A Cellular Ground Truth to Develop MRI Signatures in Glioma Models by Correlative Light Sheet Microscopy and Atlas-Based Coregistration.

Glioblastoma is the most common malignant primary brain tumor with poor overall survival. Magnetic resonance imaging (MRI) is the main imaging modality for glioblastoma but has inherent shortcomings. The molecular and cellular basis of MR signals is incompletely understood. We established a ground truth-based image analysis platform to coregister MRI and light sheet microscopy (LSM) data to each other and to an anatomic reference atlas for quantification of 20 predefined anatomic subregions. Our pipeline also includes a segmentation and quantification approach for single myeloid cells in entire LSM datasets. This method was applied to three preclinical glioma models in male and female mice (GL261, U87MG, and S24), which exhibit different key features of the human glioma. Multiparametric MR data including T2-weighted sequences, diffusion tensor imaging, T2 and T2* relaxometry were acquired. Following tissue clearing, LSM focused on the analysis of tumor cell density, microvasculature, and innate immune cell infiltration. Correlated analysis revealed differences in quantitative MRI metrics between the tumor-bearing and the contralateral hemisphere. LSM identified tumor subregions that differed in their MRI characteristics, indicating tumor heterogeneity. Interestingly, MRI signatures, defined as unique combinations of different MRI parameters, differed greatly between the models. The direct correlation of MRI and LSM allows an in-depth characterization of preclinical glioma and can be used to decipher the structural, cellular, and, likely, molecular basis of tumoral MRI biomarkers. Our approach may be applied in other preclinical brain tumor or neurologic disease models, and the derived MRI signatures could ultimately inform image interpretation in a clinical setting.SIGNIFICANCE STATEMENT We established a histologic ground truth-based approach for MR image analyses and tested this method in three preclinical glioma models exhibiting different features of glioblastoma. Coregistration of light sheet microscopy to MRI allowed for an evaluation of quantitative MRI data in histologically distinct tumor subregions. Coregistration to a mouse brain atlas enabled a regional comparison of MRI parameters with a histologically informed interpretation of the results. Our approach is transferable to other preclinical models of brain tumors and further neurologic disorders. The method can be used to decipher the structural, cellular, and molecular basis of MRI signal characteristics. Ultimately, information derived from such analyses could strengthen the neuroradiological evaluation of glioblastoma as they enhance the interpretation of MRI data.

Assessment of Tumor Cell Invasion and Radiotherapy Response in Experimental Glioma by Magnetic Resonance Elastography.

BACKGROUND: Gliomas are highly invasive brain neoplasms. MRI is the most important tool to diagnose and monitor glioma but has shortcomings. In particular, the assessment of tumor cell invasion is insufficient. This is a clinical dilemma, as recurrence can arise from MRI-occult glioma cell invasion. HYPOTHESIS: Tumor cell invasion, tumor growth and radiotherapy alter the brain parenchymal microstructure and thus are assessable by diffusion tensor imaging (DTI) and MR elastography (MRE). STUDY TYPE: Experimental, animal model. ANIMAL MODEL: Twenty-three male NMRI nude mice orthotopically implanted with S24 patient-derived glioma cells (experimental mice) and 9 NMRI nude mice stereotactically injected with 1 µL PBS (sham-injected mice). FIELD STRENGTH/SEQUENCE: 2D and 3D T2-weighted rapid acquisition with refocused echoes (RARE), 2D echo planar imaging (EPI) DTI, 2D multi-slice multi-echo (MSME) T2 relaxometry, 3D MSME MRE at 900 Hz acquired at 9.4 T (675 mT/m gradient strength). ASSESSMENT: Longitudinal 4-weekly imaging was performed for up to 4 months. Tumor volume was assessed in experimental mice (n = 10 treatment-control, n = 13 radiotherapy). The radiotherapy subgroup and 5 sham-injected mice underwent irradiation (3 × 6 Gy) 9 weeks post-implantation/sham injection. MRI-/MRE-parameters were assessed in the corpus callosum and tumor core/injection tract. Imaging data were correlated to light sheet microscopy (LSM) and histology. STATISTICAL TESTS: Paired and unpaired t-tests, a P-value ≤0.05 was considered significant. RESULTS: From week 4 to 8, a significant callosal stiffening (4.44 ± 0.22 vs. 5.31 ± 0.29 kPa) was detected correlating with LSM-proven tumor cell invasion. This was occult to all other imaging metrics. Histologically proven tissue destruction in the tumor core led to an increased T2 relaxation time (41.65 ± 0.34 vs. 44.83 ± 0.66 msec) and ADC (610.2 ± 12.27 vs. 711.2 ± 13.42 × 10-6 mm2/s) and a softening (5.51 ± 0.30 vs. 4.24 ± 0.29 kPa) from week 8 to 12. Radiotherapy slowed tumor progression. DATA CONCLUSION: MRE is promising for the assessment of key glioma characteristics. EVIDENCE LEVEL: NA TECHNICAL EFFICACY: Stage 2.

