Complex mosaic structural variations in human fetal brains

Somatic mosaicism, manifesting as single nucleotide variants (SNVs), mobile element insertions, and structural changes in the DNA, is a common phenomenon in human brain cells, with potential functional consequences. Using a clonal approach, we previously detected 200–400 mosaic SNVs per cell in three human fetal brains (15–21 wk postconception). However, structural variation in the human fetal brain has not yet been investigated. Here, we discover and validate four mosaic structural variants (SVs) in the same brains and resolve their precise breakpoints. The SVs were of kilobase scale and complex, consisting of deletion(s) and rearranged genomic fragments, which sometimes originated from different chromosomes. Sequences at the breakpoints of these rearrangements had microhomologies, suggesting their origin from replication errors. One SV was found in two clones, and we timed its origin to ∼14 wk postconception. No large scale mosaic copy number variants (CNVs) were detectable in normal fetal human brains, suggesting that previously reported megabase-scale CNVs in neurons arise at later stages of development. By reanalysis of public single nuclei data from adult brain neurons, we detected an extrachromosomal circular DNA event. Our study reveals the existence of mosaic SVs in the developing human brain, likely arising from cell proliferation during mid-neurogenesis. Although relatively rare compared to SNVs and present in ∼10% of neurons, SVs in developing human brain affect a comparable number of bases in the genome (∼6200 vs. ∼4000 bp), implying that they may have similar functional consequences.

Somatic mosaicism, the presence of more than one genotype in the somatic cells of an individual, is a prominent phenomenon in the human central nervous system. Forms of mosaicism include aneuploidies and smaller copy number variants (CNVs), structural variants (SVs), mobile element insertions, indels, and single nucleotide variants (SNVs). The developing human brain exhibits high levels of aneuploidy compared to other tissues, generating genetic diversity in neurons (Pack et al. 2005; Yurov et al. 2007; Bushman and Chun 2013). Such aneuploidy was suggested to be a natural feature of neurons, rather than a distinctive feature of neurodegeneration. However, the frequency of aneuploidy in neurons has been debated, with a separate study suggesting that aneuploidies occur in only about 2.2% of mature adult neurons (Knouse et al. 2014). They hence infer that such aneuploidy could have adverse effects at the cellular and organismal levels. Additionally, analysis of single cells from normal and pathological human brains identified large, private, and likely clonal somatic CNVs in both normal and diseased brains (Gole et al. 2013; McConnell et al. 2013; Cai et al. 2014; Knouse et al. 2016; Chronister et al. 2019; Perez-Rodriguez et al. 2019), with 3%–25% of human cerebral cortical nuclei carrying megabase-scale CNVs (Chronister et al. 2019) and deletions being twice as common as duplications (McConnell et al. 2013). Given that CNVs often arise from nonhomologous recombination and replication errors, their likely time of origin is during brain development. However, when CNVs first arise in human brain development has not yet been investigated. The present work is the first to examine this question using clonal populations of neuronal progenitor cells (NPCs) obtained from fetal human brains.Detection of CNVs in single neurons is challenging, given the need to amplify DNA. Such amplification may introduce artifacts that could, in turn, be misinterpreted as CNVs. In order to address this technical limitation, Hazen et al. reprogrammed adult postmitotic neurons using somatic cell nuclear transfer (SCNT) of neuronal nuclei into enucleated oocytes (Hazen et al. 2016). These oocytes then made sufficient copies of the neuronal genome allowing for whole-genome sequencing (WGS), thus eliminating the need for amplification in vitro. Using this method, they identified a total of nine structural variants in six neurons from mice, three of which were complex rearrangements. However, it is not possible to extend such studies to humans, given the ethical issues involved, besides the technical challenges in obtaining and cloning adult neurons. To circumvent the need of single-cell DNA amplification or nuclear cloning, we examined clonal cell populations obtained from neural progenitor cells from the frontal region of the cerebral cortex (FR), parietal cortex (PA) and basal ganglia (BG) and describe here the discovery and analysis of mosaic SVs in these NPCs (Bae et al. 2018). These clones were sequenced at 30× coverage (much higher than most previous single-cell studies), allowing identification of SVs other than large deletions and duplications as well as precise breakpoint resolution.     

