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Nucleic acid drugs are attracting significant attention as prospective therapeutics. However, their efficacy is hindered by challenges in penetrating cell membranes and reaching target tissues, limiting their applications. Nucleotidyl lipids, with their specific intermolecular interactions such as H-bonding and π-π stacking, offer a promising solution as gene delivery vehicles. In this study, a novel series of nucleotide-based amphiphiles were synthesized. These lipid molecules possess the ability to self-assemble into spherical vesicles of appropriate size and zeta potential in aqueous solution. Furthermore, their complexes with oligonucleotides demonstrated favorable biocompatibility and exhibited antiproliferative effects against a broad range of cancer cells. Additionally, when combined with the cationic lipid CLD, these complexes displayed promising in vitro performance and in vivo efficacy. By incorporating DSPE-PEGylated cRGD into the formulation, targeted accumulation of siG12D in pancreatic cancer cells increased from approximately 6% to 18%, leading to effective treatment outcomes (intravenous administration, 1 mg/kg). This finding holds significant importance for the liposomal delivery of nucleic acid drugs to extrahepatic tissues.

Abstract Aged livers have shown aggravated liver ischemia and reperfusion (IR) injury. Timely efferocytosis of apoptotic cells is a key mechanism for avoiding excessive inflammation and tissue injury. Here, we investigated the alteration of efferocytosis by aged macrophages and its role in regulating macrophage STING (stimulator of interferon genes) signaling and liver IR injury. Aged and young mice were subjected to liver partial IR model. Liver injury and inflammation were measured. Efferocytosis by aged macrophages and the underlying regulatory mechanism were analyzed as well. Aged macrophages exhibited impaired efferocytosis with decreased MerTK (c-mer proto-oncogene tyrosine kinase) activation, which was reversed by treatment of the MerTK CRISPR activation plasmid. Increased MerTK cleavage by ADAM17 (a disintegrin and metalloproteinase 17) due to enhanced ROS (reactive oxygen species) levels contributed to defective efferocytosis by aged macrophages. MerTK activation by suppressing ADAM17 or ROS improved aged macrophage efferocytosis, leading to reduced inflammatory liver injury. Moreover, increased apoptotic hepatocytes, DNA accumulation, and macrophage STING activation were observed in aged ischemic livers. Improvement in efferocytosis by aged macrophages via MerTK activation suppressed STING activation and inflammatory liver injury. Our study demonstrates that aging suppresses MerTK- mediated macrophage efferocytosis to promote macrophage STING activation and inflammatory liver IR injury, suggesting a new mechanism and potential therapy to promote inflammation resolution and efferocytosis in aged livers.

Antisense oligonucleotides (ASOs) are a class of therapeutics targeting mRNAs or genes that have attracted much attention. However, effective delivery and optimal accumulation in target tissues in vivo are still challenging issues. CT102 is an ASO that targets IGF1R mRNA and induces cell apoptosis. Herein, a detailed exploration of the tissue distribution of ASOs delivered by liposomes was carried out. A formulation that resulted in increased hepatic accumulation was identified based on multiple intermolecular interactions between DCP (cytidinyl/cationic lipid DNCA/CLD and DSPE-PEG) and oligonucleotides, including hydrogen bonding, π-π stacking, and electrostatic interactions. The structurally optimized CT102s present a novel strategy for the treatment of hepatocellular carcinoma. The gapmer CT102MOE5 and conjugate Glu-CT102MOE5 showed superior antiproliferation and IGF1R mRNA suppression effects at 100 nM in vitro and achieved greater efficacy at a lower dose and administration frequency in vivo. Combined transcriptome and proteome analyses revealed that additional associated targets and functional regulations might simultaneously exist in ASO therapy. These results showed that a combination of lipid encapsulation and structural optimization in the delivery of oligonucleotide drugs has favorable prospects for clinical application.

