Mining biosynthetic gene clusters in Paenibacillus genomes to discover novel antibiotics.

BACKGROUND: Bacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied. RESULTS: We conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin. CONCLUSIONS: This study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics.

Infrared thermographic changes after decompression surgery in patients with carpal tunnel syndrome.

BACKGROUND: Digital infrared thermal imaging (DITI), which detects infrared rays emitted from body surface to create a body heat map, has been utilized at various musculocutaneous conditions. Notably, DITI can demonstrate autonomic vasomotor activity in the nerve-innervated area, and thus may be of use in carpal tunnel syndrome (CTS). In this study, we compared DITI findings before and after carpal tunnel release (CTR) surgery in patients with unilateral CTS to investigate the corresponding neurophysiological changes. METHODS: In this retrospective cohort study, DITI parameters such as the temperature differences between the median and ulnar nerve territories and median nerve-innervated digital anisometry were measured. Subjective symptom duration, pain scale, and ultrasonographic findings were also compared before and after CTR. Patients were evaluated before and 6 weeks after CTR, respectively. RESULTS: A total of 27 patients aged 59.0 ± 11.2 years were finally included. After CTR, median nerve-innervated thermal anisometry was improved (2.55 ± 0.96 °C to 1.64 ± 1.34 °C; p = 0.003). The temperature differences between the median and ulnar nerve territories were not significantly changed. Subjective pain, the Simovic Weinberg Clinical Scale, and palmar bowing of the flexor retinaculum were also significantly improved (p <  0.001 for all comparisons). CONCLUSIONS: Our results demonstrated that DITI findings could reflect an improvement in autonomic function after CTR. Therefore, DITI can be an objective method to assess pre- and post-operative neurophysiologic changes in CTS.

Mesoporous Silica Nanoparticles as a Potential Nanoplatform: Therapeutic Applications and Considerations.

With advances in nanotechnology, nanoparticles have come to be regarded as carriers of therapeutic agents and have been widely studied to overcome various diseases in the biomedical field. Among these particles, mesoporous silica nanoparticles (MSNs) have been investigated as potential nanocarriers to deliver drug molecules to various target sites in the body. This review introduces the physicochemical properties of MSNs and synthesis procedures of MSN-based nanoplatforms. Moreover, we focus on updating biomedical applications of MSNs as a carrier of therapeutic or diagnostic cargo and review clinical trials using silica-nanoparticle-based systems. Herein, on the one hand, we pay attention to the pharmaceutical advantages of MSNs, including nanometer particle size, high surface area, and porous structures, thus enabling efficient delivery of high drug-loading content. On the other hand, we look through biosafety and toxicity issues associated with MSN-based platforms. Based on many reports so far, MSNs have been widely applied to construct tissue engineering platforms as well as treat various diseases, including cancer, by surface functionalization or incorporation of stimuli-responsive components. However, even with the advantageous aspects that MSNs possess, there are still considerations, such as optimizing physicochemical properties or dosage regimens, regarding use of MSNs in clinics. Progress in synthesis procedures and scale-up production as well as a thorough investigation into the biosafety of MSNs would enable design of innovative and safe MSN-based platforms in biomedical fields.

Bacillus integrative plasmid system combining a synthetic gene circuit for efficient genetic modifications of undomesticated Bacillus strains.

