Benefit-finding profiles and comparison of caregiving ability among informal caregivers of patients with lung cancer: A latent profile analysis.

Objective: This study aimed to explore the benefit finding (BF) profiles among informal caregivers of patients with lung cancer, identify demographic and disease characteristics, and analyze differences in caregiving ability between profiles. Methods: This cross-sectional study utilized convenience sampling to select 272 informal caregivers of patients with lung cancer from a tertiary care hospital in Guangzhou, China. The research instruments used included the Demographic and Disease Characteristics Questionnaire, the revised version of the BF Scale, and the Chinese version of the Family Caregiver Task Inventory. Data analysis was performed using latent profile analysis, chi-square test, Fisher's exact probability test, Kruskal-Wallis test, and multivariate logistic regression. Results: (1) BF can be divided into three profiles: "high benefit-family and personal growth" (Profile 1, 7.7%), "moderate benefit-unclear perception" (Profile 2, 44.9%), and "low benefit-coping ability deficient" (Profile 3, 47.4%). (2) Having a cocaregiver and a disease duration of 6-12 months were more likely to belong to Profile 1; caregivers of patients aged 40-60 years tended to belong to Profile 2; caregivers of older patients with disease duration > 12 months and clinical stage II or III were more likely to belong to Profile 3. (3) There were significant differences in the total score of caregiving ability and the scores of each dimension among the different BF profiles (P < 0.001), and the caregiving abilities of Profile 1 and Profile 2 were higher than those of Profile 3. Conclusions: There was heterogeneity in BF among informal caregivers of patients with lung cancer. Healthcare professionals can identify the key profiles of lung-cancer caregivers based on characteristics such as age, clinical stage, disease duration, and cocaregiver status and enhance their caregiving ability through targeted nursing guidance.

An intranasal live-attenuated SARS-CoV-2 vaccine limits virus transmission.

The development of effective SARS-CoV-2 vaccines has been essential to control COVID-19, but significant challenges remain. One problem is intramuscular administration, which does not induce robust mucosal immune responses in the upper airways-the primary site of infection and virus shedding. Here we compare the efficacy of a mucosal, replication-competent yet fully attenuated virus vaccine, sCPD9-ΔFCS, and the monovalent mRNA vaccine BNT162b2 in preventing transmission of SARS-CoV-2 variants B.1 and Omicron BA.5 in two scenarios. Firstly, we assessed the protective efficacy of the vaccines by exposing vaccinated male Syrian hamsters to infected counterparts. Secondly, we evaluated transmission of the challenge virus from vaccinated and subsequently challenged male hamsters to naïve contacts. Our findings demonstrate that the live-attenuated vaccine (LAV) sCPD9-ΔFCS significantly outperformed the mRNA vaccine in preventing virus transmission in both scenarios. Our results provide evidence for the advantages of locally administered LAVs over intramuscularly administered mRNA vaccines in preventing infection and reducing virus transmission.

SEPTIN2 suppresses an IFN-γ-independent, proinflammatory macrophage activation pathway.

Interferon-gamma (IFN-γ) signaling is necessary for the proinflammatory activation of macrophages but IFN-γ-independent pathways, for which the initiating stimuli and downstream mechanisms are lesser known, also contribute. Here we identify, by high-content screening, SEPTIN2 (SEPT2) as a negative regulation of IFN-γ-independent macrophage autoactivation. Mechanistically, endoplasmic reticulum (ER) stress induces the expression of SEPT2, which balances the competition between acetylation and ubiquitination of heat shock protein 5 at position Lysine 327, thereby alleviating ER stress and constraining M1-like polarization and proinflammatory cytokine release. Disruption of this negative feedback regulation leads to the accumulation of unfolded proteins, resulting in accelerated M1-like polarization, excessive inflammation and tissue damage. Our study thus uncovers an IFN-γ-independent macrophage proinflammatory autoactivation pathway and suggests that SEPT2 may play a role in the prevention or resolution of inflammation during infection.