Abstract Mixed-lineage kinase domain-like protein (MLKL)-mediated necroptosis has been implicated in aggravating liver ischemia and reperfusion (IR) injury. However, the precise role and mechanism of MLKL in regulating oxidative DNA damage of hepatocytes and subsequent activation of macrophage stimulator of interferon genes (STING) signaling remains unclear. In this study, we investigated the role of MLKL in regulating the interplay between hepatocyte injury and macrophage pro-inflammatory responses during liver IR injury. We found that IR increased MLKL expression in liver tissues of wild type (WT) mice. MLKL knockout (KO) attenuated liver IR injury and suppressed the activation of cGAS-STING signaling in intrahepatic macrophages, which was abrogated by STING activation with its agonist. Mechanistically, IR induced oxidative DNA damage in hepatocytes, leading to cGAS-STING activation in macrophages, which was suppressed by MLKL KO. Moreover, increased PTEN-induced kinase 1 (PINK1)-mediated mitophagy contributed to reduced oxidative DNA damage in hepatocytes and subsequent decreased activation of STING signaling in macrophages in MLKL KO mice. Our findings demonstrated a non-canonical role of MLKL in the pathogenesis of liver IR. MLKL deficiency significantly promoted PINK1-mediated mitophagy activation to inhibit oxidative DNA damage in hepatocytes, which in turn suppressed macrophage cGAS-STING activation and inflammatory liver IR injury.

Abstract Background HCFC1 encodes transcriptional co‐regulator HCF‐1, which undergoes an unusual proteolytic maturation at a centrally located proteolysis domain. HCFC1 variants were associated with X‐linked cobalamin metabolism disorders and mental retardation‐3. This study aimed to explore the role of HCFC1 variants in common epilepsy and the mechanism underlying phenotype heterogeneity. Methods Whole‐exome sequencing was performed in a cohort of 313 patients with idiopathic partial (focal) epilepsy. Functional studies determined the effects of the variants on the proteolytic maturation of HCF‐1, cell proliferation and MMACHC expression. The role of HCFC1 variants in partial epilepsy was validated in another cohort from multiple centers. Results We identified seven hemizygous HCFC1 variants in 11 cases and confirmed the finding in the validation cohort with additional 13 cases and six more hemizygous variants. All patients showed partial epilepsies with favorable outcome. None of them had cobalamin disorders. Functional studies demonstrated that the variants in the proteolysis domain impaired the maturation by disrupting the cleavage process with loss of inhibition of cell growth but did not affect MMACHC expression that was associated with cobalamin disorder. The degree of functional impairment was correlated with the severity of phenotype. Further analysis demonstrated that variants within the proteolysis domain were associated with common and mild partial epilepsy, whereas those in the kelch domain were associated with cobalamin disorder featured by severe and even fatal epileptic encephalopathy, and those in the basic and acidic domains were associated with mainly intellectual disability. Conclusion HCFC1 is potentially a candidate gene for common partial epilepsy with distinct underlying mechanism of proteolysis dysfunction. The HCF‐1 domains played distinct functional roles and were associated with different clinical phenotypes, suggesting a sub‐molecular effect. The distinct difference between cobalamin disorders and idiopathic partial epilepsy in phenotype and pathogenic mechanism, implied a clinical significance in early diagnosis and management.

BackgroundRecessive SZT2 variants are reported to be associated with developmental and epileptic encephalopathy 18 (DEE-18) and occasionally neurodevelopment abnormalities (NDD) without seizures. This study aims to explore the phenotypic spectrum of SZT2 and the genotype-phenotype correlation.MethodsTrios-based whole-exome sequencing was performed in patients with epilepsy. Previously reported SZT2 mutations were systematically reviewed to analyze the genotype-phenotype correlations.ResultsSZT2 variants were identified in six unrelated cases with heterogeneous epilepsy, including one de novo null variant and five pairs of biallelic variants. These variants had no or low frequencies in controls. All missense variants were predicted to alter the hydrogen bonds with surrounding residues and/or protein stability. The three patients with null variants exhibited DEE. The patients with biallelic null mutations presented severe DEE featured by frequent spasms/tonic seizures and diffuse cortical dysplasia/periventricular nodular heterotopia. The three patients with biallelic missense variants presented mild partial epilepsy with favorable outcomes. Analysis of previously reported cases revealed that patients with biallelic null mutations presented significantly higher frequency of refractory seizures and earlier onset age of seizure than those with biallelic non-null mutations or with biallelic mutations containing one null variant.SignificanceThis study suggested that SZT2 variants were potentially associated with partial epilepsy with favorable outcomes without NDD, expanding the phenotypic spectrum of SZT2. The genotype-phenotype correlation helps in understanding the underlying mechanism of phenotypic variation.