BACKGROUND: Owing to CRISPR-Cas9 and derivative technologies, genetic studies on microorganisms have dramatically increased. However, the CRISPR-Cas9 system is still difficult to utilize in many wild-type Bacillus strains owing to Cas9 toxicity. Moreover, less toxic systems, such as cytosine base editors, generate unwanted off-target mutations that can interfere with the genetic studies of wild-type strains. Therefore, a convenient alternative system is required for genetic studies and genome engineering of wild-type Bacillus strains. Because wild-type Bacillus strains have poor transformation efficiencies, the new system should be based on broad-host-range plasmid-delivery systems. RESULTS: Here, we developed a Bacillus integrative plasmid system in which plasmids without the replication initiator protein gene (rep) of Bacillus are replicated in a donor Bacillus strain by Rep proteins provided in trans but not in Bacillus recipients. The plasmids were transferred to recipients through a modified integrative and conjugative element, which is a wide host range plasmid-delivery system. Genetic mutations were generated in recipients through homologous recombination between the transferred plasmid and the genome. The system was improved by adding a synthetic gene circuit for efficient screening of the desired mutations by double crossover recombination in recipient strains. The improved system exhibited a mutation efficiency of the target gene of approximately 100% in the tested wild-type Bacillus strains. CONCLUSION: The Bacillus integrative plasmid system developed in this study can generate target mutations with high efficiency when combined with a synthetic gene circuit in wild-type Bacillus strains. The system is free of toxicity and unwanted off-target mutations as it generates the desired mutations by traditional double crossover recombination. Therefore, our system could be a powerful tool for genetic studies and genome editing of Cas9-sensitive wild-type Bacillus strains.

Effects of taxol on neuronal differentiation of postnatal neural stem cells cultured from mouse subventricular zone.

Taxol (paclitaxel), a chemotherapeutic agent for several cancers, can adversely affect the peripheral nervous system. Recently, its negative impact on cognitive function in cancer patients has become evident. In rodents, taxol impaired learning and memory, with other possible negative effects on the brain. In this study, we investigated the effects of taxol on cultured neural stem cells (NSCs) from the mouse neurogenic region, the subventricular zone (SVZ). Taxol significantly decreased both proliferation and neuronal differentiation of NSCs. Transient treatment with taxol for one day during a 4-day differentiation greatly decreased neurogenesis along with an abnormal cell cycle progression. Yet, taxol did not kill differentiated Tuj1+ neurons and those neurons had longer neurites than neurons under control conditions. For glial differentiation, taxol significantly reduced oligodendrogenesis as observed by immunostaining for Olig2 and O4. However, differentiation of astrocytes was not affected by taxol. In contrast, differentiated oligodendrocytes were extremely sensitive to taxol. Almost no Olig2-positive cells were observed after three days of treatment with taxol. Taxol has distinct effects on neurons and glial cells during their production through differentiation from NSCs as well as post-differentiation. Thus, we suggest that taxol might interfere with neurogenesis of NSCs possibly through a disturbance in the cell cycle and may eliminate differentiated oligodendrocytes.

N-Acetylcysteine extends lifespan of Drosophila via modulating ROS scavenger gene expression.

N-Acetylcysteine (NAc) has been shown to play a diversity of favorable health-related roles (e.g., antioxidant, paracetamol antidote, mucolytics, neuroprotective agent). Having said that, here in this study, we evaluate the health-promoting properties of NAc, particularly its ability to modulate organismal longevity. We note that 1 mg/ml NAc prolonged the lifespan of Drosophila. Furthermore, it was observed that NAc increased the capability of these flies to resist environmental stresses measured by starvation and paraquat stress assays. In an effort to reveal cellular mechanisms behind this interesting phenomenon, qPCR was performed, uncovering that transcript levels of catalase and phospholipid hydroperoxide glutathione peroxidase-key enzymes to fend off reactive oxygen species (ROS) assaults, were up-regulated. Correspondingly, enzyme activities of catalase and glutathione peroxidase were increased as well. Combined, we hope our research helps broaden the spectrum of clinical application for NAc so that one may eventually determine if NAc is a potentially useful anti-aging agent by encouraging others to scrutinize the hidden health benefits of NAc.

Jujube (Ziziphus Jujuba Mill.) fruit feeding extends lifespan and increases tolerance to environmental stresses by regulating aging-associated gene expression in Drosophila.

