The role of glia in the dysregulation of neuronal spinogenesis in Ube3a-dependent ASD.

Overexpression of the Ube3a gene and the resulting increase in Ube3a protein are linked to autism spectrum disorder (ASD). However, the cellular and molecular processes underlying Ube3a-dependent ASD remain unclear. Using both male and female mice, we find that neurons in the somatosensory cortex of the Ube3a 2× Tg ASD mouse model display reduced dendritic spine density and increased immature filopodia density. Importantly, the increased gene dosage of Ube3a in astrocytes alone is sufficient to confer alterations in neurons as immature dendritic protrusions, as observed in primary hippocampal neuron cultures. We show that Ube3a overexpression in astrocytes leads to a loss of astrocyte-derived spinogenic protein, thrombospondin-2 (TSP2), due to a suppression of TSP2 gene transcription. By neonatal intraventricular injection of astrocyte-specific virus, we demonstrate that Ube3a overexpression in astrocytes in vivo results in a reduction in dendritic spine maturation in prelimbic cortical neurons, accompanied with autistic-like behaviors in mice. These findings reveal an astrocytic dominance in initiating ASD pathobiology at the neuronal and behavior levels. SIGNIFICANCE STATEMENT: Increased gene dosage of Ube3a is tied to autism spectrum disorders (ASDs), yet cellular and molecular alterations underlying autistic phenotypes remain unclear. We show that Ube3a overexpression leads to impaired dendritic spine maturation, resulting in reduced spine density and increased filopodia density. We find that dysregulation of spine development is not neuron autonomous, rather, it is mediated by an astrocytic mechanism. Increased gene dosage of Ube3a in astrocytes leads to reduced production of the spinogenic glycoprotein thrombospondin-2 (TSP2), leading to abnormalities in spines. Astrocyte-specific Ube3a overexpression in the brain in vivo confers dysregulated spine maturation concomitant with autistic-like behaviors in mice. These findings indicate the importance of astrocytes in aberrant neurodevelopment and brain function in Ube3a-depdendent ASD.

Pseudocyclic Arylbenziodoxaboroles as Water-Triggered Aryne Precursors in Reactions with Organic Sulfides.

Pseudocyclic arylbenziodoxaboroles are unique aryne precursors under neutral aqueous conditions that selectively react with organic sulfides, forming the corresponding sulfonium salts. This reaction is compatible with various substituents (alkyl, halogen, CN, NO2, CHO, and cyclopropyl) in the aromatic ring or alkyl group of the sulfide. Similar reactions of sulfoxides afford o-hydroxy-substituted sulfonium salts. The structures of key products were confirmed by X-ray analysis.

Synthesis and Biological Evaluation of Telmisartan Alkylamine Derivatives as Potential Anticancer Agents.

BACKGROUND/AIM: Telmisartan is an angiotensin II receptor type 1 (AT1) antagonist with anticancer properties against solid and hematological cancer cell lines. Using telmisartan as a template, we developed alkylamine derivatives with reduced AT1 activity but increased anticancer activity. MATERIALS AND METHODS: Synthesis of candidate compounds was carried out via hexafluorophosphate benzotriazole tetramethyl uronium coupling reaction, then their inhibition of cell proliferation was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and colony-formation assay was carried out on the lead candidate compound 8 Cell death via apoptosis or necrosis by compound 8 was determined by flow cytometry using annexin V and propidium iodide, tolerability dosing was carried out in ICR mice, and tumor-reduction properties were determined in an MDA-MB-231 xenograft model. RESULTS: Some of the synthesized candidates exhibited good inhibition of cell proliferation with low micromolar half maximal effective concentrations in triple-negative breast cancer cell lines MDA-MB-231 and 4T1. Compound 8 exhibited lower affinity towards AT1 than parent telmisartan, inhibition of colony formation, and cell-cycle analysis revealed apoptosis as potentially important in causing cell death. In vivo evaluation with compound 8 indicated that it was well tolerated at high concentrations in healthy mice. Additionally, compound 8 showed higher growth inhibition in the MDA-MB-231 tumor xenograft mouse model compared to telmisartan. CONCLUSION: Our study indicated that alkylamine derivatives of telmisartan exhibited good solubility and higher inhibition of cancer cell proliferation than telmisartan. Compound 8 was found to be a good lead compound, with potential for development as an anticancer agent.

Carbon-13 chemical shift tensor measurements for nitrogen-dense compounds.

This paper reports the principal values of the 13 C chemical shift tensors for five nitrogen-dense compounds (i.e., cytosine, uracil, imidazole, guanidine hydrochloride, and aminoguanidine hydrochloride). Although these are all fundamentally important compounds, the majority do not have 13 C chemical shift tensors reported in the literature. The chemical shift tensors are obtained from 1 H→13 C cross-polarization magic-angle spinning (CP/MAS) experiments that were conducted at a high field of 18.8 T to suppress the effects of 14 N-13 C residual dipolar coupling. Quantum chemical calculations using density functional theory are used to obtain the 13 C magnetic shielding tensors for these compounds. The best agreement with experiment arises from calculations using the hybrid functional PBE0 or the double-hybrid functional PBE0-DH, along with the triple-zeta basis sets TZ2P or pc-3, respectively, and intermolecular effects modeled using large clusters of molecules with electrostatic embedding through the COSMO approach. These measurements are part of an ongoing effort to expand the catalog of accurate 13 C chemical shift tensor measurements, with the aim of creating a database that may be useful for benchmarking the accuracy of quantum chemical calculations, developing nuclear magnetic resonance (NMR) crystallography protocols, or aiding in applications involving machine learning or data mining. This work was conducted at the National High Magnetic Field Laboratory as part of a 2-week school for introducing undergraduate students to practical laboratory experience that will prepare them for scientific careers or postgraduate studies.

