Preclinical safety assessment of the suction-assisted intradermal injection of the SARS-CoV-2 DNA vaccine candidate pGO-1002 in white rabbit.

pGO-1002, a non-viral DNA vaccine that expresses both spike and ORF3a antigens of SARS-CoV-2, is undergoing phase 1 and phase 2a clinical trials in Korea and the US. A preclinical repeated-dose toxicity study in New Zealand white rabbits in compliance with Good Laboratory Practice (GLP) was conducted to assess the potential toxicity, local tolerance, and immunogenicity of the vaccine and GeneDerm suction device. The dose rate was 1.2 mg/head pGO-1002, and this was administered intradermally to a group of animals (eight animals/sex/group) three times at 2-week intervals, followed by a 4-week recovery period. After each administration, suction was applied to the injection site using the GeneDerm device. Mortality, clinical signs, body weight, food consumption, skin irritation, ophthalmology, body temperature, urinalysis, and clinical pathology were also monitored. Gross observations and histopathological evaluation were performed. Overall, pGO-1002 administration-related changes were confined to minor damage or changes at the injection site, increased spleen weight and minimal increased cellularity in white pulp. All changes of injection site were considered local inflammatory changes or pharmacological actions due to the vaccine with the changes in spleen considered consistent with vaccine-induced immune activation. All findings showed reversibility during the 4-week recovery period. Animals vaccinated with pGO-1002, administered by intradermal injection and followed by application of suction with GeneDerm, developed humoral and cellular responses against the SARS-CoV-2 antigens consistent with prior studies in rats. Collectively, it was concluded that the pGO-1002 vaccine was safe and effective under these experimental conditions and these data supported future human study of the vaccine, now known as GLS-5310, for clinical trial use.

Tumorigenicity Assessment of Human Cancer Cell Lines Xenografted on Immunodeficient Mice as Positive Controls of Tumorigenicity Testing.

Recent advances in human pluripotent stem cell (hPSC)-derived cell therapies and genome editing technologies such as CRISPR/Cas9 make regenerative medicines promising for curing diseases previously thought to be incurable. However, the possibility of off-target effects during genome editing and the nature of hPSCs, which can differentiate into any cell type and infinitely proliferate, inevitably raises concerns about tumorigenicity. Tumorigenicity acts as a major obstacle to the application of hPSC-derived and gene therapy products in clinical practice. Thus, regulatory authorities demand mandatory tumorigenicity testing as a key pre-clinical safety step for the products. In the tumorigenicity testing, regulatory guidelines request to include human cancer cell line injected positive control group (PC) animals, which must form tumors. As the validity of the whole test is determined by the tumor-forming rates (typically above 90%) of PC animals, establishing the stable tumorigenic condition of PC animals is critical for successful testing. We conducted several studies to establish the proper positive control conditions, including dose, administration routes, and the selection of cell lines, in compliance with Good Laboratory Practice (GLP) regulations and/or guidelines, which are essential for pre-clinical safety tests of therapeutic materials. We expect that our findings provide insights and practical information to create a successful tumorigenicity test and its guidelines.

Matrix Stiffening Enhances DNCB-Induced IL-6 Secretion in Keratinocytes Through Activation of ERK and PI3K/Akt Pathway.

Matrix stiffness, a critical physical property of the cellular environment, is implicated in epidermal homeostasis. In particular, matrix stiffening during the pathological progression of skin diseases appears to contribute to cellular responses of keratinocytes. However, it has not yet elucidated the molecular mechanism underlying matrix-stiffness-mediated signaling in coordination with chemical stimuli during inflammation and its effect on proinflammatory cytokine production. In this study, we demonstrated that keratinocytes adapt to matrix stiffening by increasing cell-matrix adhesion via actin cytoskeleton remodeling. Specifically, mechanosensing and signal transduction are coupled with chemical stimuli to regulate cytokine production, and interleukin-6 (IL-6) production is elevated in keratinocytes on stiffer substrates in response to 2,4-dinitrochlorobenzene. We demonstrated that ß1 integrin and focal adhesion kinase (FAK) expression were enhanced with increasing stiffness and activation of ERK and the PI3K/Akt pathway was involved in stiffening-mediated IL-6 production. Collectively, our results reveal the critical role of matrix stiffening in modulating the proinflammatory response of keratinocytes, with important clinical implications for skin diseases accompanied by pathological matrix stiffening.

The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity.

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

Yeast Nuak1 phosphorylates histone H3 threonine 11 in low glucose stress by the cooperation of AMPK and CK2 signaling.

