Cremastranone-Derived Homoisoflavanes Suppress the Growth of Breast Cancer Cells via Cell Cycle Arrest and Caspase-Independent Cell Death.

Breast cancer is the most common cancer and a frequent cause of cancer-related deaths among women wordlwide. As therapeutic strategies for breast cancer have limitations, novel chemotherapeutic reagents and treatment strategies are needed. In this study, we investigated the anti-cancer effect of synthetic homoisoflavane derivatives of cremastranone on breast cancer cells. Homoisoflavane derivatives, SH-17059 and SH-19021, reduced cell proliferation through G2/M cell cycle arrest and induced caspase-independent cell death. These compounds increased heme oxygenase-1 (HO-1) and 5-aminolevulinic acid synthase 1 (ALAS1), suggesting downregulation of heme. They also induced reactive oxygen species (ROS) generation and lipid peroxidation. Furthermore, they reduced expression of glutathione peroxidase 4 (GPX4). Therefore, we suggest that the SH-17059 and SH-19021 induced the caspase-independent cell death through the accumulation of iron from heme degradation, and the ferroptosis might be one of the potential candidates for caspase-independent cell death.

Polarization-sensitive intensity diffraction tomography.

Optical anisotropy, which is an intrinsic property of many materials, originates from the structural arrangement of molecular structures, and to date, various polarization-sensitive imaging (PSI) methods have been developed to investigate the nature of anisotropic materials. In particular, the recently developed tomographic PSI technologies enable the investigation of anisotropic materials through volumetric mappings of the anisotropy distribution of these materials. However, these reported methods mostly operate on a single scattering model, and are thus not suitable for three-dimensional (3D) PSI imaging of multiple scattering samples. Here, we present a novel reference-free 3D polarization-sensitive computational imaging technique-polarization-sensitive intensity diffraction tomography (PS-IDT)-that enables the reconstruction of 3D anisotropy distribution of both weakly and multiple scattering specimens from multiple intensity-only measurements. A 3D anisotropic object is illuminated by circularly polarized plane waves at various illumination angles to encode the isotropic and anisotropic structural information into 2D intensity information. These information are then recorded separately through two orthogonal analyzer states, and a 3D Jones matrix is iteratively reconstructed based on the vectorial multi-slice beam propagation model and gradient descent method. We demonstrate the 3D anisotropy imaging capabilities of PS-IDT by presenting 3D anisotropy maps of various samples, including potato starch granules and tardigrade.

Synthetic Homoisoflavane Derivatives of Cremastranone Suppress Growth of Colorectal Cancer Cells through Cell Cycle Arrest and Induction of Apoptosis.

Colorectal cancer is diagnosed as the third most prevalent cancer; thus, effective therapeutic agents are urgently required. In this study, we synthesized six homoisoflavane derivatives of cremastranone and investigated their cytotoxic effects on the human colorectal cancer cell lines HCT116 and LoVo. We further examined the related mechanisms of action using two of the potent compounds, SH-19027 and SHA-035. They substantially reduced the cell viability and proliferation in a dose-dependent manner. Treatment with SH-19027 and SHA-035 induced cell cycle arrest at the G2/M phase and increased expression of p21 both of which are implicated in cell cycle control. In addition, the apoptotic cell population and apoptosis-associated marker expression were accordingly increased. These results suggest that the synthesized cremastranone derivatives have anticancer effects through the suppression of cell proliferation and induction of apoptosis. Therefore, the synthesized cremastranone derivatives could be applied as novel therapeutic agents against colorectal cancer.

HDAC inhibitor and proteasome inhibitor induce cleavage and exosome-mediated secretion of HSP90 in mouse pluripotent stem cells.

Heat shock protein 90 (HSP90), one of the molecular chaperones, stabilizes several proteins necessary to maintain pluripotency of embryonic stem (ES) cells. Recently, we reported that HDAC inhibitors and proteasome inhibitors down-regulate HSP90 activity through HSP90 cleavage induced by reactive oxygen species (ROS) generation and caspase 10 activation in various cancer cells. In this study, we investigated HSP90 cleavage in mouse ES cells. HDAC inhibitors and proteasome inhibitors induced HSP90 cleavage in the mouse ES cell line R1, and the cleaved HSP90 was barely found in the cells and instead secreted out of the cells through the exosome. The HSP90 cleavage was associated with ROS generation and caspase 10 activation. In addition, HDAC inhibitor and proteasome inhibitor induced Fas expression, and the inhibition of caspase 8, a downstream molecule of Fas, blocked HSP90 cleavage. Therefore, HDAC inhibitor- and proteasome inhibitor-mediated HSP90 cleavage was induced by ROS generation and Fas expression. We observed similar results in mouse induced pluripotent stem (iPS) cells. Taken together, HSP90 cleavage was induced in mouse pluripotent cells similarly to cancer cells but differently regulated through Fas expression and exosomal secretion. These findings will be helpful in elucidating the regulation of HSP90 upon stress in pluripotent stem cells.