Jujube (Ziziphus Jujuba Mill.) fruit has been utilized as an adjunct to alleviate medical symptoms including, but not limited to, anorexia, fatigue, anxiety and mild diarrhea for centuries. Despite a growing body of literature revealing jujube fruit's health promoting properties such as anti-cancer, anti-inflammation and anti-oxidant effects (shown with cell-based in vitro platforms), there is a paucity of studies systemically examining its impact on whole organisms or throughout the entire course of life utilizing in vivo model systems. Thus, here in this study, we conducted a series of experiments to investigate if jujube fruit can modify an organism's longevity using a live Drosophila model. We found that jujube fruit feeding extended not only lifespan but also healthspan examined by stress assays such as starvation and paraquat treatment. In an effort to shed light on the mechanisms of these jujube-related benefits at the molecular level, we report that messenger RNA (mRNA) levels of 14-3-3ε, a negative FoxO (Forkhead box O transcription factor) regulator, was dramatically diminished while the abundance of d4E-BP mRNA transcript (drosophila eukaryotic translation initiation factor 4E binding protein), a FoxO target gene, was increased, suggesting enhanced FoxO activity with jujube fruit feeding. In conclusion, we hope our results will lead to multidisciplinary research to investigate the potential benefit of jujube fruit as a novel anti-aging agent.

Nerve growth factor (NGF) regulates activity of nuclear factor of activated T-cells (NFAT) in neurons via the phosphatidylinositol 3-kinase (PI3K)-Akt-glycogen synthase kinase 3ß (GSK3ß) pathway.

The Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT) plays an important role in regulating many neuronal functions, including excitability, axonal growth, synaptogenesis, and neuronal survival. NFAT can be activated by action potential firing or depolarization that leads to Ca(2+)/calcineurin-dependent dephosphorylation of NFAT and its translocation to the nucleus. Recent data suggest that NFAT and NFAT-dependent functions in neurons can also be potently regulated by NGF and other neurotrophins. However, the mechanisms of NFAT regulation by neurotrophins are not well understood. Here, we show that in dorsal root ganglion sensory neurons, NGF markedly facilitates NFAT-mediated gene expression induced by mild depolarization. The effects of NGF were not associated with changes in [Ca(2+)]i and were independent of phospholipase C activity. Instead, the facilitatory effect of NGF depended on activation of the PI3K/Akt pathway downstream of the TrkA receptor and on inhibition of glycogen synthase kinase 3ß (GSK3ß), a protein kinase known to phosphorylate NFAT and promote its nuclear export. Knockdown or knockout of NFATc3 eliminated this facilitatory effect. Simultaneous monitoring of EGFP-NFATc3 nuclear translocation and [Ca(2+)]i changes in dorsal root ganglion neurons indicated that NGF slowed the rate of NFATc3 nuclear export but did not affect its nuclear import rate. Collectively, our data suggest that NGF facilitates depolarization-induced NFAT activation by stimulating PI3K/Akt signaling, inactivating GSK3ß, and thereby slowing NFATc3 export from the nucleus. We propose that NFAT serves as an integrator of neurotrophin action and depolarization-driven calcium signaling to regulate neuronal gene expression.

Cytotoxicity of amine-modified polystyrene MPs and NPs on neural stem cells cultured from mouse subventricular zone.

Microplastics (MPs) and nanoplastics (NPs) are found in various environments such as aquatic, terrestrial, and aerial areas. Once ingested and inhaled, these tiny plastic debris damaged the digestive and respiratory organ systems in animals. In humans, the possible connection between MPs and various diseases such as lung diseases has been raised. Yet, the impact of MPs on the human nervous system has been unclear. Previous research using animals and cultured cells showed possible neurotoxicity of MPs and NPs. In this study, we used neural stem cells cultured from mouse subventricular zone to examine the effects of polystyrene (PS) NPs and MPs with sizes of 0.1 µm, 1 µm, and 2 µm on the cell proliferation and differentiation. We observed that only positively charged NPs and MPs, but not negatively charged ones, decreased cell viability and proliferation. These amine-modified NPs and MPs decreased both neurogenesis and oligodendrogenesis. Finally, fully differentiated neurons and oligodendrocytes were damaged and removed by the application of NPs and MPs. All these effects varied among different sizes of NPs and MPs, with the greatest effects from 1 µm and the least effects from 2 µm. These results clearly demonstrate the cytotoxicity and neurotoxicity of PS-NPs and MPs.

Mitochondria and plasma membrane Ca2+-ATPase control presynaptic Ca2+ clearance in capsaicin-sensitive rat sensory neurons.