Synthesis of Two-Dimensional (Cu-S)n Metal-Organic Framework Nanosheets Applied as Peroxidase Mimics for Detection of Glutathione.

Facilely synthesized peroxidase-like nanozymes with high catalytic activity and stability may serve as effective biocatalysts. The present study synthesizes peroxidase-like nanozymes with multinuclear active sites using two-dimensional (2D) metal-organic framework (MOF) nanosheets and evaluates them for their practical applications. A simple method involving a one-pot bottom-up reflux reaction is developed for the mass synthesis of (Cu-S)n MOF 2D nanosheets, significantly increasing production quantity and reducing reaction time compared to traditional autoclave methods. The (Cu-S)n MOF 2D nanosheets with the unique coordination of Cu(I) stabilized in Cu-based MOFs demonstrate impressive activity in mimicking natural peroxidase. The active sites of the peroxidase-like activity of (Cu-S)n MOF 2D nanosheets were predominantly verified as Cu(I) rather than Cu(II) of other Cu-based MOFs. The cost-effective and long-term stability of (Cu-S)n MOF 2D nanosheets make them suitable for practical applications. Furthermore, the inhibition of the peroxidase-like activity of (Cu-S)n MOF nanosheets by glutathione (GSH) could provide a simple strategy for colorimetric detection of GSH against other amino acids. This work remarkably extends the utilization of (Cu-S)n MOF 2D nanosheets in biosensing, revealing the potential for 2D (Cu-S)n MOFs.

The NSP4 T492I mutation increases SARS-CoV-2 infectivity by altering non-structural protein cleavage.

The historically dominant SARS-CoV-2 Delta variant and the currently dominant Omicron variants carry a T492I substitution within the non-structural protein 4 (NSP4). Based on in silico analyses, we hypothesized that the T492I mutation increases viral transmissibility and adaptability, which we confirmed with competition experiments in hamster and human airway tissue culture models. Furthermore, we showed that the T492I mutation increases the replication capacity and infectiveness of the virus and improves its ability to evade host immune responses. Mechanistically, the T492I mutation increases the cleavage efficiency of the viral main protease NSP5 by enhancing enzyme-substrate binding, which increases production of nearly all non-structural proteins processed by NSP5. Importantly, the T492I mutation suppresses viral-RNA-associated chemokine production in monocytic macrophages, which may contribute to the attenuated pathogenicity of Omicron variants. Our results highlight the importance of NSP4 adaptation in the evolutionary dynamics of SARS-CoV-2.

Annao Pingchong decoction alleviate the neurological impairment by attenuating neuroinflammation and apoptosis in intracerebral hemorrhage rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Intracerebral hemorrhage (ICH) is a central nervous system disease that causes severe disability or death. Even though Annao Pingchong decoction (ANPCD), a traditional Chinese decoction, has been used clinically to treat ICH in China, its molecular mechanism remains unclear. AIM OF THE STUDY: To study whether the neuroprotective effect of ANPCD on ICH rats is achieved by alleviating neuroinflammation. This paper mainly explored whether inflammation-related signaling pathways (HMGB1/TLR4/NF-κB P65) plays a role in ANPCD treatment of ICH rats. MATERIALS AND METHODS: Liquid chromatography-tandem mass spectrometry was used to analyze the chemical composition of ANPCD. ICH models were established by injecting autologous whole blood into the left caudate nucleus of Sprague-Dawley (SD) rats. Modified neurological severity scoring (mNSS) was used to assess the neurological deficits. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 were analyzed using enzyme-linked immunosorbent assay (ELISA). Pathological changes in the rat brains were observed using hematoxylin-eosin, Nissl, and TUNEL staining. The protein levels of HMGB1, TLR4, NF-κB p65, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) were measured by western blotting and immunofluorescence analysis. RESULTS: Ninety-three ANPCD compounds were identified, including 48 active plasma components. Treatment with ANPCD effectively improved the outcome, as observed by the neurological function scores analysis and brain histopathology. Our results showed that ANPCD exerts its anti-inflammatory effects by significantly downregulating the expression of HMGB1, TLR4, NF-κB p65, TNF-α, IL-1ß, and IL-6. ANPCD also exerted anti-apoptotic effects by significantly decreasing the apoptosis rate and Bax/Bcl-2 ratio. CONCLUSION: We found that ANPCD had neuroprotective effect in clinical work. Here, we also found that the action mechanism of ANPCD might be related to attenuate neuroinflammation and apoptosis. These effects were achieved by inhibiting the expression of HMGB1, TLR4 and NF-κB p65.

