Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a.

CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro, and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a ( = 0.

Massively parallel profiling of RNA-targeting CRISPR-Cas13d.

CRISPR-Cas13d cleaves RNA and is used in vivo and for diagnostics. However, a systematic understanding of its RNA binding and cleavage specificity is lacking. Here, we describe an RNA Chip-Hybridized Association-Mapping Platform (RNA-CHAMP) for measuring the binding affinity for > 10,000 RNAs containing structural perturbations and other alterations relative to the CRISPR RNA (crRNA).

Massively Parallel Profiling of RNA-targeting CRISPR-Cas13d.

Type VI CRISPR enzymes cleave target RNAs and are widely used for gene regulation, RNA tracking, and diagnostics. However, a systematic understanding of their RNA binding specificity and cleavage activation is lacking. Here, we describe RNA chip-hybridized association-mapping platform (RNA-CHAMP), a massively parallel platform that repurposes next-generation DNA sequencing chips to measure the binding affinity for over 10,000 RNA targets containing structural perturbations, mismatches, insertions, and deletions relative to the CRISPR RNA (crRNA).

Antibody escape and cryptic cross-domain stabilization in the SARS-CoV-2 Omicron spike protein.

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the repeated emergence of variants of concern. For the Omicron variant, sub-lineages BA.1 and BA.

Structure-based design of prefusion-stabilized human metapneumovirus fusion proteins.

The human metapneumovirus (hMPV) fusion (F) protein is essential for viral entry and is a key target of neutralizing antibodies and vaccine development. The prefusion conformation is thought to be the optimal vaccine antigen, but previously described prefusion F proteins expressed poorly and were not well stabilized. Here, we use structures of hMPV F to guide the design of 42 variants containing stabilizing substitutions.

Stabilized coronavirus spike stem elicits a broadly protective antibody.

Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive β-CoV antibody responses and protects mice against lethal MERS-CoV challenge.

Expression and characterization of SARS-CoV-2 spike proteins.

The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells.

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes.

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.

Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area.

We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently.

Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area.

We sequenced the genomes of 5,085 SARS-CoV-2 strains causing two COVID-19 disease waves in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston, and an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently.

Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes.

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike.

Synthesis of the extended phenacene molecules, [10]phenacene and [11]phenacene, and their performance in a field-effect transistor.

The [10]phenacene and [11]phenacene molecules have been synthesized using a simple repetition of Wittig reactions followed by photocyclization. Sufficient amounts of [10]phenacene and [11]phenacene were obtained, and thin-film FETs using these molecules have been fabricated with SiO and ionic liquid gate dielectrics. These FETs operated in p-channel.

Induction of c-Cbl contributes to anti-cancer effects of HDAC inhibitor in lung cancer.

Here we found loss of c-Cbl, an E3 ligase, expression in non-small cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens. HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl. Ectopic expression of c-Cbl induced decreased EGFR, inhibited growth in NSCLC cells.

A MedRadio-band low-energy-per-bit 4-Mbps CMOS OOK receiver for implantable medical devices.

A 4-Mbps 400-MHz On-Off Keying (OOK) receiver implemented in 0.18-um CMOS technology for implantable epilepsy sense-and-stimulation devices is presented. The proposed receiver is composed of a new current-mode full-wave envelope detector and differential cascaded gain amplifiers which is operated at MedRadio band.

HDAC inhibition decreases the expression of EGFR in colorectal cancer cells.

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase which promotes cell proliferation and survival, is abnormally overexpressed in numerous tumors of epithelial origin, including colorectal cancer (CRC). EGFR monoclonal antibodies have been shown to increase the median survival and are approved for the treatment of colorectal cancer. Histone deacetylases (HDACs), frequently overexpressed in colorectal cancer and several malignancies, are another attractive targets for cancer therapy.

TNF-alpha suppressed TGF-beta-induced CTGF expression by switching the binding preference of p300 from Smad4 to p65.

TGF-beta regulates diverse biologic effects including cell growth, cell death or apoptosis, cell differentiation, and extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF), induced by TGF-beta has been reported to mediate stimulatory action of TGF-beta-induced ECM. Although TNF-alpha was reported to suppress theTGF-beta-induced CTGF gene expression, the molecular mechanism is not well clarified.

HDAC inhibition upregulates the expression of angiostatic ADAMTS1.

HDAC inhibitors are promising anticancer agents that induce cell cycle arrest and apoptosis. However, the role of HDACs in cancer progression, such as angiogenesis and metastasis, remains largely unexplored. Among various HDAC inhibitors, we demonstrate that TSA and SAHA upregulated the expression of angiostatic ADAMTS1 in A549 cells.

Proteasome inhibitors enhance TRAIL-induced apoptosis through the intronic regulation of DR5: involvement of NF-kappa B and reactive oxygen species-mediated p53 activation.

Manipulation of TRAIL receptor 2 (DR5) pathway is a promising therapeutic strategy to overcome TRAIL-resistant lung cancer cells. Preclinical studies have shown that proteasome inhibitors enhance TRAIL-induced apoptosis in lung cancer cells, but the underlying mechanism has not been fully elucidated. In this study, we demonstrated the enhancement of TRAIL-mediated apoptosis in human alveolar epithelial cells by proteasome inhibitors that up-regulate DR5 expression.

Statins increase p21 through inhibition of histone deacetylase activity and release of promoter-associated HDAC1/2.

Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors broadly used for the control of hypercholesterolemia. Recently, they are reported to have beneficial effects on certain cancers. In this study, we show that statins inhibited the histone deacetylase (HDAC) activity and increased the accumulation of acetylated histone-H3 and the expression of p21(WAF/CIP) in human cancer cells.