Targeting the Interaction Between Spike Protein and Nucleocapsid Protein for Suppression and Detection of Human Coronavirus OC43.

Human coronavirus OC43 (HCoV-OC43) is the coronavirus most associated with "common colds", infections of the upper respiratory tract. Previously, we reported that direct interactions of nucleocapsid (N) protein and C-terminal domain of Spike protein (Spike CD) are essential for replication of SARS-CoV-2 and MERS-CoV. Thus, we developed a novel ELISA-based strategy targeting these specific interactions to detect SARS-CoV-2 and MERS-CoV. Here, we investigated whether the same principles apply to HCoV-OC43. We discovered that the S protein of HCoV-OC43 interacts with N protein and that cell penetrating Spike CD peptide inhibits virus protein expression and replication of HCoV-OC43. The interaction between HCoV-OC43 S and N proteins were recapitulated with a recombinant HCoV-OC43 Spike CD fusion protein and a recombinant HCoV-OC43 N fusion protein in vitro. By producing an anti-HCoV-OC43 N protein-specific monoclonal antibody, we established a virus detection system based on the interaction between recombinant Spike CD and N protein of HCoV-OC43. We suggest that the interaction between Spike CD and N protein is conserved in coronaviruses and therefore could be a target for therapeutics against both novel coronavirus and its variants.

Production of a Monoclonal Antibody to the Nucleocapsid Protein of SARS-CoV-2 and Its Application to ELISA-Based Detection Methods with Broad Specificity by Combined Use of Detector Antibodies.

The coronavirus disease 2019 pandemic, elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is ongoing. Currently accessible antigen-detecting rapid diagnostic tests are limited by their low sensitivity and detection efficacy due to evolution of SARS-CoV-2 variants. Here, we produced and characterized an anti-SARS-CoV-2 nucleocapsid (N) protein-specific monoclonal antibody (mAb), 2A7H9. Monoclonal antibody 2A7H9 and a previously developed mAb, 1G10C4, have different specificities. The 2A7H9 mAb detected the N protein of S clade, delta, iota, and mu but not omicron, whereas the 1G10C4 antibody recognized the N protein of all variants under study. In a sandwich enzyme-linked immunosorbent assay, recombinant N protein bound to the 1G10C4 mAb could be detected by both 1G10C4 and 2A7H9 mAbs. Similarly, N protein bound to the 2A7H9 mAb was detected by both mAbs, confirming the existence of dimeric N protein. While the 1G10C4 mAb detected omicron and mu with higher efficiency than S clade, delta, and iota, the 2A7H9 mAb efficiently detected all the strains except omicron, with higher affinity to S clade and mu than others. Combined use of 1G10C4 and 2A7H9 mAb resulted in the detection of all the strains with considerable sensitivity, suggesting that antibody combinations can improve the simultaneous detection of virus variants. Therefore, our findings provide insights into the development and improvement of diagnostic tools with broader specificity and higher sensitivity to detect rapidly evolving SARS-CoV-2 variants.

Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein.

SARS-CoV-2 infections continue to spread quickly by human-to-human transmission around the world. Therefore, developing methods to rapidly detect SARS-CoV-2 with high sensitivity are still urgently needed. We produced a monoclonal antibody that specifically detects the N protein of SARS-CoV-2 and recognizes N protein in cell lysates of SARS-CoV-2-infected Vero cells but not in cell lysates of MERS-CoV- or HCoV-OC43-infected Vero cells. This antibody recognized N protein in SARS-CoV-2 clades S, GR, and GH and recognized N protein in all the SARS-CoV-2 clades to ∼300 pfu. Previously, we reported that the coronavirus N protein interacts with the C-terminal domain of the spike protein (Spike CD). In this study, we developed an ELISA-based "bait and prey" system to confirm the interaction between SARS-CoV-2 Spike CD and N protein using recombinant fusion proteins. Furthermore, this system can be modified to quantitatively detect SARS-CoV-2 in culture media of infected cells by monitoring the interaction between the recombinant Spike CD fusion protein and the viral N protein, which is captured by the N protein-specific antibody. Therefore, we conclude that our N protein-specific monoclonal antibody and our ELISA-based bait and prey system could be used to diagnose SARS-CoV-2 infections.

