Cold atmospheric plasma stabilizes mismatch repair for effective, uniform treatment of diverse colorectal cancer cell types.

Mismatch Repair (MMR) mechanisms play a pivotal role in rectifying DNA replication errors and maintaining the stability of DNA microsatellite structure. Colorectal cancer (CRC) can be characterized into microsatellite stability (MSS) and microsatellite instability (MSI) subtypes based on the functionality of MMR. MSI CRC notably exhibits enhanced chemotherapy resistance, attributable to diminished MMR-related protein expression. Cold atmospheric plasma (CAP) has emerged as a promising treatment modality, demonstrating efficacy in inducing apoptosis in various cancer cells. However, the therapeutic impact of CAP on MSI colorectal cancer, and the underlying mechanisms remain elusive. In this study, we investigated the effects of CAP on MSI (MC38, HCT116, and LOVO) and MSS (CT26 and HT29) CRC cell lines. We are probing into the products of CAP treatment. Our findings indicate that CAP treatment induces comparable effects on apoptosis, reactive oxygen species (ROS), and reactive nitrogen species (RNS), as well as the expression of apoptosis-related proteins in both MSI and MSS cells. Mechanistically, CAP treatment led to an elevation in the expression of mismatch repair proteins (MLH1 and MSH2), particularly in MSI cells, which notably have been proven to facilitate the activation of apoptosis-related proteins. Collectively, our study reveals that CAP enhances apoptotic signaling and induces apoptosis in MSI colorectal cancer cells by upregulating the expression of MMR-related proteins, thereby reinforcing MMR stabilization.

Dose-Dependent Effects in Plasma Oncotherapy: Critical In Vivo Immune Responses Missed by In Vitro Studies.

Cold atmospheric plasma (CAP) generates abundant reactive oxygen and nitrogen species (ROS and RNS, respectively) which can induce apoptosis, necrosis, and other biological responses in tumor cells. However, the frequently observed different biological responses to in vitro and in vivo CAP treatments remain poorly understood. Here, we reveal and explain plasma-generated ROS/RNS doses and immune system-related responses in a focused case study of the interactions of CAP with colon cancer cells in vitro and with the corresponding tumor in vivo. Plasma controls the biological activities of MC38 murine colon cancer cells and the involved tumor-infiltrating lymphocytes (TILs). In vitro CAP treatment causes necrosis and apoptosis in MC38 cells, which is dependent on the generated doses of intracellular and extracellular ROS/RNS. However, in vivo CAP treatment for 14 days decreases the proportion and number of tumor-infiltrating CD8+T cells while increasing PD-L1 and PD-1 expression in the tumors and the TILs, which promotes tumor growth in the studied C57BL/6 mice. Furthermore, the ROS/RNS levels in the tumor interstitial fluid of the CAP-treated mice are significantly lower than those in the MC38 cell culture supernatant. The results indicate that low doses of ROS/RNS derived from in vivo CAP treatment may activate the PD-1/PD-L1 signaling pathway in the tumor microenvironment and lead to the undesired tumor immune escape. Collectively, these results suggest the crucial role of the effect of doses of plasma-generated ROS and RNS, which are generally different in in vitro and in vivo treatments, and also suggest that appropriate dose adjustments are required upon translation to real-world plasma oncotherapy.

A bispecific nanobody dimer broadly neutralizes SARS-CoV-1 & 2 variants of concern and offers substantial protection against Omicron via low-dose intranasal administration.

