Biology subjects
Publications and source records attributed to Kim, C. H..
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Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed treatment for individuals experiencing major depressive disorder (MDD). The therapeutic mechanisms that take place before, during, or after SSRIs bind the serotonin transporter (SERT) are poorly understood, partially because no studies exist of the cellular and subcellular pharmacokinetic properties of SSRIs in living cells. We studied escitalopram and fluoxetine using new intensity- based drug-sensing fluorescent reporters ("iDrugSnFRs") targeted to the plasma membrane (PM), cytoplasm, or endoplasmic reticulum (ER) of cultured neurons and mammalian cell lines. We also employed chemical detection of drug within cells and phospholipid membranes. The drugs attain equilibrium in neuronal cytoplasm and ER, at approximately the same concentration as the externally applied solution, with time constants of a few s (escitalopram) or 200-300 s (fluoxetine). Simultaneously, the drugs accumulate within lipid membranes by [≥] 18-fold (escitalopram) or 180-fold (fluoxetine), and possibly by much larger factors. Both drugs leave cytoplasm, lumen, and membranes just as quickly during washout. We synthesized membrane-impermeant quaternary amine derivatives of the two SSRIs. The quaternary derivatives are substantially excluded from membrane, cytoplasm, and ER for > 2.4 h. They inhibit SERT transport-associated currents 6- or 11-fold less potently than the SSRIs (escitalopram or fluoxetine derivative, respectively), providing useful probes for distinguishing compartmentalized SSRI effects. Although our measurements are orders of magnitude faster than the "therapeutic lag" of SSRIs, these data suggest that SSRI-SERT interactions within organelles or membranes may play roles during either the therapeutic effects or the "antidepressant discontinuation syndrome". SIGNIFICANCE STATEMENTSelective serotonin reuptake inhibitors stabilize mood in several disorders. In general, these drugs bind to the serotonin (5-hydroxytryptamine) transporter (SERT), which clears serotonin from CNS and peripheral tissues. SERT ligands are effective and relatively safe; primary care practitioners often prescribe them. However, they have several side effects and require 2 to 6 weeks of continuous administration until they act effectively. How they work remains perplexing, contrasting with earlier assumptions that the therapeutic mechanism involves SERT inhibition followed by increased extracellular serotonin levels. This study establishes that two SERT ligands, fluoxetine and escitalopram, enter neurons within minutes, while simultaneously accumulating in many membranes. Such knowledge will motivate future research, hopefully revealing where and how SERT ligands "engage" their therapeutic target(s).
The adolescent social experience is essential for the maturation of the prefrontal cortex in mammalian species. However, it still needs to be determined which cortical circuits mature with such experience and how it shapes adult social behaviors in a sex-specific manner. Here, we examined social approaching behaviors in male and female mice after post-weaning social isolation (PWSI), which deprives social experience during adolescence. We found that the PWSI, particularly isolation during late adolescence, caused an abnormal increase in social approaches (hypersociability) only in female mice. We further found that the PWSI female mice showed reduced parvalbumin (PV) expression in the left orbitofrontal cortex (OFCL). When we measured neural activity in the female OFCL, a substantial number of neurons showed higher activity when mice sniffed other mice (social sniffing) than when they sniffed an object (object sniffing). Interestingly, the PWSI significantly reduced both the number of activated neurons and the activity level during social sniffing in female mice. Similarly, the CRISPR/Cas9-mediated knock-down of PV in the OFCL during late adolescence enhanced sociability and reduced the social sniffing-induced activity in adult female mice via decreased excitability of PV+ neurons and reduced synaptic inhibition in the OFCL. Moreover, optogenetic activation of excitatory neurons or optogenetic inhibition of PV+ neurons in the OFCL enhanced sociability in female mice. Our data demonstrate that the adolescent social experience is critical for the maturation of PV+ inhibitory circuits in the OFCL; this maturation shapes female social behavior via enhancing social representation in the OFCL. Significance StatementAdolescent social isolation often changes adult social behaviors in mammals. Yet, we do not fully understand the sex-specific effects of social isolation and the brain areas and circuits that mediate such changes. Here, we found that adolescent social isolation causes three abnormal phenotypes in female but not male mice: hypersociability, decreased PV+ neurons in the OFCL, and decreased socially evoked activity in the OFCL. Moreover, PV deletion in the OFCL in vivo caused the same phenotypes in female mice by increasing excitation compared with inhibition within the OFCL. Our data suggest that adolescent social experience is required for PV maturation in the OFCL, which is critical for evoking OFCL activity that shapes social behaviors in female mice.