Bacteria and macrophages in the tumor microenvironment.

Cancer and microbial infections are significant worldwide health challenges. Numerous studies have demonstrated that bacteria may contribute to the emergence of cancer. In this review, we assemble bacterial species discovered in various cancers to describe their variety and specificity. The relationship between bacteria and macrophages in cancer is also highlighted, and we look for ample proof to establish a biological basis for bacterial-induced macrophage polarization. Finally, we quickly go over the potential roles of metabolites, cytokines, and microRNAs in the regulation of the tumor microenvironment by bacterially activated macrophages. The complexity of bacteria and macrophages in cancer will be revealed as we gain a better understanding of their pathogenic mechanisms, which will lead to new therapeutic approaches for both inflammatory illnesses and cancer.

Functional studies of HLA and its role in SARS-CoV-2: Stimulating T cell response and vaccine development.

In the biological immune process, the major histocompatibility complex (MHC) plays an indispensable role in the expression of HLA molecules in the human body when viral infection activates the T-cell response to remove the virus. Since the first case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in 2019, how to address and prevent SARS-CoV-2 has become a common problem facing all mankind. The T-cell immune response activated by MHC peptides is a way to construct a defense line and reduce the transmission and harm of the virus. Presentation of SARS-CoV-2 antigen is associated with different types of HLA phenotypes, and different HLA phenotypes induce different immune responses. The prediction of SARS-CoV-2 mutation information and the design of vaccines based on HLAs can effectively activate autoimmunity and cope with virus mutations, which can provide some references for the prevention and treatment of SARS-CoV-2.

Leonurine Regulates Hippocampal Nerve Regeneration in Rats with Chronic and Unpredictable Mild Stress by Activating SHH/GLI Signaling Pathway and Restoring Gut Microbiota and Microbial Metabolic Homeostasis.

Depression is a highly prevalent and heterogeneous disorder that requires new strategies to overcome depression. In this study, we aimed to investigate whether leonurine modulated hippocampal nerve regeneration in chronic and unpredictable mild stress (CUMS) rats through the SHH/GLI signaling pathway and restoring gut microbiota and microbial metabolic homeostasis. The CUMS rat model was constructed and treated with leonurine. The body weight of rats was recorded, and a series of tests were performed. Western blot was utilized to measure the expression of BDNF and 5-HT in the hippocampus. Then the expression of SHH, GLI, PTCH, and SMO were measured by qRT-PCR and western blot. The colocalization of BrdU+DCX and BrdU+NeuN was evaluated by IF. 16S rDNA high-throughput sequencing was applied to detect the composition and distribution of gut microbiota. The differential metabolites were analyzed by untargeted metabolomics. The correlation between gut microbiota and microbial metabolites was analyzed by Pearson correlation coefficient. After CUMS modeling, the body weight of rats was decreased, and the expression of BDNF and 5-HT were decreased, while the body weight was recovered, and the expression of BDNF and 5-HT were increased after leonurine treatment. Leonurine reversed the reduction in the colocalization of BrdU+DCX and BrdU+NeuN and the reduction in the levels of SHH, GLI, PTCH, and SMO induced by CUMS modeling. Leonurine also restored gut microbiota and microbial metabolites homeostasis in CUMS rats. Furthermore, Prevotellaceae_Ga6A1_group was negatively correlated with 3-Oxocholic acid, nutriacholic acid, and cholic acid. Collectively, leonurine regulated hippocampal nerve regeneration in CUMS rats by activating the SHH/GLI signaling pathway and restoring gut microbiota and microbial metabolic homeostasis.

