Neuronal CTCF Is Necessary for Basal and Experience-Dependent Gene Regulation, Memory Formation, and Genomic Structure of BDNF and Arc

SummaryCCCTC-binding factor (CTCF) is an organizer of higher-order chromatin structure and regulates gene expression. Genetic studies have implicated mutations in CTCF in intellectual disabilities. However, the role of CTCF-mediated chromatin structure in learning and memory is unclear. We show that depletion of CTCF in postmitotic neurons, or depletion in the hippocampus of adult mice through viral-mediated knockout, induces deficits in learning and memory. These deficits in learning and memory at the beginning of adulthood are correlated with impaired long-term potentiation and reduced spine density, with no changes in basal synaptic transmission and dendritic morphogenesis and arborization. Cognitive disabilities are associated with downregulation of cadherin and learning-related genes. In addition, CTCF knockdown attenuates fear-conditioning-induced hippocampal gene expression of key learning genes and loss of long-range interactions at the BDNF and Arc loci. This study thus suggests that CTCF-dependent gene expression regulation and genomic organization are regulators of learning and memory

Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cellfree cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development

Whole-Genome Amplification—Surveying Yield, Reproducibility, and Heterozygous Balance, Reported by STR-Targeting MIPs

Whole-genome amplification is a crucial first step in nearly all single-cell genomic analyses, with the following steps focused on its products. Bias and variance caused by the whole-genome amplification process add numerous challenges to the world of single-cell genomics. Short tandem repeats are sensitive genomic markers used widely in population genetics, forensics, and retrospective lineage tracing. A previous evaluation of common whole-genome amplification targeting ~1000 non-autosomal short tandem repeat loci is extended here to ~12,000 loci across the entire genome via duplex molecular inversion probes. Other than its improved scale and reduced noise, this system detects an abundance of heterogeneous short tandem repeat loci, allowing the allelic balance to be reported. We show here that while the best overall yield is obtained using RepliG-SC, the maximum uniformity between alleles and reproducibility across cells are maximized by Ampli1, rendering it the best candidate for the comparative heterozygous analysis of single-cell genomes

Whole-Genome Amplification—Surveying Yield, Reproducibility, and Heterozygous Balance, Reported by STR-Targeting MIPs

Whole-genome amplification is a crucial first step in nearly all single-cell genomic analyses, with the following steps focused on its products. Bias and variance caused by the whole-genome amplification process add numerous challenges to the world of single-cell genomics. Short tandem repeats are sensitive genomic markers used widely in population genetics, forensics, and retrospective lineage tracing. A previous evaluation of common whole-genome amplification targeting ~1000 non-autosomal short tandem repeat loci is extended here to ~12,000 loci across the entire genome via duplex molecular inversion probes. Other than its improved scale and reduced noise, this system detects an abundance of heterogeneous short tandem repeat loci, allowing the allelic balance to be reported. We show here that while the best overall yield is obtained using RepliG-SC, the maximum uniformity between alleles and reproducibility across cells are maximized by Ampli1, rendering it the best candidate for the comparative heterozygous analysis of single-cell genomes

Lesion location impact on functional recovery of the hemiparetic upper limb.

The effect of stroke topography on the recovery of hemiparetic upper limb (HUL) function is unclear due to limitations in previous studies-examination of lesion effects only in one point of time, or grouping together patients with left and right hemispheric damage (LHD, RHD), or disregard to different lesion impact on proximal and distal operations. Here we used voxel-based lesion symptom mapping (VLSM) to investigate the impact of stroke topography on HUL function taking into consideration the effects of (a) assessment time (subacute, chronic phases), (b) side of damaged hemisphere (left, right), (c) HUL part (proximal, distal). HUL function was examined in 3 groups of patients-Subacute (n = 130), Chronic (n = 66), and Delta (n = 49; patients examined both in the subacute and chronic phases)-using the proximal and distal sub-divisions of the Fugl-Meyer (FM) and the Box and Blocks (B&B) tests. HUL function following LHD tended to be affected in the subacute phase mainly by damage to white matter tracts, the putamen and the insula. In the chronic phase, a similar pattern was shown for B&B performance, whereas FM performance was affected by damage only to the white matter tracts. HUL function following RHD was affected in both phases, mainly by damage to the basal ganglia, white matter tracts and the insula, along with a restricted effect of damage to other cortical structures. In the chronic phase HUL function following RHD was affected also by damage to the thalamus. In the small Delta groups the following trends were found: In LHD patients, delayed motor recovery, captured by the B&B test, was affected by damage to the sensory-motor cortex, white matter association fibers and parts of the perisilvian cortex. In the RHD patients of the Delta group, delayed motor recovery was affected by damage to white matter projection fibers. Proximal and distal HUL functions examined in LHD patients (both in the subacute and chronic phases) tended to be affected by similar structures-mainly white matter projection tracts. In RHD patients, a distinction between proximal and distal HUL functions was found in the subacute but not in the chronic phase, with proximal and distal HUL functions affected by similar subcortical and cortical structures, except for an additional impact of damage to the superior temporal cortex and the retro-lenticular internal capsule only on proximal HUL function. The current study suggests the existence of important differences between the functional neuroanatomy underlying motor recovery following left and right hemisphere damage. A trend for different lesion effects was shown for residual proximal and distal HUL motor control. The study corroborates earlier findings showing an effect of the time after stroke onset (subacute, chronic) on the results of VLSM analyses. Further studies with larger sample size are required for the validation of these results

