Protein language models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing.

Current base editors (BEs) use DNA deaminases, including cytidine deaminase in cytidine BE (CBE) or adenine deaminase in adenine BE (ABE), to facilitate transition nucleotide substitutions. Combining CBE or ABE with glycosylase enzymes can induce limited transversion mutations. Nonetheless, a critical demand remains for BEs capable of generating alternative mutation types, such as T>G corrections. In this study, we leveraged pre-trained protein language models to optimize a uracil-N-glycosylase (UNG) variant with altered specificity for thymines (eTDG). Notably, after two rounds of testing fewer than 50 top-ranking variants, more than 50% exhibited over 1.5-fold enhancement in enzymatic activities. When eTDG was fused with nCas9, it induced programmable T-to-S (G/C) substitutions and corrected db/db diabetic mutation in mice (up to 55%). Our findings not only establish orthogonal strategies for developing novel BEs but also demonstrate the capacities of protein language models for optimizing enzymes without extensive task-specific training data.

Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase.

RNA splicing is a critical mechanism by which to modify transcriptome, and its dysregulation is the underlying cause of many human diseases. It remains challenging, however, to genetically modulate a splicing event in its native context. Here, we demonstrate that a CRISPR-guided cytidine deaminase (i.e., targeted-AID mediated mutagenesis [TAM]) can efficiently modulate various forms of mRNA splicing. By converting invariant guanines to adenines at either 5' or 3' splice sites (SS), TAM induces exon skipping, activation of alternative SS, switching between mutually exclusive exons, or targeted intron retention. Conversely, TAM promotes downstream exon inclusion by mutating cytidines into thymines at the polypyrimidine tract. Applying this approach, we genetically restored the open reading frame and dystrophin function of a mutant DMD gene in patient-derived induced pluripotent stem cells (iPSCs). Thus, the CRISPR-guided cytidine deaminase provides a versatile genetic platform to modulate RNA splicing and to correct mutations associated with aberrant splicing in human diseases.

Direct measuring of single-heterogeneous bubble nucleation mediated by surface topology.

Heterogeneous bubble nucleation is one of the most fundamental interfacial processes ranging from nature to technology. There is excellent evidence that surface topology is important in directing heterogeneous nucleation; however, deep understanding of the energetics by which nanoscale architectures promote nucleation is still challenging. Herein, we report a direct and quantitative measurement of single-bubble nucleation on a single silica nanoparticle within a microsized droplet using scanning electrochemical cell microscopy. Local gas concentration at nucleation is determined from finite element simulation at the corresponding faradaic current of the peak-featured voltammogram. It is demonstrated that the criteria gas concentration for nucleation first drops and then rises with increasing nanoparticle radius. An optimum nanoparticle radius around 10 nm prominently expedites the nucleation by facilitating the special topological nanoconfinements that consequently catalyze the nucleation. Moreover, the experimental result is corroborated by our theoretical calculations of free energy change based on the classic nucleation theory. This study offers insights into the impact of surface topology on heterogenous nucleation that have not been previously observed.

The cerebral lymphatic drainage system and its implications in epilepsy.

The central nervous system has long been thought to lack a clearance system similar to the peripheral lymphatic system. Therefore, the clearance of metabolic waste in the central nervous system has been a subject of great interest in neuroscience. Recently, the cerebral lymphatic drainage system, including the parenchymal clearance system and the meningeal lymphatic network, has attracted considerable attention. It has been extensively studied in various neurological disorders. Solute accumulation and neuroinflammation after epilepsy impair the blood-brain barrier, affecting the exchange and clearance between cerebrospinal fluid and interstitial fluid. Restoring their normal function may improve the prognosis of epilepsy. However, few studies have focused on providing a comprehensive overview of the brain clearance system and its significance in epilepsy. Therefore, this review addressed the structural composition, functions, and methods used to assess the cerebral lymphatic system, as well as the neglected association with epilepsy, and provided a theoretical basis for therapeutic approaches in epilepsy.

Solutal Marangoni force controls lateral motion of electrolytic gas bubbles.

