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Mutations in PLP1, the gene that encodes proteolipid protein (PLP), result in failure of myelination and neurological dysfunction in the X-chromosome-linked leukodystrophy Pelizaeus–Merzbacher disease (PMD)1,2. Most PLP1 mutations, including point mutations and supernumerary copy variants, lead to severe and fatal disease. Patients who lack PLP1 expression, and Plp1-null mice, can display comparatively mild phenotypes, suggesting that PLP1 suppression might provide a general therapeutic strategy for PMD1,3–5. Here we show, using CRISPR–Cas9 to suppress Plp1 expression in the jimpy (Plp1jp) point-mutation mouse model of severe PMD, increased myelination and restored nerve conduction velocity, motor function and lifespan of the mice to wild-type levels. To evaluate the translational potential of this strategy, we identified antisense oligonucleotides that stably decrease the levels of Plp1 mRNA and PLP protein throughout the neuraxis in vivo. Administration of a single dose of Plp1-targeting antisense oligonucleotides in postnatal jimpy mice fully restored oligodendrocyte numbers, increased myelination, improved motor performance, normalized respiratory function and extended lifespan up to an eight-month end point. These results suggest that PLP1 suppression could be developed as a treatment for PMD in humans. More broadly, we demonstrate that oligonucleotide-based therapeutic agents can be delivered to oligodendrocytes in vivo to modulate neurological function and lifespan, establishing a new pharmaceutical modality for myelin disorders. In a mouse model of the leukodystrophy Pelizaeus–Merzbacher disease, myelination, motor performance, respiratory function and lifespan are improved by suppressing proteolipid protein expression, suggesting PLP1 as a therapeutic target for human patients with this disease and, more broadly, antisense oligonucleotides as a pharmaceutical modality for treatment of myelin disorders.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate iPSC lines and deeply characterised their genetic properties using whole genome sequencing, their genomic stability upon CRISPR/Cas9-based gene editing, and their phenotypic properties including differentiation to commonly-used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and hundreds of its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.SummaryThe authors of this collaborative study deeply characterized human induced pluripotent stem cell (iPSC) lines to rationally select a clonally-derived cell line that performs well across multiple modalities. KOLF2.1J was identified as a candidate reference cell line based on single-cell analysis of its gene expression in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and the efficiency with which it differentiated into multiple target cell populations. Since it is deeply characterized and can be readily acquired, KOLF2.1J is an attractive reference cell line for groups working with iPSCs.Graphical abstract

Summary: Pelizaeus-Merzbacher disease (PMD) is a fatal X-linked disorder caused by loss of myelinating oligodendrocytes and consequent hypomyelination. The underlying cellular and molecular dysfunctions are not fully defined, but therapeutic enhancement of oligodendrocyte survival could restore functional myelination in patients. Here we generated pure, scalable quantities of induced pluripotent stem cell-derived oligodendrocyte progenitor cells (OPCs) from a severe mouse model of PMD, Plp1jimpy. Temporal phenotypic and transcriptomic studies defined an early pathological window characterized by endoplasmic reticulum (ER) stress and cell death as OPCs exit their progenitor state. High-throughput phenotypic screening identified a compound, Ro 25–6981, which modulates the ER stress response and rescues mutant oligodendrocyte survival in jimpy, in vitro and in vivo, and in human PMD oligocortical spheroids. Surprisingly, increasing oligodendrocyte survival did not restore subsequent myelination, revealing a second pathological phase. Collectively, our work shows that PMD oligodendrocyte loss can be rescued pharmacologically and defines a need for multifactorial intervention to restore myelination. : Tesar and colleagues interrogate the severe genetic myelin disorder Pelizaeus-Merzbacher disease (PMD) using iPSC-derived oligodendrocyte progenitor cells (OPCs). Using high-throughput phenotypic screening, they rescue cell death of mutant oligodendrocytes immediately after initiation of differentiation with chemical modulators, including Ro 25–6981. Interestingly oligodendrocyte restoration did not lead to widespread myelination, unmasking a new phase of the disease. Keywords: iPSC disease modeling, high-throughput screening, oligodendrocyte progenitor cells, oligodendrocytes, myelin, Pelizaeus-Merzbacher disease, proteolipid protein 1, endoplasmic reticulum stress, PLP1, rare disease

