Deletion Of Site Research Articles

Function alteration of engineered heart tissues carrying a shorter filamin C protein induced by CRISPR/Cas9

Dilated cardiomyopathy (DCM) is characterized by ventricular dilation and systolic dysfunction beginning in young adults and leading to heart failure with few treatment options. Loss-of-function mutations in the FLNC gene, encoding Filamin C, including splicing mutations, have been strongly associated with an arrhythmogenic DMC phenotype. FLNC-related cardiomyopathies are autosomal dominant disease and the mechanisms leading to the phenotype are not fully understood. In vitro models are needed to improve knowledge of pathogenic mechanisms. A control iPSc line (ICAN-403.3) was established from healthy donor fibroblasts and a mutated FLNC subclone (ICAN-FLNC-42.1) was derived using CRISPR/Cas9 technology. The iPSc cells were then differentiated into cardiomyocytes and beating pseudo-tissues in order to study the functional effects of the mutation. We generated two iPS clones, one derived from the other after mutation induced by non-homologous CRISPR/Cas9-dependent DNA repair. The clones were characterized for their pluripotency (immunoflurescence and qPCR of genes expressed in the pluripotent state, alkaline phosphatase test), their genomic integrity (exome, karyotype) and for their capacity for differentiation in the 3 layers (ScoreCard). More specifically, these cells can be differentiated into beating cardiomyocytes (> 85% of cells express troponin T) and exhibit sarcomeric striation by immunostaining of actinin-2. Sequencing shows that the mutation induced by CRISPR/Cas9 is a deletion of the 3′ terminal of exon 42 of the FLNC gene found in the homozygous state [Chr7(GRCh38):g.128854898_128854915del] and lead to the deletion of the consensus donor splice site of exon 42. Additionally, no off-target in cardiac expressed genes has been detected by exome sequencing. Analysis by RT-PCR and immunoblot shows that the mutation induces a exon 42 skipping and reduced expression of a protein of lower molecular weight. Cardiac tissues records for 3 weeks [engineered heart tissue (EHT) generated from the two iPS clones differentiated into cardiomyocytes] show, for the mutant, a lower beat frequency and a higher arrhythmia index. This model will make it possible to understand the pathophysiological mechanism of DCM due to FLNC mutations. These models are also useful for testing potential therapeutic pathways.

Nearly Optimal Planar $k$ Nearest Neighbors Queries under General Distance Functions

We study the $k$ nearest neighbors problem in the plane for general, convex, pairwise disjoint sites of constant description complexity such as line segments, disks, and quadrilaterals under a general family of distance functions including the $L_p$ norms and additively weighted Euclidean distances. We compose a static data structure for this general setting with nearly optimal $O(n\log\log n)$ space, optimal $O(\log n+k)$ query time, and optimal expected $O(n\log n)$ preprocessing time. We also devise a dynamic data structure (that allows insertions and deletions of sites) with $O(n\log n)$ space, $O(\log^2n+k\log n)$ query time, and expected amortized $O(\log^2 n)$ insertion time and $O(\log^4n)$ deletion time, matching the best known time complexities for point sites in the Euclidean metric and improving many applications such as dynamic minimum spanning trees in a general planar metric and dynamic connectivity in disk intersection graphs. Our results, to some extent, indicate that for the $k$ nearest neighbors problem, general distance functions may share the same time complexities with point sites in the Euclidean metric. To achieve this progress, we design vertical shallow cuttings of linear size for general distance functions. Vertical shallow cuttings are a key technique to tackle the $k$ nearest neighbors problem for point sites in the Euclidean metric, while existing generalizations to general distance functions are either not vertical or not of linear size. Our innovation is a new random sampling technique for the analysis of geometric structures, and since this technique provides a new way to develop geometric algorithms, we believe it is of independent interest.