Current SARS-CoV-2 Omicron subvariants impose a heavy burden on global health systems by evading immunity from most developed neutralizing antibodies and vaccines. Here, we identified a nanobody (aSA3) that strongly cross-reacts with the receptor binding domain (RBD) of both SARS-CoV-1 and wild-type (WT) SARS-CoV-2. The dimeric construct of aSA3 (aSA3-Fc) tightly binds and potently neutralizes both SARS-CoV-1 and WT SARS-CoV-2. Based on X-ray crystallography, we engineered a bispecific nanobody dimer (2-3-Fc) by fusing aSA3-Fc to aRBD-2, a previously identified broad-spectrum nanobody targeting an RBD epitope distinct from aSA3. 2-3-Fc exhibits single-digit ng/mL neutralizing potency against all major variants of concerns including BA.5. In hamsters, a single systemic dose of 2-3-Fc at 10 mg/kg conferred substantial efficacy against Omicron infection. More importantly, even at three low doses of 0.5 mg/kg, 2-3-Fc prophylactically administered through the intranasal route drastically reduced viral RNA loads and completely eliminated infectious Omicron particles in the trachea and lungs. Finally, we discovered that 2(Y29G)-3-Fc containing a Y29G substitution in aRBD-2 showed better activity than 2-3-Fc in neutralizing BA.2.75, a recent Omicron subvariant that emerged in India. This study expands the arsenal against SARS-CoV-1, provides potential therapeutic and prophylactic candidates that fully cover major SARS-CoV-2 variants, and may offer a simple preventive approach against Omicron and its subvariants.

Evaluation and Comparison of Serological Methods for COVID-19 Diagnosis.

The worldwide pandemic of COVID-19 has become a global public health crisis. Various clinical diagnosis methods have been developed to distinguish COVID-19-infected patients from healthy people. The nucleic acid test is the golden standard for virus detection as it is suitable for early diagnosis. However, due to the low amount of viral nucleic acid in the respiratory tract, the sensitivity of nucleic acid detection is unsatisfactory. As a result, serological screening began to be widely used with the merits of simple procedures, lower cost, and shorter detection time. Serological tests currently include the enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), and chemiluminescence immunoassay (CLIA). This review describes various serological methods, discusses the performance and diagnostic effects of different methods, and points out the problems and the direction of optimization, to improve the efficiency of clinical diagnosis. These increasingly sophisticated and diverse serological diagnostic technologies will help human beings to control the spread of COVID-19.

ASC deglutathionylation is a checkpoint for NLRP3 inflammasome activation.

Activation of NLRP3 inflammasome is precisely controlled to avoid excessive activation. Although multiple molecules regulating NLRP3 inflammasome activation have been revealed, the checkpoints governing NLRP3 inflammasome activation remain elusive. Here, we show that activation of NLRP3 inflammasome is governed by GSTO1-promoted ASC deglutathionylation in macrophages. Glutathionylation of ASC inhibits ASC oligomerization and thus represses activation of NLRP3 inflammasome in macrophages, unless GSTO1 binds ASC and deglutathionylates ASC at ER, under control of mitochondrial ROS and triacylglyceride synthesis. In macrophages expressing ASCC171A, a mutant ASC without glutathionylation site, activation of NLRP3 inflammasome is GSTO1 independent, ROS independent, and signal 2 less dependent. Moreover, AscC171A mice exhibit NLRP3-dependent hyperinflammation in vivo. Our results demonstrate that glutathionylation of ASC represses NLRP3 inflammasome activation, and GSTO1-promoted ASC deglutathionylation at ER, under metabolic control, is a checkpoint for activating NLRP3 inflammasome.

Pulling-Force Spinning Top for Serum Separation Combined with Paper-Based Microfluidic Devices in COVID-19 ELISA Diagnosis.

The spread of Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), resulting in a global pandemic with around four million deaths. Although there are a variety of nucleic acid-based tests for detecting SARS-CoV-2, these methods have a relatively high cost and require expensive supporting equipment. To overcome these limitations and improve the efficiency of SARS-CoV-2 diagnosis, we developed a microfluidic platform that collected serum by a pulling-force spinning top and paper-based microfluidic enzyme-linked immunosorbent assay (ELISA) for quantitative IgA/IgM/IgG measurements in an instrument-free way. We further validated the paper-based microfluidic ELISA analysis of SARS-CoV-2 receptor-binding domain (RBD)-specific IgA/IgM/IgG antibodies from human blood samples as a good measurement with higher sensitivity compared with traditional IgM/IgG detection (99.7% vs 95.6%) for early illness onset patients. In conclusion, we provide an alternative solution for the diagnosis of SARS-CoV-2 in a portable manner by this smart integration of pulling-force spinning top and paper-based microfluidic immunoassay.