Electrochemical gas-evolving reactions have been widely used for industrial energy conversion and storage processes. Gas bubbles form frequently at the electrode surface due to a small gas solubility, thereby reducing the effective reaction area and increasing the over-potential and ohmic resistance. However, the growth and motion mechanisms for tiny gas bubbles on the electrode remains elusive. Combining molecular dynamics (MD) and fluid dynamics simulations (CFD), we show that there exists a lateral solutal Marangoni force originating from an asymmetric distribution of dissolved gas near the bubble. Both MD and CFD simulations deliver a similar magnitude of the Marangoni force of ∼0.01 nN acting on the bubble. We demonstrate that this force may lead to lateral bubble oscillations and analyze the phenomenon of dynamic self-pinning of bubbles at the electrode boundary.

Use of machine learning approaches to predict transition of retention in care among people living with HIV in South Carolina: a real-world data study.

Maintaining retention in care (RIC) for people living with HIV (PLWH) helps achieve viral suppression and reduce onward transmission. This study aims to identify the best machine learning model that predicts the RIC transition over time. Extracting from the enhanced HIV/AIDS reporting system, this study included 9765 PLWH from 2005 to 2020 in South Carolina. Transition of RIC was defined as the change of RIC status in each two-year time window. We applied seven classifiers, such as Random Forest, Support Vector Machine, eXtreme Gradient Boosting and Long-short-term memory, for each lagged response to predict the subsequent year's RIC transition. Classification performance was assessed using balanced prediction accuracy, the area under the curve (AUC), recall, precision and F1 scores. The proportion of the four categories of RIC transition was 13.59%, 29.78%, 9.06% and 47.57%, respectively. Support Vector Machine was the best approach for every lag model based on both the F1 score (0.713, 0.717 and 0.719) and AUC (0.920, 0.925 and 0.928). The findings could facilitate the risk augment of PLWH who are prone to follow-up so that clinicians and policymakers could come up with more specific strategies and relocate resources for intervention to keep them sustained in HIV care.

Parental Rejection and School-aged Children's Externalizing Behavior Problems in China: The Roles of Executive Function and Callous-unemotional Traits.

This study examined the mediating role of children's executive function (EF) in the relation between parental rejection and children's externalizing behavior problems and whether this mediation varies depending on their callous-unemotional (CU) trait levels. Two hundred and eighty-four Chinese school-aged children and their fathers and mothers participated. Both fathers and mothers reported on parental rejection, children's externalizing behavior problems, EF, and CU traits. The results showed that EF mediated the association between parental rejection and externalizing behavior problems. Moreover, the negative link between EF and externalizing behavior problems was moderated by CU traits; in particular, the combination of higher-level CU traits and lower-level EF predicted more externalizing behavior problems. Our findings point to the importance of considering family context and multiple personal factors simultaneously to decrease children's behavior problems.

Upregulation of TRPC5 in hippocampal excitatory synapses improves memory impairment associated with neuroinflammation in microglia knockout IL-10 mice.

BACKGROUND: Members of the transient receptor potential canonical (TRPC) protein family are widely distributed in the hippocampus of mammals and exert respective and cooperative influences on the functions of neurons. The relationship between specific TRPC subtypes and neuroinflammation is receiving increasing attention. METHODS: Using Cx3cr1CreERIL-10-/- transgenic mice and their littermates to study the relationship between TRPC channels and memory impairment. RESULTS: We demonstrated that Cx3cr1CreERIL-10-/- mice displayed spatial memory deficits in object location recognition (OLR) and Morris water maze (MWM) tasks. The decreased levels of TRPC4 and TRPC5 in the hippocampal regions were verified via reverse transcription polymerase chain reaction, western blotting, and immunofluorescence tests. The expression of postsynaptic density protein 95 (PSD95) and synaptophysin in the hippocampus decreased with an imbalance in the local inflammatory environment in the hippocampus. The number of cells positive for ionized calcium-binding adaptor molecule 1 (Iba1), a glial fibrillary acidic protein (GFAP), increased with the high expression of interleukin 6 (IL-6) in Cx3cr1CreERIL-10-/- mice. The nod-like receptor protein 3 (NLRP3) inflammasome was also involved in this process, and the cytokines IL-1ß and IL-18 activated by NLRP3 were also elevated by western blotting. The co-localization of TRPC5 and calmodulin-dependent protein kinase IIα (CaMKIIα) significantly decreased TRPC5 expression in excitatory neurons. AAV9-CaMKIIα-TRPC5 was used to upregulate TRPC5 in excitatory neurons in the hippocampus. CONCLUSIONS: The results showed that the upregulation of TRPC5 improved the memory performance of Cx3cr1CreERIL-10-/- mice related to inhibiting NLRP3 inflammasome-associated neuroinflammation.