The central processes of primary nociceptors form synaptic connections with the second-order nociceptive neurons located in the dorsal horn of the spinal cord. These synapses gate the flow of nociceptive information from the periphery to the CNS, and plasticity at these synapses contributes to centrally mediated hyperalgesia and allodynia. Although exocytosis and synaptic plasticity are controlled by Ca(2+) at the release sites, the mechanisms underlying presynaptic Ca(2+) signalling at the nociceptive synapses are not well characterized. We examined the presynaptic mechanisms regulating Ca(2+) clearance following electrical stimulation in capsaicin-sensitive nociceptors using a dorsal root ganglion (DRG)/spinal cord neuron co-culture system. Cytosolic Ca(2+) concentration ([Ca(2+)]i) recovery following electrical stimulation was well approximated by a monoexponential function with a ∼2 s. Inhibition of sarco-endoplasmic reticulum Ca(2+)-ATPase did not affect presynaptic [Ca(2+)]i recovery, and blocking plasmalemmal Na(+)/Ca(2+) exchange produced only a small reduction in the rate of [Ca(2+)]i recovery (∼12%) that was independent of intracellular K(+). However, [Ca(2+)]i recovery in presynaptic boutons strongly depended on the plasma membrane Ca(2+)-ATPase (PMCA) and mitochondria that accounted for ∼47 and 40%, respectively, of presynaptic Ca(2+) clearance. Measurements using a mitochondria-targeted Ca(2+) indicator, mtPericam, demonstrated that presynaptic mitochondria accumulated Ca(2+) in response to electrical stimulation. Quantitative analysis revealed that the mitochondrial Ca(2+) uptake is highly sensitive to presynaptic [Ca(2+)]i elevations, and occurs at [Ca(2+)]i levels as low as ∼200-300 nm. Using RT-PCR, we detected expression of several putative mitochondrial Ca(2+) transporters in DRG, such as MCU, Letm1 and NCLX. Collectively, this work identifies PMCA and mitochondria as the major regulators of presynaptic Ca(2+) signalling at the first sensory synapse, and underlines the high sensitivity of the mitochondrial Ca(2+) uniporter in neurons to cytosolic Ca(2+).

Distinct activation properties of the nuclear factor of activated T-cells (NFAT) isoforms NFATc3 and NFATc4 in neurons.

The Ca(2+)/calcineurin-dependent transcription factor NFAT (nuclear factor of activated T-cells) is implicated in regulating dendritic and axonal development, synaptogenesis, and neuronal survival. Despite the increasing appreciation for the importance of NFAT-dependent transcription in the nervous system, the regulation and function of specific NFAT isoforms in neurons are poorly understood. Here, we compare the activation of NFATc3 and NFATc4 in hippocampal and dorsal root ganglion neurons following electrically evoked elevations of intracellular Ca(2+) concentration ([Ca(2+)](i)). We find that NFATc3 undergoes rapid dephosphorylation and nuclear translocation that are essentially complete within 20 min, although NFATc4 remains phosphorylated and localized to the cytosol, only exhibiting nuclear localization following prolonged (1-3 h) depolarization. Knocking down NFATc3, but not NFATc4, strongly diminished NFAT-mediated transcription induced by mild depolarization in neurons. By analyzing NFATc3/NFATc4 chimeras, we find that the region containing the serine-rich region-1 (SRR1) mildly affects initial NFAT translocation, although the region containing the serine-proline repeats is critical for determining the magnitude of NFAT activation and nuclear localization upon depolarization. Knockdown of glycogen synthase kinase 3ß (GSK3ß) significantly increased the depolarization-induced nuclear localization of NFATc4. In contrast, inhibition of p38 or mammalian target of rapamycin (mTOR) kinases had no significant effect on nuclear import of NFATc4. Thus, electrically evoked [Ca(2+)](i) elevation in neurons rapidly and strongly activates NFATc3, whereas activation of NFATc4 requires a coincident increase in [Ca(2+)](i) and suppression of GSK3ß, with differences in the serine-proline-containing region giving rise to these distinct activation properties of NFATc3 and NFATc4.