Insertion/deletion (Indel) variant of the goat RORA gene is associated with growth traits

RAR related orphan receptor A (RORA), which encodes the retinoid-acid-related orphan receptor alpha (RORα), is a clock gene found in skeletal muscle. Several studies have shown that RORα plays an important role in bone formation, suggesting that RORA gene may take part in the regulation of growth and development. The purpose of this research is to study the insertion/deletion (indel) variations of the RORA gene and investigate the relationship with the growth traits of Shaanbei white cashmere (SBWC) goats. Herein, the current study identified that the P4-11-bp and P11-28-bp deletion sites are polymorphic among 12 pairs of primers within the RORA gene in the SBWC goats (n = 641). Moreover, the P11-28-bp deletion locus was significantly related to the body height (p = 0.046), height at hip cross (p = 0.012), and body length (p = 0.003). Both of P4-11-bp and P11-28-bp indels showed the moderate genetic diversity (0.25<PIC < 0.5). Thus, the study results indicated that the P11-28-bp deletion site should be used as a molecular marker to select excellent goat individuals with superior growth performance.

The transcription factor Atf1 lowers the transition barrier for nucleosome-mediated establishment of heterochromatin.

Transcription factors can exert opposite effects depending on the chromosomal context. The fission yeast transcription factor Atf1 both activates numerous genes in response to stresses and mediates heterochromatic gene silencing in the mating-type region. Investigating this context dependency, we report here that the establishment of silent heterochromatin in the mating-type region occurs at a reduced rate in the absence of Atf1 binding. Quantitative modeling accounts for the observed establishment profiles by a combinatorial recruitment of histone-modifying enzymes: locally by Atf1 at two binding sites and over the whole region by dynamically appearing heterochromatic nucleosomes, a source of which is the RNAi-dependent cenH element. In the absence of Atf1 binding, the synergy is lost, resulting in a slow rate of heterochromatin formation. The system shows how DNA-binding proteins can influence local nucleosome states and thereby potentiate long-range positive feedback on histone-modification reactions to enable heterochromatin formation over large regions in a context-dependent manner.

Abstract 518: Erk5 S496 Phosphorylation, But Not Erk5 Kinase Or Transcriptional Activity, Is Responsible For Promoting Macrophage Inflammation And Mitochondrial Dysfunction Via Upregulating Novel Site Of Nrf2 K518 Sumoylation

ERK5 is a dual kinase-transcription factor, which contains two transcriptional transactivation domains in the C-terminus and a kinase domain in the N-terminus. Many ERK5 kinase inhibitors have been developed, and are being tested in clinical studies for cancer and inflammatory diseases. Recent data has raised questions regarding the functional role of these ERK5 kinase inhibitors. Specifically, the possible link between blockade of pro-inflammatory ERK5 S496 phosphorylation and the anti-inflammatory effects of ERK5-specific kinase inhibitors has largely been neglected. In this study, we aimed to study the role and regulatory mechanisms of ERK5 S496 phosphorylation on macrophage inflammation and the impact of ERK5-specific kinase inhibitors. ATP binding site deletion mutant of ERK5b (a kinase-dead mutant) inhibited KLF2 induction but not oxidized LDL (oxLDL)-induced ERK5 S496 phosphorylation and TNFα expression. In contrast, both specific ERK5 kinase inhibitors (AX15836 and XMD8-92) and a dual phosphorylation site mutant of ERK5 (AEF) inhibited not only KLF2 but also oxLDL-induced ERK5 S496 phosphorylation and TNFα induction. These data suggested that ERK5 S496 phosphorylation, but not ERK5 kinase activity, plays a crucial role in ERK5-mediated pro-inflammatory effects. We also discovered a key effect of ERK5 S496 phosphorylation on SUMOylation at a novel site of NRF2 (i.e., K518), which inhibited NRF2 transcriptional activity without affecting ERK5 kinase activity, and antagonized oxLDL-induced macrophage inflammation. The role of NRF2 activation on the efficiency of oxidative phosphorylation (OXPHOS) and ATP synthesis had previously been reported, and we found that both ERK5 S496A and NRF2 K518R mutants abolished oxLDL-induced reduction of OXPHOS, ATP, and NAD + levels. In summary, we discovered a novel mechanism in which ERK5 S496 phosphorylation directly inhibited NRF2 activity via SUMOylation of NRF2 at K518 and thereby induced macrophage inflammation and mitochondrial dysfunction. The often-neglected role of ERK S496 signaling should be carefully considered in the interpretation of prior reports of ERK5 knockdown and pharmacological kinase inhibition relative to cellular inflammation and mitochondrial dysfunction.

