Cell-targeted gene modification by delivery of CRISPR-Cas9 ribonucleoprotein complexes in pseudotyped lentivirus-derived nanoparticles.

To fully utilize the potential of CRISPR-Cas9-mediated genome editing, time-restricted and targeted delivery is crucial. By modulating the pseudotype of engineered lentivirus-derived nanoparticles (LVNPs), we demonstrate efficient cell-targeted delivery of Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes, supporting gene modification in a defined subset of cells in mixed cell populations. LVNPs pseudotyped with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein resulted in angiotensin-converting enzyme 2 (ACE2)-dependent insertion or deletion (indel) formation in an ACE2+/ACE2- population of cells, whereas Nipah virus glycoprotein pseudotyping resulted in Ephrin-B2/B3-specific gene knockout. Additionally, LVNPs pseudotyped with Edmonston strain measles virus glycoproteins (MV-H/F) delivered Cas9/sgRNA RNPs to CD46+ cells with and without additional expression of SLAM (signaling lymphocytic activation molecule; CD150). However, an engineered SLAM-specific measles virus pseudotype (measles virus-hemagglutinin/fusion [MV-H/F]-SLAM) efficiently targeted LVNPs to SLAM+ cells. Lentiviral vectors (LVs) pseudotyped with MV-H/F-SLAM efficiently transduced >80% of interleukin (IL)-4/IL-21-stimulated primary B cells cultured on CD40 ligand (CD40L)-expressing feeder cells. Notably, LVNPs pseudotyped with MV-H/F and MV-H/F-SLAM reached indel rates of >80% and >60% in stimulated primary B cells, respectively. Collectively, our findings demonstrate the modularity of LVNP-directed delivery of ready-to-function Cas9/sgRNA complexes. Using a panel of different pseudotypes, we provide evidence that LVNPs can be engineered to induce effective indel formation in a subpopulation of cells defined by the expression of surface receptors.

Higher thermal plasticity in flowering phenology increases flowering output.

Ongoing climate change poses an increasing threat to biodiversity. To avoid decline or extinction, species need to either adjust or adapt to new environmental conditions or track their climatic niches across space. In sessile organisms such as plants, phenotypic plasticity can help maintain fitness in variable and even novel environmental conditions and is therefore likely to play an important role in allowing them to survive climate change, particularly in the short term. Understanding a species' response to rising temperature is crucial for planning well-targeted and cost-effective conservation measures. We sampled seeds of three Hypericum species (H. maculatum, H. montanum, and H. perforatum), from a total of 23 populations originating from different parts of their native distribution areas in Europe. We grew them under four different temperature regimes in a greenhouse to simulate current and predicted future climatic conditions in the distribution areas. We measured flowering start, flower count, and subsequent seed weight, allowing us to study variations in the thermal plasticity of flowering phenology and its relation to fitness. Our results show that individuals flowered earlier with increasing temperature, while the degree of phenological plasticity varied among species. More specifically, the plasticity of H. maculatum varied depending on population origin, with individuals from the leading range edge being less plastic. Importantly, we show a positive relationship between higher plasticity and increased flower production, indicating adaptive phenological plasticity. The observed connection between plasticity and fitness supports the idea that plasticity may be adaptive. This study underlines the need for information on plasticity for predicting species' potential to thrive under global change and the need for studies on whether higher phenotypic plasticity is currently being selected as natural populations experience a rapidly changing climate.

Dual intron-targeted CRISPR-Cas9-mediated disruption of the AML RUNX1-RUNX1T1 fusion gene effectively inhibits proliferation and decreases tumor volume in vitro and in vivo.

Oncogenic fusion drivers are common in hematological cancers and are thus relevant targets of future CRISPR-Cas9-based treatment strategies. However, breakpoint-location variation in patients pose a challenge to traditional breakpoint-targeting CRISPR-Cas9-mediated disruption strategies. Here we present a new dual intron-targeting CRISPR-Cas9 treatment strategy, for targeting t(8;21) found in 5-10% of de novo acute myeloid leukemia (AML), which efficiently disrupts fusion genes without prior identification of breakpoint location. We show in vitro growth rate and proliferation reduction by 69 and 94% in AML t(8;21) Kasumi-1 cells, following dual intron-targeted disruption of RUNX1-RUNX1T1 compared to a non t(8;21) AML control. Furthermore, mice injected with RUNX1-RUNX1T1-disrupted Kasumi-1 cells had in vivo tumor growth reduction by 69 and 91% compared to controls. Demonstrating the feasibility of RUNX1-RUNX1T1 disruption, these findings were substantiated in isolated primary cells from a patient diagnosed with AML t(8;21). In conclusion, we demonstrate proof-of-principle of a dual intron-targeting CRISPR-Cas9 treatment strategy in AML t(8;21) without need for precise knowledge of the breakpoint location.