Depression-like behavior associated with E/I imbalance of mPFC and amygdala without TRPC channels in mice of knockout IL-10 from microglia.

Depression has a growing impact on public health. Accumulating evidence supports an association between depression and increased immune system activity. IL-10 is a key cytokine that inhibits excessive inflammatory responses and is related to the anti-inflammatory and protective functions of the central nervous system (CNS). Cx3cr1CreERIL-10-/- mice were used in our study. We aimed to identify the role of IL-10 in microglia in depression and anxiety-like behavior. We performed a series of behavioral tests on the mice; the Cx3cr1CreERIL-10-/- male mice showed depression- and anxiety-like behavior compared with the littermates. The expression of transient receptor potential canonical 5 (TRPC5) decreased in both the medial prefrontal cortex (mPFC) and amygdala regions. The cytokines IL-1ß and IL-6 increased, and IL-10 was decreased by western blotting. The knockout mice showed different trends in the effects of synaptic proteins. In the mPFC, IL-10 knockout induced a decrease in NR2B and synaptophysin; in the amygdala region, there was a significant increase in NR2B and PSD95. IL-10 knockout from microglia induced a decrease in GAD67 and parvalbumin (Pv) in the mPFC, but not in the amygdala. Our results showed enhanced depression and anxiety-like behavior in the Cx3cr1CreER IL-10-/- mice, which could be related to an imbalance in local excitatory and inhibitory transmission, as well as neuroinflammation in the mPFC and amygdala. This imbalance was associated with increased local inflammation. Although many studies have demonstrated the role of TRPC channels in emotional responses, our study showed that TRPC was not involved in this process in Cx3cr1CreERIL-10-/- mice.

Dynamic Equilibrium Model for Surface Nanobubbles in Electrochemistry.

Gas bubbles are ubiquitous in electrochemical processes, particularly in water electrolysis. Due to the development of gas-evolving electrocatalysis and energy conversion technology, a deep understanding of gas bubble behaviors at the electrode surface is highly desirable. In this work, by combining theoretical analysis and molecular simulations, we study the behaviors of a single nanobubble electrogenerated at a nanoelectrode. With the dynamic equilibrium model, the stability criteria for stationary surface nanobubbles are established. We show theoretically that a slight change in either the gas solubility or solute concentration results in various nanobubble dynamic states at a nanoelectrode: contact line pinning in aqueous and ethylene glycol solutions, oscillation of pinning states in dimethyl sulfoxide, and mobile nanobubbles in methanol. The above complex nanobubble behavior at the electrode/electrolyte interface is explained by the competition between gas influx into the nanobubble and outflux from the nanobubble.

Cucurbitacin IIb improved active chromatin-induced systemic lupus erythematosus via balancing the percentage of Th17 and Treg cells.

The pathogenesis of systemic lupus erythematosus (SLE) is closely associated with aberrant immune system. Here, the aim of our study was to explore the regulation of cucurbitacin IIb (CuIIb) to Th17/Treg cells in SLE. Compared with normal mice, the percentage of Treg cells was downregulated in SLE mouse model, and Th17 was upregulated. Meantime, the production of Treg-related transcription factor (foxp3) in SLE model mouse was reduced, and the production of Th17-related transcription factor (RORγt) was increased. After treatment with CuIIb, the percentage of Treg cells in SLE mice was partly upregulated, and Th17 cells percentage was downregulated. The expression of foxp3 and RORγt in SLE mice were promoted and inhibited by CuIIb treatment, respectively. SLE-induced kidney injury also was improved by CuIIb treatment. In vitro, we demonstrated again that CuIIb upregulated the percentage of Treg cells in lymphocytes from SLE mice, and downregulated the percentage of Th17 cells. Highly expressed IL-6 and IL17, and lowly expressed IL-10 and TGF-ß in lymphocytes from SLE mice were repressed and facilitated by CuIIb treatment, respectively. Overall, our data proved that CuIIb improved kidney injury in SLE mice through balancing the percentage of Th17 and Treg cells. Our data provided a reliable evidence to support the potential of CuIIb in SLE treatment.

Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells.

A large number of genetic variants have been associated with human diseases. However, the lack of a genetic diversification approach has impeded our ability to interrogate functions of genetic variants in mammalian cells. Current screening methods can only be used to disrupt a gene or alter its expression. Here we report the fusion of activation-induced cytidine deaminase (AID) with nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) for efficient genetic diversification, which enabled high-throughput screening of functional variants. Guided by single guide (sg)RNAs, dCas9-AID-P182X (AIDx) directly changed cytidines or guanines to the other three bases independent of AID hotspot motifs, generating a large repertoire of variants at desired loci. Coupled with a uracil-DNA glycosylase inhibitor, dCas9-AIDx converted targeted cytidines specifically to thymines, creating specific point mutations. By targeting BCR-ABL with dCas9-AIDx, we efficiently identified known and new mutations conferring imatinib resistance in chronic myeloid leukemia cells. Thus, targeted AID-mediated mutagenesis (TAM) provides a forward genetic tool to screen for gain-of-function variants at base resolution.

Arachidonic acid induces macrophage cell cycle arrest through the JNK signaling pathway.

BACKGROUND: Arachidonic acid (AA) has potent pro-apoptotic effects on cancer cells at a low concentration and on macrophages at a very high concentration. However, the effects of AA on the macrophage cell cycle and related signaling pathways have not been fully investigated. Herein we aim to observe the effect of AA on macrophages cell cycle. RESULTS: AA exposure reduced the viability and number of macrophages in a dose- and time-dependent manner. The reduction in RAW264.7 cell viability was not caused by apoptosis, as indicated by caspase-3 and activated caspase-3 detection. Further research illustrated that AA exposure induced RAW264.7 cell cycle arrested at S phase, and some cell cycle-regulated proteins were altered accordingly. Moreover, JNK signaling was stimulated by AA, and the stimulation was partially reversed by a JNK signaling inhibitor in accordance with cell cycle-related factors. In addition, nuclear and total Foxo1/3a and phosphorylated Foxo1/3a were elevated by AA in a dose- and time-dependent manner, and this elevation was suppressed by the JNK signaling inhibitor. CONCLUSION: Our study demonstrated that AA inhibits macrophage viability by inducing S phase cell cycle arrest. The JNK signaling pathway and the downstream FoxO transcription factors are involved in AA-induced RAW264.7 cell cycle arrest.

Multiple genes, especially immune-regulating genes, contribute to disease susceptibility in systemic sclerosis.

PURPOSE OF REVIEW: Systemic sclerosis (SSc) is a complex autoimmune disorder that occurs in a genetically susceptible host. Genetic studies of SSc in recent years have defined or suggested a number of new genes with polymorphisms conferring susceptibility to or protection against SSc. RECENT FINDINGS: Although not all genes fall neatly into one functional category, the major genes with polymorphisms associated with SSc are those involved in immune regulation and inflammation, especially T-cell differentiation, proliferation, activation, B-cell signaling, and innate immunity. SUMMARY: Understanding the functions of SSc-associated genes will provide important new insights in future studies to explore the pathogenesis of SSc, as well as to develop targeted therapies for SSc.

Botulinum toxin A in the treatment of trigeminal neuralgia.

AIMS: The aims of this study were to investigate the clinical effects and safety of botulinum toxin A (BTX-A) in treating trigeminal neuralgia and its influences on accompanied depression, anxiety, sleep disorders, and quality of life. METHODS AND MATERIAL: Eighty-seven patients with one-branch classical trigeminal neuralgia were injected with BTX-A in the pain area. The visual analogic scale score, sleep interference score, Hamilton Anxiety Scale score, Hamilton Depression Scale score, and side effects were assessed at 1 week prior to and 8 weeks after treatment, respectively. RESULTS: The effective rates after 1, 2, 4, and 8 weeks of treatment were 48.28%, 66.67%, 78.16%, and 80.46%, respectively. The effective rates of anxiety and depression were 90.32% and 96.77%, respectively. When compared to that before treatment, the quality of life was significantly better in terms of role-physical, bodily pain, general health, vitality, social functioning, role-emotional, and mental health (all P < 0.01), while physical function was not significantly improved (P = 0.317). CONCLUSION: BTX-A treatment can significantly relieve the pain in trigeminal neuralgia patients; improve anxiety, depression, and sleep; and increase the quality of life. BTX-A treatment is a safe and effective method to treat classical trigeminal neuralgia.