Breakthrough Gastrointestinal COVID-19 and Intrahost Evolution Consequent to Combination Monoclonal Antibody Prophylaxis.

Breakthrough gastrointestinal COVID-19 was observed after experimental SARS-CoV-2 upper mucosal infection in a rhesus macaque undergoing low-dose monoclonal antibody prophylaxis. High levels of viral RNA were detected in intestinal sites contrasting with minimal viral replication in upper respiratory mucosa. Sequencing of virus recovered from tissue in 3 gastrointestinal sites and rectal swab revealed loss of furin cleavage site deletions present in the inoculating virus stock and 2 amino acid changes in spike that were detected in 2 colon sites but not elsewhere, suggesting compartmentalized replication and intestinal viral evolution. This suggests suboptimal antiviral therapies promote viral sequestration in these anatomies.

The SETD8/ELK1/bach1 complex regulates hyperglycaemia-mediated EndMT in diabetic nephropathy

BackgroundDiabetic nephropathy (DN), the most common microvascular complication in patients with diabetes, induces kidney failure. Previous research showed that endothelial-to-mesenchymal transition (EndMT) of human glomerular endothelial cells (HGECs) is involved in the progression of DN. Moreover, SET domain-containing protein 8 (SETD8), ETS-domain containing protein (ELK1) and BTB and CNC homology 1 (bach1) all participate in endothelial injury. In this study, we hypothesize that the SETD8/ELK1/bach1 functional axis is involved in mediating EndMT in diabetic nephropathy.MethodsImmunohistochemistry, Western blotting and qPCR were performed to determine the protein and mRNA levels of genes in HGECs and the kidney tissues of participants and rats. Immunofluorescence, Co-IP and GST pulldown assays were performed to verify the direct interaction between SETD8 and ELK1. ChIP and dual-luciferase assays were performed to determine the transcriptional regulation of bach1 and Snail. AVV-SETD8 injection in rat kidney was used to verify the potential protective effect of SETD8 on DN.ResultsOur current study showed that hyperglycaemia triggered EndMT by increasing Snail expression both in vitro and in vivo. Moreover, high glucose increased bach1 expression in HGECs, positively regulating Snail and EndMT. As a transcription factor, ELK1 was augmented and participated in hyperglycaemia-induced EndMT via modulation of bach1 expression. Moreover, ELK1 was found to associate with SETD8. Furthermore, SETD8 negatively regulated EndMT by cooperating with bach1 to regulate Snail transcription. Furthermore, histone H4-Lys-20 monomethylation (H4K20me1), which is downstream of SETD8, was accompanied by ELK1 localization at the same promoter region of bach1. ELK1 overexpression enhanced bach1 promoter activity, which disappeared after specific binding site deletion. Mutual inhibition between ELK1 and SETD8 was found in HGECs. In vivo, SETD8 overexpression decreased ELK1 and bach1 expression, as well as EndMT. Moreover, SETD8 overexpression improved the renal function of rats with DN.ConclusionsSETD8 cooperates with ELK1 to regulate bach1 transcription, thus participating in the progression of DN. In addition, SETD8 interacts with bach1 to modulate Snail transcription, thus inducing EndMT in DN. SETD8 plays a core role in the SETD8/ELK1/bach1 functional axis, which participates in hyperglycaemia-mediated EndMT in DN, and SETD8 may be a potential therapeutic target for DN.Trial registration ChiCTR, ChiCTR2000029425. 2020/1/31, http://www.chictr.org.cn/showproj.aspx?proj=48548

Chitosan-based formulation of hemagglutinin antigens for oculo-nasal booster vaccination of chickens against influenza viruses