Intra-individual variation in Galium odoratum is affected by experimental drought and shading.

BACKGROUND AND AIMS: Climate-change induced warmer spring temperatures advance tree leaf-out and result in earlier shading of the forest floor. Climate change also leads to more frequent droughts. Forest understorey herbs may respond to these environmental changes by varying traits at different hierarchical levels of organization. While trait mean variation at the inter-individual level in response to environmental changes is well-studied, little is known about how variation at the intra-individual level responds. METHODS: We sampled genets of the forest understorey herb Galium odoratum from 21 populations in three regions in Germany, varying in microclimatic conditions. The genets were transplanted into a common garden, where we applied shading and drought treatments. We measured plant height and leaf length and width, and calculated the coefficient of variation (CV) at different hierarchical levels: intra-population, intra-genet, intra-ramet and intra-shoot. KEY RESULTS: Variance partitioning showed that intra-shoot CV represented most of the total variation, followed by intra-ramet CV. We found significant variation in CV of plant height and leaf width among populations of origin, indicating that CV is at least partly genetically based. The soil temperature at populations' origins correlated negatively with CV in plant height, suggesting adaptation to local conditions. Furthermore, we observed that early shade led to increased intra-ramet CV in leaf length, while drought reduced intra-shoot CV in leaf width. Finally, intra-shoot leaf width mean and CV were independent under control conditions but correlated under drought. CONCLUSIONS: Our experimental results reveal correlations of intra-individual variation with soil temperature, indicating that intra-individual variation can evolve and may be adaptive. Intra-individual variation responded plastically to drought and shading, suggesting functional changes to improve light capture and reduce evapotranspiration. In conclusion, intra-individual variation makes up the majority of total trait variation in this species and can play a key role in plant adaptation to climatic change.

SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate.

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here, we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, 4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2.

Author Correction: SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

[Abdominal ectopic pregnancy].

This review summarises the knowledge of abdominal ectopic pregnancy (AEP), which is a rare condition with higher morbidity and mortalilty than other types of ectopic pregnancies. The condition can be primary, if the pregnancy implants directly on to an abdominal site, or it can be secondary after a tubar abortion. AEP differs from tubal pregnancies by a normal level of human chorionic gonadotropin and rare vaginal bleeding, which causes a diagnostic delay. In an early pregnancy the treatment is laparoscopic removal, but in second and third trimester pregnancies laparotomy is preferred, if possible preceded by MRI for mapping of vascular involvement and location of placenta.

Reliability and validity of The Danish pediatric voice handicap index.

BACKGROUND: The pediatric voice handicap index (pVHI) questionnaire was developed in 2006 to provide parental information regarding the impact of a voice disorder on their child's life. OBJECTIVES: The aim of this study was to make a Danish version of the original American pVHI and to validate the Danish pVHI by evaluating its internal consistency and reliability. MATERIALS AND METHODS: The original version of the pVHI was translated into Danish. Nineteen parents of dysphonic children, diagnosed in a tertiary otolaryngology hospital department, and 43 parents of children without known voice disorder (control group) completed the questionnaire. The internal consistency, content validity including comparisons of the scores in the two groups and the test-retest reliability were assessed through statistical analysis. RESULTS: The total pVHI scores significantly differed between the group of parents with dysphonic children and the group of parents with children without known voice disorder (p < 0.001). The internal consistency showed an excellent consistency (Chronbach's α > 0.9) of the three subdomains score and the total pVHI score. The test-re-test reliability of the total pVHI score was "strong" with a Pearson's correlation coefficient of 0.97. CONCLUSIONS AND SIGNIFICANCE: The Danish pVHI is a valid and reliable instrument to assess the parents' perception of the impact of a voice disorder on a child's physical, social and emotional well-being.

Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling.

The adaptor molecule stimulator of IFN genes (STING) is central to production of type I IFNs in response to infection with DNA viruses and to presence of host DNA in the cytosol. Excessive release of type I IFNs through STING-dependent mechanisms has emerged as a central driver of several interferonopathies, including systemic lupus erythematosus (SLE), Aicardi-Goutières syndrome (AGS), and stimulator of IFN genes-associated vasculopathy with onset in infancy (SAVI). The involvement of STING in these diseases points to an unmet need for the development of agents that inhibit STING signaling. Here, we report that endogenously formed nitro-fatty acids can covalently modify STING by nitro-alkylation. These nitro-alkylations inhibit STING palmitoylation, STING signaling, and subsequently, the release of type I IFN in both human and murine cells. Furthermore, treatment with nitro-fatty acids was sufficient to inhibit production of type I IFN in fibroblasts derived from SAVI patients with a gain-of-function mutation in STING. In conclusion, we have identified nitro-fatty acids as endogenously formed inhibitors of STING signaling and propose for these lipids to be considered in the treatment of STING-dependent inflammatory diseases.

