Potent Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain.

Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, the fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL (∼0.043 nM) and 3.18 ng/mL (∼0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnostic reagents for COVID-19.ImportanceTo date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics, including SARS-CoV-2 targeting antibodies, remains critical. Due to their small size (13-15 kDa), high solubility, and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multivalent formats than the conventional antibody. Here, we report a series of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.

FreeHi-C spike-in simulations for benchmarking differential chromatin interaction detection.

High-throughput genome-wide chromatin conformation capture assay (Hi-C) is routinely used to profile long-range genomic interactions and three-dimensional organization of genomes. A key application of Hi-C is the comparative analysis of genomic interactions across different time points, cellular conditions, or multiple stimuli. While operating characteristics of methods for Hi-C data processing such as normalization, pairwise interaction and higher-order organization detection have been relatively well studied, properties of methods for differential chromatin interaction detection are less investigated. We have recently developed FreeHi-C to enable data-driven non-parametric simulations from Hi-C experiments. Here, we extend FreeHi-C with a user/data-driven spike-in module to facilitate comparisons of differential chromatin interaction detection methods where the ground truth differential chromatin interactions are known under a wide variety of settings. We use FreeHi-C to benchmark four differential chromatin interaction detection methods, namely HiCcompare, multiHiCcompare, diffHic, and Selfish, using three comparative analysis settings with different sequencing depths and spike-in proportions. This comparison reveals distinguished performances in terms of the standard metrics such as the false discovery rate control, detection power, significance order, precision-recall curve, and receiver operating characteristic curve as well as overall genomic properties of the types of differential chromatin interactions detectable by each method. Furthermore, it highlights the lack of power for all methods in small replication settings.

A 40-50 GHz RF Front-End with Integrated Local Oscillator Leakage Calibration.

This article presents a transmitter (TX) front-end operating at frequencies covering 40-50 GHz, including a differential quadrature mixer with integrated amplitude and phase imbalance tuning, a power amplifier, and a detection mixer (DM) that supports local oscillator (LO) leakage signal or image signal calibration. Benefiting from the amplitude and phase imbalance tuning network of the in-phase quadrature (IQ) signal generator at the LO input, the TX exhibits more than 30 dBc image signal rejection over the full frequency band without any post-calibration. Based on the LO leakage signal fed back by the DM integrated at the RF output, the LO leakage of the TX has been improved by more than 10 dB through the LO leakage calibration module integrated in the quadrature mixer. When the intermediate frequency (IF) signal is fixed at 1 GHz, the TX's 1 dB compressed output power (OP1 dB) is higher than 13.5 dBm over the operating band. Thanks to the LO leakage signal calibration unit and the IQ signal generator, the TX is compliant with the error vector magnitude (EVM) requirement of the IEEE 802.11aj standard up to the 64-quadrature amplitude modulation (QAM) operating mode.

A 77 GHz Power Amplifier with 19.1 dBm Peak Output Power in 130 nm SiGe Process.

This article reports a two-stage differential structure power amplifier based on a 130 nm SiGe process operating at 77 GHz. By introducing a tunable capacitor for amplitude and phase balance at the center tap of the secondary coil of the traditional Marchand balun, the balun achieves amplitude imbalance less than 0.5 dB and phase imbalance less than 1 degree within the operating frequency range of 70-85 GHz, which enables the power amplifier to exhibit comparable output power over a wide operating frequency band. The power amplifier, based on a designed 3-bit digital analog convertor (DAC)-controlled base bias current source, exhibits small signal gain fluctuation of less than 5 dB and saturation output power fluctuation of less than 2 dB near the 80 GHz frequency point when the ambient temperature varies in the range of -40 °C to 125 °C. Benefiting from the aforementioned design, the tested single-path differential power amplifier exhibits a small signal gain exceeding 16 dB, a saturation output power exceeding 18 dBm, and a peak saturation output power of 19.1 dBm in the frequency band of 70-85 GHz.

Phase 1 dose-escalation study of SEA-CD40: a non-fucosylated CD40 agonist, in advanced solid tumors and lymphomas.