Changes in available nutrients are inevitable events for most living organisms. Upon nutritional stress, several signaling pathways cooperate to change the transcription program through chromatin regulation to rewire cellular metabolism. In budding yeast, histone H3 threonine 11 phosphorylation (H3pT11) acts as a marker of low glucose stress and regulates the transcription of nutritional stress-responsive genes. Understanding how this histone modification 'senses' external glucose changes remains elusive. Here, we show that Tda1, the yeast ortholog of human Nuak1, is a direct kinase for H3pT11 upon low glucose stress. Yeast AMP-activated protein kinase (AMPK) directly phosphorylates Tda1 to govern Tda1 activity, while CK2 regulates Tda1 nuclear localization. Collectively, AMPK and CK2 signaling converge on histone kinase Tda1 to link external low glucose stress to chromatin regulation.

Bundling of Collagen Fibrils Using Sodium Sulfate for Biomimetic Cell Culturing.

Collagen is the most abundant extracellular matrix protein. The concentrations, structural arrangement, and directionality of collagen depend on the type of tissue. Thick fibril bundles of collagen are observed in most collagenous tissues, including connective tissues, bones, and tendons, indicating that they play a critical role in many cell functions. In this study, we developed a new method to regulate collagen bundling without altering the protein concentration, temperature, or pH by using sodium sulfate to replicate bundled collagen fibrils found in vivo. Microstructure analysis revealed that both the thickness of the fibril bundles and the pore size of the matrix increased with the amount of sodium sulfate. In contrast, there was no significant change in the bulk mechanical stiffness of the collagen matrix. The modified collagen bundle matrix was used to investigate the responses of human cervical cancer cells by mimicking the extracellular environments of a tumor. Compared to the normal collagen matrix, cells on the collagen bundle matrix exhibited significant changes in morphology, with a reduced cell perimeter and aspect ratio. The cell motility, which was analyzed in terms of the speed of migration and mean squared displacement, decreased for the collagen bundle matrix. Additionally, the critical time taken for the peak turning angle to converge to 90° decreased, indicating that the migration direction was regulated by geometric cues provided by collagen bundles rather than by the intrinsic cell persistence. The experimental results imply that collagen bundles play an important role in determining the magnitude and direction in cancer cell migration. The proposed method of extracellular matrix modification can be applied to investigate various cellular behaviors in both physiological and pathological environments.

When histones are under glucose starvation.

Under nutritional stress, cells undergo metabolic rewiring that results in changes of various cellular processes that include gene transcription. This transcriptional regulation requires dynamic chromatin remodeling that involves histone post-translational modifications. There are several histone marks that may act as switches upon starvation for stress-response pathways.

Rewritable, Printable Conducting Liquid Metal Hydrogel.

The development of high-performance printable electrical circuits, particularly based on liquid metals, is fundamental for device interconnection in flexible electronics, motivating numerous attempts to develop a variety of alloys and their composites. Despite their great potential, rewritable and printable electronic circuits based on liquid metals are still manufactured on demand. In this study, we demonstrate liquid metal-based hydrogels suitable for rewritable, printable electrical circuits. Our liquid metal hydrogels are based on sedimentation-induced composites of eutectic gallium-indium (EGaIn) particles in poly(ethylene glycol) diacrylate (PEGDA). The EGaIn particles are vertically phase-segregated in the PEGDA. When a composite surface with high EGaIn content is gently scratched, the surface covering PEGDA is removed, followed by the rupture of the native oxide layers of the particles, and the exposed EGaIn becomes conductive. The subsequent water-driven swelling of PEGDA on the scratched surface completely erases the conductive circuit, causing the system to reset. Our friction-responsive liquid metal hydrogel exhibits writing-erasing endurance for 20 cycles, with a dramatic change in the electrical resistance from metal (∼1 Ω) to insulator (∼107 Ω). By employing surface friction pen printing, we demonstrate mechanically flexible, rewritable, printable electrical conductors suitable for displays.

Enhanced lateral resolution in continuous wave stimulated emission depletion microscopy using tightly focused annular radially polarized excitation beam.

The lateral resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy is enhanced about 12% by applying annular-shaped amplitude modulation to the radially polarized excitation beam. A focused annularly filtered radially polarized excitation beam provides a more condensed point spread function (PSF), which contributes to enhance effective STED resolution of CW STED microscopy. Theoretical analysis shows that the FWHM of the effective PSF on the detection plane is smaller than for conventional CW STED. Simulation shows the donut-shaped PSF of the depletion beam and confocal optics suppress undesired PSF sidelobes. Imaging experiments agree with the simulated resolution improvement.

Effect of Mechanical Stretch on the DNCB-induced Proinflammatory Cytokine Secretion in Human Keratinocytes.