Small molecule Z363 co-regulates TAF10 and MYC via the E3 ligase TRIP12 to suppress tumour growth.

BACKGROUND: The MYC oncoprotein, also known as the master regulator of genes, is a transcription factor that regulates numerous physiological processes, including cell cycle control, apoptosis, protein synthesis and cell adhesion, among others. MYC is overexpressed in approximately 70% of human cancers. Given its pervasive role in cancer biology, MYC down-regulation has become an attractive cancer treatment strategy. METHODS: The CRISPR/Cas9 method was used to produce KO cell models. Western blot was used to analyzed the expressions of MYC and TATA-binding proteinassociated factors 10 (TAF10) in cancer cells (MCF7, A549, HepG2 cells) Cell culture studies were performed to determine the mechanisms by which small molecules (Z363119456, Z363) affects MYC and TAF10 expressions and functions. Mouse studies were carried out to investigate the impact of Z363 regulation on tumor growth. RESULTS: Z363 activate Thyroid hormone Receptor-interacting Protein 12 (TRIP12), which phosphorylates MYC at Thr58, resulting in MYC ubiquitination and degradation and thereby regulating MYC target genes. Importantly, TRIP12 also induces TAF10 degradation, which reduces MYC protein levels. TRIP12, an E3 ligase, controls MYC levels both directly and indirectly by inhibiting MYC or TAF10 activity. CONCLUSIONS: In summary,these results demonstrate the anti-cancer properties of Z363, a small molecule that is co-regulated by TAF10 and MYC.

Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection.

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. Conventionally secreted ORF8 is glycosylated and loses the ability to recognize interleukin 17 receptor A of macrophages, possibly due to the steric hindrance imposed by N-glycosylation at Asn78. However, unconventionally secreted ORF8 does not undergo glycosylation without experiencing the ER-Golgi trafficking, thereby activating the downstream NF-κB signaling pathway and facilitating a burst of cytokine release. Furthermore, we show that ORF8 deletion in SARS-CoV-2 attenuates inflammation and yields less lung lesions in hamsters. Our data collectively highlights a role of ORF8 protein in the development of cytokine storms during SARS-CoV-2 infection.

Fauna access outweighs litter mixture effect during leaf litter decomposition.

Decomposition rates of litter mixtures reflect the combined effects of litter species diversity, litter quality, decomposers, their interactions with each other and with the environment. The outcomes of those interactions remain ambiguous and past studies have reported conflicting results (e.g., litter mixture richness effects). To date, how litter diversity and soil fauna interactions shape litter mixture decomposition remains poorly understood. Through a sixteen month long common garden litter decomposition experiment, we tested these interaction effects using litterbags of three mesh sizes (micromesh, mesomesh, and macromesh) to disentangle the contributions of different fauna groups categorized by their size at Wuhan botanical garden (subtropical climate). We examined the decomposition of five single commonly available species litters and their full 26 mixtures combination spanning from 2 to 5 species. In total, 2325 litterbags were incubated at the setup of the experiment and partly harvested after 1, 3, 6, 9, and 16 months after exposure to evaluate the mass loss and the combined effects of soil fauna and litter diversity. We predicted that litter mixture effects should increase with increased litter quality dissimilarity, and soil fauna should enhance litter (both single species litter and litter mixtures) decomposition rate. Litter mass loss ranged from 26.9 % to 87.3 %. Soil fauna access to litterbags accelerated mass loss by 29.8 % on average. The contribution of soil mesofauna did not differ from that of soil meso- and macrofauna. Incubation duration and its interactions with litter quality dissimilarities together with soil fauna determined the litter mixture effect. Furthermore, the litter mixture effect weakened as the decomposition progresses. Faunal contribution was broadly additive to the positive mixture effect irrespective of litter species richness or litter dissimilarity. This implies that combining the dissimilarity of mixture species and contributions of different soil fauna provides a more comprehensive understanding of mixed litter decomposition.

The role of the Notch signalling pathway in the pathogenesis of ulcerative colitis: from the perspective of intestinal mucosal barrier.

