CircRNA (circ)_0007823 Contributes to Triple-Negative Breast Cancer Progression and Cisplatin Resistance via the miR-182-5p/FOXO1 Pathway.

Cisplatin (DDP) is used for the clinical management of triple-negative breast cancer (TNBC). However, the development of drug resistance limits its therapeutic efficacy. Circular RNAs (circRNAs) are known to be involved in tumor DDP resistance. In our previous study, we reported that circ_0007823 expression is downregulated and correlated with adverse prognosis in TNBC. However, its association with DDP resistance remains unclear. This study aimed to determine the role of circ_0007823 and miR-182-5p in DDP-resistant TNBC and explore the underlying mechanisms. First, expression profiles circ_0007823, microRNA (miR)-182-5p, and forkhead box O1 (FOXO1) in TNBC cells were determined. Additionally, biological characteristics of cells, including apoptosis, cell cycle, proliferation, and migration, were analyzed using various assays. Luciferase reporter and rescue assays were used to determine the correlations among circ_0007823, miR-182-5p, and FOXO1 expression. MiR-182-5p was overexpressed in DDP-resistant TNBC cells. MiR-182-5p knockdown suppressed the invasiveness and increased the apoptosis of drug-resistant cells, contributing to G1 arrest and S phase reduction. Mechanistically, circ_0007823 targeted miR-182-5p, and its overexpression drastically reduced the promotional effects of the miR-182-5p mimic on the aggression and transfer ability of drug-resistant cells. Furthermore, FOXO1 overexpression increased the sensitivity of cells to DDP and reduced their malignant progression. Therefore, FOXO1 was established as the downstream target of miR-182-5p that may be used to treat DDP-resistant TNBC. In summary, circ_0007823 overexpression attenuated DDP resistance in TNBC via the miR-182-5p-FOXO1 axis, indicating the therapeutic potential of circ_0007823 DDP-resistant TNBC treatment.

Effect of thyroid function on offspring neurodevelopment in people receiving ART therapy: a prospective cohort study.

CONTEXT: Adequate maternal thyroid hormone is vital for fetal neurodevelopment. Abnormal thyroid function can cause developmental defects in offspring from spontaneous pregnancies; however, research in assisted reproduction is lacking. OBJECTIVES: To investigate the association between thyroid disorders and offspring neurodevelopment from assisted reproduction. DESIGN: Prospective cohort study. SETTING AND PARTICIPANTS: In this prospective and longitudinal birth cohort study (Jiangsu, China), we included 729 women who had their thyroid function tested before ART cycle and delivered liveborn babies between November 2015 and June 2020. MAIN OUTCOME MEASURES: Maternal thyroid function was assessed by measuring thyroid antibodies, free thyroxine, and serum thyroid-stimulating hormone. The third edition Bayley Scales of Infant and Toddler Development screening test (Bayley-III screening test) is used to assess the infant's neurodevelopment. RESULTS: In multivariate corrected linear regression analysis, infants of women with subclinical hypothyroidism demonstrated a significantly lower receptive communication score (ß = -0.63, 95% CI [-1.12, -0.14], P = 0.013), with stratified analysis showing a significant association among female offspring (ß = -0.87, 95% CI [-1.59, -0.15], P = 0.018) but null association among male offspring (ß = -0.44, 95% CI [-1.03, 0.15], P = 0.145). No significant differences were found in assisted pregnancy population with normal thyroid function and positive antibodies according to the diagnostic cut-offs applied to normal pregnant women. CONCLUSIONS: Subclinical hypothyroidism in assisted pregnancies correlates with lower communication scores in 1-year-olds, especially in girls. Recommending medication for subclinical hypothyroidism throughout, regardless of thyroid autoantibody status.

Radon exhalation behavior and determination of diffusion migration parameters in spherical porous emanation media.

There is a significant impact of the radon diffusion coefficient and the free radon production rate on the exhalation of radon from porous materials that can be regarded as spheres, hexahedrons, or cylinders. To understand this effect, the radon exhalation rules of spherical porous media with different radii were studied according to the radon diffusion migration theory. A specialized method for simultaneous determination of the radon diffusion coefficient and the free radon production rate of the spherical porous media was proposed, and applied to determine the above two parameters for two hemispherical test blocks with different radii. The results show that:(1) For spherical porous media with a certain radon diffusion length (Ld), as the radius (r0) of the sphere increases, the radon exhalation rate first increases, and tends to stabilize at r0≥6Ld; The free radon release share gradually decreases from approximately 1, and drops to a steady state at r0≥18Ld. (2) Compared with conventional methods, the relative error of the free radon production rate determined by the proposed method is within 3.9%, which verifies the reliability of the new method.