Skin is exposed to various physico-chemical cues. Keratinocytes, a major component of the skin epidermis, directly interact with the surrounding extracellular matrix, and thus, biochemical and biophysical stimulations from the matrix regulate the function of keratinocytes. Although it was reported that inflammatory responses of skin were altered by an applied mechanical force, understanding how the keratinocytes sense the mechanical stimuli and regulate a cytokine secretion remains unclear. Here, we designed a device that is able to apply chemo-mechanical cues to keratinocytes and assess their proinflammatory cytokine IL-6 production. We showed that when chemical stimuli were applied with mechanical stimuli simultaneously, the IL-6 production markedly increased compared to that observed with a single stimulus. Quantitative structural analysis of cellular components revealed that the applied mechanical stretch transformed the cell morphology into an elongated shape, increased the cell size, and dictated the distribution of focal adhesion complex. Our results suggest that the mechanical cue-mediated modulation of focal adhesion proteins and actin cytoskeleton translates into intracellular signaling associated with the IL-6 production particularly in skin sensitization. Our study can be applied to understand proinflammatory responses of skin under altered biophysical environments of the skin.

Histone H3 threonine 11 phosphorylation by Sch9 and CK2 regulates chronological lifespan by controlling the nutritional stress response.

Upon nutritional stress, the metabolic status of cells is changed by nutrient signaling pathways to ensure survival. Altered metabolism by nutrient signaling pathways has been suggested to influence cellular lifespan. However, it remains unclear how chromatin regulation is involved in this process. Here, we found that histone H3 threonine 11 phosphorylation (H3pT11) functions as a marker for nutritional stress and aging. Sch9 and CK2 kinases cooperatively regulate H3pT11 under stress conditions. Importantly, H3pT11 defective mutants prolonged chronological lifespan (CLS) by altering nutritional stress responses. Thus, the phosphorylation of H3T11 by Sch9 and CK2 links a nutritional stress response to chromatin in the regulation of CLS.

Determination of the molecular assembly of actin and actin-binding proteins using photoluminescence.

Actin, the most abundant protein in cells, polymerizes into filaments that play key roles in many cellular dynamics. To understand cell dynamics and functions, it is essential to examine the cytoskeleton structure organized by actin and actin-binding proteins. Here, we pave the way for determining the molecular assembly of the actin cytoskeleton using direct photonic in situ analysis, providing the photoluminescence characteristics of actin as a function of filament length and bundling, without labeling. In experiments for monomeric and filamentous actin reconstituted in vitro, structural forms of actin are identified from the peak positions and intensities of photoluminescence. Actin monomers exhibited small intensity emission peaks at 334 nm, whereas filamentous and bundled actin showed a shifted peak at 323 nm with higher intensity. The peak shift was found to be proportional to the length of the actin filament. With probing structural change of actin in various cells in vivo, our study provides an efficient and precise analytical in situ tool to examine the cytoskeleton structure. It will be beneficial for elucidating the mechanism of various cellular functions such as cell migration, differentiation, cytokinesis and adhesion. Furthermore, our technique can be applied to detect the alterations in the cell cytoskeleton that can occur during many pathological processes.

Dot1 regulates nucleosome dynamics by its inherent histone chaperone activity in yeast.

Dot1 (disruptor of telomeric silencing-1, DOT1L in humans) is the only known enzyme responsible for histone H3 lysine 79 methylation (H3K79me) and is evolutionarily conserved in most eukaryotes. Yeast Dot1p lacks a SET domain and does not methylate free histones and thus may have different actions with respect to other histone methyltransferases. Here we show that Dot1p displays histone chaperone activity and regulates nucleosome dynamics via histone exchange in yeast. We show that a methylation-independent function of Dot1p is required for the cryptic transcription within transcribed regions seen following disruption of the Set2-Rpd3S pathway. Dot1p can assemble core histones to nucleosomes and facilitate ATP-dependent chromatin-remodeling activity through its nucleosome-binding domain, in vitro. Global analysis indicates that Dot1p appears to be particularly important for histone exchange and chromatin accessibility on the transcribed regions of long-length genes. Our findings collectively suggest that Dot1p-mediated histone chaperone activity controls nucleosome dynamics in transcribed regions.

Comparison of alveolar ridge preservation methods using three-dimensional micro-computed tomographic analysis and two-dimensional histometric evaluation.

