Effects of Subchronic Buspirone Treatment on Depressive Profile in Socially Isolated Rats: Implication of Early Life Experience on 5-HT1A Receptor-Related Depression.

The heterogeneity of etiology may serve as a crucial factor in the challenges of treatment, including the low response rate and the delay in establishing therapeutic effect. In the present study, we examined whether social experience since early life is one of the etiologies, with the involvement of the 5-HT1A receptors, and explored the potentially therapeutic action of the subchronic administration of buspirone, a partial 5-HT1A agonist. Rats were isolation reared (IR) since their weaning, and the depressive profile indexed by the forced-swim test (FST) was examined in adulthood. Nonspecific locomotor activity was used for the IR validation. Buspirone administration (1 mg/kg/day) was introduced for 14 days (week 9-11). The immobility score of the FST was examined before and after the buspirone administration. Tissue levels of serotonin (5-HT) and its metabolite 5-HIAA were measured in the hippocampus, the amygdala, and the prefrontal cortex. Efflux levels of 5-HT, dopamine (DA), and norepinephrine (NE) were detected in the hippocampus by brain dialysis. Finally, the full 5-HT1A agonist 8-OH-DPAT (0.5 mg/kg) was acutely administered in both behavioral testing and the dialysis experiment. Our results showed (i) increased immobility time in the FST for the IR rats as compared to the social controls, which could not be reversed by the buspirone administration; (ii) IR-induced FST immobility in rats receiving buspirone was corrected by the 8-OH-DPAT; and (iii) IR-induced reduction in hippocampal 5-HT levels can be reversed by the buspirone administration. Our data indicated the 5-HT1A receptor-linked early life social experience as one of the mechanisms of later life depressive mood.

An evolved AAV variant enables efficient genetic engineering of murine T cells.

Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knockins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens avenues in experimental T cell immunology.

High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails.

Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80-90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46-62%) and yields (>1.5 × 109 modified cells) exceeding those of conventional approaches.

Interferon-independent STING signaling promotes resistance to HSV-1 in vivo.

The Stimulator of Interferon Genes (STING) pathway initiates potent immune responses upon recognition of DNA. To initiate signaling, serine 365 (S365) in the C-terminal tail (CTT) of STING is phosphorylated, leading to induction of type I interferons (IFNs). Additionally, evolutionary conserved responses such as autophagy also occur downstream of STING, but their relative importance during in vivo infections remains unclear. Here we report that mice harboring a serine 365-to-alanine (S365A) mutation in STING are unexpectedly resistant to Herpes Simplex Virus (HSV)-1, despite lacking STING-induced type I IFN responses. By contrast, resistance to HSV-1 is abolished in mice lacking the STING CTT, suggesting that the STING CTT initiates protective responses against HSV-1, independently of type I IFNs. Interestingly, we find that STING-induced autophagy is a CTT- and TBK1-dependent but IRF3-independent process that is conserved in the STING S365A mice. Thus, interferon-independent functions of STING mediate STING-dependent antiviral responses in vivo.

Understanding patients' and caregivers' perspectives and educational needs in Parkinson's disease: a multi-ethnic Asian study.

INTRODUCTION: Little is known regarding the educational needs and perspectives of people living with Parkinson's disease (PD), particularly in Asia. OBJECTIVE: To assess knowledge and perceptions regarding PD in a large multiethnic urban Asian cohort of patients and caregivers. METHODS: We conducted a survey at a university hospital neurology clinic, using a novel Knowledge and Perception of Parkinson's Disease Questionnaire (KPPDQ). RESULTS: The KPPDQ had satisfactory psychometric properties among patients and caregivers. Five hundred subjects were recruited with a 97% response rate (211 patients, 273 caregivers). Non-motor symptoms such as urinary problems, visual hallucinations and pain were relatively poorly recognized. Many (≈ 50-80%) respondents incorrectly believed that all PD patients experience tremor, that PD is usually familial, and that there is a cure for PD. About one-half perceived PD to be caused by something the patient had done in the past, and that PD medications were likely to cause internal organ damage. Issues of stigma/shame were relevant to one-third of patients, and 70% of patients perceived themselves to be a burden to others. Two-thirds of participants felt that PD imposed a heavy financial toll. Participants were about equally divided as to whether they would consider treatment with deep brain stimulation, tube feeding or invasive ventilation. Over three-quarters of patients expressed a preference to die at home. CONCLUSIONS: Important knowledge gaps, misperceptions and perspectives on PD were identified, highlighting the need for further efforts to raise awareness and provide accurate information regarding PD, and to address patient's and caregivers' needs and preferences.

Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field-effect transistor.

