Background

Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of motor neurons (MNs), leading to paralysis, respiratory failure and death within 2-5 years of diagnosis. The exact mechanisms of sporadic ALS, which comprises 90% of all cases, remain unknown. In familial ALS, mutations in superoxide dismutase (SOD1) cause 10% of cases.

Methods

ALS patient-derived human-induced pluripotent stem cells (ALS hiPSCs, harboring the SOD1^AV4 mutation), were differentiated to MNs (ALS-MNs). The neuroprotective effects of conditioned medium (CM) of hESCs (H9), wt hiPSCs (WTC-11) and the ALS iPSCs, on MN apoptosis and viability, formation and maintenance of neurites, mitochondrial activity and expression of inflammatory genes, were examined. For in vivo studies, 200 μl of CM from the ALS iPSCs (CS07 and CS053) was injected subcutaneously into the ALS model mice (transgenic for the human SOD1^G93A mutation). Animal agility and strength, muscle innervation and mass, neurological score, onset of paralysis and lifespan of the ALS mice were assayed. After observing significant disease-modifying effects, the CM was characterized biochemically by fractionation, comparative proteomics, and epigenetic screens for the dependence on pluripotency. CM of fibroblasts that were differentiated from the wt hiPSCs lacked any neuroprotective activity and was used as a negative control throughout the studies.

Results

The secretome of PSCs including the ALS patient iPSCs was neuroprotective in the H₂O₂ model. In the model with pathogenic SOD1 mutation, ALS iPSC-CM attenuated all examined hallmarks of ALS pathology, rescued human ALS-MNs from denervation and death, restored mitochondrial health, and reduced the expression of inflammatory genes. The ALS iPSC-CM also improved neuro-muscular health and function, and delayed paralysis and morbidity in ALS mice. Compared side by side, cyclosporine (CsA), a mitochondrial membrane blocker that prevents the leakage of mitochondrial DNA, failed to avert the death of ALS-MNs, although CsA and ALS iPSC-CM equally stabilized MN mitochondria and attenuated inflammatory genes. Biochemical characterization, comparative proteomics, and epigenetic screen all suggested that it was the interactome of several key proteins from different fractions of PSC-CM that delivered the multifaceted neuroprotection.

Conclusions

This work introduces and mechanistically characterizes a new biologic for treating ALS and other complex neurodegenerative diseases.

The use of biomaterials has substantially contributed to both our understanding of tumorigenesis and our ability to identify and capture tumour cells in vitro and in vivo. Natural and synthetic biomaterials can be applied as models to recapitulate key features of the tumour microenvironment in vitro, including architectural, mechanical and biological functions. Engineered biomaterials can further mimic the spatial and temporal properties of the surrounding tumour niche to investigate the specific effects of the environment on disease progression, offering an alternative to animal models for the testing of cancer cell behaviour. Biomaterials can also be used to capture and detect cancer cells in vitro and in vivo to monitor tumour progression. In this Review, we discuss the natural and synthetic biomaterials that can be used to recreate specific features of tumour microenvironments. We examine how biomaterials can be applied to capture circulating tumour cells in blood samples for the early detection of metastasis. We highlight biomaterial-based strategies to investigate local regions adjacent to the tumour and survey potential applications of biomaterial-based devices for diagnosis and prognosis, such as the detection of cellular deformability and the non-invasive surveillance of tumour-adjacent stroma.

Abstract TopHat is a popular spliced aligner for RNA-sequence (RNA-seq) experiments. In this paper, we describe TopHat2, which incorporates many significant enhancements to TopHat. TopHat2 can align reads of various lengths produced by the latest sequencing technologies, while allowing for variable-length indels with respect to the reference genome. In addition to de novo spliced alignment, TopHat2 can align reads across fusion breaks, which can occur after genomic translocations. TopHat2 combines the ability to identify novel splice sites with direct mapping to known transcripts, producing sensitive and accurate alignments, even for highly repetitive genomes or in the presence of pseudogenes. TopHat2 is available at http://ccb.jhu.edu/software/tophat.

Flow cytometry (FCM) is an analytic technique that is capable of detecting and recording the emission of fluorescence and light scattering of cells or particles (that are collectively called "events") in a population¹. A typical FCM experiment can produce a large array of data making the analysis computationally intensive². Current FCM data analysis platforms (FlowJo³, etc.), while very useful, do not allow interactive data processing online due to the data size limitations. Here we report a more effective way to analyze FCM data on the web. Freecyto is a free and intuitive Python-flask-based web application that uses a weighted k-means clustering algorithm to facilitate the interactive analysis of flow cytometry data. A key limitation of web browsers is their inability to interactively display large amounts of data. Freecyto addresses this bottleneck through the use of the k-means algorithm to quantize the data, allowing the user to access a representative set of data points for interactive visualization of complex datasets. Moreover, Freecyto enables the interactive analyses of large complex datasets while preserving the standard FCM visualization features, such as the generation of scatterplots (dotplots), histograms, heatmaps, boxplots, as well as a SQL-based sub-population gating feature². We also show that Freecyto can be applied to the analysis of various experimental setups that frequently require the use of FCM. Finally, we demonstrate that the data accuracy is preserved when Freecyto is compared to conventional FCM software.

This work extrapolates to humans the previous animal studies on blood heterochronicity and establishes a novel direct measurement of biological age. Our results support the hypothesis that, similar to mice, human aging is driven by age-imposed systemic molecular excess, the attenuation of which reverses biological age, defined in our work as a deregulation (noise) of 10 novel protein biomarkers. The results on biological age are strongly supported by the data, which demonstrates that rounds of therapeutic plasma exchange (TPE) promote a global shift to a younger systemic proteome, including youthfully restored pro-regenerative, anticancer, and apoptotic regulators and a youthful profile of myeloid/lymphoid markers in circulating cells, which have reduced cellular senescence and lower DNA damage. Mechanistically, the circulatory regulators of the JAK-STAT, MAPK, TGF-beta, NF-κB, and Toll-like receptor signaling pathways become more youthfully balanced through normalization of TLR4, which we define as a nodal point of this molecular rejuvenation. The significance of our findings is confirmed through big-data gene expression studies.

Many patients with COVID-19 experience a range of debilitating symptoms months after being infected, a syndrome termed long-haul COVID. A 68-year-old male presented with lung opacity, fatigue, physical and cognitive weaknesses, loss of smell and lymphocytopenia. After rounds of therapeutic plasma exchange (TPE), the patient returned to normal activities and work. Mechanistically in the patient's peripheral blood mononuclear cells (PBMCs), markers of inflammatory macrophages diminished and markers of lymphocytes, including natural killer (NK) cells and cytotoxic CD8 T-cells, increased. Circulating inflammatory proteins diminished, while positive regulators of tissue repair increased. This case study suggests that TPE has the capacity to treat long-haul COVID.

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.