Drosophila melanogaster as an emerging translational model of human nephrolithiasis.

PURPOSE: The limitations imposed by human clinical studies and mammalian models of nephrolithiasis have hampered the development of effective medical treatments and preventive measures for decades. The simple but elegant Drosophila melanogaster is emerging as a powerful translational model of human disease, including nephrolithiasis. It may provide important information essential to our understanding of stone formation. We present the current state of research using D. melanogaster as a model of human nephrolithiasis. MATERIALS AND METHODS: We comprehensively reviewed the English language literature using PubMed®. When necessary, authoritative texts on relevant subtopics were consulted. RESULTS: The genetic composition, anatomical structure and physiological function of Drosophila malpighian tubules are remarkably similar to those of the human nephron. The direct effects of dietary manipulation, environmental alteration and genetic variation on stone formation can be observed and quantified in a matter of days. Several Drosophila models of human nephrolithiasis have been developed, including genetically linked and environmentally induced stones. A model of calcium oxalate stone formation is among the most recent fly models of human nephrolithiasis. CONCLUSIONS: The ability to readily manipulate and quantify stone formation in D. melanogaster models of human nephrolithiasis presents the urological community with a unique opportunity to increase our understanding of this enigmatic disease.

Elicitation of Innate Immunity by a Bacterial Volatile 2-Nonanone at Levels below Detection Limit in Tomato Rhizosphere.

Bacterial volatile compounds (BVCs) exert beneficial effects on plant protection both directly and indirectly. Although BVCs have been detected in vitro, their detection in situ remains challenging. The purpose of this study was to investigate the possibility of BVCs detection under in situ condition and estimate the potentials of in situ BVC to plants at below detection limit. We developed a method for detecting BVCs released by the soil bacteria Bacillus velezensis strain GB03 and Streptomyces griseus strain S4-7 in situ using solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME-GC-MS). Additionally, we evaluated the BVC detection limit in the rhizosphere and induction of systemic immune response in tomato plants grown in the greenhouse. Two signature BVCs, 2-nonanone and caryolan-1-ol, of GB03 and S4-7 respectively were successfully detected using the soil-vial system. However, these BVCs could not be detected in the rhizosphere pretreated with strains GB03 and S4-7. The detection limit of 2-nonanone in the tomato rhizosphere was 1 µM. Unexpectedly, drench application of 2-nonanone at 10 nM concentration, which is below its detection limit, protected tomato seedlings against Pseudomonas syringae pv. tomato. Our finding highlights that BVCs, including 2-nonanone, released by a soil bacterium are functional even when present at a concentration below the detection limit of SPME-GC-MS.

Cell Factory Engineering of Undomesticated Bacillus Strains Using a Modified Integrative and Conjugative Element for Efficient Plasmid Delivery.

A large number of Bacillus strains have been isolated from various environments and many of them have great potential as cell factories. However, they have been rarely developed as cell factories due to their poor transformation efficiency. In this study, we developed a highly efficient plasmid delivery system for undomesticated Bacillus strains using a modified integrative and conjugative element (MICE), which was designed to be activated by an inducer, prevent self-transfer, and deliver desired plasmids to the recipient cells. The MICE system was demonstrated to successfully introduce a gfp-containing plasmid into all 41 undomesticated Bacillus subtilis strains tested and eight other Bacillus species. The MICE was used to deliver a cytosine base editor (CBE)-based multiplex genome-editing tool for the cell factory engineering of the Bacillus species. The introduced CBE enabled one-step inactivation of the major extracellular protease genes of the tested strains. The engineered strains were used as hosts for heterologous expression of nattokinase, which resulted in various enzyme expression levels. The results suggested that the MICE and CBE systems can be powerful tools for genetic engineering of undomesticated Bacillus strains, and greatly contribute to the expansion of the Bacillus cell factory.

Cytosine Base Editor-Mediated Multiplex Genome Editing to Accelerate Discovery of Novel Antibiotics in Bacillus subtilis and Paenibacillus polymyxa.