Most methods for the detection of nucleic acids require many reagents and expensive and bulky instrumentation. Here, we report the development and testing of a graphene-based field-effect transistor that uses clustered regularly interspaced short palindromic repeats (CRISPR) technology to enable the digital detection of a target sequence within intact genomic material. Termed CRISPR-Chip, the biosensor uses the gene-targeting capacity of catalytically deactivated CRISPR-associated protein 9 (Cas9) complexed with a specific single-guide RNA and immobilized on the transistor to yield a label-free nucleic-acid-testing device whose output signal can be measured with a simple handheld reader. We used CRISPR-Chip to analyse DNA samples collected from HEK293T cell lines expressing blue fluorescent protein, and clinical samples of DNA with two distinct mutations at exons commonly deleted in individuals with Duchenne muscular dystrophy. In the presence of genomic DNA containing the target gene, CRISPR-Chip generates, within 15 min, with a sensitivity of 1.7 fM and without the need for amplification, a significant enhancement in output signal relative to samples lacking the target sequence. CRISPR-Chip expands the applications of CRISPR-Cas9 technology to the on-chip electrical detection of nucleic acids.

Effects of 5HT1A Activation on Depression Profile Following 5-HT Depletion in Rats Lacking Social Attachment Since Weanling.

OBJECTIVE: Post weanling isolation-reared (IR) rats are featured with depressive phenotype, yet its mechanism is not clearly defined particularly in terms of the involvement of central 5-HT1A receptors. The present study aims to examine the effects of 5HT1A activation on forced swim test (FST) in IR rats following 5-HT depletion. METHODS: Social control (SOC) and IR rats received an intracerebraoventricular (ICV) injection of 5-HT depletion agent, 5,7-DHT. 14 days after the surgery, rats were assessed their performance in FST with or without the challenge with a 5-HT1A agonist, 8-OH-DPAT. Rats were then sacrificed for analyzing their 5-HT tissue levels and the expressions of their 5-HA1A receptors in prefrontal cortex (PFC), hippocampus (HPX), and amygdala (AMY). RESULTS: 5,7-DHT decreased the tissue concentration of 5-HT in both IR and SOC rats. IR rats were more immobile and less sensitive to the lesion-induced immobility, however this effect was reversed by acute challenge of 8-OH-DPAT. 5,7-DHT lesion increased the expression of PFC 5-HT1A receptors. CONCLUSION: The integrity of central 5-HT system is developmentally crucial for the 5-HT1A-relevant depression profile in rats of social isolation.

Effects of 5HT1A Activation on Gating Profile Following 5HT Depletion in Rats Lacking Social Attachment Since Weanling.

OBJECTIVE: Central 5-HT1A receptor is involved in the modulation of sensorimotor gating function. However, its precise role is not clearly defined in developmentally social deprived (isolation rearing, IR) rats featured with impaired sensorimotor gating ability. We therefore aimed to examine the effects of 5HT1A activation on acoustic startle response (ASR) and prepulse inhibition (PPI) in IR rats in a condition of compromised presynaptic 5-HT functions. METHODS: Social control (SOC) and IR rats received an intracerebraoventricular (ICV) injection of 5-HT depletor, 5,7-DHT. Seven days later rats entered a protocol of 8-OH-DPAT, a 5-HT1A agonist, in which locomotor activity, ASR and PPI and their tissue levels of 5-HT were measured. RESULTS: Our results found that both IR and 5,7-DHT decreased the tissue concentration of 5-HT. IR-induced hyperactivity and gating impairment were unaffected by 5-HT depletion. 8-OH-DPAT strengthened the ASR in IR but not SOC rats and the drug-reduced PPI could be adjusted by 5,7-DHT pretreatment. 8-OH-DPAT at 100 µg/kg enhanced PPI in 5-HT-depleted SOC rats. However for IR rats, 8-OH-DPAT strengthened PPI in sham rats but downgraded it in depletion condition. CONCLUSION: The integrity of central 5-HT system is important to 5-HT1A-modulated sensorimotor gating in isolation-reared rats.

A Cleavage-Responsive Stem-Loop Hairpin for Assaying Guide RNA Activity.

The scope of the CRISPR-Cas9 technology now reaches far beyond genomic engineering. While significant efforts are driving the evolution of this revolutionary biomedical tool, the in vitro cleavage assay remains the standard method implemented to validate the guide RNA that directs endonuclease Cas9 to a desired genomic target. Here, we report the development of an alternative guide RNA validation system called GUIDER. GUIDER features a hairpin loop structure with a proximal guanosine-rich unit, a distal fluorophore unit, and a gRNA-targeting stem component. Cleavage of GUIDER by its complementary RNA-guided Cas9 endonuclease complex yields a fluorescent emission at 525 nm, signaling effective cleavage of the hairpin structure. GUIDER was validated using the model gene target mpcsk9, and it was able to identify the gRNA that could most efficiently cleave the target mpcsk9 gene. The modular design of GUIDER should allow it to have broad applicability in validating gRNAs, and its fluorescent signal output offers a rapid, simple, and quantitative measure of Cas9-mediated DNA cleavage.