Design and Synthesis of a Series of l-trans-4-Substituted Prolines as Selective Antagonists for the Ionotropic Glutamate Receptors Including Functional and X-ray Crystallographic Studies of New Subtype Selective Kainic Acid Receptor Subtype 1 (GluK1) Antagonist (2S,4R)-4-(2-Carboxyphenoxy)pyrrolidine-2-carboxylic Acid.

Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxyphenoxy)pyrrolidine-2-carboxylic acid (1b), for cloned homomeric kainic acid receptors subtype 1 (GluK1) was attained (Ki = 4 µM). In a functional assay, 1b displayed full antagonist activity with IC50 = 6 ± 2 µM. A crystal structure was obtained of 1b when bound in the ligand binding domain of GluK1. A domain opening of 13-14° was seen compared to the structure with glutamate, consistent with 1b being an antagonist. A structure-activity relationship study showed that the chemical nature of the tethering atom (C, O, or S) linking the pyrrolidine ring and the phenyl ring plays a key role in the receptor selectivity profile and that substituents on the phenyl ring are well accommodated by the GluK1 receptor.

Structure-Activity Relationship Study of Ionotropic Glutamate Receptor Antagonist (2S,3R)-3-(3-Carboxyphenyl)pyrrolidine-2-carboxylic Acid.

Herein we describe the first structure-activity relationship study of the broad-range iGluR antagonist (2S,3R)-3-(3-carboxyphenyl)pyrrolidine-2-carboxylic acid (1) by exploring the pharmacological effect of substituents in the 4, 4', or 5' positions and the bioisosteric substitution of the distal carboxylic acid for a phosphonic acid moiety. Of particular interest is a hydroxyl group in the 4' position 2a which induced a preference in binding affinity for homomeric GluK3 over GluK1 (Ki = 0.87 and 4.8 µM, respectively). Two X-ray structures of ligand binding domains were obtained: 2e in GluA2-LBD and 2f in GluK1-LBD, both at 1.9 Å resolution. Compound 2e induces a D1-D2 domain opening in GluA2-LBD of 17.3-18.8° and 2f a domain opening in GluK1-LBD of 17.0-17.5° relative to the structures with glutamate. The pyrrolidine-2-carboxylate moiety of 2e and 2f shows a similar binding mode as kainate. The 3-carboxyphenyl ring of 2e and 2f forms contacts comparable to those of the distal carboxylate in kainate.

Binding mode of an α-amino acid-linked quinoxaline-2,3-dione analogue at glutamate receptor subtype GluK1.

Two α-amino acid-functionalized quinoxalines, 1a (CNG-10301) and 1b (CNG-10300), of a quinoxaline moiety coupled to an amino acid moiety were designed, synthesized, and characterized pharmacologically. While 1a displayed low affinity at native AMPA, KA, and NMDA receptors, and at homomeric GluK1,3 receptors, the affinity for GluK2 was in the midmicromolar range (Ki = 136 µM), 1b displayed low to midmicromolar range binding affinity at all the iGluRs (Ki = 9-126 µM). In functional experiments (outside-out patches excised from transfected HEK293T cells), 100 µM 1a partially blocked GluK1 (33% peak response), while GluK2 was unaffected (96% peak response). Furthermore, 1a was shown not to be an agonist at GluK1 and GluK2 at 100 µM. On the other hand, 100 µM 1b fully antagonized GluK1 (8% peak response) but only partially blocked GluK2 (33% peak response). An X-ray structure at 2.3 Å resolution of 1b in the GluK1-LBD (ligand-binding domain) disclosed an unexpected binding mode compared to the predictions made during the design phase; the quinoxaline moiety remains to act as an amino acid bioisostere, but the amino acid moiety is oriented into a new area within the GluK1 receptor. The structure of the GluK1-LBD with 1b showed a large variation in domain openings of the three molecules from 25° to 49°, demonstrating that the GluK1-LBD is capable of undergoing major domain movements.