Avian influenza viruses (AIVs) and especially highly pathogenic (HP) AIVs of the H5 and H7 subtypes are of both veterinary and public health concern worldwide. In response to the demand for effective vaccines against H5N1 HPAIVs, we produced recombinant protein based on hemagglutinin (HA), a protective viral antigen. A fragment of the HA ectodomain, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. Finally, the protein was formulated in Tris-HCl buffer of pH 8.0 or PBS of pH 7.4 to obtain antigens denoted rH5–1 and rH5–2, respectively. The systemic prime and boost immunizations proved that rH5–1 adsorbed to aluminum hydroxide induces anti-H5 HA neutralizing antibodies and protective immune responses against H5N1 HPAIVs in chickens. The present studies were aimed at stimulating immune responses via the mucosal routes using the systemic prime-mucosal boost strategy. Efficacy trials were performed in commercial layer chickens. For systemic and mucosal immunizations, H5 HA antigens were adjuvanted with aluminum hydroxide and chitosan glutamate, respectively. The first dose of rH5–2 was administered subcutaneously, while its second dose was administered subcutaneously, intraocularly, oculo-nasally, or intranasally. rH5–1 was delivered to the subcutaneously primed chickens by the intranasal route. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition tests. Intraocularly and oculo-nasally delivered rH5–2 mixed with chitosan glutamate was capable of stimulating anti-H5 HA IgY antibody responses in the subcutaneously primed chickens; however, it was ineffective when administered by the intranasal route. Efficient intranasal boosting was achieved using rH5–1. The enhanced production of antigen-specific antibodies was reflected in the development of H5-subtype specific and hemagglutination inhibiting antibodies. Conclusively, the subcutaneous prime and oculo-nasal boost vaccination is proposed as the target strategy for future optimization.

Reduced Virulence and Enhanced Host Adaption during Antibiotics Therapy: a Story of a Within-Host Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 11 Evolution in a Patient with a Serious Scrotal Abscess.

ABSTRACTCarbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated globally and threatened human life. The sequence type (ST) 11 CRKP is a dominant clone in Asia, but how this clone evolves in vivo then adapts to the host and facilitates dissemination remains largely unknown. Here, the genomic dynamics of 4 ST11-CRKP isolates, which were sequentially collected from the urine of a patient with initial serious scrotal abscess and finally recovered without effective medication, were analyzed. Genomic differences were identified and their implications for pathogenesis and host adaptation were investigated. The related transcriptional pathways were further explored by RNA-Seq. Genomic analysis identified 4 to 24 mutations, among which 94% to 100% of them were synonymous or intergenic mutations. During 47 days of antibiotics therapy, CRKP underwent adaptive evolution, including tigecycline resistance and virulence attenuation. Tigecycline resistance was caused by a deletion within the ramR ribosomal binding site, which has been described by us previously. On the other hand, mutations associated with two genes, acyltransferase (act) and ompK26, resulted in the attenuation phenotype of ST11-CRKP. act deficiency reduced the capsular polysaccharide (CPS) production, enhanced biofilm formation, weakened capsular protection, and decreased induction of proinflammatory cytokines. Further RNA-Seq analysis revealed that act influenced the expression of ldhA, bglX, mtnK, and metE which likely participate in capsular synthesis and biofilm formation. ompK26 affected the virulence by its overexpression caused by the deletion of the upstream repressor binding site. This study presents a within-host adaption of ST11-CRKP and suggests an important role of CPS in the adaptive evolution of virulence and persistence of CRKP.IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated worldwide and can cause life-threatening infections, including pneumonia, bloodstream infections, urinary tract infections, intraabdominal infection, liver abscess, and meningitis. CRKP infection is the leading cause of high mortality in hospitals. The sequence type (ST) 11 CRKP is a dominant clone and accounts for 60% of CRKP infections in China. Recently, the ST11-CRKP with high transmissibility is increasingly identified. Understanding how this clone has evolved is crucial for developing strategies to control its further dissemination. The significance of our research is the identification of the in vivo genomic dynamics of ST11-CRKP and the genetic basis for ST11-CRKP that facilitate persistence and dissemination. Furthermore, our study also highlights the importance of monitoring the within-host evolution of pathogens during the treatment and developing interventions to minimize the potential impact of host adaptation on human health.

NHX-Type Na+/H+ Antiporter Gene Expression Under Different Salt Levels and Allelic Diversity of HvNHX in Wild and Cultivated Barleys.

