in vitro and in vivo.

[Efficacy, Moderators and Mediators of Manualized Family-Based Treatments in Adolescents with Eating Disorders: A Systematic Review]. / Wirksamkeit, Moderatoren und Mediatoren manualisierter familienbasierter Therapie bei Jugendlichen mit Essstörungen: Eine systematische Übersichtsarbeit.

Family-based therapy (FBT) is currently the most evidence-based treatment for adolescents with eating disorders. The aim of this review is to summarize previous research results regarding the efficacy of the manualized FBT according to Lock and Le Grange and to report on moderators and mediators. In 5 randomized controlled trials (RCTs) in anorexia nervosa (N=560) remission rates were between 21.2-42% at end of treatment, between 21.8-40% at 6-month follow-up, and between 29-49% at 12-month follow-up. Remission rates for patients with bulimia nervosa (2 RCTs, N=210) were 39%, 29-44% and 49% respectively. It would be desirable to replicate these results through independent working groups and in other countries. In addition, it would also be important to evaluate FBT in comparison to cognitive behavioral therapy and psychodynamic therapy, and to further explore strategies for non-responders.

Luminescent and fluorescent triple reporter plasmid constructs for Wnt, Hedgehog and Notch pathway.

Tracking the activity of signalling pathways is a fundamental method for basic science, as well as in cancer- and pharmaceutical research. The developmental pathways Wnt, Hedgehog and Notch are frequently deregulated in cancers and represent a valuable target for the discovery of novel anticancer compounds. Here we present reporter systems for tracking activity of these pathways by using specific promoter elements driving the expression of either sensitive luciferases or fluorescent proteins. A high level of sensitivity was obtained using the luciferase reporter genes for firefly (FLuc), secreted Gaussia (GLuc) and synthetic NanoLuc (NLuc). As fluorescent reporter proteins, mTurqouise2, tdTomato and iRFP720 were chosen. Specificity of pathway activity was validated by co-transfection with pathway activating genes, showing significant response to induction. In addition, multi-gene plasmids were cloned, allowing the detection of all three pathways by one vector. By using the multi-gene vector 3P-Luc (wnt-NLuc, hedgehog-FLuc, Notch-GLuc), we could unambiguously demonstrate the crosstalk between pathways, while excluding cross reactivity of luciferase substrates. First studies with synthetic compounds confirmed the applicability of the system for future drug screening approaches.

4-Methylumbelliferone improves the thermogenic capacity of brown adipose tissue.

Therapeutic increase of brown adipose tissue (BAT) thermogenesis is of great interest as BAT activation counteracts obesity and insulin resistance. Hyaluronan (HA) is a glycosaminoglycan, found in the extracellular matrix, which is synthesized by HA synthases (Has1/Has2/Has3) from sugar precursors and accumulates in diabetic conditions. Its synthesis can be inhibited by the small molecule 4-methylumbelliferone (4-MU). Here, we show that the inhibition of HA-synthesis by 4-MU or genetic deletion of Has2/Has3 improves BAT`s thermogenic capacity, reduces body weight gain, and improves glucose homeostasis independently from adrenergic stimulation in mice on diabetogenic diet, as shown by a magnetic resonance T2 mapping approach. Inhibition of HA synthesis increases glycolysis, BAT respiration and uncoupling protein 1 expression. In addition, we show that 4-MU increases BAT capacity without inducing chronic stimulation and propose that 4-MU, a clinically approved prescription-free drug, could be repurposed to treat obesity and diabetes.

A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells.

Single-domain antibodies have emerged as highly versatile nanoprobes for advanced cellular imaging. For real-time visualization of endogenous antigens, fluorescently labelled nanobodies (chromobodies, CBs) are introduced as DNA-encoded expression constructs in living cells. Commonly, CB expression is driven from strong, constitutively active promoters. However, high expression levels are sometimes accompanied by misfolding and aggregation of those intracellular nanoprobes. Moreover, stable cell lines derived from random genomic insertion of CB-encoding transgenes bear the risk of disturbed cellular processes and inhomogeneous CB signal intensities due to gene positioning effects and epigenetic silencing. In this study we propose a strategy to generate optimized CB expressing cell lines. We demonstrate that expression as ubiquitin fusion increases the fraction of intracellularly functional CBs and identified the elongation factor 1α (EF1-α) promoter as highly suited for constitutive CB expression upon long-term cell line cultivation. Finally, we applied a CRISPR/Cas9-based gene editing approach for targeted insertion of CB expression constructs into the adeno-associated virus integration site 1 (AAVS1) safe harbour locus of human cells. Our results indicate that this combinatorial approach facilitates the generation of fully functional and stable CB cell lines for quantitative live-cell imaging of endogenous antigens.

Chromobodies to Quantify Changes of Endogenous Protein Concentration in Living Cells.

Understanding cellular processes requires the determination of dynamic changes in the concentration of genetically nonmodified, endogenous proteins, which, to date, is commonly accomplished by end-point assays in vitro Molecular probes such as fluorescently labeled nanobodies (chromobodies, CBs) are powerful tools to visualize the dynamic subcellular localization of endogenous proteins in living cells. Here, we employed the dependence of intracellular levels of chromobodies on the amount of their endogenous antigens, a phenomenon, which we termed antigen-mediated CB stabilization (AMCBS), for simultaneous monitoring of time-resolved changes in the concentration and localization of native proteins. To improve the dynamic range of AMCBS we generated turnover-accelerated CBs and demonstrated their application in visualization and quantification of fast reversible changes in antigen concentration upon compound treatment by quantitative live-cell imaging. We expect that this broadly applicable strategy will enable unprecedented insights into the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.