Ganoderma microsporum immunomodulatory protein acts as a multifunctional broad-spectrum antiviral against SARS-CoV-2 by interfering virus binding to the host cells and spike-mediated cell fusion.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus that has caused over 6 million fatalities. SARS-CoV-2 variants with spike mutations are frequently endowed with a strong capability to escape vaccine-elicited protection. Due to this characteristic, a broad-spectrum inhibitor against SARS-CoV-2 infection is urgently demanded. Ganoderma microsporum immunomodulatory protein (GMI) was previously reported to alleviate infection of SARS-CoV-2 through ACE2 downregulation whereas the impact of GMI on virus itself was less understood. Our study aims to determine the effects of GMI on SARS-CoV-2 pseudovirus and the more detailed mechanisms of GMI inhibition against SARS-CoV-2 pseudovirus infection. METHODS: ACE2-overexpressing HEK293T cells (HEK293T/ACE2) and SARS-CoV-2 pseudoviruses carrying spike variants were used to study the effects of GMI in vitro. Infectivity was evaluated by fluorescence microscopy and flow cytometry. Fusion rate mediated by SARS-CoV-2 spike protein was examined with split fluorescent protein /luciferase systems. The interactions of GMI with SARS-CoV-2 pseudovirus and ACE2 were investigated by immunoprecipitation and immunoblotting. RESULTS: GMI broadly blocked SARS-CoV-2 infection in various cell lines. GMI effectively inhibited the infection of pseudotyped viruses carrying different emerged spike variants, including Delta and Omicron strains, on HEK293T/hACE2 cells. In cell-free virus infection, GMI dominantly impeded the binding of spike-bearing pseudotyped viruses to ACE2-expressing cells. In cell-to-cell fusion model, GMI could efficiently inhibit spike-mediated syncytium without the requirement of ACE2 downregulation. CONCLUSIONS: GMI, an FDA-approved dietary ingredient, acts as a multifunctional broad-spectrum antiviral against SARS-CoV-2 and could become a promising candidate for preventing or treating SARS-CoV-2 associated diseases.

Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) Regulates Osteoclast Differentiation via the Ca2+ /NFATc1 Axis.

DC-STAMP is a multi-pass transmembrane protein essential for cell-cell fusion between osteoclast precursors during osteoclast (OC) development. DC-STAMP-/- mice have mild osteopetrosis and form mononuclear cells with limited resorption capacity. The identification of an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) on the cytoplasmic tail of DC-STAMP suggested a potential signaling function. The absence of a known DC-STAMP ligand, however, has hindered the elucidation of downstream signaling pathways. To address this problem, we engineered a light-activatable DC-STAMP chimeric molecule in which light exposure mimics ligand engagement that can be traced by downstream Ca2+ signaling. Deletion of the cytoplasmic ITIM resulted in a significant elevation in the amplitude and duration of intracellular Ca2+ flux. Decreased NFATc1 expression in DC-STAMP-/- cells was restored by DC-STAMP over-expression. Multiple biological phenotypes including cell-cell fusion, bone erosion, cell mobility, DC-STAMP cell surface distribution, and NFATc1 nuclear translocation were altered by deletion of the ITIM and adjacent amino acids. In contrast, mutations on each of the tyrosine residues surrounding the ITIM showed no effect on DC-STAMP function. Collectively, our results suggest that the ITIM on DC-STAMP is a functional motif that regulates osteoclast differentiation through the NFATc1/Ca2+ axis. J. Cell. Physiol. 232: 2538-2549, 2017. © 2016 Wiley Periodicals, Inc.

DC-STAMP: A Key Regulator in Osteoclast Differentiation.

Osteoimmunology research is a new emerging research field that investigates the links between the bone and immune responses. Results from osteoimmunology studies suggest that bone is not only an essential component of the musculoskeletal system, but is also actively involved in immune regulation. Many important factors involved in immune regulation also participate in bone homeostasis. Bone homeostasis is achieved by a coordinated action between bone-synthesizing osteoblasts and bone-degrading osteoclasts. An imbalanced action between osteoblasts and osteoclasts often results in pathological bone diseases: osteoporosis is caused by an excessive osteoclast activity, whereas osteopetrosis results from an increased osteoblast activity. This review focuses on dendritic cell-specific transmembrane protein (DC-STAMP), an important protein currently considered as a master regulator of osteoclastogenesis. Of clinical relevance, the frequency of circulating DC-STAMP+ cells is elevated during the pathogenesis of psoriatic diseases. Intriguingly, recent results suggest that DC-STAMP also plays an imperative role in bone homeostasis by regulating the differentiation of both osteoclasts and osteoblasts. This article summarizes our current knowledge on DC-STAMP by focusing on its interacting proteins, its regulation on osteoclastogenesis-related genes, its possible involvement in immunoreceptor tyrosine-based inhibitory motif (ITIM)-mediated signaling cascade, and its potential of developing therapeutics for clinical applications. J. Cell. Physiol. 231: 2402-2407, 2016. © 2016 Wiley Periodicals, Inc.