Salinity tolerance is a multifaceted trait attributed to various mechanisms. Wild barley is highly specialized to grow under severe environmental conditions of Tibet and is well-known for its diverse germplasm with high tolerance to abiotic stresses. The present study focused on determining the profile of the expression of isoforms of the HvNHX gene in 36 wild and two cultivated barley under salt stress. Our findings revealed that in leaves and roots, expression of HvNHX1 and HvNHX3 in XZ16 and CM72 was upregulated at all times as compared with sensitive ones. The HvNHX2 and HvNHX4 isoforms were also induced by salt stress, although not to the same extent as HvNHX1 and HvNHX3. Gene expression analysis revealed that HvNHX1 and HvNHX3 are the candidate genes that could have the function of regulators of ions by sequestration of Na+ in the vacuole. HvNHX1 and HvNHX3 showed a wide range of sequence variations in an amplicon, identified via single-nucleotide polymorphisms (SNPs). Evaluation of the sequencing data of 38 barley genotypes, including Tibetan wild and cultivated varieties, showed polymorphisms, including SNPs, and small insertion and deletion (INDEL) sites in the targeted genes HvNHX1 and HvNHX3. Comprehensive analysis of the results revealed that Tibetan wild barley has distinctive alleles of HvNHX1 and HvNHX3 which confer tolerance to salinity. Furthermore, less sodium accumulation was observed in the root of XZ16 than the other genotypes as visualized by CoroNa-Green, a sodium-specific fluorophore. XZ16 is the tolerant genotype, showing least reduction of root and leaf dry weight under moderate (150 mM) and severe (300 mM) NaCl stress. Evaluation of genetic variation and identification of salt tolerance mechanism in wild barley could be promoting approaches to unravel the novel alleles involved in salinity tolerance.

SMRT and Illumina RNA-Seq Identifies Potential Candidate Genes Related to the Double Flower Phenotype and Unveils SsAP2 as a Key Regulator of the Double-Flower Trait in Sagittaria sagittifolia.

Double flowers are one of the important objectives of ornamental plant breeding. Sagittaria sagittifolia is an aquatic herb in the Alismataceae family that is widely used as an ornamental plant in gardens. However, the reference genome has not been published, and the molecular regulatory mechanism of flower formation remains unclear. In this study, single molecule real-time (SMRT) sequencing technology combined with Illumina RNA-Seq was used to perform a more comprehensive analysis of S. sagittifolia for the first time. We obtained high-quality full-length transcripts, including 53,422 complete open reading frames, and identified 5980 transcription factors that belonged to 67 families, with many MADS-box genes involved in flower formation being obtained. The transcription factors regulated by plant hormone signals played an important role in the development of double flowers. We also identified an AP2 orthologous gene, SsAP2, with a deletion of the binding site for miR172, that overexpressed SsAP2 in S. sagittifolia and exhibited a delayed flowering time and an increased number of petals. This study is the first report of a full-length transcriptome of S. sagittifolia. These reference transcripts will be valuable resources for the analysis of gene structures and sequences, which provide a theoretical basis for the molecular regulatory mechanism governing the formation of double flowers.

Abstract P4-02-11: A SIM2s/SEMA7A switch drives therapeutic resistance in ER+ breast cancer