Ticks are blood-feeding ectoparasites that also transmit various pathogens, posing severe risks to human and animal health. DNA viruses play a crucial role in the microbial ecology of ticks, but their distribution and ecological significance remain largely undetermined. Here, we assembled an extensive catalog encompassing 4320 viral operational taxonomic units (vOTUs) from six main dominant tick species in China, of which 94.8% have not been found in any other environment. To bridge the knowledge gap in tick DNA virus research and provide a crucial resource platform, we developed the Tick DNA Virus Database. This database includes the vOTUs that are known to cause diseases. Most of the predicted vOTUs are associated with dominant bacterial and archaeal phyla. We identified 105 virus-encoded putative auxiliary metabolic genes (AMGs) that are involved in host metabolism and environmental adaptation, potentially influencing ticks through both top-down and bottom-up mechanisms. The identification of microbial communities and antibiotic resistance in wild tick species suggests that wild ticks are reservoirs of antibiotic resistance and potential spreaders of antibiotic resistance. These findings reveal the potential role of tick viruses in ecosystems, highlighting the importance of monitoring tick microbiomes to address global public health challenges. [ABSTRACT FROM AUTHOR]

Small interfering RNAs (siRNAs) are widely studied for their highly specific gene silencing activity. However, obstacles remain to the clinical application of siRNAs. Attaching conjugates to siRNAs can improve their stability and broaden their application, and most functional conjugates of siRNAs locate at the 3′-terminus of the sense or antisense strand. In this work, we found that conjugating a group at the 5′-terminus of the antisense strand via phosphodiester was practicable, especially when the group was a flexible moiety such as an alkyl linker. When conjugating a bulky ligand, such as cRGD, the length of the 5′-phosphodiester linker between the ligand and the 5′-terminus of the antisense strand was the key in terms of RNA interference (RNAi). With a relative longer linker, the conjugates showed potency similar to siRNA. A highly efficient transfection system composed of a neutral cytidinyl lipid (DNCA) and a gemini-like cationic lipid (CLD) was employed to deliver siRNAs or their conjugates. The cRGD conjugates showed superior targeting delivery and antitumor efficacy in vivo and also selective cellular uptake in vitro. This unity of encapsulation and conjugation strategy may provide potential strategies for siRNA-based gene therapy.

IntroductionPRRT2 is a major causative gene for self-limited familial neonatal-infantile epilepsy, paroxysmal kinesigenic dyskinesia, and paroxysmal kinesigenic dyskinesia with infantile convulsions. Voluntary movement trigger is prominent in adolescence and adulthood, but the triggers are unknown in infants.MethodsA gene panel designed for targeted next-generation sequencing (NGS) was used to screen genetic abnormalities in a cohort of 45 cases with infantile convulsions. The copy number variation was detected by a computational method based on the normalized depth of coverage and validated by a quantitative real-time polymerase chain reaction (RT-qPCR) method. The genotype-phenotype correlation of the PRRT2 mutation gene was analyzed.ResultsA de novo heterozygous PRRT2 deletion was identified in a child who had infantile convulsions induced by vigorous sucking. Seizures happened during the change of feeding behavior from breast to formula, which led to hungry and vigorous sucking. Ictal electroencephalograms recorded seizures with focal origination, which provided direct evidence of epileptic seizures in infants with PRRT2 mutations. Seizures stopped soon after the feeding behavior was changed by reducing feeding interval time and extending feeding duration. Data reanalysis on our previously reported cases with PRRT2 mutations showed that six of 18 (33.3%) patients had infantile convulsions or infantile non-convulsion seizures during feeding. The mutations included two truncating mutations (c.579dupA/p.Glu194Argfs*6, and c.649dupC/p.Arg217Profs*8) that were identified in each of the three affected individuals.ConclusionsThis study suggests that feeding, especially vigorous sucking, is potentially a trigger and highlights the significance of feeding behavior in preventing seizures in infants with PRRT2 mutations. Identification of PRRT2 haploinsufficiency mutations in the patients with infantile convulsions induced by sucking suggested a potential genotype-phenotype correlation.

Single-stranded siRNA (ss-siRNA) refers to the antisense strand of siRNA, which plays the role of gene silencing. Since single-stranded RNA is unstable, the present study employed a homemade neutral cytidinyl/cationic lipid delivery system and chemical modifications to improve its stability. The results showed that with the aid of mixed lipids, ss-siRNA could knock down 40% of target mRNA at 25 nM. With 2ʹ-F/2ʹ-OMe, phosphorothioate and 5ʹ-terminal phosphorylation, the optimized ss-siRNA could knock down 80% of target mRNA at 25 nM. Further knocking down AGO2, the ss-siRNAs could not effectively silence target mRNAs. Analysis of the biodistribution in vivo showed that ss-siRNA was less durable than siRNA, but spread more quickly to tissues. This study provides a safe and efficient ss-siRNA delivery and modification strategy, which lays the foundation for future works.