Monoclonal antibodies cross-reacting with fibroblasts of myocardial interstitial connective tissue and with group a streptococcal cell wall protein antigens

N. F. Gamaleya Research Institute of Epidemiology and Microbiology. Institute of Rheumatology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR, P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 7, pp. 74–76, July, 1989.     

Inducible nitric oxide synthase expression and cardiomyocyte dysfunction during sustained moderate ischemia in pigs

In acute myocardial ischemia, regional blood flow and function are proportionally reduced. With prolongation of ischemia, function further declines at unchanged blood flow. We studied the involvement of an inflammatory signal cascade in such progressive dysfunction and whether dysfunction is intrinsic to cardiomyocytes. In 10 pigs, ischemia was induced by adjusting inflow into the cannulated left anterior coronary artery to reduce coronary arterial pressure to 45 mm Hg (ISCH); 4 pigs received the inducible nitric oxide synthase (iNOS) inhibitors aminoguanidine or L-N(6)-(1-iminoethyl)-lysine during ISCH (ISCH+iNOS-Inhib); 6 pigs served as controls (SHAM). Anterior (AW) and posterior (PW) systolic wall thickening (sonomicrometry) were measured. After 6 hours, nitric oxide (NO) synthase (NOS) protein expression, NOS activity, and NO metabolites (nitrite/nitrate/nitroso species) were quantified in biopsies isolated from AW and PW. Cardiomyocyte shortening and intracellular calcium (Indo-1 acetoxymethyl ester) were measured without and with the NOS substrate L-arginine (100 micromol/L). In ISCH, AW wall thickening decreased from 42+/-4% (baseline) to 16+/-3% (6 hours). Wall thickening remained unchanged in ISCH-PW and SHAM-AW/PW. NOS2 (iNOS) protein expression and activity, but not NOS3 (endothelial NO synthase), were increased in ISCH-AW and ISCH-PW. iNOS expression correlated with increased nitrite contents. Cardiomyocyte shortening was reduced in ISCH-AW versus SHAM-AW (4.4+/-0.3% versus 5.6+/-0.3%). L-Arginine reduced cardiomyocyte shortening further in ISCH-AW (to 2.8+/-0.2%) and ISCH-PW (3.4+/-0.4% versus 5.4+/-0.4%) but not in SHAM or in ISCH+iNOS-Inhib; intracellular [Ca(2+)] remained unchanged. With L-arginine, in vitro AW cardiomyocyte shortening correlated with in vivo AW wall thickening (r=0.72). In conclusion, sustained regional ischemia induces myocardial iNOS expression in pigs, which contributes to contractile dysfunction at the cardiomyocyte level.     