Temperature impacts on the growth of hydrogen bubbles during ultrasonic vibration-enhanced hydrogen generation.

To improve the hydrogen precipitation performance on the surface of the catalytic layer of the proton exchange membrane (PEM) hydrogen cathode, ultrasonic vibration was employed to accelerate the detachment of hydrogen bubbles on the surface of the catalytic layer. Based on the energy and mechanical analyses of nano and microbubbles, the hydrogen bubble generation mechanism and the effect of temperature on bubble parameters during the evolution process when the ultrasonic field is coupled with the electric field are investigated. The nucleation frequency of the hydrogen bubbles, the relationship between the pressure and temperature and the operating temperature during the generation and detachment of bubbles as well as the detachment radius of bubbles under the action of the ultrasonic field are obtained. The effects of ultrasound and temperature on hydrogen production were verified by visual experiments. The results show that the operating temperature affects the nucleation, growth, and detachment processes of hydrogen bubbles. The effect of temperature on the nucleation frequency of bubbles mainly comes from the Gibbs free energy required for the electrolysis reaction. The bubble radius and growth rate are both related to the temperature to the power of one-third. Ultrasonic waves enhance the separation of hydrogen bubbles from the catalyst surface by acoustic cavitation and impact effects. An increase in the working temperature reduces the activation energy barriers to be overcome for the electrolysis reaction of water, which together with a decrease in the Gibbs free energy and the surface tension coefficient, leads to an increase in the nucleation frequency of the catalytic layer and a decrease in the radius of bubble detachment, and thus improves the hydrogen precipitation performance. Visualization experiments show that in actual PEM hydrogen production, ultrasonic intensification can promote the formation of nucleation sites. The ultrasonic induced fine bubble flow not only has a drag effect on the bubble, but also intensifies the polymerization growth of the bubble due to the impact of the fine bubble flow, thus speeding up the detachment of the bubble, shortening the covering time of the hydrogen bubble on the surface of the catalytic electrode, reducing the activation voltage loss and improve the hydrogen production efficiency of PEM. The experimental results show that when the electrolyte is 60°C, the maximum hydrogen production efficiency of ultrasound is increased by 7.34%, and the average hydrogen production efficiency is increased by 5.83%.

The ratio of lymphocyte/red blood cells and platelets/lymphocytes are predictive biomarkers for lymph node metastasis in patients with breast cancer.

BACKGROUND: Axillary lymph node metastasis (LNM) affects the progression of breast cancer. However, it is difficult to preoperatively diagnose axillary lymph node status with high sensitivity. Therefore, we hypothesized that platelets/lymphocytes ratio (PLR) and lymphocytes/ red blood cells ratio (LRR) might help in the prognosis of lymph node metastasis in T1-T2 breast cancer. METHODS: 166 patients (Chang Ning Maternity & Infant Health Institute) were included in our study, and the associations of PLR and LPR with lymph node metastasis were investigated. Peripheral blood was collected one week before the surgery, and the patients were divided into different categories based on their PLR and LRR. RESULTS: The incidence of LNM was significantly increased in the high PLR group (p= 0.002) compared with the low PLR group; LNM was also significantly increased in the low LRR group (p= 0.036) compared with the high LPR group. Further, our study revealed that high PLR (p< 0.001, OR = 4.397, 95% CI = 2.005-9.645), low LRR (p= 0.017, OR = 0.336, 95%CI = 0.136-0.825) and high clinical T stage (p< 0.001, OR = 3.929, 95%CI = 1.913-8.071) are independent predictors of LNM. CONCLUSIONS: PLR and LRR could be identified as predictors of LNM in patients with T1/T2 breast cancer.

Structure-based discovery of dual pathway inhibitors for SARS-CoV-2 entry.