PURPOSE: This study evaluated the efficacy of alveolar ridge preservation methods with and without primary wound closure and the relationship between histometric and micro-computed tomographic (CT) data. MATERIALS AND METHODS: Porcine hydroxyapatite with polytetrafluoroethylene membrane was implanted into a canine extraction socket. The density of the total mineralized tissue, remaining hydroxyapatite, and new bone was analyzed by histometry and micro-CT. The statistical association between these methods was evaluated. RESULTS: Histometry and micro-CT showed that the group which underwent alveolar preservation without primary wound closure had significantly higher new bone density than the group with primary wound closure (P<0.05). However, there was no significant association between the data from histometry and micro-CT analysis. CONCLUSION: These results suggest that alveolar ridge preservation without primary wound closure enhanced new bone formation more effectively than that with primary wound closure. Further investigation is needed with respect to the comparison of histometry and micro-CT analysis.

Hrp3 controls nucleosome positioning to suppress non-coding transcription in eu- and heterochromatin.

The positioning of the nucleosome by ATP-dependent remodellers provides the fundamental chromatin environment for the regulation of diverse cellular processes acting on the underlying DNA. Recently, genome-wide nucleosome mapping has revealed more detailed information on the chromatin-remodelling factors. Here, we report that the Schizosaccharomyces pombe CHD remodeller, Hrp3, is a global regulator that drives proper nucleosome positioning and nucleosome stability. The loss of Hrp3 resulted in nucleosome perturbation across the chromosome, and the production of antisense transcripts in the hrp3Δ cells emphasized the importance of nucleosome architecture for proper transcription. Notably, perturbation of the nucleosome in hrp3 deletion mutant was also associated with destabilization of the DNA-histone interaction and cell cycle-dependent alleviation of heterochromatin silencing. Furthermore, the effect of Hrp3 in the pericentric region was found to be accomplished via a physical interaction with Swi6, and appeared to cooperate with other heterochromatin factors for gene silencing. Taken together, our data indicate that a well-positioned nucleosome by Hrp3 is important for the spatial-temporal control of transcription-associated processes.

A lysine-rich region in Dot1p is crucial for direct interaction with H2B ubiquitylation and high level methylation of H3K79.

Dot1p is involved in maintenance of the heterochromatin boundary, the DNA damage response, and transcriptional regulation in yeast and animals. Dot1p is a histone H3 lysine 79 (H3K79) methyltransferase, but H3K79 trimethylation (H3K79me3) by Dot1p requires histone H2B monoubiquitylation (H2Bub) as a pre-requisite. The underlying mechanism for H2Bub requirement has not been well elucidated. In this work, we found that nucleosomes containing H2Bub stimulate the yeast Dot1p to produce H3K79me3. A pulldown assay showed that the yeast Dot1p directly binds to ubiquitin. In addition, we demonstrate that a lysine-rich region (aa 101-140) in the first half of DNA binding domain of the Dot1p is critical in interaction with ubiquitin as well as binding to nucleosome core. Consistent with this, either deletion or point mutation of the lysine-rich region resulted in defect in global H3K79me3 accumulation and subtelomeric gene silencing in vivo. Taken together, our results indicate that a direct interaction between the lysine-rich region of Dot1p and the ubiquitin of H2Bub is required for H2Bub-mediated trans-tail regulation.

A case of polymicrobial endocarditis caused by anaerobic organisms in an injection drug user.

Endocarditis is a serious complication of injection drug use most commonly caused by Staphylococcus aureus. We report a case of tricuspid valve polymicrobial bacterial endocarditis in an injection drug user from 3 oral anaerobes: Actinomyces odontolytica, Veillonella species, and Prevotella melaninogenica. The patient was believed to have acquired these organisms from his habit of licking the needle in order to gauge the strength of the cocaine prior to injection. The patient was successfully treated with a 6-week course of penicillin G and metronidazole. This case demonstrates the importance of a detailed history in designing empiric therapy.

Management of postburn recalcitrant knee ulcers in pediatric patients.

The sequelae of severe lower-extremity burn injuries in children include ulcerations and unstable scars of the anterior knee. Although the weight-bearing and ambulatory demands on this joint predispose the ulcers to chronicity, recalcitrance to treatment in the absence of systemic factors may be indicative of the presence of less-than-optimal local factors mitigating against healing. In our experience, excessive skin tightness around the knee joint has played a key role in this respect. This retrospective study on 10 patients with 16 recalcitrant knee ulcers demonstrated the inadequacy of the traditional treatment approach of ulcer excision and grafting. However, incisional release of tight skin above the knee joint and resurfacing the defect with split-thickness skin graft (mean size, 118.26 +/- 35.32 cm2) eliminated excessive tension and allowed the ulcers to heal spontaneously and permanently. We found this approach useful in select patients, and we are favorably disposed to additional releasing incision and grafting if the need arises in the growing child.