Channelrhodopsin-2–XXL,a powerful optogenetic tool for low-light applications

Channelrhodopsin-2 (ChR2) has provided a breakthrough for the optogenetic control of neuronal activity. In adult Drosophila melanogaster, however, its applications are severely constrained. This limitation in a powerful model system has curtailed unfolding the full potential of ChR2 for behavioral neuroscience. Here, we describe the D156C mutant, termed ChR2-XXL (extra high expression and long open state), which displays increased expression, improved subcellular localization, elevated retinal affinity, an extended open-state lifetime, and photocurrent amplitudes greatly exceeding those of all heretofore published ChR variants. As a result, neuronal activity could be efficiently evoked with ambient light and even without retinal supplementation. We validated the benefits of the variant in intact flies by eliciting simple and complex behaviors. We demonstrate efficient and prolonged photostimulation of monosynaptic transmission at the neuromuscular junction and reliable activation of a gustatory reflex pathway. Innate male courtship was triggered in male and female flies, and olfactory memories were written through light-induced associative training.Identifying causal relationships between neuronal activity and animal behavior is a fundamental goal of neuroscience. Crucially, this task requires testing whether defined neuronal populations are sufficient for eliciting behavioral modules. The development of light-gated ion channels that can be genetically targeted to specific cells has provided a unique solution to this challenge. In pioneering work, such optogenetic effectors or actuators were originally used as multicomponent approaches (1–3). The introduction of Channelrhodopsin-1 (ChR1) (4) and especially ChR2 as a light-sensitive cation channel (5) dramatically advanced the field by providing an efficient and straightforward single-component strategy for stimulating neuronal activity (6, 7).Besides cell-specific targeting of appropriate effector elements, precise neuronal control by optogenetics demands efficient light delivery to the neurons of interest. For behavioral studies, photostimulation is ideally accomplished in intact, freely moving organisms and accompanied by functional readouts. The combination of a rich, well-characterized behavioral repertoire and elegant molecular genetics has contributed to Drosophila’s strong impact on behavioral neurogenetics (8, 9). However, low light transmission through the pigmented cuticle presupposes high light intensities for using ChR2 in flies. This obstacle greatly complicates the experimental setup for freely moving animals, and the required light energies can cause heat damage when stimulation is applied over extended time periods. Moreover, limited cellular availability of all-trans-retinal (hereafter retinal for short) demands adding high retinal concentrations as a dietary supplement. If optical access to target cells is not provided by a translucent body wall (e.g., as in nematodes, zebrafish, and Drosophila larvae), an alternative solution is the implantation of an optical fiber directly into the brain. Although this approach has been used successfully in mammals (10), such an invasive procedure is infeasible for the study of intact small organisms.Due to these restrictions in Drosophila, ChR2 has not reached the popularity attained in other organisms, and instead the field has turned mainly to thermogenetic neuronal stimulation (11–13). As with all techniques, there are also drawbacks to using temperature as a stimulus, such as undesired background activity and a multitude of temperature-sensitive cellular processes and behavioral responses. Photo-liberation of caged ATP, combined with genetic targeting of ATP-gated ion channels, has been introduced as a different optogenetic technique in Drosophila (3, 14). However, its applications are constrained by invasive, time-consuming procedures for injection of caged ATP and a limited experimental time window.Here, we introduce improved ChR2 variants as an alternative approach to address these shortcomings in Drosophila. Compared with wild-type ChR2 (ChR2-wt), expression of these mutants in target cells led to strongly enhanced photocurrents. We provide the first report, to our knowledge, of ChR2-T159C (15, 16) in flies and describe a ChR2 variant, ChR2-XXL (extra high expression and long open state), that is characterized by an extended open-state lifetime, elevated cellular expression, enhanced axonal localization, and reduced dependence on retinal addition. As a consequence, this mutant does not require dietary retinal supplementation to depolarize cells, evoke synaptic transmission, and activate neuronal networks at very low irradiance. These features enabled behavioral photostimulation in freely moving flies using diffuse low-intensity light.     

Regulatory element copy number differences shape primate expression profiles

Gene expression differences are shaped by selective pressures and contribute to phenotypic differences between species. We identified 964 copy number differences (CNDs) of conserved sequences across three primate species and examined their potential effects on gene expression profiles. Samples with copy number different genes had significantly different expression than samples with neutral copy number. Genes encoding regulatory molecules differed in copy number and were associated with significant expression differences. Additionally, we identified 127 CNDs that were processed pseudogenes and some of which were expressed. Furthermore, there were copy number-different regulatory regions such as ultraconserved elements and long intergenic noncoding RNAs with the potential to affect expression. We postulate that CNDs of these conserved sequences fine-tune developmental pathways by altering the levels of RNA.     