BC15-31 is a DNA aptamer that targets heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which plays a crucial role in the process of pre-RNA maturation and is also essential for the rapid proliferation of tumor cells. In this research, we modified BC15-31 with a phosphorothioate (PS) backbone, LNA, and 2-O-MOE to enhance its stability and target affinity. In addition, a neutral cytidinyl lipid (DNCA) and a cationic lipid (CLD) were mixed to encapsulate modified aptamers with the aim of improving their cell permeability with low toxicity. Under the DNCA/CLD package, aptamers are mainly distributed in the nucleus. A modified sequence WW-24 showed an excellent selective anti-melanoma (A375 cells, ∼25 nM, 80%) activity, targeted to both hnRNP A1 and hnRNP A2/B1 found by the BLI experiment, and induced more early and late apoptosis in vitro, which also showed stronger antitumor effect and longer accumulation time in vivo. These results provide a new strategy for further clinical applications.

To characterize human leukocyte antigen (HLA) loci as risk factors in aromatic antiepileptic drug-induced maculopapular exanthema (AED-MPE). A case-control study was performed to investigate HLA loci involved in AED-MPE in a southern Han Chinese population. Between January 2007 and June 2019, 267 patients with carbamazepine (CBZ), oxcarbazepine (OXC), or lamotrigine (LTG) associated MPE and 387 matched drug-tolerant controls from six centers were enrolled. HLA-A/B/C/DRB1 genotypes were determined using sequence-based typing. Potential risk alleles were validated by meta-analysis using data from different populations and in silico analysis of protein-drug interactions. HLA-DRB1*04:06 was significantly associated with OXC-MPE (p = 0.002, pc = 0.04). HLA-B*38:02 was associated with CBZ-MPE (p = 0.03). When pooled, HLA-A*24:02, HLA-A*30:01, and HLA-B*35:01 additionally revealed significant association with AED-MPE. Logistic regression analysis showed a multiplicative interaction between HLA-A*24:02 and HLA-B*38:02 in CBZ-MPE. Meta-analysis of data from different populations revealed that HLA-24*:02 and HLA-A*30:01 were associated with AED-MPE (p = 0.02 and p = 0.04, respectively). In silico analysis of protein-drug interaction demonstrated that HLA-A*24:02 and HLA-A*30:01 had higher affinities with the three aromatic AEDs than the risk-free HLA-A allele. HLA-DRB1*04:06 showed relatively specific high affinity with S-monohydroxy derivative of OXC. HLA-DRB1*04:06 is a specific risk allele for OXC-induced MPE in the Southern Han Chinese. HLA-A*24:02, possibly HLA-A*30:01, are common risk factors for AED-MPE. The multiplicative risk potential between HLA-A*24:02 and HLA-B*38:02 suggests that patients with two risk alleles are at greater risk than those with one risk allele. Inclusion of these HLA alleles in pre-treatment screening would help estimating the risk of AED-MPE.

Abstract Background Cryptosporidium parvum is a globally distributed zoonotic parasite and an important opportunistic pathogen in immunocompromised patients. Little is known on the metabolic dynamics of the parasite, and study is hampered by the lack of molecular and genetic tools. Here we report the development of the first Agilent microarray for C. parvum (CpArray15K) that covers all predicted ORFs in the parasite genome. Global transcriptome analysis using CpArray15K coupled with real-time qRT-PCR uncovered a number of unique metabolic features in oocysts, the infectious and environmental stage of the parasite. Results Oocyst stage parasites were found to be highly active in protein synthesis, based on the high transcript levels of genes associated with ribosome biogenesis, transcription and translation. The proteasome and ubiquitin associated components were also highly active, implying that oocysts might employ protein degradation pathways to recycle amino acids in order to overcome the inability to synthesize amino acids de novo. Energy metabolism in oocysts was featured by the highest level of expression of lactate dehydrogenase (LDH) gene. We also studied parasite responses to UV-irradiation, and observed complex and dynamic regulations of gene expression. Notable changes included increased transcript levels of genes involved in DNA repair and intracellular trafficking. Among the stress-related genes, TCP-1 family members and some thioredoxin-associated genes appear to play more important roles in the recovery of UV-induced damages in the oocysts. Our observations also suggest that UV irradiation of oocysts results in increased activities in cytoskeletal rearrangement and intracellular membrane trafficking. Conclusions CpArray15K is the first microarray chip developed for C. parvum, which provides the Cryptosporidium research community a needed tool to study the parasite transcriptome and functional genomics. CpArray15K has been successfully used in profiling the gene expressions in the parasite oocysts as well as their responses to UV-irradiation. These observations shed light on how the parasite oocysts might adapt and respond to the hostile external environment and associated stress such as UV irradiation.