BACKGROUND: SEA-CD40 is an investigational, non-fucosylated, humanized monoclonal IgG1 antibody that activates CD40, an immune-activating tumor necrosis factor receptor superfamily member. SEA-CD40 exhibits enhanced binding to activating FcγRIIIa, possibly enabling greater immune stimulation than other CD40 agonists. A first-in-human phase 1 trial was conducted to examine safety, pharmacokinetics, and pharmacodynamics of SEA-CD40 monotherapy in patients with advanced solid tumors and lymphoma. METHODS: SEA-CD40 was administered intravenously to patients with solid tumors or lymphoma in 21-day cycles with standard 3+3 dose escalation at 0.6, 3, 10, 30, 45, and 60 µg/kg. An intensified dosing regimen was also studied. The primary objectives of the study were to evaluate the safety and tolerability and identify the maximum tolerated dose of SEA-CD40. Secondary objectives included evaluation of the pharmacokinetic parameters, antitherapeutic antibodies, pharmacodynamic effects and biomarker response, and antitumor activity. RESULTS: A total of 67 patients received SEA-CD40 including 56 patients with solid tumors and 11 patients with lymphoma. A manageable safety profile was observed, with predominant adverse events of infusion/hypersensitivity reactions (IHRs) reported in 73% of patients. IHRs were primarily ≤grade 2 with an incidence associated with infusion rate. To mitigate IHRs, a standardized infusion approach was implemented with routine premedication and a slowed infusion rate. SEA-CD40 infusion resulted in potent immune activation, illustrated by dose dependent cytokine induction with associated activation and trafficking of innate and adaptive immune cells. Results suggested that doses of 10-30 µg/kg may result in optimal immune activation. SEA-CD40 monotherapy exhibited evidence of antitumor activity, with a partial response in a patient with basal cell carcinoma and a complete response in a patient with follicular lymphoma. CONCLUSIONS: SEA-CD40 was tolerable as monotherapy and induced potent dose dependent immune cell activation and trafficking consistent with immune activation. Evidence of monotherapy antitumor activity was observed in patients with solid tumors and lymphoma. Further evaluation of SEA-CD40 is warranted, potentially as a component of a combination regimen. TRIAL REGISTRATION NUMBER: NCT02376699.

A 66-76 GHz Wide Dynamic Range GaAs Transceiver for Channel Emulator Application.

In this study, we developed a single-channel channel emulator module with an operating frequency covering 66-67 GHz, including a 66-76 GHz wide dynamic range monolithic integrated circuit designed based on 0.1 µm pHEMT GaAs process, a printed circuit board (PCB) power supply bias network, and low-loss ridge microstrip line to WR12 (60-90 GHz) waveguide transition structure. Benefiting from the on-chip multistage band-pass filter integrated at the local oscillator (LO) and radio frequency (RF) ends, the module's spurious components at the RF port were greatly suppressed, making the module's output power dynamic range over 50 dB. Due to the frequency-selective filter integrated in the LO chain, each clutter suppression in the LO chain exceeds 40 dBc. Up and down conversion loss of the module is better than 14 dB over the 66-67 GHz band, the measured IF input P1 dB is better than 10 dBm, and the module consumes 129 mA from a 5 V low dropout supply. A low-loss ridged waveguide ladder transition was designed (less than 0.4 dB) so that the output interface of the module is a WR12 waveguide interface, which is convenient for direct connection with an instrument with E-band (60-90 GHz) waveguide interface.

microRNA-449a suppresses non-small cell lung cancer.

This study was set to determine the expression of microRNA-449a in cancer tissue and serum of non-small cell lung cancer (NSCLC) patients and explore the underlying mechanisms of its tumor suppressor functions. We selected 50 NSCLC patients in our hospital as the lung cancer group and 50 healthy volunteers as the control group. RT-PCR was performed to detect the expression levels of microRNA-449a in NSCLC tissue and the plasma of NSCLC patients. Further, we transfected microRNA-449a mimic and inhibitor in lung cancer cell line A549, and used western blot to determine the expression level of apoptosis-related molecules Bcl-2 and p53. Compared with the surrounding tissue, microRNA-449a exhibited significantly reduced mRNA expression, which was statistically different (P < 0.05). microRNA-449a exhibited significantly lower expression in NSCLC patients' plasma than the healthy volunteers, which was statistically different (P < 0.05). Spearman correlation analysis showed that microRNA-449a expression levels in NSCLC tissue and plasma of NSCLC patients were reversely correlated with lung cancer differentiation (P < 0.05). But microRNA-449a expression in the surrounding tissue was not significantly correlated with lung cancer differentiation (P > 0.05). Compared with negative control, cell proliferation and p53 and Bcl-2 expression significantly decreased after microRNA-449a mimic transfection (P < 0.05). However, after transfection of microRNA-449a inhibitor, cell proliferation and p53 and Bcl-2 expression significantly increased after microRNA-449a mimic transfection (P < 0.05). microRNA-449a expression levels in NSCLC are significantly lower than those in the surrounding tissue, and its expression levels in NSCLC patients are also lower than those in healthy volunteers. The tumor suppression role of microRNA-449a could be due to its promotion of tumor cell proliferation and its inhibition of tumor cell apoptosis.