We report a reagentless, intensity-based S-methadone fluorescent sensor, iS-methadoneSnFR, consisting of a circularly permuted GFP inserted within the sequence of a mutated bacterial periplasmic binding protein (PBP). We evolved a previously reported nicotine-binding PBP to become a selective S-methadone-binding sensor, via three mutations in the PBPs second shell and hinge regions. iS-methadoneSnFR displays the necessary sensitivity, kinetics, and selectivity - notably enantioselectivity against R-methadone - for biological applications. Robust iS-methadoneSnFR responses in human sweat and saliva and mouse serum enable diagnostic uses. Expression and imaging in mammalian cells demonstrate that S-methadone enters at least two organelles and undergoes acid trapping in the Golgi apparatus, where opioid receptors can signal. This work shows a straightforward strategy in adapting existing PBPs to serve real-time applications ranging from subcellular to personal pharmacokinetics.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a deoxyribonucleic acid (DNA)-cleaving endonuclease and a crispr ribonucleic acid (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared to the wild type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 7.41 and 7.60 times respectively. In our study, when the chimeric DNA-RNA guided en-Cas12a effector was used, the gene editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.
BackgroundThe healing environment within the posterolateral lumbar spine is one of the most clinically challenging bone healing environments in all of orthopaedics due to a lack of a contained space and the need to form de novo bone in a non-bony environment. Our group has previously published data that suggests that sclerostin in expressed locally at high levels throughout the process of a spinal fusion mass maturing. MethodsWe computationally identified multiple FDA-approved drugs, as well as a novel drug, for their ability to disrupt the interaction between sclerostin and its receptor, LRP5/6. The drugs were tested in several in vitro biochemical assays using murine MC3T3 and MSCs, assessing their ability to (1) enhance canonical Wnt signaling, (2) promote the accumulation of the active (non-phosphorylated) form of {beta}-catenin, and (3) enhance the intensity and signaling duration of BMP signaling. These drugs were then tested subcutaneously in rats as standalone osteoinductive agents on plain collagen sponges. Finally, the top drug candidates (called VA1 and C07) were tested in a rabbit posterolateral spine fusion model for their ability to achieve a successful fusion. ResultsWe show that by controlling GSK3b phosphorylation, these SMIs simultaneously enhance canonical Wnt signaling and potentiate canonical BMP signaling intensity and duration. We also demonstrate that the SMIs produce dose-dependent ectopic mineralization in vivo in rats as well as significantly increase posterolateral spine fusion rates in rabbits in vivo, both as standalone osteogenic drugs and in combination with autologous iliac crest bone graft. ConclusionsFew if any osteogenic small molecules have been described that possess the osteoinductive potency of BMP itself - that is, the ability to form de novo ectopic bone as a standalone agent, particularly in stringent in vivo environments. Herein, we describe two such SMIs that have this unique ability and thus may have potential application as novel cost-effective biologic bone graft substitutes for achieving consistent spinal fusion or even or critical-sized fracture defects. FundingThis work was supported by a Veteran Affairs Career Development Award (IK2-BX003845).
Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase (RT) after nick formation by CRISPR nickase. In this study, we developed a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas 9 [FnCas9(H969A)] nickase module, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing was dramatically extended, and accurate prime editing was induced specifically for the target genes.
CD19-targeting chimeric antigen receptor (CAR) T cells have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, recent clinical data indicate that a significant portion of patients still do not benefit from the therapy owing to various resistance mechanisms, including high expression of multiple inhibitory immune checkpoint receptors on activated CAR T cells. Here, we report a lentiviral two-in-one CAR T approach in which two checkpoint receptors are downregulated simultaneously by a dual short-hairpin RNA (shRNA) cassette integrated into a CAR vector. Using this system, we evaluated CD19-targeting CAR T cells in the context of four different checkpoint combinations--PD-1/TIM-3, PD-1/LAG-3, PD-1/CTLA-4 and PD-1/TIGIT--and found that CAR T cells with PD-1/TIGIT downregulation uniquely exerted synergistic antitumor effects in mouse xenograft models compared with PD-1 single downregulation, and maintained cytolytic and proliferative capacity upon repeated antigen exposure. Importantly, functional and phenotypic analyses of CAR T cells as well as analyses of transcriptomic profiles suggested that downregulation of PD-1 enhances short-term effector function, whereas downregulation of TIGIT is primarily responsible for maintaining a less-differentiated/exhausted state, providing a potential mechanism for the observed synergy. The PD-1/TIGIT-downregulated CAR T cells generated from diffuse large B-cell lymphoma patient-derived T cells using a clinically applicable manufacturing process also showed robust antitumor activity and significantly improved persistence in vivo compared with conventional CD19-targeting CAR T cells. Overall, our results demonstrate that the cell-intrinsic PD-1/TIGIT dual downregulation strategy may prove effective in overcoming immune checkpoint-mediated resistance in CAR T therapy.