Since 2019, SARS-CoV-2 has evolved rapidly and gained resistance to multiple therapeutics targeting the virus. Development of host-directed antivirals offers broad-spectrum intervention against different variants of concern. Host proteases, TMPRSS2 and CTSL/CTSB cleave the SARS-CoV-2 spike to play a crucial role in the two alternative pathways of viral entry and are characterized as promising pharmacological targets. Here, we identify compounds that show potent inhibition of these proteases and determine their complex structures with their respective targets. Furthermore, we show that applying inhibitors simultaneously that block both entry pathways has a synergistic antiviral effect. Notably, we devise a bispecific compound, 212-148, exhibiting the dual-inhibition ability of both TMPRSS2 and CTSL/CTSB, and demonstrate antiviral activity against various SARS-CoV-2 variants with different viral entry profiles. Our findings offer an alternative approach for the discovery of SARS-CoV-2 antivirals, as well as application for broad-spectrum treatment of viral pathogenic infections with similar entry pathways.

Crystal structure of mRNA cap (guanine-N7) methyltransferase E12 subunit from monkeypox virus and discovery of its inhibitors.

In July 2022, the World Health Organization announced monkeypox as a public health emergency of international concern (PHEIC), and over 85,000 global cases have been reported currently. However, preventive and therapeutic treatments for the monkeypox virus (MPXV) remain limited. MPXV mRNA cap N7 methyltransferase (MTase) is composed of two subunits (E1 C-terminal domain (E1CTD) and E12) which are essential for the replication of MPXV. Here, we solved a 2.16 Å crystal structure of E12. We also docked the D1CTD of the vaccinia virus (VACV) corresponding to the E1CTD in MPXV with E12 and found critical residues at their interface. These residues were further used for drug screening. After virtual screening, the top 347 compounds were screened out and a list of top 20 potential MPXV E12 inhibitors were discovered, including Rutin, Quercitrin, Epigallocatechin, Rosuvastatin, 5-hydroxy-L-Tryptophan, and Deferasirox, etc., which were potential E12 inhibitors. Taking the advantage of the previously unrecognized special structure of MPXV MTase composing of E1CTD and E12 heterodimer, we screened for inhibitors targeting MTase for the first time based on the interface between the heterodimer of MPXV MTase. Our study may provide insights into the development of anti-MPXV drugs.

Hsa_circ_0007823 Overexpression Suppresses the Progression of Triple-Negative Breast Cancer via Regulating miR-182-5p-FOXO1 Axis.

Background: This study aimed to analyze the specific expression of hsa_circ_0007823 in triple-negative breast cancer (TNBC) and explore the roles and related molecular mechanisms of hsa_circ_0007823 in TNBC. Materials and Methods: Relative hsa_circ_0007823 levels in TNBC tissues and cell lines were examined by reverse transcription-quantitative polymerase chain reaction. The value of hsa_circ_0007823 levels was evaluated in patients' clinicopathological characteristics and prognostic prediction. A dual-luciferase reporter assay was used to determine the relationship between hsa_circ_0007823, miR-182-5p, and FOXO1. The effect of circ_0007823 overexpression on the growth of TNBC cells was investigated in vitro and in vivo. Results: Lower levels of hsa_circ_0007823 were found in TNBC tissues and cell lines and were closely associated with lymph node metastasis, poorer overall and disease-free survival rates. MiR-182-5p was significantly up-regulated, whereas FOXO1 was down-regulated in TNBC cell lines. The miR-182-5p inhibition up-regulated FOXO1 in TNBC cells. Dual-luciferase reporter assays showed that hsa_circ_0007823, miR-182-5p, and FOXO1 interacted with each other. Overexpression of circ_0007823 significantly inhibited the viability, migration, and invasion of TNBC cell lines, but promoted apoptosis. In vivo experiments showed that circ_0007823 overexpression inhibited tumor growth and down-regulated miR-182-5p and up-regulated FOXO1. Conclusion: Hsa_circ_0007823 overexpression could suppress the growth, invasion, and migration of TNBC cells, and inhibit tumor growth by regulating miR-182-5p/FOXO1.

Molecular mechanisms of SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir is a specific antiviral drug that targets the main protease (Mpro) of SARS-CoV-2 and has been approved to treat COVID-191,2. As an RNA virus characterized by high mutation rates, whether SARS-CoV-2 will develop resistance to nirmatrelvir is a question of concern. Our previous studies have shown that several mutational pathways confer resistance to nirmatrelvir, but some result in a loss of viral replicative fitness, which is then compensated for by additional alterations3. The molecular mechanisms for this observed resistance are unknown. Here we combined biochemical and structural methods to demonstrate that alterations at the substrate-binding pocket of Mpro can allow SARS-CoV-2 to develop resistance to nirmatrelvir in two distinct ways. Comprehensive studies of the structures of 14 Mpro mutants in complex with drugs or substrate revealed that alterations at the S1 and S4 subsites substantially decreased the level of inhibitor binding, whereas alterations at the S2 and S4' subsites unexpectedly increased protease activity. Both mechanisms contributed to nirmatrelvir resistance, with the latter compensating for the loss in enzymatic activity of the former, which in turn accounted for the restoration of viral replicative fitness, as observed previously3. Such a profile was also observed for ensitrelvir, another clinically relevant Mpro inhibitor. These results shed light on the mechanisms by which SARS-CoV-2 evolves to develop resistance to the current generation of protease inhibitors and provide the basis for the design of next-generation Mpro inhibitors.