Local blood coagulation drives cancer cell arrest and brain metastasis in a mouse model.

Clinically relevant brain metastases (BMs) frequently form in cancer patients, with limited options for effective treatment. Circulating cancer cells must first permanently arrest in brain microvessels to colonize the brain, but the critical factors in this process are not well understood. Here, in vivo multiphoton laser-scanning microscopy of the entire brain metastatic cascade allowed unprecedented insights into how blood clot formation and von Willebrand factor (VWF) deposition determine the arrest of circulating cancer cells and subsequent brain colonization in mice. Clot formation in brain microvessels occurred frequently (>95%) and specifically at intravascularly arrested cancer cells, allowing their long-term arrest. An extensive clot embedded ∼20% of brain-arrested cancer cells, and those were more likely to successfully extravasate and form a macrometastasis. Mechanistically, the generation of tissue factor-mediated thrombin by cancer cells accounted for local activation of plasmatic coagulation in the brain. Thrombin inhibition by treatment with low molecular weight heparin or dabigatran and an anti-VWF antibody prevented clot formation, cancer cell arrest, extravasation, and the formation of brain macrometastases. In contrast, tumor cells were not able to directly activate platelets, and antiplatelet treatments did reduce platelet dispositions at intravascular cancer cells but did not reduce overall formation of BMs. In conclusion, our data show that plasmatic coagulation is activated early by intravascular tumor cells in the brain with subsequent clot formation, which led us to discover a novel and specific mechanism that is crucial for brain colonization. Direct or indirect thrombin and VWF inhibitors emerge as promising drug candidates for trials on prevention of BMs.

Supporting the planning of urban blue-green infrastructure for biodiversity: A multi-scale prioritisation framework.

Primary considerations for urban blue-green infrastructure (BGI) encompass sustainable stormwater/urban heat management while biodiversity conservation is often considered an inherent benefit rather than a core planning requirement. However, ecological function of BGI as 'stepping stones' or linear corridors for otherwise fragmented habitats is undisputed. While quantitative approaches for modelling ecological connectivity in conservation planning are well established, mismatches in scope and scale with models that support the planning of BGI makes their adoption and integration difficult across disciplines. Technical complexities have led to ambiguity around circuit and network-based approaches, focal node placement, spatial extents, and resolution. Furthermore, these approaches are often computationally intensive, and considerable gaps remain in their use for identifying local-scale critical "pinch-points" that urban planners may respond to with the integration of BGI interventions that address biodiversity enhancement among other ecosystem services. Here, we present a framework that simplifies and integrates the merits of regional connectivity assessments with a focus on urban areas to prioritise BGI planning interventions while reducing computational demands. Our framework facilitates: (1) modelling potential ecological corridors at a coarse regional scale, (2) prioritising local-scale BGI interventions based on the relative contribution of individual nodes in this regional network, and (3) inferring connectivity hot- and cold-spots for local-scale BGI interventions. We illustrate this in the Swiss lowlands, demonstrating how, compared to previous work, we are able to identify and rank different priority locations across the region for BGI interventions in support of biodiversity enhancement and how their local-scale functional design may be benefited by addressing specific environmental variables.

Use of pulsatile gonadotropin-releasing hormone (GnRH) in patients with functional hypothalamic amenorrhea (FHA) results in monofollicular ovulation and high cumulative live birth rates: a 25-year cohort.