[A large abdomen can camouflage the symptoms of an ovarian tumour]. / Svær adipositas kan sløre symptomerne ved ovarietumorer

Mucinous cystadenomas of the ovaries are benign tumours which can grow to sometimes gigantic proportions. Symptoms may be vague and due to complications hence the tumour can go undiagnosed for several years. This is a case report of a 48-year-old woman with a 40-kg ovarian tumour developed through 19 years. In spite of discomfort and complaints of the very large abdomen she had not received thorough examination previously. A large abdomen and discomfort should always lead to transabdominal ultrasound when transvaginal ultrasound - as in this case - does not diagnose the condition.

Low prevalence of oral and nasal human papillomavirus in employees performing CO2-laser evaporation of genital warts or loop electrode excision procedure of cervical dysplasia.

Risk of human papillomavirus (HPV) transmission during laser vaporisation of genital warts or loop electrode excision procedure is controversial. An oral rinse, a nasal swabs, history of HPV related diseases and data on HPV exposure were collected from 287 employees at departments of dermato-venerology and gynaecology in Denmark. A mucosal HPV type was found among 5.8% of employees with experience of laser treatment of genital warts as compared to 1.7% of those with no experience (p = 0.12). HPV prevalence was not higher in employees participating in electrosurgical treatment or cryotherapy of genital warts, or loop electrode excision procedure compared with those who did not. HPV 6 or 11 were not detected in any samples. Hand warts after the age of 24 years was more common among dermatology than among non-dermatology personnel (18% vs. 8.0%, p = 0.03). Mucosal HPV types are infrequent in the oral and nasal cavity of health care personnel, however, employees at departments of dermato-venereology are at risk of acquiring hand warts.

Diagnostic methods for fast-track identification of endometrial cancer in women with postmenopausal bleeding and endometrial thickness greater than 5 mm.

OBJECTIVE: This study aims to evaluate the diagnostic efficiency of pattern recognition by transvaginal ultrasonography (TVS) and gel infusion sonography (GIS) for identifying endometrial pathology and to compare this setup with a standard setup of endometrial sampling (ES), hysteroscopy with pattern evaluation (HY(pattern)), or magnetic resonance imaging (MRI). METHODS: This study used a prospective cohort of 174 women with postmenopausal bleeding and endometrial thickness of 5 mm or greater. Resectoscopic biopsy (hysteroscopy with biopsy) samples or hysterectomy served as reference standard. Malignant and benign endometrial patterns were evaluated with TVS, GIS and HY(pattern) were then added. The efficiency of each diagnostic strategy, including ES and MRI findings (n = 83), was compared and evaluated against the reference standard. RESULTS: ES, TVS, GIS, and HY(pattern) had high diagnostic efficiency (area under the curve) for malignancy diagnosis (ES, 0.90; TVS, 0.88; GIS, 0.92; HY(pattern), 0.91). When insufficient samples were incorporated, ES was less efficient than the other techniques. ES was not more efficient in the subgroup of women without localized lesions than in the subgroup of women with localized lesions. MRI and HY(pattern) added limited efficiency, whereas hysteroscopy with biopsy was most efficient. CONCLUSIONS: As a first-line technique, pattern recognition on TVS, GIS, and HY(pattern) correctly identifies 9 of 10 women with malignancy and is superior to pattern recognition on ES when insufficient samples are included. Endometrial pattern evaluated with TVS and GIS is a fast and efficient first-line diagnostic tool that outperforms ES in women with or without localized lesions. Malignant patterns on TVS/GIS should warrant fast-track evaluation, whereas women with benign patterns may be selected for office or operative hysteroscopy. A fast-track diagnostic setup based on pattern recognition is presented.

[Better coordination between primary care, community settings and diabetes outpatient clinic for patients with type 2 diabetes].

Integrated care programmes for patients with type 2 diabetes can be successfully implemented by planning the programmes in coordination between the sectors primary care, community settings and diabetes outpatient clinic, and with involvement of leaders and employees. Our project has resulted in: 1) more patients with type 2 diabetes receiving diabetes management courses, 2) improved diabetes management of primary care, and 3) improved confidence and respect between sectors involved in diabetes care.

[Mobile health initiative for foreign prostitutes in the Region of Central Jutland and the Region of North Jutland].

Some foreign prostitutes are victims of traffiquing, do not speak Danish and are not familiar with the services offered in the Danish Health Service. The mobile health initiative localises foreign prostitutes and offers health checks and help in case of illness or pregnancy. The article reports method and findings from the first two-year period with this initiative.

Preoperative staging of endometrial cancer using TVS, MRI, and hysteroscopy.