Lateral Patellar Facetectomy and Medial Reefing in Patients With Lateral Facet Syndrome After Patellar-Retaining Total Knee Arthroplasty

We analyzed clinical outcomes of partial lateral patellar facetectomy and medial reefing in patients with lateral patellar facet syndrome with painful patellar-retaining total knee arthroplasty. 34 patients were followed for a mean of 40 months. All 34 patients were matched with those having secondary patellar resurfacing without facetectomy. Both groups experienced significant pain relief and range of motion improvement. The facetectomy group had higher Kujala scores than those in patellar resurfacing group. Patients with facetectomy had significantly less pain postoperatively. There were significant differences in postoperative lateral patellar tilt and congruency angle in both groups. The mid-term results for LPF with medial reefing are promising to resolve pain in patients with lateral patellar facet syndrome in patellar-retaining TKA. Therapeutic level III (retrospective comparative study).     

Mechanisms of ATP- and glutamate-mediated calcium signaling in white matter astrocytes

Neurotransmitters released at synapses mediate Ca2+ signaling in astrocytes in CNS grey matter. Here, we show that ATP and glutamate evoke these Ca2+ signals in white matter astrocytes of the mouse optic nerve, a tract that contains neither neuronal cell bodies nor synapses. We further demonstrate that action potentials along white matter axons trigger the release of ATP and the intercellular propagation of astroglial Ca2+ signals. These mechanisms were studied in astrocytes in intact optic nerves isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the human glial fibrillary acidic protein promoter (GFAP) by Fura-2 ratiometric Ca2+ imaging. ATP evoked astroglial Ca2+ signals predominantly via metabotropic P2Y1 and ionotropic P2X7 purinoceptors. Glutamate acted on both AMPA- and NMDA-type receptors, as well as on group I mGlu receptors to induce an increase in astroglial [Ca2+]i. The direct Ca2+ signal evoked by glutamate was small, and the main action of glutamate was to trigger the release of the "gliotransmitter" ATP by a mechanism involving P2X7 receptors; propagation of the glutamate-mediated Ca2+ signal was significantly reduced in P2X7 knock-out mice. Furthermore, axonal action potentials and mechanical stimulation of astrocytes both induced the release of ATP, to propagate Ca2+ signals in astrocytes and neighboring EGFP-negative glia. Our data provide a model of multiphase axon-glial signaling in the optic nerve as follows: action potentials trigger axonal release of ATP, which evokes further release of ATP from astrocytes, and this acts by amplifying the initiating signal and by transmitting an intercellular Ca2+ wave to neighboring glia.     

Microarray method for multiplex and serial latex agglutination tests with digital image registration

A microarray analytical system for performing tests of latex agglutination reaction in microformat with digital image registration was developed. The system allows the application of latex microdrops to the surface of the carrier in the form of a regular microarray and mixing of the latex droplets with the individual samples in each droplet of the microarray. The reaction is performed in a total mixture volume of about 1 microl for each of 30 samples simultaneously with video registration and interpretation of the results using a scanning device and specially developed software. The results of the semi-quantitative determination of C-Reactive Protein, Rheumatoid Factor and Anti-Streptolysin O concentrations by traditional macro- and proposed micro-arrayagglutination method were compared with the immunoturbidimetric measurements used as reference method. It was concluded that the suggested method for performing latex agglutination reactions on the basis of a microarray approach with digital image evaluation of results can provide a high throughput and reliable results and also offers significant advantages to the traditional latex agglutination tests with visual interpretation. Comprehensive documentation and objectification of readouts show a siginificant improvement to the present methodology.     

Extraorganic Hepatic Artery Aneurysm: Failure of Transcatheter Embolization

Hepatic artery aneurysm (HAA) was diagnosed in a 62-year-old man who was a poor candidate for surgery because of severe liver cirrhosis and diabetes mellitus. Two attempts to occlude the HAA by transcatheter embolization failed because of recanalization of the aneurysm. Moreover, aneurysmal dilatation of the superior mesenteric artery and the left renal artery developed and progressed. Both the literature and the present case show that an individual approach to treatment of extraorganic HAA should be chosen in dependantan location and anatomy of the lesion.