PURPOSE: To analyze outcomes of pulsatile administration of gonadotropin-releasing hormone (GnRH) in infertile women diagnosed with functional hypothalamic amenorrhea (FHA). METHODS: A single-center retrospective cohort study was conducted from 1996 to 2020. Sixty-six patients with the diagnosis FHA that underwent therapy using the pulsatile GnRH pump for conception were included and analyzed. The primary outcome was the live birth rate (LBR). Secondary outcomes were the number of dominant follicles, ovulation rate, biochemical pregnancy rate (BPR), clinical pregnancy rate (CPR), miscarriage rate, and multiple pregnancy rate. A matched control group was selected to compare the birth weight of newborn children. RESULTS: During the study period, 66 patients with FHA underwent 82 treatments (14 of 66 patients had more than one treatment) and a total of 212 cycles (ovulation induction attempts) using pulsatile GnRH. The LBR per treatment was 65.9%. The ovulation rate per cycle was 96%, and monofollicular ovulation was observed in 75% of cycles. The BPR per treatment was 80.5%, and the cumulative CPR per treatment was 74.4%. The miscarriage rate was 11.5%. One dizygotic twin pregnancy was observed (1.6%). Average newborn birth weight (NBW) from patients with FHA was comparable to the control group. CONCLUSION(S): In patients with FHA, excellent pregnancy rates were achieved using the subcutaneous GnRH pump. The high cumulative LBR with normal NBW as well as low rates of multiple gestation indicate that the pulsatile GnRH pump represents a safer and more physiologic alternative to ovulation induction with injectable gonadotropins. TRIAL REGISTRATION: Ethics Committee Northwest and Central Switzerland (Ethikkommission Nordwest- und Zentralschweiz - EKNZ) - Project-ID 2020-01612.

Socio-technical networks of infrastructure management: Network concepts and motifs for studying digitalization, decentralization, and integrated management.

Networked infrastructure systems - including energy, transportation, water, and wastewater systems - provide essential services to society. Globally, these services are undergoing major transformative processes such as digitalization, decentralization, or integrated management. Such processes not only depend on technical changes in infrastructure systems but also include important social and socio-technical dimensions. In this article, we propose a socio-technical network perspective to study the ensemble of social actors and technical elements involved in an infrastructure system, and their complex relations. We conceptualize structurally explicit socio-technical networks of networked infrastructure systems based on methodological considerations from network analysis and draw on concepts from socio-technical system theories and social-ecological network studies. Based on these considerations, we suggest analytical methods to study basic network concepts such as density, reciprocity, and centrality in a socio-technical network. We illustrate socio-technical motifs, i.e., meaningful sub-structures in socio-technical networks of infrastructure management. Drawing on these, we describe how infrastructure systems can be analyzed in terms of digitalization, decentralization, and integrated management from a socio-technical network perspective. Using the example of urban wastewater systems, we illustrate an empirical application of our approach. The results of an empirical case study in Switzerland demonstrate the potential of socio-technical networks to promote a deeper understanding of complex socio-technical relations in networked infrastructure systems. We contend that such a deeper understanding could improve management practices of infrastructure systems and is becoming even more important for enabling future data-driven, decentralized, and more integrated infrastructure management.

In vivo nanoparticle imaging of innate immune cells can serve as a marker of disease severity in a model of multiple sclerosis.