Abstract BACKGROUND: Estrogen Receptor (ER) + breast cancers (BC) comprise over 70% of BC cases and can be targeted via ER modulated therapies. Despite this, ER+BC patients can experience recurrence within 20 years and the majority of BC related deaths can be attributed to metastatic ER+BC. These distant metastases commonly become diagnosed as endocrine therapy resistant. Thus, there is an unmet need to identify novel biomarkers for treating ER+ patients with metastasis. We have identified a tumor suppressor, Singleminded 2s (SIM2s), expressed in breast epithelial cells that inhibits EMT and metastasis, and is downregulated in the progression of breast disease. Our previous studies found that loss of SIM2s expression results in downregulation of ESR1 and increased basal markers in the MCF7 (ER+BC) cell line. Likewise, Semaphorin 7a (SEMA7A) expression is associated with decreased overall survival in ER+BC patients. Patients with SEMA7Ahi tumors treated with Tamoxifen exhibit shortened relapse free survival. SEMA7A is a downstream target of both NFKB and PTGS2/COX-2; and our previously published results establish a cross-talk between NFKB and SIM2s to regulate PTGS2 via interactions with the AKT pathway. Moreover, COX-2 inhibition is associated with better prognosis for breast cancer patients. Therefore, we are investigating how SIM2s may repress AKT signaling in SEMA7A driven ER resistance and metastasis. Methods: We performed in silico analysis of the SEMA7A promoter using the ConTra web server. For in vitro assays, we examined estrogen dependent differences in SIM2s, ER, and SP1 binding utilizing luciferase reporter assays using intact promoter and SIM2s site deletion mutants (Invitrogen) in MCF7 cells. Using immunoblotting and qPCR analysis we also assessed expression of SEMA7A, ESR1, PI3K/AKT signaling genes and EMT associated genes in Crispr/Cas9 SIM2s knockout (SIM2KO) MCF7 cells. Treatment of MCF7 cells with exogenous SEMA7A was used to observe possible changes in SIM2s expression and SIM2s promoter activity. For in vivo assays, we used immunohistochemistry (IHC) to assess SIM2s in SEMA7A low and high expressing MCF7 xenografts. To examine the relationship between SIM2s and ER, SEMA7A-/- (KO) mice were crossed with C57/BL6 MMTV-PYMT mice. Results: In silico analysis of the SEMA7A promoter via ConTra revealed four potential SIM2 response elements containing the SIM2s central midline elements (CME) binding motif and highly conserved estrogen response element half-sites adjacent to two specificity protein 1 consensus bindings sites. Luciferase reporter assays in MCF7 cells confirmed SIM2s represses basal and estrogen induced-SEMA7A promoter activity. Moreover, SIM2s was unable to repress the SEMA7A promoter activity with CME mutation. MCF7 SIM2KO cells exhibit increased SEMA7A promoter activity, SEMA7A, AKT signaling, EMT signatures and decreased ESR1, CDH1, and PTEN protein and mRNA expression. Both SIM2s expression and SIM2s promoter activity are significantly decreased with the addition of exogenous SEMA7A. Moreover, MCF7 xenografts reveal reciprocal expression of SIM2s and SEMA7A. Additionally in MMTV-PyMT mice, SIM2s expression is lost as tumors transition from pre-malignant to invasive phenotypes, yet we observe maintenance of SIM2s and ER in SEMA7A-/-;PyMT mice. Conclusion: These findings establish a regulatory relationship between SIM2s and SEMA7A in ER+BC. SIM2s functions to downregulate SEMA7A mediated pro-tumor signaling and maintain ER expression, but can be turned off to allow for resistance in ER+BC. Given the observed reciprocal expression, our study suggests a SIM2s/SEMA7A switch which may confer resistance in ER+BC via AKT signaling through downregulation of PTEN. Thus, a SIM2s/SEMA7A switch may act as a prognostic indicator to provide therapeutic advantages in resistant ER+BC metastasis. Citation Format: Garhett L Wyatt, Lyndsey S Crump, Traci R Lyons, Weston W Porter. A SIM2s/SEMA7A switch drives therapeutic resistance in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-11.

Dynamic Runx1 chromatin boundaries affect gene expression in hematopoietic development

The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increase in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells is associated with the formation of tissue-specific sub-TADs over Runx1, a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletion of promoter-proximal CTCF sites at the sub-TAD boundaries has no obvious effects on E-P interactions but leads to partial loss of domain structure, mildly affects gene expression, and delays hematopoietic development. Together, our analysis of gene regulation at a large multi-promoter developmental gene reveals that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.

Gene manipulation in the Mucorales fungus Rhizopus oryzae using TALENs with exonuclease overexpression.

The Mucorales fungal genus Rhizopus is used for the industrial production of organic acids, enzymes and fermented foods. The metabolic engineering efficiency of Rhizopus could be improved using gene manipulation; however, exogenous DNA rarely integrates into the host genome. Consequently, a genetic tool for Mucorales fungi needs to be developed. Recently, programmable nucleases that generate DNA double-strand breaks (DSBs) at specific genomic loci have been used for genome editing in various organisms. In this study, we examined gene disruption in Rhizopus oryzae using transcription activator-like effector nucleases (TALENs), with and without exonuclease overexpression. TALENs with an overexpressing exonuclease induced DSBs, followed by target site deletions. Although DSBs are repaired mainly by nonhomologous end joining in most organisms, our results suggested that in R. oryzae microhomology-mediated end joining was the major DSB repair system. Our gene manipulation method using TALENs coupled with exonuclease overexpression contributes to basic scientific knowledge and the metabolic engineering of Rhizopus.