Ulcerative colitis is a common digestive disorder worldwide, with increasing incidence in recent years. It is an urgent problem to be solved, as it seriously affects and threatens the health and life of the global population. Studies have shown that dysfunction of the intestinal mucosal barrier is a critical pathogenic factor and molecular basis of ulcerative colitis, and some scholars have described it as a "barrier organ disease." While the Notch signalling pathway affects a series of cellular processes, including proliferation, differentiation, development, migration, and apoptosis. Therefore, it can regulate intestinal stem cells, CD4+ T cells, innate lymphoid cells, macrophages, and intestinal microbiota and intervene in the chemical, physical, immune, and biological mucosal barriers in cases of ulcerative colitis. The Notch signalling pathway associated with the pathogenesis of ulcerative colitis has distinct characteristics, with good regulatory effects on the mucosal barrier. However, research on ulcerative colitis has mainly focused on immune regulation, anti-inflammatory activity, and antioxidant stress; therefore, the study of the Notch signalling pathway suggests the possibility of understanding the pathogenesis of ulcerative colitis from another perspective. In this article we explore the role and mechanism of the Notch signalling pathway in the pathogenesis of ulcerative colitis from the perspective of the intestinal mucosal barrier to provide new targets and theoretical support for further research on the pathogenesis and clinical treatment of ulcerative colitis.

MicroRNAs as immune regulators and biomarkers in tuberculosis.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is one of the most lethal infectious disease worldwide, and it greatly affects human health. Some diagnostic and therapeutic methods are available to effectively prevent and treat TB; however, only a few systematic studies have described the roles of microRNAs (miRNAs) in TB. Combining multiple clinical datasets and previous studies on Mtb and miRNAs, we state that pathogens can exploit interactions between miRNAs and other biomolecules to avoid host mechanisms of immune-mediated clearance and survive in host cells for a long time. During the interaction between Mtb and host cells, miRNA expression levels are altered, resulting in the changes in the miRNA-mediated regulation of host cell metabolism, inflammatory responses, apoptosis, and autophagy. In addition, differential miRNA expression can be used to distinguish healthy individuals, patients with TB, and patients with latent TB. This review summarizes the roles of miRNAs in immune regulation and their application as biomarkers in TB. These findings could provide new opportunities for the diagnosis and treatment of TB.

Folic acid Ameliorates the Declining Quality of Sodium Fluoride-Exposed Mouse Oocytes through the Sirt1/Sod2 Pathway.

Excessive sodium fluoride (NaF) intake interferes with reproductive function in humans and animals; however, strategies to prevent these effects are still underexplored. Here, we showed that in vivo and in vitro supplementation of folic acid (FA) efficaciously improved the quality of NaF-exposed oocytes. FA supplementation not only increased ovulation of oocytes from NaF-treated mice but also enhanced oocyte meiotic competency and fertilization ability by restoring the spindle/chromosome structure. Moreover, FA supplementation could exert a beneficial effect on NaF- exposed oocytes by restoring mitochondrial function, eliminating reactive oxygen species accumulation to suppress apoptosis. We also found that FA supplementation restored the defective phenotypes in oocytes through a Sirt1/Sod2-dependent mechanism. Inhibition of Sirt1 with EX527 abolished the FA-mediated improvement in NaF-exposed oocyte quality. Collectively, our data indicated that FA supplementation is a feasible approach to protect oocytes from NaF-related deterioration.

ISOC1 Modulates Inflammatory Responses in Macrophages through the AKT1/PEX11B/Peroxisome Pathway.

Inflammation underlies a variety of physiological and pathological processes and plays an essential role in shaping the ensuing adaptive immune responses and in the control of pathogens. However, its physiological functions are not completely clear. Using a LPS-treated RAW264.7 macrophage inflammation model, we found that the production of inflammatory cytokines in ISOC1-deficient cells was significantly higher than that in the control group. It was further proved that ISOC1 deficiency could activate AKT1, and the overactivation of AKT1 could reduce the stability of PEX11B through protein modification, thereby reducing the peroxisome biogenesis and thus affecting inflammation. In this study, we reported for the first time the role of ISOC1 in innate immunity and elucidated the mechanism by which ISOC1 regulates inflammation through AKT1/PEX11B/peroxisome. Our results defined a new role of ISOC1 in the regulatory mechanism underlying the LPS-induced inflammatory response.