Synthesis and antioxidative activity of 2-substituted phenyl-5-(3'-indolyl)-oxazole derivatives.

AIM: To study the synthesis of 5-(3'-indolyl)-oxazoles and their antioxidative activity. METHODS: The amides were prepared from tryptophan and different acid derivatives by the catalytic dehydration of dicyclohexyl carbodiimide (DCC). The characteristic heterocyclic ring system of 5-(3'-indolyl)-oxazoles was constructed by oxidative cyclization of amide, using dicholorodicyanoquinone (DDQ). Their antioxidative activity in vitro was tested using DPPH system. RESULTS: Eleven 2-substituted phenyl-5-(3'-indolyl)-oxazoles were prepared, the compounds 21 and 22 have shown antioxidative activity 3-4 times stronger than that of Vit E, and the compound 29 showed antioxidative activity almost as same as Vit E. CONCLUSION: Three 5-(3'-indoyl)-oxazole compounds synthesized showed potent antioxidative effect and they would be a good antioxidants.

Characterization of odor-active compounds in cooked meat of farmed obscure puffer (Takifugu obscurus) using gas chromatography-mass spectrometry-olfactometry.

The volatile and odor-active compounds in cooked meat of farmed obscure puffer (Takifugu obscurus) were analyzed by gas chromatography-mass spectrometry-olfactometry (GC-MS-O). The volatile compounds were extracted by the simultaneous distillation-extraction (SDE) method, then separated and identified by GC-MS. Odor-active compounds in the SDE extract were characterized by GC-MS-O. A total of 68 volatile compounds were found, including 23 aldehydes, 10 alcohols, nine ketones, 17 N- or S-containing compounds and aromatics, three acids, three alkanes, and three esters. Of these, 31 odor-active compounds were detected and identified. Trimethylamine (fishy), octanal (grassy, leafy, green), (E)-2-octenal (roast, fatty), 1-octen-3-ol (fishy, fatty, mushroom, grassy), 2-ethyl-1-hexanethiol (cooked fish), (E,E)-2,4-octadienal (cooked meat, sweet), 2-acetylthiazole (meaty, roast, nutty, sulfur), 2-acetylpyrrole (nutty, walnut, bread) were identified as the key odorants in the cooked meat of farmed obscure puffer based on posterior intensity and time-intensity methods.

Antimicrobial effect of chitooligosaccharides produced by chitosanase from Pseudomonas CUY8.

The aim of this study was to investigate the antimicrobial effect of chitooligosaccharides prepared by chitosanase from Pseudomonas CUY8. Antimicrobial activities of different degrees of deacetylation (DD) and polymerization (DP) of chitooligosaccharides against various species of bacteria and fungi were measured. The antimicrobial effects of chitooligosaccharides compared with chitosan and chitosanase were evaluated. Inhibitory diameter of chitooligosaccharides at the concentration of 0.1% with DP 4 was 19 +/- 0.20mm, and inhibitory activity with DD 90% was 79+/- 2.1%, which were higher than other DP and DD, respectively. The results showed that antimicrobial activities of chitooligosaccharides increased with increase of DD, but decreased with increase of DP. Chitooligosaccharides, chitosan and chitosanase all showed significantly stronger antimicrobial activities against bacteria than fungi (p<0.001). Antimicrobial activities of chitooligosaccharides were significantly higher than that of chitosan (p<0.05), but insignificantly lower than that of chitosanase (p>0.05).