GATA3 Truncation Mutants Alter EMT Related Gene Expression via Partial Motif Recognition in Luminal Breast Cancer Cells.

GATA3 is known to be one of the most frequently mutated genes in breast cancer. More than 10% of breast tumors carry mutations in this gene. However, the functional consequence of GATA3 mutations is still largely unknown. Clinical data suggest that different types of GATA3 mutations may have distinct roles in breast cancer characterization. In this study, we have established three luminal breast cancer cell lines that stably express different truncation mutants (X308 splice site deletion, C321 frameshift, and A333 frameshift mutants) found in breast cancer patients. Transcriptome analysis identified common and distinct gene expression patterns in these GATA3 mutant cell lines. In particular, the impacts on epithelial-to-mesenchymal transition (EMT) related genes are similar across these mutant cell lines. Chromatin localization of the mutants is highly overlapped and exhibits non-canonical motif enrichment. Interestingly, the A333 frameshift mutant expressed cells displayed the most significant impact on the GATA3 binding compared to X308 splice site deletion and C321fs mutants expressed cells. Our results suggest the common and different roles of GATA3 truncation mutations during luminal breast cancer development.

A novel mutation at the AMEL primer binding region on the Y chromosome in AMELY negative male

Gender identification in forensic DNA typing is an important tool for criminal investigation as well as STR typing. Most methods are based on a size difference of amelogenin X and Y (AMELX and AMELY). There have been some reports that the method by amelogenin (AMEL) incorrectly typed some males as females because the AMELY allele is not detected. AMELY allele dropout is often caused by deletions encompassing AMELY on Yp11.2 and accompanied with Y-STR allele dropout (especially DYS458). However, an unusual deletion was found in our laboratory. The AMELY allele could be recovered by using another commercial kit and alternative AMEL primer sets, and the Y-STR markers resulted in a complete profile. Sequencing results showed that there was an 8bp deletion in AMELY at the position corresponding to 41-48bp downstream from the 3' end of the 6bp deletion site in AMELX. This is considered a novel mutation at a primer binding region.

Visualization of copper nanoclusters for SARS-CoV-2 Delta variant detection based on rational primers design.

Here, based on the design of rational primers and copper nanoclusters (CuNCs), we present a method for the accurate detection of the SARS-CoV-2 Delta variant, which is capable of distinguishing the Delta variant with its single nucleotide polymorphism from the ‘wild type’ coronavirus (NC_045512.2), and realizing visualization signal out. Specifically, we show that dual priming oligonucleotide (DPO) primers and AT primers can be used to distinguish between wild types and mutations of this virus by polymerase chain reaction (PCR) analysis and that visualization can be achieved via the red fluorescence of CuNCs in ultraviolet radiation. Among the results, it was found that the R-1-down (DPO)-6I and F-1-30 AT, with the single nucleotide deletion site designed at the 3′ end of the downstream primer, showed the best specificity towards the Delta variant. Moreover, the use of AT primers increased the AT contents of the PCR products, thus meeting the template requirements generated by the CuNCs. It was also found that the AT primers could assist with improving detection specificity. Finally, we demonstrate that the visualization of the CuNCs-based detection assay exhibited a linear relationship in 0.5 pg μL−1–50 ng μL−1, with a limit of quantitation (LOQ) of 0.5 pg μL−1.