In this study, chemical modification of 2′-deoxyinosine (2′-dI) and D-/L-isothymidine (D-/L-isoT) was performed on AS1411. They could promote the nucleotide-protein interaction by changing the local conformation. Twenty modified sequences were obtained, FCL-I and FCL-II showed the most noticeable activity improvement. They stabilized the G-quadruplex, remained highly resistant to serum degradation and specificity for nucleolin, further inhibited tumor cell growth, exhibited a stronger ability to influence the different phases of the tumor cell cycle, induced S-phase arrest, promoted the inhibition of DNA replication, and suppressed the unwound function of a large T antigen as powerful as AS1411. The microarray analysis and TaqMan PCR results showed that FCL-II can upregulate the expression of four breast-cancer-related, lowly expressed miRNAs and downregulate the expression of three breast-cancer-related, highly expressed miRNAs (>2.5-fold). FCL-II resulted in enhanced treatment effects greater than AS1411 in animal experiments (p < 0.01). The computational results further proved that FCL-II exhibits more structural advantages than AS1411 for binding to the target protein nucleolin, indicating its great potential in antitumor therapy.

Purpose: Gene therapy, stem cell therapy, and low-energy extracorporeal shockwave therapy (ESWT) have been investigated as treatments for refractory erectile dysfunction (ED), but inconclusive evidence has been obtained. We investigated the effect of a next-generation electromagnetic cylinder ESWT device on an animal model of ED. Materials and Methods: Diabetes mellitus (DM)-induced rats were divided into 3 groups: group 1, control; group 2, DM; and group 3, DM+ESWT. Rats were treated with ESWT 3 times a week for 2 weeks. After the treatment course, intracavernous pressure was measured and the corpus cavernosum and cavernous nerve were evaluated. Results: In the DM group, all parameters predicted to be significantly lower in the ED model had statistically significantly decreased (p＜0.01). As a measurement of erectile function, intracavernous pressure was evaluated. The DM+ESWT group exhibited significantly restored erectile function compared to the DM group (p＜0.05). Moreover, ESWT treatment restored smooth muscle content, as assessed by Masson’s trichrome staining (p＜0.05). Finally, corporal tissue and the dorsal nerve were evaluated by immunohistochemistry, Western blotting, and ELISA. After ESWT treatment, vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), platelet endothelial cell adhesion molecule-1, cyclic guanosine monophosphate, and neuronal nitric oxide synthase (nNOS) expression levels were restored to levels in the DM group (p＜0.05). Conclusions: Electromagnetic cylinder ESWT device resulted in increased VEGF, nNOS, and eNOS expression; reduced smooth muscle atrophy; and increased endothelial cell regeneration in a DM-associated ED model. Our data suggest that safe and effective application could be possible in future clinical studies.

Cu-based tandem catalysis provides an efficient route to boost production of C2+ products for electrochemical carbon dioxide reduction (CO2RR), but colloidally synthesized Cu NPs are still less explored in tandem electrocatalyst design. A Cu-based tandem catalyst was constructed in hexane by assembling colloidal Cu NPs and Ni–N–C nanocarbon with tunable Cu NPs/Ni–N–C mass ratios, of which colloidal Cu NPs are homogeneously distributed over Ni–N–C nanocarbon. The Cu NPs/Ni–N–C tandem catalysts have been fully characterized by TEM, PXRD and XPS. In the H-type cell, the CO2RR contrast experiments verified that the optimized Cu NPs/Ni–N–C composite produced tandem effect in generating C2H4, benefiting from which a fourfold promotion in faradaic efficiency, and fivefold enhancement in partial current density were achieved compared to the reference Cu NPs/Vulcan carbon. Finally, the post-CO2RR Cu NPs/Ni–N–C catalysts were also measured by CV, EIS, TEM and XPS to further understand the change of tandem catalyst during CO2RR. An efficient Cu-based tandem catalyst was designed by compositing colloidal Cu NPs and Ni–N–C nanocarbon, which boosts cascade production of C2H4 from electrochemical carbon dioxide reduction. [ABSTRACT FROM AUTHOR]