Microparticle entrapment for drug release from porous-surfaced bone implants.

Metallic bone implants face interfacial concerns, such as infection and insufficient bone formation. Combination of drug-loaded microparticles with the implant surface is a promising approach to reducing the concerns. The present study reports a simple method for this purpose. Drug-loaded chitosan and alginate microparticles were separately prepared by emulsion methods. Dry microparticles were introduced into porous titanium (Ti) coatings on Ti discs, and induced to agglomerate in pores by wetting with water. Agglomerates were stably entrapped in the pores: 77-82% retained in the coating after immersion in a water bath for 7 d. Discs carrying drug-loaded microparticles showed a rapid release within 6 h and a subsequent slow release up to 1 d. After coculture with Staphylococcus epidermidis for 24 h, the discs formed inhibition zones, confirming antibacterial properties. These suggest that the microparticle entrapment-based method is a promising method for reducing some of the bone-implant interfacial concerns.

Inactivation of the tumor suppressor WTX in a subset of pediatric tumors.

WTX is a tumor suppressor gene expressed during embryonic development and inactivated in 20-30% of cases of Wilms tumor, the most common pediatric kidney cancer. WTX has been implicated in several cellular processes including Wnt signaling, WT1 transcription, NRF2 degradation, and p53 function. Given that WTX is widely expressed during embryonic development and has been recently shown to regulate mesenchymal precursor cells in several organs, we tested for the potential involvement of WTX in a panel of pediatric tumors and adult sarcomas. A total of 353 tumors were screened for WTX deletions by fluorescence in situ hybridization (FISH). Discrete somatic WTX deletions were identified in two cases, one hepatoblastoma and one embryonal rhabdomyosarcoma, and confirmed by array comparative genomic hybridization. Direct sequencing of the full WTX open reading frame in 24 hepatoblastomas and 21 embryonal rhabdomyosarcomas did not identify additional mutations in these tumor types. The presence of WTX mRNA was confirmed in hepatoblastomas and embryonal rhabdomyosarcomas without WTX deletions by RNA-in situ hybridization. Notably, tumors with evidence of WTX inactivation, Wilms tumor, hepatoblastoma and rhabdomyosarcoma, are primitive tumors that resemble undifferentiated precursor cells and are linked to overgrowth syndromes. These results indicate that WTX inactivation occurs in a wider variety of tumor types than previously appreciated and point to shared pathogenic mechanisms between a subset of pediatric malignancies.

Complete Freund's adjuvant-induced acute inflammatory pain could be attenuated by triptolide via inhibiting spinal glia activation in rats.

BACKGROUND: Inflammatory pain is one of the most common clinical symptoms, mechanical allodynia and thermal hypersensitivities are associated with proinflammatory cytokines, and proinflammatory cytokine antagonists could alleviate the hypersensitivity. Previous studies showed that a traditional Chinese medicine ingredient, triptolide could inhibit inflammatory cytokines; however, it was still unknown whether triptolide had beneficial effects on treating inflammatory pain. MATERIALS AND METHODS: The effects of triptolide on Complete Freund's Adjuvant-induced acute inflammatory pain were investigated using behavioral tests. The activation of spinal glia was morphologically observed by immunofluorescent histochemistry. The levels of OX42, glia fibrillary acidic protein, and phosphorylated extracellular signal-regulated kinase in the spinal cord were detected by Western blot, and the messenger RNA levels of interleukin 1ß, interleukin 6, and tumor necrosis factor alpha were detected by real-time polymerase chain reaction. RESULTS: These results demonstrate that the triptolide effectively attenuates inflammatory pain induced by Complete Freund's Adjuvant, the underlying mechanism may regulate the phosphorylated extracellular signal-regulated kinase signaling pathway and inhibit the spinal glia activation, and then downregulate the proinflammatory cytokines; the triptolide may be clinically useful as a drug of anti-inflammatory pain. CONCLUSIONS: In the present study, we first reported that repeated systemic administration of triptolide could safely prevent and reverse inflammatory pain. The triptolide may serve as a new potential compound for developing safe therapeutics for patients suffering inflammatory pain.