Regeneration of myelin is mediated by oligodendrocyte progenitor cells (OPCs), an abundant stem cell population in the CNS and the principal source of new myelinating oligodendrocytes. Loss of myelin-producing oligodendrocytes in the central nervous system (CNS) underlies a number of neurological diseases, including multiple sclerosis (MS) and diverse genetic diseases1–3. Using high throughput chemical screening approaches, we and others have identified small molecules that stimulate oligodendrocyte formation from OPCs and functionally enhance remyelination in vivo4–10. Here we show a broad range of these pro-myelinating small molecules function not through their canonical targets but by directly inhibiting CYP51 (cytochrome P450, family 51), TM7SF2, or EBP (emopamil binding protein), a narrow range of enzymes within the cholesterol biosynthesis pathway. Subsequent accumulation of the 8,9-unsaturated sterol substrates of these enzymes is a key mechanistic node that promotes oligodendrocyte formation, as 8,9-unsaturated sterols are effective when supplied to OPCs in purified form while analogous sterols lacking this structural feature have no effect. Collectively, our results define a unifying sterol-based mechanism-of-action for most known small-molecule enhancers of oligodendrocyte formation and highlight specific targets to propel the development of optimal remyelinating therapeutics.

Organoid technologies provide an accessible system to examine cellular composition, interactions, and organization in the developing human brain, but previously have lacked oligodendrocytes, the myelinating glia of the central nervous system. Here we reproducibly generate oligodendrocytes and myelin in human pluripotent stem cell-derived “oligocortical spheroids”. Transcriptional, immunohistochemical, and electron microscopy analyses demonstrate molecular features consistent with maturing oligodendrocytes by 20 weeks in culture, including expression of MYRF, PLP1, and MBP proteins and initial myelin wrapping of axons, with maturation to longitudinal wrapping and compact myelin by 30 weeks. Promyelinating drugs enhance the rate and extent of oligodendrocyte generation and myelination, while oligocortical spheroids generated from patients with a genetic myelin disorder recapitulate human disease phenotypes. Oligocortical spheroids provide a versatile platform to observe and dissect the complex interactions required for myelination of the developing central nervous system and offer new opportunities for disease modeling and therapeutic development in human tissue.

Glia are increasingly appreciated for the varied and critical roles they play in the health and disease of the nervous system. Damage or dysfunction of myelinating glia, oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system, is the underlying cause of numerous neurological disorders. In this chapter, we highlight the variety of insults—inflammatory, genetic, and environmental—which lead to disorders of myelin and describe the current clinical understanding of the major myelin diseases. We also highlight the current advances in myelin disease research that are moving us steadily closer to meaningful therapies for patients.

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients face progressive disability due to failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue and are associated with white matter microstructure. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development.

Summary Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. Video Abstract

Mutations in proteolipid protein 1 (PLP1) result in failure of myelination and severe neurological dysfunction in the X-linked pediatric leukodystrophy Pelizaeus-Merzbacher disease (PMD). The majority of PLP1 variants, including supernumerary copies and various point mutations, lead to early mortality. However, PLP1-null patients and mice display comparatively mild phenotypes, suggesting that reduction of aberrant PLP1 expression might provide a therapeutic strategy across PMD genotypes. Here we show, CRISPR-Cas9 mediated germline knockdown of Plp1 in the severe jimpy (Plp1jp) point mutation mouse model of PMD rescued myelinating oligodendrocytes, nerve conduction velocity, motor function, and lifespan to wild-type levels, thereby validating PLP1 suppression as a therapeutic approach. To evaluate the therapeutic potential of Plp1 suppression in postnatal PMD mice, we tested antisense oligonucleotides (ASOs) that stably decrease mouse Plp1 mRNA and protein in vivo. Administration of a single intraventricular dose of Plp1-targeted ASOs to postnatal jimpy mice increased myelination, improved motor behavior, and extended lifespan through an 8-month endpoint. Collectively, these results support the development of PLP1 suppression as a disease-modifying therapy for most PMD patients. More broadly, we demonstrate that RNA therapeutics can be delivered to oligodendrocytes in vivo to modulate neurological function and lifespan, opening a new treatment modality for myelin disorders.