Genome-based identification of new antibiotics is emerging as an alternative to traditional methods. However, uncovering hidden antibiotics under the background of known antibiotics remains a challenge. To over this problem using a quick and effective genetic approach, we developed a multiplex genome editing system using a cytosine base editor (CBE). The CBE system achieved simultaneous double, triple, quadruple, and quintuple gene editing with efficiencies of 100, 100, 83, and 75%, respectively, as well as the 100% editing efficiency of single targets in Bacillus subtilis. Whole-genome sequencing of the edited strains showed that they had an average of 8.5 off-target single-nucleotide variants at gRNA-independent positions. The CBE system was used to simultaneously knockout five known antibiotic biosynthetic gene clusters to leave only an uncharacterized polyketide biosynthetic gene cluster in Paenibacillus polymyxa E681. The polyketide showed antimicrobial activities against gram-positive bacteria, but not gram-negative bacteria and fungi. Therefore, our findings suggested that the CBE system might serve as a powerful tool for multiplex genome editing and greatly accelerating the unraveling of hidden antibiotics in Bacillus and Paenibacillus species.

Diagnostic Role of Flexion-extension Central Motor Conduction Time in Cervical Spondylotic Myelopathy.

STUDY DESIGN: Retrospective study. OBJECTIVE: The purpose of this study was to assess the diagnostic usefulness of flexion-extension central motor conduction time(CMCT) for patients with cervical spondylotic myelopathy (CSM). SUMMARY OF BACKGROUND DATA: Previous reports have suggested that cervical cord compression can be aggravated by neck motions. Thus, the importance of dynamic magnetic resonance imaging (MRI) has been emphasized. However, authors of this study found no reports conducted at the time of this research on whether flexion-extension CMCT was useful for detecting myelopathy. METHODS: We enrolled 227 patients with CSM for this study. We acquired CMCT recorded from the abductor pollicis brevis muscle. All patients underwent a dynamic CMCT study during neck flexion and extension as well as a static study during neutral neck. Static and dynamic MRIs were also scanned. We read all MR images using Muhle classification (MC). RESULTS: CMCT was significantly delayed with flexion (P < 0.01) and extension (P < 0.01) compared to neutral neck position. Patients with MC grade 1 and 2 showed significant lag in CMCT during flexion and extension. No significant lag by neck motion was observed for those in the MC grade 3. We also evaluated the amount of CMCT variation according to MC grade change (G0, G1, G2) by neck motion. Delta-CMCT of both G1 and G2 were significantly larger than those of G0 in both flexion and extension. In neutral neck, the CMCT showed significant difference between MC grades 1 and 3. They also displayed significant delay with delay with high signal intensity on T2 MRI. More than one-third of the patients whose CMCT was within normal range in neutral neck presented abnormal CMCT in neck flexion (35.3%) and extension (37.8%). CONCLUSION: CMCT is significantly slower in both neck flexion and neck extension than in the neutral neck position. These findings reflect the dynamic cervical cord impingement.Level of Evidence: 4.

A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein.

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

Mitochondrial Ca2+ cycling facilitates activation of the transcription factor NFAT in sensory neurons.

Ca(2+)-dependent gene regulation controls many aspects of neuronal plasticity. Significant progress has been made toward understanding the roles of voltage- and ligand-gated Ca(2+) channels in triggering specific transcriptional responses. In contrast, the functional importance of Ca(2+) buffers and Ca(2+) transporters in neuronal gene regulation is less clear despite their critical contribution to the spatiotemporal control of Ca(2+) signals. Here we examined the role of mitochondrial Ca(2+) uptake and release in regulating the Ca(2+)-dependent transcription factor NFAT (nuclear factor of activated T-cells), which has been implicated in synaptic plasticity, axonal growth, and neuronal survival. Intense stimulation of sensory neurons by action potentials or TRPV1 agonists induced rapid activation and nuclear import of NFAT. Nuclear translocation of NFAT was associated with a characteristic prolonged [Ca(2+)](i) elevation (plateau) that resulted from Ca(2+) uptake by, and its subsequent release from, mitochondria. Measurements using a mitochondrial Ca(2+) indicator, mtPericam, showed that this process recruited mitochondria throughout the cell body, including the perinuclear region. [Ca(2+)](i) levels attained during the plateau phase were similar to or higher than those required for NFAT activation (200-300 nm). The elimination of the [Ca(2+)](i) plateau by blocking either mitochondrial Ca(2+) uptake via the uniporter or Ca(2+) release via the mitochondrial Na(+)/Ca(2+) exchanger strongly reduced nuclear import of NFAT. Furthermore, preventing Ca(2+) mobilization via the mitochondrial Na(+)/Ca(2+) exchanger diminished NFAT-mediated transcription. Collectively, these data implicate activity-induced Ca(2+) uptake and prolonged release from mitochondria as a novel regulatory mechanism in neuronal excitation-transcription coupling.