Differential Signaling and Virus Production in Calu-3 Cells and Vero Cells upon SARS-CoV-2 Infection.

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is responsible for the current coronavirus disease 2019 (COVID-19) pandemic. Signaling pathways that are essential for virus production have potential as therapeutic targets against COVID-19. In this study, we investigated cellular responses in two cell lines, Vero and Calu-3, upon SARS-CoV-2 infection and evaluated the effects of pathway-specific inhibitors on virus production. SARS-CoV-2 infection induced dephosphorylation of STAT1 and STAT3, high virus production, and apoptosis in Vero cells. However, in Calu-3 cells, SARS-CoV-2 infection induced long-lasting phosphorylation of STAT1 and STAT3, low virus production, and no prominent apoptosis. Inhibitors that target STAT3 phosphorylation and dimerization reduced SARS-CoV-2 production in Calu-3 cells, but not in Vero cells. These results suggest a necessity to evaluate cellular consequences upon SARS-CoV-2 infection using various model cell lines to find out more appropriate cells recapitulating relevant responses to SARS-CoV-2 infection in vitro.

Cleavage of HSP90ß induced by histone deacetylase inhibitor and proteasome inhibitor modulates cell growth and apoptosis.

HSP90, one of the molecular chaperones, contributes to protein stability in most living organisms. Previously, we found cleavage of HSP90 by caspase 10 in response to treatment with histone deacetylase inhibitor or proteasome inhibitor in leukemic cell lines. In this study, we investigated this phenomenon in various cell lines and found that HSP90 was cleaved by treatment with SAHA or MG132 in 6 out of 16 solid tumor cell lines. To further investigate the effects of HSP90 cleavage on cells, we introduced mutations to the potential cleavage sites of HSP90ß and found that the 294th aspartic acid residue of the protein was mainly cleaved. In the K562 and Mia-PaCa-2 cell lines expressing HSP90ß D294A, the cleavage of HSP90 by the treatment with SAHA or MG132 was reduced compared with the K562 and Mia-PaCa-2 cell lines expressing HSP90ß WT. Accordingly, cell growth and survival were enhanced by HSP90ß D294A expression. Therefore, we suggest that HSP90 cleavage widely occurs in several cell lines, and cleavage of HSP90 may have a potential for one of the mechanisms involved in the anti-tumor effects of known drugs and novel anti-tumor drug candidates.

Inhibition of MUC1-C Increases ROS and Cell Death in Mouse Embryonic Stem Cells.

BACKGROUND AND OBJECTIVES: Embryonic stem (ES) cells have the capacity to self-renew and generate all types of cells. MUC1-C, a cytoplasmic subunit of MUC1, is overexpressed in various carcinomas and mediates signaling pathways to regulate intracellular metabolic processes and gene expression involved in the maintenance of cancer cells. However, the functional role of MUC1-C in ES cells is not well understood. In this study, we investigated the role of MUC1-C on growth, survival, and differentiation of mouse ES (mES) cells. METHODS AND RESULTS: Undifferentiated mES cells expressed the MUC1-C protein and the expression level was decreased during differentiation. Inhibition of MUC1-C, by the specific inhibitor GO201, reduced proliferation of mES cells. However, there was no prominent effect on pluripotent markers such as Oct4 expression and STAT3 signaling, and MUC1-C inhibition did not induce differentiation. Inhibition of MUC1-C increased the G1 phase population, decreased the S phase population, and increased cell death. Furthermore, inhibition of MUC1-C induced disruption of the ROS balance in mES cells. CONCLUSIONS: These results suggest that MUC1-C is involved in the growth and survival of mES cells.

Microarrays Incorporating Gold Grid Patterns for Protein Quantification.

Protein microarrays are miniaturized two-dimensional arrays, incorporating thousands of immobilized proteins, typically printed in minute amounts on functionalized solid substrates, which can be analyzed in a high-throughput fashion. Irreproducibility of the printing techniques adopted, resulting in inconsistently and nonuniformly deposited microscopic spots, nonuniform signal intensities from the printed microspots, and significantly high background noise are some of the critical issues that affect protein analysis using traditional protein microarrays. To overcome such issues, in this study, we introduced a novel gold grid pattern-based protein microarray. The grid patterns incorporated in our microarray are equivalent to the spots used for protein analysis in conventional protein microarrays. We utilized the signal intensities from the grid patterns acting as spots for quantifying the protein concentration levels. To demonstrate the utility of our novel design concept, we quantified as low as 66.7 ng/mL of bovine serum albumin using our gold grid pattern-based protein microarray. Our grid pattern-based design concept for protein quantification overcame the signal nonuniformity issues and ensured that the dominance of any distorted signal from a single spot did not affect the overall protein quantification results as encountered in conventional protein microarrays.

