Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens.

Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes-such as transcriptional profiles-at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays.

Influence of electron donor and acceptor substituents on tuning the nonlinear optical properties of zinc phthalocyanine derivatives.

Four phthalocyanine derivatives with different electron donor and acceptor substituents (B1, B2, B3, and B4) were synthesized by a solid-phase melting method. The influence of substituent type on the nonlinear optical properties of the materials was investigated in detail. In the case of similar conjugated structures, B3, which has amino electron-donor groups, presents high intramolecular charge transfer, a low energy gap (2.05 eV), and good nonlinear optical properties. Compared with B3, B4 has a larger π-conjugated structure and its energy gap is 0.04 eV smaller. Moreover, B4 has stronger reverse saturation absorption (7 × 10-12 m/W) and optical limiting performance. The four phthalocyanine derivatives exhibit third harmonic generation (THG) characteristics. Furthermore, the third harmonic strengths of B1, B2, B3, and B4 are 4 times, 9 times, 11 times, and 21 times that of SiO2, respectively. So, B4 has the best application potential in laser protection and frequency conversion.

Mass Spectrometry-Based Proteomics Analysis Unveils PTPRS Inhibits Proliferation and Inflammatory Response of Keratinocytes in Psoriasis.

In this study, we used data-independent acquisition-mass spectrometry (DIA-MS) to analyze the serum proteome in psoriasis vulgaris (PsO). The serum proteomes of seven healthy controls and eight patients with PsO were analyzed using DIA-MS. Weighted gene co-expression network analysis was used to identify differentially expressed proteins (DEPs) that were closely related to PsO. Hub proteins of PsO were also identified. The Proteomics Drug Atlas 2023 was used to predict candidate hub protein drugs. To confirm the expression of the candidate factor, protein tyrosine phosphatase receptor S (PTPRS), in psoriatic lesions and the psoriatic keratinocyte model, immunohistochemical staining, quantitative real-time polymerase chain reaction, and western blotting were performed. A total of 129 DEPs were found to be closely related to PsO. The hub proteins for PsO were PVRL1, FGFR1, PTPRS, CDH2, CDH1, MCAM, and THY1. Five candidate hub protein drugs were identified: encorafenib, leupeptin, fedratinib, UNC 0631, and SCH 530348. PTPRS was identified as a common pharmacological target for these five drugs. PTPRS knockdown in keratinocytes promoted the proliferation and expression of IL-1α, IL-1ß, IL-23A, TNF-α, MMP9, CXCL8, and S100A9. PTPRS expression was decreased in PsO, and PTPRS negatively regulated PsO. PTPRS may be involved in PsO pathogenesis through the inhibition of keratinocyte proliferation and inflammatory responses and is a potential treatment target for PsO.

Nonlinear Optical Properties of Pyrene Derivatives Based on a Donor-Acceptor Structure and Its Polyurethane Composites.

Two pyrenyl Schiff base derivatives with π conjugated structures (B2 and B3) were designed and synthesized. Then, B2 and B3 were added into polyurethane to obtain doped and bonded polyurethane nonlinear optical materials (B2/PU and B3/PU), respectively. The synthesized B2, B3, and polyurethane nonlinear optical materials were tested by a nanosecond (ns) and picosecond (ps) pulse Z-scan at a 532 nm wavelength. Due to the two-photon absorption-induced excited state absorption (TPA-ESA), B2, B3, and polyurethane nonlinear optical materials show reverse saturable absorption (RSA). From a quantum chemistry calculation, it can be concluded that the RSA of B2 and B3 comes from the large π conjugated system and intramolecular charge transfer. Furthermore, B2, B3, and the polyurethane nonlinear optical materials show good optical limiting. B2/PU and B3/PU not only have excellent nonlinear optical properties but also have good transmittance, thermal stability, and processability of polyurethane materials. The combination of pyrenyl Schiff base derivatives and polyurethane materials greatly improves the application of nonlinear small molecules in the field of optical limiting and all-optical switching.

Two polyketide synthase-encoding genes are required for biosynthesis of the polyketide virulence factor, T-toxin, by Cochliobolus heterostrophus.