Two drugs, miconazole and clobetasol, have functions that modulate differentiation of oligodendrocyte progenitor cells directly, enhance remyelination, and significantly reduce disease severity in mouse models of multiple sclerosis. Multiple sclerosis is characterized by an autoimmune response and failure of remyelination in the brain due to defects in differentiation of myelin-producing cells from oligodendrocyte progenitor cells. Most current treatments target the immune system. Paul Tesar and colleagues screened for compounds that can enhance oligodendrocyte maturation from mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitors. They found two drugs — miconazole (an antifungal) and clobetasol (a steroid) — that enhance myelin production in vivo in mouse models of multiple sclerosis and enhanced the differentiation of human oligodendrocytes progenitors in vitro. Mechanistically, these compounds appear to target both the immune response and oligodendrocyte progenitor cells. Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes1. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention2. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells3,4,5. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.

A circular-scan streak tube designed for use in a laser ranging system is described. Electrostatic focusing is used between the photocathode and a microchannel plate, and electrons from the microchannel plate are proximity focused onto an output phosphor screen. Electron beam deflection is achieved by driving two orthogonal sets of deflection plate assemblies in phase quadrature at a frequency of 200 MHz. The light intensity in the output beam trace is measured by using a circular Reticon array of 720 photodiodes, which is fiber-optically coupled to the output phosphor screen of the tube. Sample measurements of frequency-doubled Nd:YAG laser-pulse waveforms are given. Also the output beamwidth has been measured at the 200-MHz deflection frequency. The results suggest a limiting resolution of 33 psec for the circular-scan streak tube used for these tests.