A premature stop codon in BrFLC2 transcript results in early flowering in oilseed-type Brassica rapa plants

Nonsense-mediated mRNA decay (NMD)-mediated degradation of BrFLC2 transcripts is the main cause of rapid flowering of oilseed-type B. rapa 'LP08' plants. Many Brassica species require vernalization (long-term winter-like cooling) for transition to the reproductive stage. In the past several decades, scientific efforts have been made to discern the molecular mechanisms underlying vernalization in many species. Thus, to identify the key regulators required for vernalization in Brassica rapa L., we constructed a linkage map composed of 7833 single nucleotide polymorphism markers using the late-flowering Chinese cabbage (B. rapa L. ssp. pekinensis) inbred line 'Chiifu' and the early-flowering yellow sarson (B. rapa L. ssp. trilocularis) line 'LP08' and identified a single major QTL on the upper-arm of the chromosome A02. In addition, we compared the transcriptomes of the lines 'Chiifu' and 'LP08' at five vernalization time points, including both non-vernalized and post-vernalization conditions. We observed that BrFLC2 was significantly downregulated in the early flowering 'LP08' and had two deletion sites (one at 4th exon and the other at 3' downstream region) around the BrFLC2 genomic region compared with the BrFLC2 genomic region in 'Chiifu'. Large deletion at 3' downstream region did not significantly affect transcription of both sense BrFLC2 transcript and antisense transcript, BrFLC2as along vernalization time course. However, the other deletion at 4th exon of BrFLC2 resulted in the generation of premature stop codon in BrFLC2 transcript in LP08 line. Cycloheximide treatment of LP08 line showed the de-repressed level of BrFLC2 in LP08, suggesting that low transcript level of BrFLC2 in LP08 might be caused by nonsense-mediated mRNA decay removing the nonsense transcript of BrFLC2. Collectively, this study provides a better understanding of the molecular mechanisms underlying floral transition in B. rapa.

Insertion-deletion systems with substitutions I

In this paper, we discuss the addition of substitutions as a further type of operations to (in particular, context-free) insertion-deletion systems, i.e., in addition to insertions and deletions we allow single letter replacements to occur. We investigate the effect of the addition of substitution rules on the context dependency of such systems, thereby also obtaining new characterizations of and even normal forms for context-sensitive (CS) and recursively enumerable (RE) languages and their phrase-structure grammars. More specifically, we prove that for each RE language, there is a system generating this language that only inserts and deletes strings of length two without considering the context of the insertion or deletion site, but which may change symbols (by a substitution operation) by checking a single symbol to the left of the substitution site. When we allow checking left and right single-letter context in substitutions, even context-free insertions and deletions of single letters suffice to reach computational completeness. When allowing context-free insertions only, checking left and right single-letter context in substitutions gives a new characterization of CS. This clearly shows the power of this new type of rules.

Fusion of the HMGA2 and BNC2 Genes in Uterine Leiomyoma With t(9;12)(p22;q14).

The translocation t(9;12) (p22;q14~15) has been reported in lipomas, pleomorphic adenomas, a myolipoma, two chondroid hamartomas, and two uterine leiomyomas. In lipomas and pleomorphic adenomas, the translocation fuses HMGA2 (12q14) with the NFIB gene from 9p22; in myolipoma, it fuses HMGA2 with C9orf92 from 9p22; and in chondroid hamartomas, fluorescence in situ hybridization (FISH) investigations showed the chromosomal aberration to cause intragenic rearrangement of HMGA2. The translocation's molecular consequence in a uterine leiomyoma is described here. A typical leiomyoma was investigated using banding cytogenetics, FISH, RNA sequencing, reverse transcription polymerase chain reaction and Sanger sequencing. A single translocation, t(9;12)(p22;q14) leading to an HMGA2::BNC2 chimera, was found in tumor cells. A sequence of the untranslated part of exon 5 of HMGA2 (nucleotide 1035 in the NCBI reference sequence NM_003483.4) had fused with a sequence from the untranslated part of exon 7 of BNC2 from 9p22 (nucleotide 9284 in reference sequence NM_017637.6). At the molecular level, the t(9;12)(p22;q14~15) found in several benign tumors appears to be heterogeneous fusing HMGA2 with either BNC2, C9orf92 or NFIB which all three map close to one another within a 3 Mbp region in 9p22. Because the fusion point in HMGA2 in the present tumor lays downstream from the first Let-7 miRNA consensus binding site, we conclude that deletion of the first Let-7 miRNA binding site is not important for the transcriptional upregulation of HMGA2 caused by the genomic rearrangement.