OBJECTIVES: To evaluate the accuracy of different preoperative modalities for staging of endometrial cancer to restrict extensive surgery to patients at high risk of metastatic disease. SETTING: Aarhus University Hospital. POPULATION: 156 women referred in 2006-2011 because of atypical endometrial hyperplasia (G0) or endometrial cancer. METHODS: Patients were offered preoperative transvaginal ultrasonography (TVS), magnetic resonance imaging (MRI), and hysteroscopic-directed biopsies from the uterine tumor and cervix. Final pathology of the removed uterus was the reference standard. Patients were divided into low risk (<50% myometrial invasion, and grades 0, 1, 2, and no cervical invasion) or high risk (all others). MAIN OUTCOME MEASURES: Accuracy, sensitivity, specificity, positive/negative predictive value. RESULTS: Patients were aged 32-88 years, with a mean body mass index of 29. At final pathology 81% had cancer and 19% G0 or no residual tumor; 54% were high risk. Hysteroscopy-directed biopsies had a higher accuracy (92%) than endometrial biopsy (58%) for differentiating G0 from cancer (p < 0.001); grade 3 tumor identification had similar accuracy (93 vs. 92%). Deep myometrial invasion was estimated with higher accuracy by MRI (82%) than TVS (74%) (p < 0.02). For cervical involvement, hysteroscopy-directed biopsies had higher accuracy (94%) than MRI (84%,) and TVS (80%) (p < 0.02). Accuracy for identifying high-risk women was highest (83%) using a combination of MRI and hysteroscopic-directed biopsies, compared with TVS and endometrial biopsy (72%) (p < 0.05). CONCLUSION: Preoperative staging with MRI and hysteroscopy-directed biopsy can identify eight of 10 women with high risk of lymph node metastases and spare eight of 10 low-risk women extended surgery.

Better treatment of outpatients with type 1 diabetes after introduction of continuous subcutaneous insulin infusion.

INTRODUCTION: Continuous subcutaneous insulin infusion (CSII) was introduced in the outpatient diabetes clinic in Fredericia, Denmark, in 2005. The aim of this study was to evaluate the quality of metabolic control and patient satisfaction in type 1 diabetic patients treated with CSII. MATERIAL AND METHODS: In 2009-2010, a database with registration of metabolic variables and patient satisfaction was established. The collected material is a combination of retrospective and prospective data. Patient satisfaction was measured by use of the Diabetes Treatment Satisfaction Questionnaire Status (DTSQs) and change (DTSQc) versions. RESULTS: By 31 December 2010, the database contained data from 68 active patients. Compared with before the initiation of CSII, glycohaemoglobin (HbA1c) had decreased significantly from 8.0% (5.8-13.7%) to 7.6% (6.1-9.5%). The improved glycaemic control was maintained each year until ≤ 4 years after initiation of CSII (p < 0.01).The fraction of patients with an HbA1c ≤ 7% had increased from 13% to 24%, the fraction of patients with an HbA1c > 9% had decreased from 18% to 3%, and the number of serious attacks of hypoglycaemia had decreased (p < 0.05). Only three episodes of ketoacidosis were observed. The DTSQs and DTSQc showed a higher patient satisfaction during CSII treatment (p < 0.01) than before its introduction. Compared with before the introduction of CSII, the patient satisfaction score had increased from 19 (12-33) to 34.5 (27-36) (p < 0.01). CONCLUSION: Type 1 diabetes patients who were changed from treatment with multi-injection therapy to CSII showed improved glycaemic control, a reduced number of hypoglycaemic attacks and improved and very high levels of patient satisfaction.

Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches.

Using insulin as a model protein for binding of oxaliplatin to proteins, various mass spectrometric approaches and techniques were compared. Several different platinum adducts were observed, e.g. addition of one or two diaminocyclohexane platinum(II) (Pt(dach)) molecules. By top-down analysis and fragmentation of the intact insulin-oxaliplatin adduct using nano-electrospray ionisation quadrupole time-of-flight mass spectrometry (nESI-Q-ToF-MS), the major binding site was assigned to histidine5 on the insulin B chain. In order to simplify the interpretation of the mass spectrum, the disulphide bridges were reduced. This led to the additional identification of cysteine6 on the A chain as a binding site along with histidine5 on the B chain. Digestion of insulin-oxaliplatin with endoproteinase Glu-C (GluC) followed by reduction led to the formation of five peptides with Pt(dach) attached. Identification of several of the binding sites was obtained using matrix-assisted laser desorption/ionization (MALDI)-ToF-ToF-MS and liquid chromatography-nESI-Q-ToF-MS. Upon comparing the top-down and bottom-up approaches, the suitability of the bottom-up approach for determining binding sites was questioned, as the release and possible re-association of Pt(dach) were demonstrated upon enzymatic digestion. The associated advantages and disadvantages of ESI and MALDI were also pointed out.