Implementation of laser acupuncture with lifting-thrusting through the use of mechanically immobile components.

Lifting and thrusting constitute an important manipulation method in traditional Chinese acupuncture. Lifting and thrusting enables the implementation of various features, such as reinforcement and reduction, which enhance acupuncture effectiveness. Laser acupuncture stimulates acupoints through laser light, which is a noninvasive treatment, but can still achieve effects similar to those obtained from traditional acupuncture. Lifting and thrusting can be achieved by moving the laser focal point back and forth, thus concentrating the energy, as does the tip of the acupuncture needle when it is moved upward and downward in the acupoint. This article presents a novel optical design of a laser acupuncture device, in which a focus-tunable lens is used to move the position of the focused light in order to achieve the lifting and thrusting mechanism through programmable changes to the control current of the focus-tunable lens. The device employs an infrared laser with a wavelength of 808 nm and a maximum power of 150 mW. The focus-tunable lens used in this study had a diopter of -10 to +5. The results revealed that by controlling the lens diopter, the focused light can be moved from 4.5 to 9.5 cm. Therefore, the range of the lift and thrust for the laser acupuncture device was 5 cm. The area of the focal point was approximately 6×10(-3) mm2, which is comparable to that of the commonly used traditional acupuncture needle tip. Because the components are immobile, no additional space is required for the moving lens. Therefore, the size of the laser acupuncture head can be minimized, and the effectiveness of focus tuning can be improved.

Emulated laser-acupuncture system.

A novel laser-acupuncture system was developed that can be used to implement the manipulation methods of traditional acupuncture, such as lifting and thrusting. A 780 nm laser diode with a maximum power of 90 mW was used as the light source. The focus point of the laser beam was adjustable by changing the position of the lens, facilitating the implementation of the lifting and thrusting methods of traditional Chinese medicine and achieving various stimulation depths at the acupuncture point. The images for the light spots from the outlet of the emulated laser acupuncture were captured at various distances and their sizes were calculated. The result showed that the diameter of the focused light spot (i.e., at the focus point) was 0.11 mm, which is close to the diameter of commonly used needles (with diameters of approximately 0.22 mm). The area of the light spot 1 cm from the focus point was approximately 50 times larger, indicating that the unit power might be 1/50 of the power of the focus point. To study the effect of emulated laser acupuncture on human meridians, after stimulating the Shenmen point (HT7) of five subjects and obtaining their Ryodoraku values of the heart meridian and the small-intestine meridian, a paired t test showed that the laser stimulation incorporating lifting and thrusting was significantly higher than the laser stimulation without lifting and thrusting (p<0.05). The result is consistent with traditional acupuncture in that acupuncture incorporating lift and thrust is more effective than that without lift and thrust.

Regulation of human osteoclast development by dendritic cell-specific transmembrane protein (DC-STAMP).

Osteoclasts (OC) are bone-resorbing, multinucleated cells that are generated via fusion of OC precursors (OCP). The frequency of OCP is elevated in patients with erosive inflammatory arthritis and metabolic bone diseases. Although many cytokines and cell surface receptors are known to participate in osteoclastogenesis, the molecular mechanisms underlying the regulation of this cellular transformation are poorly understood. Herein, we focused our studies on the dendritic cell-specific transmembrane protein (DC-STAMP), a seven-pass transmembrane receptor-like protein known to be essential for cell-to-cell fusion during osteoclastogenesis. We identified an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail of DC-STAMP, and developed an anti-DC-STAMP monoclonal antibody 1A2 that detected DC-STAMP expression on human tumor giant cells, blocked OC formation in vitro, and distinguished four patterns of human PBMC with a positive correlation to OC potential. In freshly isolated monocytes, DC-STAMP(high) cells produced a higher number of OC in culture than DC-STAMP(low) cells and the surface expression of DC-STAMP gradually declined during osteoclastogenesis. Importantly, we showed that DC-STAMP is phosphorylated on its tyrosine residues and physically interacts with SHP-1 and CD16, an SH2-domain-containing tyrosine phosphatase and an ITAM-associated protein, respectively. Taken together, these data show that DC-STAMP is a potential OCP biomarker in inflammatory arthritis. Moreover, in addition to its effect on cell fusion, DC-STAMP dynamically regulates cell signaling during osteoclastogenesis.

Biomarkers to Diagnose Early Arthritis in Patients With Psoriasis.