Mechanisms of prolonged presynaptic Ca2+ signaling and glutamate release induced by TRPV1 activation in rat sensory neurons.

Transient receptor potential vanilloid receptor 1 (TRPV1)-mediated release of neuroactive peptides and neurotransmitters from the peripheral and central terminals of primary sensory neurons can critically contribute to nociceptive processing at the periphery and in the CNS. However, the mechanisms that link TRPV1 activation with Ca2+ signaling at the release sites and neurosecretion are poorly understood. Here we demonstrate that a brief stimulation of the receptor using either capsaicin or the endogenous TRPV1 agonist N-arachidonoyl-dopamine induces a prolonged elevation of presynaptic [Ca2+](i) and a concomitant enhancement of glutamate release at sensory synapses. Initiation of this response required Ca2+ entry, primarily via TRPV1. The sustained phase of the response was independent of extracellular Ca2+ and was prevented by inhibitors of mitochondrial Ca2+ uptake and release mechanisms. Measurements using a mitochondria-targeted Ca2+ indicator, mtPericam, revealed that TRPV1 activation elicits a long-lasting Ca2+ elevation in presynaptic mitochondria. The concentration of TRPV1 agonist determined the duration of mitochondrial and cytosolic Ca2+ signals in presynaptic boutons and, consequently, the period of enhanced glutamate release and action potential firing by postsynaptic neurons. These data suggest that mitochondria control vanilloid-induced neurotransmission by translating the strength of presynaptic TRPV1 stimulation into duration of the postsynaptic response.

Expansion of antibacterial spectrum of xanthorrhizol against Gram-negatives in combination with PMBN and food-grade antimicrobials.

Xanthorrhizol (XTZ), isolated from Curcuma xanthorrhiza, has potent antifungal and antibacterial activity. It shows very strong activity against Gram-positive bacteria, such as Streptococcus mutans and Staphylococcus aureus, but is generally not active against Gram-negative bacteria. In this study, we explored the possibility of using a combination strategy for expanding the antimicrobial spectrum of XTZ against Gram-negative bacteria. To take advantage of XTZ being a food-grade material, 10 food-grade or generally recognized as safe (GRAS) antimicrobial compounds with low toxicities were selected for combination therapy. In addition, polymyxin B nonapeptide (PMBN), which is less toxic than polymyxin B, was also selected as an outer membrane permeabilizer. The antibacterial activity of various double or triple combinations with or without XTZ were assayed in vitro against four Gram-negative bacterial species (Escherichia coli, Salmonella enterica serovar Typhi, Salmonella enterica serovar Typhimurium, and Vibrio cholerae), with synergistic combinations exhibiting clear activity subjected to further screening. The combinations with the greatest synergism were XTZ + PMBN + nisin, XTZ + PMBN + carvacrol, and XTZ + PMBN + thymol. These combinations also showed potent antimicrobial activity against Shigella spp., Yersinia enterocolitica, and Acinetobacter baumannii. In time-kill assays, the three combinations achieved complete killing of E. coli within 2 h, and S. Typhi and V. cholera within 15 min. This is the first report on expanding the activity spectrum of XTZ against Gram-negative bacteria through combination with PMBN and food-grade or GRAS substances, with the resulting findings being particularly useful for increasing the industrial and medical applications of XTZ.