Additional file 1 of MCProj: metacell projection for interpretable and quantitative use of transcriptional atlases

Additional file 1: Fig S1. Validation: leave-one-batch-out projections. Similar to Fig. 2A. Counting cells classified according to their reference annotated type and the type assigned to their query metacell by scARCHES. Fig S2. Validation: leave-one-batch-out projections. Similar to Fig. 2A. Counting cells classified according to their reference annotated type and the type assigned to their query metacell by Seurat. Fig S3. Validation: leave-one-batch-out projections. Similar to Fig. 2A. Counting cells classified according to their reference annotated type and the type assigned to their query metacell by scMAP. Fig S4. Validation: leave-one-type-out projections. Similar to Fig. 3A. Counting cells classified according to their reference annotated type and the type assigned to their query metacell by scARCHES. Fig S5. Validation: leave-one-type-out projections. Similar to Fig. 3A. Counting cells classified according to their reference annotated type and the type assigned to their query metacell by Seurat. Fig S6. Validation: leave-one-type-out projections. Similar to Fig. 3A. Counting cells classified according to their reference annotated type and the type assigned to their query metacell by scMAP. Fig S7. The fraction of query cells for which a synthetic technology difference was applied (and was not corrected), which were either not successfully projected, or which were assigned a type different than the expected type, for each of the methods we compared, very close to Fig. 2B

Analysis of Chronic Granulomatous Disease in the Kavkazi Population in Israel Reveals Phenotypic Heterogeneity in Patients with the Same NCF1 mutation (c.579G>A)

Chronic granulomatous disease (CGD) is an innate immune deficiency disorder of phagocytes, resulting from mutations in the components of the NADPH oxidase complex that impair the synthesis of oxygen radicals, thus rendering patients susceptible to recurrent infections and excessive hyperinflammatory responses. The most common autosomal recessive form of CGD is p47phox deficiency, which is often clinically milder than the more common X-linked recessive form. Here, we report data on genetics, clinical and biochemical findings in 17 CGD patients of Kavkazi origin with the nonsense mutation c.579G>A in the NCF1 gene, leading to p47phox deficiency. Diagnosis was based on detailed clinical evaluation, respiratory burst activity by cytochrome c reduction and dihydrorhodamine-1,2,3 (DHR) assay by flow cytometry, expression of p47phox by immunoblotting and molecular confirmation by DNA sequence analysis. Twelve male and five female patients with median age at onset of 2.5 years (range 1 day to 9 years) were included in the study. The present cohort displays an encouraging 88% overall long-term survival, with median follow-up of 17 years. Clinical manifestations varied from mild to severe expression of the disease. Correlation between genotype and phenotype is unpredictable, although the Kavkazi patients were more severely affected than other patients with p47phox deficiency. Kavkazi CGD patients harbor a common genetic mutation that is associated with a heterogeneous clinical phenotype. Early diagnosis and proper clinical management in an experienced phagocytic leukocyte center is imperative to ensure favorable patient outcome. New treatment strategies are ongoing, but results are not yet conclusiv

Heuristic for Maximizing DNA Reuse in Synthetic DNA Library Assembly

<i>De novo</i> DNA synthesis is in need of new ideas for increasing production rate and reducing cost. DNA reuse in combinatorial library construction is one such idea. Here, we describe an algorithm for planning multistage assembly of DNA libraries with shared intermediates that greedily attempts to maximize DNA reuse, and show both theoretically and empirically that it runs in linear time. We compare solution quality and algorithmic performance to the best results reported for computing DNA assembly graphs, finding that our algorithm achieves solutions of equivalent quality but with dramatically shorter running times and substantially improved scalability. We also show that the related computational problem <i>bounded-depth min-cost string production</i> (BDMSP), which captures DNA library assembly operations with a simplified cost model, is NP-hard and APX-hard by reduction from vertex cover. The algorithm presented here provides solutions of near-minimal stages and thanks to almost instantaneous planning of DNA libraries it can be used as a metric of ″manufacturability″ to guide DNA library design. Rapid planning remains applicable even for DNA library sizes vastly exceeding today’s biochemical assembly methods, future-proofing our method