GSK3 inhibition rescues growth and telomere dysfunction in dyskeratosis congenita iPSC-derived type II alveolar epithelial cells.

Dyskeratosis congenita (DC) is a rare genetic disorder characterized by deficiencies in telomere maintenance leading to very short telomeres and the premature onset of certain age-related diseases, including pulmonary fibrosis (PF). PF is thought to derive from epithelial failure, particularly that of type II alveolar epithelial (AT2) cells, which are highly dependent on Wnt signaling during development and adult regeneration. We use human induced pluripotent stem cell-derived AT2 (iAT2) cells to model how short telomeres affect AT2 cells. Cultured DC mutant iAT2 cells accumulate shortened, uncapped telomeres and manifest defects in the growth of alveolospheres, hallmarks of senescence, and apparent defects in Wnt signaling. The GSK3 inhibitor, CHIR99021, which mimics the output of canonical Wnt signaling, enhances telomerase activity and rescues the defects. These findings support further investigation of Wnt agonists as potential therapies for DC-related pathologies.

Synthesis and biological evaluation of N, N-dialkylcarboxy coumarin-NO donor conjugates as potential anticancer agents.

A series of nitric oxide (NO) donor furoxan conjugates of N, N-dialkylcarboxy coumarins have been synthesized as potential anticancer agents. The synthesized compounds have been tested for their in vitro antiproliferative activities on various cancer and noncancerous cell lines. The candidate derivatives exhibit selectivity towards cancer cells with excellent activities in low nM to µM concentrations. In vitro mechanistic studies indicate that the candidate compounds generate substantial NO, inhibit colony formation, and cause apoptosis in cancer cells. A preliminary in vivo tolerance study of the lead candidate 10 in mice indicates that it is well-tolerated, evidenced by zero mortality and normal body weight gains in treated mice. Further translation of the lead derivative 10 using MDA-MB-231 based tumor xenograft model shows good tumor growth reduction.

Synthesis and biological evaluation of novel 2-alkoxycarbonylallylester phosphonium derivatives as potential anticancer agents.

Several phosphonium derivatives have been synthesized from Baylis-Hillman (BH) reaction derived allyl bromides and aryl phosphines as mitochondria targeting anticancer agents. In vitro cell proliferation inhibition studies on various solid tumor cell lines indicate that most of the compounds exhibit IC50 values in µM concentrations. Further studies reveal that ß-substituted BH bromide derived phosphonium derivatives enhance the biological activity to low µM IC50 values. In vitrometabolic studies show that the lead candidate compound 16 inhibits the production of mitochondrial ATP, increases the proton leak within the mitochondrial membrane and abolishes the spare respiratory capacity in a concentration dependent manner.

Highly Parallel Profiling of Cas9 Variant Specificity.

Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.

RNA-guided DNA insertion with CRISPR-associated transposases.

CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.

Synthesis and biological evaluation of 2-alkoxycarbonylallyl esters as potential anticancer agents.

The reaction of carboxylic acids with Baylis-Hillman reaction derived α-bromomethyl acrylic esters readily provide 2-(alkoxycarbonyl)allyl esters in good to excellent yields. These functionalized allyl esters have been evaluated for their cell proliferation inhibition properties against breast cancer (MDA-MB-231 and 4T1) and pancreatic cancer (MIAPaCa-2) cell lines to explore their potential as anticancer agents. Several of the synthesized derivatives exhibit good potency against all three cancer cell lines. Our structure activity relationship (SAR) studies on 2-carboxycarbonyl allyl esters indicate that substituted aromatic carboxylic acids provide enhanced activity compared to substituted aliphatic carboxylic acid analogs. Di- and tri-allyl esters derived from di-and tri-carboxylic acids exhibit higher inhibition of cell proliferation than mono esters. Further SAR studies indicate that the double bond in the 2-(alkoxycarbonyl)allyl ester is required for its activity, and there is no increase in activity with increased chain length of the alkoxy group. Two lead candidate compounds have been identified from the cell proliferation inhibition studies and their preliminary mechanism of action as DNA damaging agents has been evaluated using epifluorescence and western blot analysis. One of the lead compounds has been further evaluated for its systemic toxicity in healthy CD-1 mice followed by anticancer efficacy in a triple negative breast cancer MDA-MB-231 xenograft model in NOD-SCID mice. These two in vivo studies indicate that the lead compound is well tolerated in healthy CD-1 mice and exhibits good tumor growth inhibition compared to breast cancer drug doxorubicin.