Structural biology of SARS-CoV-2 Mpro and drug discovery.

Since its outbreak in late 2019, the COVID-19 pandemic has drawn enormous attention worldwide as a consequence of being the most disastrous infectious disease in the past century. As one of the most immediately druggable targets of SARS-CoV-2, the main protease (Mpro) has been studied thoroughly. In this review, we provide a comprehensive summary of recent advances in structural studies of Mpro, which provide new knowledge about Mpro in terms of its biological function, structural characteristics, substrate specificity, and autocleavage process. We examine the remarkable strides made in targeting Mpro for drug discovery during the pandemic. We summarize insights into the current understanding of the structural features of Mpro and the discovery of existing Mpro-targeting drugs, illuminating pathways for the future development of anti-SARS-CoV-2 therapeutics.

Application effect of low-dose spiral CT on pulmonary nodules and its diagnostic value for benign and malignant nodules.

OBJECTIVE: This study was designed to determine the application effect of low-dose computed tomography (LDCT) on detecting pulmonary nodules (PNs) and its diagnostic value for benign and malignant pulmonary nodules. METHODS: Data of 432 patients with PNs admitted to Julu County Hospital between March 2018 and June 2021 in were collected and analysed retrospectively. All patients underwent LDCT and conventional-dose spiral computed tomography (CT). The detection rate and image characteristics of the two methods were compared, and the image quality and radiation dose of the two diagnostic methods were also compared. RESULTS: No significant difference was found between LDCT and conventional-dose spiral CT in the detection rate of lung cancer (P>0.05). The area under the curve of conventional-dose CT was 0.932, with a specificity and sensitivity of 93.87% and 92.45%, and the area under the curve of LDCT was 0.902, with a specificity and sensitivity of 90.80% and 89.62%. The radiation dose consumed during LDCT was greatly less than that consumed by conventional-dose CT (P<0.05). Additionally, the two methods were not different in CT image quality and superior vena cava artifact (P>0.05). No notable difference was found between LDCT and conventional-dose CT in terms of the diagnosis rate of PNs in vascular aggregation sign, pleural indentation sign, lobulation sign and spiculation sign. CONCLUSION: LDCT can clearly show the typical images of early lung cancer, with less effective radiation dose, and can thus contribute to a high detection rate, so it is worth popularizing.

Dynamic modeling and simulation of rigid-flexible coupling cable system by absolute nodal coordinate formulation.

The rigid-flexible coupling cable system under large deformation is studied, and the beam element from absolute node coordinate formulation is used to establish flexible cable body of the system. Different numerical integral algorithms are discussed for solving the rigid-flexible cable system and an integration strategy which combines Implicit Euler with Minimum Residual Method (MINRES) is proposed. The influence of the position and number of rigid components and different the lengths of the flexible elements on the system dynamics are analyzed. With constant total mass of the system, higher number of rigid components and their uniform distribution contribute to stabilization of the swing of the flexible cable body. When the total length of the cable is constant, increasing the number of beam elements enhances the nonlinear characteristics of the swing motion and damages the stability. The influence of different factors on the movement of large deformation flexible cable body is obtained through modeling and simulation of the rigid-flexible coupling cable system.

Structure-based design of anti-mycobacterial drug leads that target the mycolic acid transporter MmpL3.

New anti-tubercular agents are urgently needed to address the emerging threat of drug resistance to human tuberculosis. Here, we have used structure-assisted methods to develop compounds that target mycobacterial membrane protein large 3 (MmpL3). MmpL3 is essential for the transport of mycolic acids, an important cell-wall component of mycobacteria. We prepared compounds that potently inhibit the growth of Mycobacterium tuberculosis (Mtb) and other mycobacteria in cell culture. The cryoelectron microscopy (cryo-EM) structure of mycobacterial MmpL3 in complex with one of these compounds (ST004) was determined using lipid nanodiscs at an overall resolution of 3.36 Å. The structure reveals the binding mode of ST004 to MmpL3, with the S4 and S5 subsites of the inhibitor-binding pocket in the proton translocation channel playing vital roles. These data are a promising starting point for the development of anti-tuberculosis drugs that target MmpL3.