Vitamin C-induced competitive binding of HIF-1α and p53 to ubiquitin E3 ligase CBL contributes to anti-breast cancer progression through p53 deacetylation.

Vitamin C (VC), in regard to its effectiveness against tumors, has had a controversial history in cancer treatment. However, the anticancer mechanisms of VC are not fully understood. Here, we reported that VC exerted an anticancer effect on cancer cell and xenograft models via inhibiting HIF-1α-dependent cell proliferation and promoting p53-dependent cell apoptosis. To be specific, VC modulated the competitive binding of HIF-1α and p53 to their common E3 ubiquitin ligase CBL, thereby inhibiting tumorigenesis. Moreover, VC treatment activated SIRT1, resulting in p53 deacetylation and CBL-p53 complex dissociation, which in turn facilitated CBL recruitment of HIF-1α for ubiquitination in a proteasome-dependent manner. Altogether, our results provided a mechanistic rationale for exploring the therapeutic use of VC in cancer therapy.

A Novel ERK2 Degrader Z734 Induces Apoptosis of MCF-7 Cells via the HERC3/p53 Signaling Pathway.

Breast cancer is one of the leading causes of death worldwide, and synthetic chemicals targeting specific proteins or various molecular pathways for tumor suppression, such as ERK inhibitors and degraders, have been intensively investigated. The targets of ERK participate in the regulation of critical cellular mechanisms and underpin the progression of anticancer therapy. In this study, we identified a novel small molecule, which we named Z734, as a new mitogen-activated protein kinase 1 (ERK2) degrader and demonstrated that Z734 inhibits cell growth by inducing p53-mediated apoptotic pathways in human breast cancer cells. Treatment with Z734 resulted in the inhibition of cancer cell proliferation, colony formation and migration invasion, as well as cancer cell death via apoptosis. In addition, the Co-IP and GST pulldown assays indicated that the HECT and RLD domains containing E3 ubiquitin protein ligase 3 (HERC3) could directly interact with ERK2 through the HECT domain, promoting ERK2 ubiquitination. We also observed a strong link between HERC3 and p53 for the modulation of apoptosis. HERC3 can increase the protein and phosphorylation levels of p53, which further promotes apoptotic activity. In a xenograft mouse model, the effect was obtained in a treatment group that combined Z734 with lapatinib compared with that of the single-treatment groups. In summary, our results indicated that Z734 actively controls the development of breast cancer through apoptosis, and HERC3 may mediate ERK2 and p53 signaling, which offers new potential targets for clinical therapy.

Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts.

SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation.

Nucleocapsid mutations R203K/G204R increase the infectivity, fitness, and virulence of SARS-CoV-2.

Previous work found that the co-occurring mutations R203K/G204R on the SARS-CoV-2 nucleocapsid (N) protein are increasing in frequency among emerging variants of concern or interest. Through a combination of in silico analyses, this study demonstrates that R203K/G204R are adaptive, while large-scale phylogenetic analyses indicate that R203K/G204R associate with the emergence of the high-transmissibility SARS-CoV-2 lineage B.1.1.7. Competition experiments suggest that the 203K/204R variants possess a replication advantage over the preceding R203/G204 variants, possibly related to ribonucleocapsid (RNP) assembly. Moreover, the 203K/204R virus shows increased infectivity in human lung cells and hamsters. Accordingly, we observe a positive association between increased COVID-19 severity and sample frequency of 203K/204R. Our work suggests that the 203K/204R mutations contribute to the increased transmission and virulence of select SARS-CoV-2 variants. In addition to mutations in the spike protein, mutations in the nucleocapsid protein are important for viral spreading during the pandemic.