Previously syntactic irregularity has been most studied with chord sequences. However, the same chord may be interpreted as having different harmonic functions, implying perceptual ambiguity. Hence, syntactic irregularity and perceptual ambiguity may be processed simultaneously. We devised 3 different 5-chord sequences in which the ending chord differed with the tonic (T), submediant (SM), and supertonic (ST). In terms of syntactic regularity, T is most regular, ST is most irregular. However, in terms of perceptual ambiguity, the most irregular ST had the salient highest voice. Therefore, the SM was the most ambiguous condition. We investigated how the human brain separates syntactic irregularity and perceptual ambiguity in terms of effective connectivity in bilateral inferior frontal gyri (IFGs) and superior temporal gyri (STGs) with magnetoencephalography in 19 subjects. Correct rate was lower for the most ambiguous chord (SM) (P = 0.020) as expected. Connectivity from the right to the left IFG was enhanced for the most irregular chord (ST) (P = 0.024, false discovery rate (FDR) corrected), whereas connectivity from the right to the left STG was enhanced for the most ambiguous chord (SM) (P < 0.001, FDR corrected). The correct rate was negatively correlated with connectivity in the STG, further reflecting perceptual ambiguity (P = 0.026). We found that syntactic irregularity and perceptual ambiguity in music are dissociated in connectivity between bilateral IFGs and STGs, respectively. Significance StatementWe provide the first neurophysiological evidence of the processing of perceptual ambiguity, other than syntactic irregularity, implied in musical chords. We found that the notion of "perceptually ambiguity" is applicable to musical chord stimuli different in syntactic irregularity, and that perceptual ambiguity is separate from syntactic irregularity. Our data demonstrate that the brain interprets the three conditions of musical chords as both "from regular to irregular" and "from ambiguous to unambiguous" conditions simultaneously. This study is the first to unveil dissociation of connectivity by syntactic irregularity and perceptual ambiguity involved in musical chord stimuli.
In real music, the original melody may appear intact, with little elaboration only, or significantly modified. Since a melody is most easily perceived in music, hearing significantly modified melody may change a brain connectivity. Mozart KV 265 is comprised of a theme with an original melody of "Twinkle Twinkle Little Star" and its significant variations. We studied whether effective connectivity changes with significantly modified melody, between bilateral inferior frontal gyri (IFGs) and Heschls gyri (HGs) using magnetoencephalography (MEG). Among the 12 connectivities, the connectivity from the left IFG to the right HG was consistently increased with significantly modified melody compared to the original melody in 2 separate sets of the same rhythmic pattern with different melody (p = 0.005 and 0.034, Bonferroni corrected). Our findings show that the modification of an original melody in a real music changes the brain connectivity.
Cancer cells often heterogeneously respond to genotoxic chemotherapy, leading to fractional killing and chemoresistance1, 2, which remain as the major obstacles in cancer treatment. It is widely believed that DNA damage induces a uniform response in regulating transcription and that cell fate is passively determined by a threshold mechanism evaluating the level of transcriptional responses3. On the contrary to this assumption, here we show that a surprisingly high level of heterogeneity exists in individual cell transcriptome responses to DNA damage, and that these transcriptome variations dictate the cell fate after DNA damage. Many DNA damage response genes, including tumor suppressor p53 targets, were exclusively expressed in only a subset of cells having specific cell fate, producing unique stress responses tailored for the fate that the cells are committed to. For instance, CDKN1A, the best known p53 target inhibiting cell cycle, was specifically expressed in a subset of cells undergoing cell cycle checkpoint, while other pro-apoptotic p53 targets were expressed only in cells undergoing apoptosis. A small group of cells exhibited neither checkpoint nor apoptotic responses, but produced a unique transcriptional program that conferred strong chemoresistance to the cells. The heterogeneous transcriptome response to DNA damage was also observed at the protein level in flow cytometry. Our results demonstrate that cell fate heterogeneity after DNA damage is mediated by distinct transcriptional programs generating fate-specific gene expression landscapes. This finding provides an important insight into understanding heterogeneous chemotherapy responses of cancer cells.
We propose and experimentally validate a label-free, volumetric, and automated assessment method of immunological synapse dynamics using a combinational approach of optical diffraction tomography and deep learning-based segmentation. The proposed approach enables automatic and quantitative spatiotemporal analyses of immunological synapse kinetics regarding morphological and biochemical parameters related to the total protein densities of immune cells, thus providing a new perspective for studies in immunology.