BACKGROUND: Psoriatic arthritis is a potentially destructive, inflammatory joint disease that affects 20% to 30% of patients with psoriasis. Psoriasis precedes the onset of joint inflammation by approximately 10 years, providing a unique opportunity to intervene and prevent or delay onset of musculoskeletal manifestations. The emergence of sensitive imaging modalities and cellular biomarkers may facilitate early identification of patients with psoriasis who have subclinical joint disease and might help stratify patients with an early onset of arthritis. METHODS: The translational studies described herein are focused on the development of cellular biomarkers identified with flow cytometry and cell culture techniques in patients with psoriasis and psoriatic arthritis. RESULTS AND CONCLUSION: The combination of power Doppler ultrasound imaging and cellular biomarkers (ie, CD16 and dendritic cell specific transmembrane protein) to diagnose early psoriatic arthritis and to stratify patients with established psoriatic arthritis provides a new opportunity to optimize treatment outcomes in this potentially disabling disease.

Position effect on the directionality of silencer function in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, silencers flanking the HML and HMR loci initiate the establishment of transcriptional silencing. We demonstrate that the activity of a silencer pertaining to its potency and directionality is dependent on its genomic position. The context of the HML-E silencer is more permissive to silencer function than that of HML-I or HMR-E, despite that HML-E and HML-I are only 3.3 kb apart. The apparent strength and directionality of a silencer in a particular location is affected by other silencing elements (silencers and protosilencers) present in its context. We show that at the HML locus, at least four silencing elements engage in multiple functional interactions that contribute to the activities of the silencers. Notably, these dispersed silencing elements can synergize to silence genes located not only inside, but also outside the HML sequence that harbors them. Moreover, the relative positions and orientations of these elements are important for silencing, indicating that they belong to an intricate silencing network.

A targeted histone acetyltransferase can create a sizable region of hyperacetylated chromatin and counteract the propagation of transcriptionally silent chromatin.

Transcriptionally silent chromatin is associated with reduced histone acetylation and its propagation depends on histone hypoacetylation promoted by histone deacetylases. We show that tethered histone acetyltransferase (HAT) Esa1p or Gcn5p creates a segment of hyperacetylated chromatin that is at least 2.6 kb in size and counteracts transcriptional silencing that emanates from a silencer in yeast. Esa1p and Gcn5p counteract URA3 silencing even when they are targeted 1.7 kb downstream of the promoter and >2.0 kb from the silencer. The anti-silencing effect of a targeted HAT is strengthened by increasing the number of targeting sites, but impaired by events that enhance silencing. A tethered HAT can also counteract telomeric silencing. The anti-silencing effect of Gcn5p is abolished by a mutation that eliminated its HAT activity or by deleting the ADA2 gene encoding a structural component of Gcn5p-containing HAT complexes. These results demonstrate that a tethered HAT complex can create a sizable region of histone hyperacetylation and serve as a barrier to encroaching repressive chromatin.

Rap1p and other transcriptional regulators can function in defining distinct domains of gene expression.

Barrier elements that are able to block the propagation of transcriptional silencing in yeast are functionally similar to chromatin boundary/insulator elements in metazoans that delimit functional chromosomal domains. We show that the upstream activating sequences of many highly expressed ribosome protein genes and glycolytic genes exhibit barrier activity. Analyses of these barriers indicate that binding sites for transcriptional regulators Rap1p, Abf1p, Reb1p, Adr1p and Gcn4p may participate in barrier function. We also present evidence suggesting that Rap1p is directly involved in barrier activity, and its barrier function correlates with local changes in chromatin structure. We further demonstrate that tethering the transcriptional activation domain of Rap1p to DNA is sufficient to recapitulate barrier activity. Moreover, targeting the activation domain of Adr1p or Gcn4p also establishes a barrier to silencing. These results support the notion that transcriptional regulators could also participate in delimiting functional domains in the genome.

Multiple defects in antigen presentation and T cell development by mice expressing cytoplasmic tail-truncated CD1d.

For members of the CD1 family of beta(2)-microglobulin-associated lipid-presenting molecules, tyrosine-based motifs in the cytoplasmic tail and invariant chain (Ii) govern glycoprotein trafficking through endosomal compartments. Little is known about the intracellular pathways of CD1 trafficking and antigen presentation. However, in vitro studies with cells transfected with mutant CD1 that had a truncated cytoplasmic tail have suggested a role for these tyrosine motifs in some, but not all, antigenic systems. By introducing a deletion of the tyrosine motif into the germ line, and through homologous recombination in embryonic stem cells, we now describe knock-in mice with the CD1d cytoplasmic tail deleted. Despite adequate surface CD1d expression and the presence of Ii, these mutant mice showed multiple and selective abnormalities in intracellular trafficking, antigen presentation and T cell development, demonstrating the critical functions of the CD1d cytoplasmic tail motif in vivo.