Cochliobolus heterostrophus race T, causal agent of southern corn leaf blight, requires T-toxin (a family of C35 to C49 polyketides) for high virulence on T-cytoplasm maize. Production of T-toxin is controlled by two unlinked loci, Tox1A and Tox1B, carried on 1.2 Mb of DNA not found in race O, a mildly virulent form of the fungus that does not produce T-toxin, or in any other Cochliobolus spp. or closely related fungus. PKS1, a polyketide synthase (PKS)-encoding gene at Tox1A, and DEC1, a decarboxylase-encoding gene at Tox1B, are necessary for T-toxin production. Although there is evidence that additional genes are required for T-toxin production, efforts to clone them have been frustrated because the genes are located in highly repeated, A+T-rich DNA. To overcome this difficulty, ligation specificity-based expression analysis display (LEAD), a comparative amplified fragment length polymorphism/gel fractionation/capillary sequencing procedure, was applied to cDNAs from a near-isogenic pair of race T (Tox1+) and race O (Tox1-) strains. This led to discovery of PKS2, a second PKS-encoding gene that maps at Tox1A and is required for both T-toxin biosynthesis and high virulence to maize. Thus, the carbon chain of each T-toxin family member likely is assembled by action of two PKSs, which produce two polyketides, one of which may act as the starter unit for biosynthesis of the mature T-toxin molecule.

A novel link between the proteasome pathway and the signal transduction pathway of the bone morphogenetic proteins (BMPs).

BACKGROUND: The intracellular signaling events of the bone morphogenetic proteins (BMPs) involve the R-Smad family members Smad1, Smad5, Smad8 and the Co-Smad, Smad4. Smads are currently considered to be DNA-binding transcriptional modulators and shown to recruit the master transcriptional co-activator CBP/p300 for transcriptional activation. SNIP1 is a recently discovered novel repressor of CBP/p300. Currently, the detailed molecular mechanisms that allow R-Smads and Co-Smad to co-operatively modulate transcription events are not fully understood. RESULTS: Here we report a novel physical and functional link between Smad1 and the 26S proteasome that contributes to Smad1- and Smad4-mediated transcriptional regulation. Smad1 forms a complex with a proteasome beta subunit HsN3 and the ornithine decarboxylase antizyme (Az). The interaction is enhanced upon BMP type I receptor activation and occur prior to the incorporation of HsN3 into the mature 20S proteasome. Furthermore, BMPs trigger the translocation of Smad1, HsN3 and Az into the nucleus, where the novel CBP/p300 repressor protein SNIP1 is further recruited to Smad1/HsN3/Az complex and degraded in a Smad1-, Smad4- and Az-dependent fashion. The degradation of the CBP/p300 repressor SNIP1 is likely an essential step for Smad1-, Smad4-mediated transcriptional activation, since increased SNIP1 expression inhibits BMP-induced gene responses. CONCLUSIONS: Our studies thus add two additional important functional partners of Smad1 into the signaling web of BMPs and also suggest a novel mechanism for Smad1 and Smad4 to co-modulate transcription via regulating proteasomal degradation of CBP/p300 repressor SNIP1.

Analysis of differential gene expression by ligation specificity-based transcript profiling.

An open architecture mRNA profiling technology, LEAD (Ligation specificity-based Expression Analysis Display), was developed for studying differential gene expression. This method utilizes restriction enzymes with N(m) degeneracy in their recognition/cleavage sequences to fractionate cDNA population (N represents any one of the four possible bases; while m > or = 1, represents the number of degenerate bases). The fractionated cDNAs are subpooled by selective ligation with specific adapters, and then amplified and labeled by PCR. Fluorescent-labeled cDNA fingerprints are separated by electrophoresis as distinct bands with unique size and sequence, and quantified electronically by using the LEAD Finder program. The specificity of ligation and the uniform efficiency of PCR reaction allow precise quantification of differential gene expression among samples. Transcripts of low abundance (1/100,000 copies) can be detected, allowing the status of nearly all mRNA to be monitored. Because of its sequence independence, this technology can be used to monitor gene expression in both model and nonmodel systems lacking whole genome information. It can also be applied to separate and collect different cDNA species fingerprints to build a nonredundant EST library for microarray and other applications.

Micropreparative capillary gel electrophoresis of DNA: rapid expressed sequence tag library construction.

A capillary gel electrophoresis based automated DNA fraction collection technique was developed to support a novel DNA fragment-pooling strategy for expressed sequence tag (EST) library construction. The cDNA population is first cleaved by BsaJ I and EcoR I restriction enzymes, and then subpooled by selective ligation with specific adapters followed by polymerase chain reaction (PCR) amplification and labeling. Combination of this cDNA fingerprinting method with high-resolution capillary gel electrophoresis separation and precise fractionation of individual cDNA transcript representatives avoids redundant fragment selection and concomitant repetitive sequencing of abundant transcripts. Using a computer-controlled capillary electrophoresis device the transcript representatives were separated by their size and fractions were automatically collected in every 30 s into 96-well plates. The high resolving power of the sieving matrix ensured sequencing grade separation of the DNA fragments (i.e., single-base resolution) and successful fraction collection. Performance and precision of the fraction collection procedure was validated by PCR amplification of the collected DNA fragments followed by capillary electrophoresis analysis for size and purity verification. The collected and PCR-amplified transcript representatives, ranging up to several hundred base pairs, were then sequenced to create an EST library.