Impact of Neck Position on the Probability of Common Carotid Artery Puncture During Ultrasound-Guided Stellate Ganglion Block.

BACKGROUND: The carotid artery must be avoided during stellate ganglion block. However, information on optimal neck position during the ultrasound-guided approach is limited. OBJECTIVE: To investigate the relation between the target area of the procedure and the carotid artery in different neck positions. DESIGN: Observational study. SETTING: Tertiary university. PARTICIPANTS: A total of 30 sides of the neck from 18 healthy participants were included. METHODS: An ultrasound transducer was placed at the level of the anterior tubercle of C6 with a short-axis view for measuring the distance from the tip of the C6 anterior tubercle to the margin of the carotid artery. The participants were first examined through ultrasonography in 3 different rotational neck positions (neutral, semicontralateral rotation, and full-contralateral rotation), in the supine position. After changing to the lateral decubitus position, the measurement was performed again in the same 3 neck positions. MAIN OUTCOME MEASURES: The C6 anterior tubercle to carotid distance was measured with ultrasound. RESULTS: The C6 anterior tubercle to carotid distance was the longest with full-contralateral neck rotation (P < .05). The distance was longer in the semicontralateral neck rotation compared with the neutral neck position (P < .05). Supine or decubitus positions did not affect the distance. CONCLUSIONS: We suggest that the full-contralateral neck rotation posture in either the supine or decubitus position is most beneficial for avoiding damage to the carotid artery during the ultrasound-guided stellate ganglion block. LEVEL OF EVIDENCE: Not applicable.

Shoulder Manipulation After Distention Arthrography: Does Audible Cracking Affect Improvement in Adhesive Capsulitis? A Preliminary Study.

OBJECTIVE: To investigate whether an audible cracking sound during shoulder manipulation following distention arthrography is clinically significant in patients with adhesive capsulitis of the shoulder. METHODS: A total of 48 patients (31 women, 17 men) with primary adhesive capsulitis of the shoulder completed the study. All participants underwent C-arm-guided arthrographic distention of the glenohumeral joint with injections of a corticosteroid and normal saline. After distention, we performed flexion and abduction manipulation of the shoulder. The patients were grouped into sound and non-sound groups based on the presence or absence, respectively, of an audible cracking sound during manipulation. We assessed shoulder pain and disability based on a Numeric Rating Scale (NRS), the Shoulder Pain and Disability Index (SPADI), and passive range of motion (ROM) measurements (flexion, abduction, internal and external rotation) before the procedure and again at 3 weeks and at 6 weeks after the intervention. RESULTS: The patients were divided into two groups: 21 were included in the sound group and 27 in the non-sound group. In both groups, the results of the NRS, SPADI, and ROM assessments showed statistically significant improvements at both 3 and 6 weeks after the procedure. However, there were no significant differences between the two groups except with respect to external rotation at 6 weeks, at which time the sound group showed a significant improvement in external rotation when compared with the non-sound group (p<0.05). CONCLUSION: These findings showed that manipulation following distention arthrography was effective in decreasing pain and increasing shoulder range of motion. In addition, the presence of an audible cracking sound during manipulation, especially on external rotation, was associated with better shoulder range of motion.

Ultrasound-guided lateral femoral cutaneous nerve conduction study.

OBJECTIVE: To verify the utility of the lateral femoral cutaneous nerve (LFCN) ultrasound-guided conduction technique compared to that of the conventional nerve conduction technique. METHODS: Fifty-eight legs of 29 healthy participants (18 males and 11 females; mean age, 42.7±14.9 years) were recruited. The conventional technique was performed bilaterally. The LFCN was localized by ultrasound. Cross-sectional area (CSA) of the LFCN and the distance between the anterior superior iliac spine (ASIS) and the LFCN was measured. The nerve conduction study was repeated with the corrected cathode location. Sensory nerve action potential (SNAP) amplitudes of the LFCN were recorded and compared between the ultrasound-guided and conventional techniques. RESULTS: Mean body mass index of the participants was 23.7±3.5 kg/m(2), CSA was 4.2±1.9 mm(2), and the distance between the ASIS and LFCN was 5.6±1.7 mm. The mean amplitude values were 6.07±0.52 µV and 6.66±0.54 µV using the conventional and ultrasound-guided techniques, respectively. The SNAP amplitude of the LFCN using the ultrasound-guided technique was significantly larger than that recorded using the conventional technique. CONCLUSION: Correcting the stimulation position using the ultrasound-guided technique helped obtain increased SNAP amplitude.