Eucalyptus camaldulensis Dehn. (Kırmızı okaliptüs), Juglans regia L. (Ceviz ağacı), Melia azedarach L. (Tespih ağacı), Nerium oleander L. (Zakkum ağacı), Raphanus sativus I. (Antep turpu) ve Thymus sp. (Kekik) gibi allelopatik özelliğe sahip bitki türlerinin kültür bitkileri ile yabancıot türlerinin gelişimine etkisinin saptandığı çalışma çimlenme denemeleri, sera denemeleri ve arazi denemeleri olmak üzere üç aşamada gerçekleştirilmiştir. Çimlenme denemelerinde bu türlerin su ekstraktları, sera ve arazi koşullarında ise parçalanmış bitki atıkları toprağa karıştırılarak uygulama yapılmıştır. Sera koşullarında bu türlerin pamuk, mısır, soya ile Amaranthus retro flexus L, Portulaca oleracea L, Prosopis stephaniana Kunth. ve Xanthium strumarium L. `un gelişimine etkisi incelenmiştir. Tarla koşullarında yine aynı türlerin parçalanmış bitki atıklarının mısır ile yabancıot türlerinin gelişimine etkisi tespit edilmiştir. Yapılan çalışmalarda; Çimlenme denemelerinde; % Çimlenen tohum sayısı, sera denemelerinde; Bitki boy uzunluğu, bitki yaprak sayısı, toprak üstü ve toprak altı aksamının yaş-kuru ağırlıkları, tarla denemelerinde; m2 'deki yabancıot ve kültür bitkisi sayısı, % genel yabancıot kaplama alanları, hasat edilen kültür bitkilerinin boy uzunluğu, hasat edilen kültür bitkilerinin toprak üstü aksamının yaş ağırlığı, 1000 dane ağırlığı ve verimden elde edilen sonuçlar değerlendirilmiştir. Çimlenme denemelerinde, uygulamalardan N. oleander'i takiben M. azedarach ve Thymus sp.'nin su ekstraktlarının tohumçimlenmesini inhibe ettiği saptanmıştır. Sera koşullarında M.azedarach ve N. oleander `in parçalanmış bitki atıklarının kültür bitkilerinin gelişimini, kuru ağırlığını arttırdığı, yabancıot gelişimini azalttığı tespit edilmiştir. Tarla koşullarında kültür bitksi gelişimini olumlu etkileyen, hiç uygulama yapılmamış kontrole göre verimi arttıran E.camaldulensis, J.regia, M.azedarach, N.oleander ve R.sativus 'un parçalanmış bitki atıklarının yabancıot gelişimini azalttığı belirlenmiştir. 112 SUMMARY In this study, some plant species that had allelopathic effects were investigated in terms of allelopathic and bioherbicide effects on the growth of weed sand cultivated plants. The study was carried out in three steps, germination tests, greenhouse and field experiments, respectively. Plant extracts of these species were used for germination test, while plant parts were used for greenhouse and field experiments. The allelopathic effects of these species on the growth of cotton, corn, soybean, Amaranthus retroflexus L, Port ulaca oleracea L., Prosopis stephaniana Kunth., and Xanthium strumarium L. were also determined. In the field experiment, the some plant parts were used to investigate the allelopathic effects of them on corn and weeds. In the experiments, the following criteria were determined; -percentage of germinated seeds (for laboratory experiments) -height of plants; number of leafs; fresh and dry weight of plants (for greenhouse experiments) -number of weed and cultivated plants in 1 m2 ; percentage cover of weeds in the field; height of plants harvested; fresh weight of the cultivated plants; weight of 1000 corn seeds; (for field experiments) The extracts of N. oleander, M.azedarach and Thymus sp. inhibited the germination of seeds involved in germination tests. Plant parts of M.azedarach, N. oleander showed a positive effects on the growth and dry weight of cultivated plants, but a113 negative effect on the growth of weeds in greenhouse experiments. The same results were obtained for effects of E.camaldulensis, J.regia, M.azedarach, N.oleander, and R.sativus in the field experiments. 121

Tumor detection at bronchoscopy involves a linear visual search throughout the accessible part of the bronchial tree with biopsy at suspicious locations. We have designed, built and tested a series of small radiation detector probes that can be passed through the biopsy channel of a bronchoscope and used to locate sites of increased uptake of tumor-seeking radiopharmaceuticals. Such probes can help to locate visually obscured or submucosal tumors and can identify sites for biopsy. NaI(Tl) and CsI(Tl) scintillation detectors with flexible fiber optic light guides and a CdTe semiconductor detector have been constructed. Tumor phantom studies have successfully predicted the performance of the NaI(Tl) probe in clinical studies using 57Co-bleomycin.

New vinyl oil monomers have been prepared by combining epoxidized unsaturated oils with vinyl carboxylic acids. This is accomplished by heating the reactants in the presence of free radical inhibitors and appropriate reaction catalysts. The reaction consists of cleavage of the oxirane ring followed by addition of the vinyl acid through its carboxylic group. By varying the reactants and the conditions of reaction, a large family of related, but different, vinyl oil monomers can be prepared. Vicinal methacryloxy-hydroxy soy oil monomers are clear, yellow, medium viscosity (2000–2500 cpoise), high molecular weight (1300–1500) liquids. Chemical analyses are presented for typical preparations. The monomers readily homopolymerize by a free radical mechanism to clear, solid, thermoset resins. They are compatible with a variety of commercial vinyl monomers and form copolymers which range from highly viscous liquids through soft gels, to tough rubbery products and hard resins. Data are presented on physical properties of copolymer castings of several specific vicinal methacryloxy-hydroxy soy oil monomers.

Details of apparatus construction and refinements are presented for a micro capillary rise method of determining absolute values of surface tension. Experimental values of surface tension for benzene, n-pentadecane, and methyl laurate are recorded and compared with previous literature values. Parachors are determined. The method requires a minute amount of sample and is simple, quick, precise, and accurate.