Innate immune cells play a key role in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Current clinical imaging is restricted to visualizing secondary effects of inflammation, such as gliosis and blood-brain barrier disruption. Advanced molecular imaging, such as iron oxide nanoparticle imaging, can allow direct imaging of cellular and molecular activity, but the exact cell types that phagocytose nanoparticles in vivo and how phagocytic activity relates to disease severity is not well understood. In this study we used MRI to map inflammatory infiltrates using high-field MRI and fluorescently labeled cross-linked iron oxide nanoparticles for cell tracking. We confirmed nanoparticle uptake and MR detectability ex vivo. Using in vivo MRI, we identified extensive nanoparticle signal in the cerebellar white matter and circumscribed cortical gray matter lesions that developed during the disease course (4.6-fold increase of nanoparticle accumulation in EAE compared with healthy controls, P < 0.001). Nanoparticles showed good cellular specificity for innate immune cells in vivo, labeling activated microglia, infiltrating macrophages, and neutrophils, whereas there was only sparse uptake by adaptive immune cells. Importantly, nanoparticle signal correlated better with clinical disease than conventional gadolinium (Gd) imaging (r, 0.83 for nanoparticles vs. 0.71 for Gd-imaging, P < 0.001). We validated our approach using the Food and Drug Administration-approved iron oxide nanoparticle ferumoxytol. Our results show that noninvasive molecular imaging of innate immune responses can serve as an imaging biomarker of disease activity in autoimmune-mediated neuroinflammation with potential clinical applications in a wide range of inflammatory diseases.

Removal of DNA-fragmented spermatozoa using flow cytometry and sorting does not improve the outcome of intracytoplasmic sperm injection.

PURPOSE: The DNA fragmentation in sperm is associated with reduced outcome in assisted reproduction. Using YoPro1 as the staining dye and flow cytometry and sorting (FACS), the number of spermatozoa with DNA fragmentation can be lowered to 5%. Can the cumulative outcome of ICSI be improved using FACS? METHODS: A prospective, randomized, double-blind clinical trial was conducted in 104 infertile couples with male infertility based on abnormal conventional semen analysis results. Cumulative ongoing pregnancy rate was the primary outcome parameter. In 52 cases, semen was processed for ICSI using swim-up. In another 52 cases, spermatozoa with fragmented DNA were removed with FACS. RESULTS: The cumulative pregnancy rate at 12 weeks of gestation (51.9% versus 46.2%) and live birth rate (42.3% versus 34.6%) were higher and the miscarriage rate was lower (27.8% versus 35.3%) after FACS-sorting as compared with swim-up. An interim analysis scheduled before initiation of the study after 100 cases demonstrated that the aim of a 20% gain in pregnancy rate could not be achieved. For that reason, the prospective study was stopped prematurely. CONCLUSIONS: A trend towards consistently better results was achieved by removing spermatozoa with fragmented DNA. The fragmentation of the DNA in sperm is the end stage of apoptosis. Sorting of spermatozoa may be improved by selecting parameters of processes active more upstream of apoptosis, such as chromatin decondensation. TRIAL REGISTRATION: NCT02166567 . June 14, 2014.

Strategies in megasynthase engineering - fatty acid synthases (FAS) as model proteins.

Megasynthases are large multienzyme proteins that produce a plethora of important natural compounds by catalyzing the successive condensation and modification of precursor units. Within the class of megasynthases, polyketide synthases (PKS) are responsible for the production of a large spectrum of bioactive polyketides (PK), which have frequently found their way into therapeutic applications. Rational engineering approaches have been performed during the last 25 years that seek to employ the "assembly-line synthetic concept" of megasynthases in order to deliver new bioactive compounds. Here, we highlight PKS engineering strategies in the light of the newly emerging structural information on megasynthases, and argue that fatty acid synthases (FAS) are and will be valuable objects for further developing this field.

Drivers for and against municipal wastewater recycling: a review.

The reclamation, treatment and reuse of municipal wastewater can provide important environmental benefits. In this paper, 25 studies on this topic were reviewed and it was found that there are many (>150) different drivers acting for and against wastewater recycling. To deal with the challenge of comparing studies which entailed different research designs, a framework was developed which allowed the literature to be organized into comparable study contexts. Studies were categorized according to the level of analysis (wastewater recycling scheme, city, water utility, state, country, global) and outcome investigated (development/investment in new schemes, program implementation, percentage of wastewater recycled, percentage of water demand covered by recycled water, multiple outcomes). Findings across comparable case studies were then grouped according to the type (for or against recycling) and category of driver (social, natural, technical, economic, policy or business). The utility of the framework is demonstrated by summarizing the findings from four Australian studies at the city level. The framework offers a unique approach for disentangling the broad range of potential drivers for and against water recycling and to focus on those that seem relevant in specific study contexts. It may offer a valuable starting point for building hypotheses in future work.