Crystal Structures of Wolbachia CidA and CidB Reveal Determinants of Bacteria-induced Cytoplasmic Incompatibility and Rescue.

Cytoplasmic incompatibility (CI) results when Wolbachia bacteria-infected male insects mate with uninfected females, leading to embryonic lethality. "Rescue" of viability occurs if the female harbors the same Wolbachia strain. CI is caused by linked pairs of Wolbachia genes called CI factors (CifA and CifB). The co-evolution of CifA-CifB pairs may account in part for the incompatibility patterns documented in insects infected with different Wolbachia strains, but the molecular mechanisms remain elusive. Here, we use X-ray crystallography and AlphaFold to analyze the CI factors from Wolbachia strain wMel called CidAwMel and CidBwMel. Substituting CidAwMel interface residues with those from CidAwPip (from strain wPip) enables the mutant protein to bind CidBwPip and rescue CidBwPip-induced yeast growth defects, supporting the importance of CifA-CifB interaction in CI rescue. Sequence divergence in CidAwPip and CidBwPip proteins affects their pairwise interactions, which may help explain the complex incompatibility patterns of mosquitoes infected with different wPip strains.

Structural and mechanistic insights into the complexes formed by Wolbachia cytoplasmic incompatibility factors.

Wolbachia bacteria, inherited through the female germ line, infect a large fraction of arthropod species. Many Wolbachia strains manipulate host reproduction, most commonly through cytoplasmic incompatibility (CI). CI, a conditional male sterility, results when Wolbachia-infected male insects mate with uninfected females; viability is restored if the female is similarly infected (called "rescue"). CI is used to help control mosquito-borne viruses such as dengue and Zika, but its mechanisms remain unknown. The coexpressed CI factors CifA and CifB form stable complexes in vitro, but the timing and function of this interaction in the insect are unresolved. CifA expression in the female germ line is sufficient for rescue. We report high-resolution structures of a CI-factor complex, CinA-CinB, which utilizes a unique binding mode between the CinA rescue factor and the CinB nuclease; the structures were validated by biochemical and yeast growth analyses. Importantly, transgenic expression in Drosophila of a nonbinding CinA mutant, designed based on the CinA-CinB structure, suggests CinA expressed in females must bind CinB imported by sperm in order to rescue embryonic viability. Binding between cognate factors is conserved in an enzymatically distinct CI system, CidA-CidB, suggesting universal features in Wolbachia CI induction and rescue.

Remdesivir overcomes the S861 roadblock in SARS-CoV-2 polymerase elongation complex.

Remdesivir (RDV), a nucleotide analog with broad-spectrum features, has exhibited effectiveness in COVID-19 treatment. However, the precise working mechanism of RDV when targeting the viral RNA-dependent RNA polymerase (RdRP) has not been fully elucidated. Here, we solve a 3.0-Å structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRP elongation complex (EC) and assess RDV intervention in polymerase elongation phase. Although RDV could induce an "i+3" delayed termination in meta-stable complexes, only pausing and subsequent elongation are observed in the EC. A comparative investigation using an enterovirus RdRP further confirms similar delayed intervention and demonstrates that steric hindrance of the RDV-characteristic 1'-cyano at the -4 position is responsible for the "i+3" intervention, although two representative Flaviviridae RdRPs do not exhibit similar behavior. A comparison of representative viral RdRP catalytic complex structures indicates that the product RNA backbone encounters highly conserved structural elements, highlighting the broad-spectrum intervention potential of 1'-modified nucleotide analogs in anti-RNA virus drug development.

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis.

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

The main protease and RNA-dependent RNA polymerase are two prime targets for SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses an unprecedented global health crisis. It is particularly urgent to develop clinically effective therapies to contain the pandemic. The main protease (Mpro) and the RNA-dependent RNA polymerase (RdRP), which are responsible for the viral polyprotein proteolytic process and viral genome replication and transcription, respectively, are two attractive drug targets for SARS-CoV-2. This review summarizes up-to-date progress in the structural and pharmacological aspects of those two key targets above. Different classes of inhibitors individually targeting Mpro and RdRP are discussed, which could promote drug development to treat SARS-CoV-2 infection.