Two doses of botulinum toxin type A for the treatment of trigeminal neuralgia: observation of therapeutic effect from a randomized, double-blind, placebo-controlled trial.

BACKGROUND: In the majority of cases, trigeminal neuralgia (TN) is a unilateral condition with ultra-short stabbing pain located along one or more branches of the trigeminal nerve. Although prophylactic pharmacological treatment is first choise, considering of insufficient effect or unacceptable side effects, neurosurgical treatment or lesion treatment should be considered. In addition to all these procedures mentioned above, one approach has been based on local intradermal and/or submucosal injections of Botulinum Toxin Type A (BTX-A). METHODS: We conducted a randomized, double-blind, placebo-controlled since November 2012, and adopted local multi-point injection in 84 cases of classical TN with different doses of BTX-A. Eighty four patients were randomized into following groups: placebo (n = 28); BTX-A 25U (n = 27); BTX-A 75U (n = 29). Follow-up visits were conducted every week after the injection, and the overall duration of the study for each patient were 8 weeks to observe the pain severity, efficacy and adverse reactions at endpoint. RESULTS: The visual analogue scale (VAS) scores of 25U and 75U groups reduced significantly compared to placebo as early as week 1, and sustained until week 8 throughout the study. There was no significant difference in VAS between 25U and 75U groups throughout the study. The response rates of 25U group (70.4%) and 75U group (86.2%) were significantly higher than placebo group (32.1%) at week 8, and there was no significant difference between 25U and 75U groups. Evaluation of the Patient Global Impression of Change (PGIC) demonstrated that 66.7% (25U group) and 75.9% (75U group) of the patients reported that their pain symptoms were 'much improved' or 'very much improved' versus 32.1% of the placebo group, and there was also no significant difference between 25U and 75U groups. All adverse reactions were graded as mild or moderate. CONCLUSIONS: BTX-A injection in TN is safe and efficient. It is a useful treatment for refractory TN. Lower dose (25U) and high dose (75U) were similar in efficacy in short-term.

Therapeutic effect of Botulinum toxin-A in 88 patients with trigeminal neuralgia with 14-month follow-up.

BACKGROUND: We investigated the long-term effects and safety of botulinum toxin-A (BTX-A) for treating trigeminal neuralgia (TN). We also studied long-term maintenance of this therapeutic effect. METHODS: A visual analog scale (VAS) score, pain attack frequency per day, patient's overall response to treatment and side effects during 14-month follow-up were evaluated in 88 patients with TN receiving BTX-A. The primary endpoints were pain severity (assessed by VAS) and pain attack frequency per day. The secondary endpoint was the patient's overall response to treatment, assessed using the Patient Global Impression of Change. The influence of different doses (≤50, 50-100 and ≥100 U) on the therapeutic effect was evaluated. RESULTS: Treatment was deemed "effective" within 1 month in 81 patients and at 2 months in 88 patients (100%). The shortest period of effective treatment was 3 months, and complete control of pain was observed in a maximum of 46 patients. The therapeutic effect decreased gradually after 3 months, and the prevalence of effective treatment at 14 months was 38.6%, with complete control of pain seen in 22 patients (25%). There was no significant difference in the prevalence of effective treatment between different dose groups at identical time points (p > 0.05). Three patients showed swelling at injection sites and 10 patients showed facial asymmetry, both of which disappeared spontaneously without special treatment. CONCLUSION: Local subcutaneous injection of BTX-A for TN treatment has considerable therapeutic effects lasting several months and is safe for this indication. At least one-quarter of patients maintained complete analgesia. The maintenance period of the therapeutic effect may be related to the reduction in the VAS score after the first injection of BTX-A.