Modified human skin cell isolation protocol and its influence on keratinocyte and melanocyte culture.

Introduction: With the increasing emphasis on the use of nonanimal ingredients in clinical care, studies have proposed the use of TrypLE™ as an alternative to trypsin. However, previous research has reported insufficient cell yield and viability when using TrypLE to isolate skin cells compared to the dispase/trypsin-EDTA method. This study aimed to propose an improved method for increasing the yield and viability of cells isolated by TrypLE and to evaluate isolated keratinocytes and melanocytes. Methods: Foreskin tissues were isolated to keratinocytes and melanocytes using the trypsin-EDTA protocol and our modified TrypLE protocol. The yield and viability of freshly isolated cells were compared, the epidermal residue after cell suspension filtration was analyzed histologically, and the expression of cytokeratin 14 (CK14) and Melan-A was detected by flow cytometry. After cultivation, keratinocytes and melanocytes were further examined for marker expression and proliferation. A coculture model of melanocytes and HaCaT cells was used to evaluate melanin transfer. Results: The yield, viability of total cells and expression of the keratinocyte marker CK14 were similar for freshly isolated cells from both protocols. No differences were observed in the histologic analysis of epidermal residues. Moreover, no differences in keratinocyte marker expression or melanocyte melanin transfer function were observed after culture. However, melanocytes generated using the TrypLE protocol exhibited increased Melan-A expression and proliferation in culture. Conclusion: Our TrypLE protocol not only solved the problems of insufficient cell yield and viability in previous studies but also preserved normal cell morphology and function, which enables the clinical treatment of depigmentation diseases.

Effects of interrupting residues on DNA dumbbell structures formed by CCTG tetranucleotide repeats associated with myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is a neurogenerative disease caused by caprylic/capric triglyceride (CCTG) tetranucleotide repeat expansions in intron 1 of the cellular nucleic acid-binding protein (CNBP) gene. Non-B DNA structures formed by CCTG repeats can promote genetic instability, whereas interrupting motifs of NCTG (N = A/T/G) within CCTG repeats help to maintain genomic stability. However, whether the interrupting motifs can affect DNA structures of CCTG repeats remains unclear. Here, we report that four CCTG repeats with an interrupting 3'-A/T/G residue formed dumbbell structures, whereas a non-interrupting 3'-C residue resulted in a multi-loop structure exhibiting conformational dynamics that may contribute to a higher tendency of escaping from DNA mismatch repair and causing repeat expansions. The results provide new structural insights into the genetic instability of CCTG repeats in DM2.

Phase separation of SPIN1 through its IDR facilitates histone methylation readout and tumorigenesis.

Spindlin1 (SPIN1) is a unique multivalent histone modification reader that plays a role in ribosomal RNA transcription, chromosome segregation, and tumorigenesis. However, the function of the extended N-terminal region of SPIN1 has remained unclear. Here, we discovered that SPIN1 can form phase-separated and liquid-like condensates both in vitro and in vivo through its N-terminal intrinsically disordered region (IDR). The phase separation of SPIN1 recruits the histone methyltransferase MLL1 to the same condensates and enriches the H3K4 methylation marks. This process also facilitates the binding of SPIN1 to H3K4me3 and activates tumorigenesis-related genes. Moreover, SPIN1-IDR enhances the genome-wide chromatin binding of SPIN1 and facilitates its localization to genes associated with the MAPK signaling pathway. These findings provide new insights into the biological function of the IDR in regulating SPIN1 activity and reveal a previously unrecognized role of SPIN1-IDR in histone methylation readout. Our study uncovers the crucial role of appropriate biophysical properties of SPIN1 in facilitating gene expression and links phase separation to tumorigenesis, which provides a new perspective for understanding the function of SPIN1.

High Glucose Is a Stimulation Signal of the Salt-Tolerant Yeast Zygosaccharomyces rouxii on Thermoadaptive Growth.

The salt-tolerant yeast Zygosaccharomyces rouxii is a typical aroma-producing yeast used in food brewing, but its mechanism of high temperature tolerance is still unclear. In this study, the response mechanism of Z. rouxii to glucose under high temperature stress at 40 °C was explored, based on the total synthetic lowest-nutrient medium. The results of the growth curves and scanning electron microscopy showed that high glucose was necessary for Z. rouxii to restore growth under high temperature stress, with the biomass at 300 g/L of glucose (OD600, 120h = 2.44 ± 0.26) being 8.71 times higher than that at 20 g/L (OD600, 120h = 0.28 ± 0.08). The results of the transcriptome analysis, combined with RT-qPCR, showed that the KEGG analysis of differentially expressed genes was enriched in pathways related to glucose metabolism, and high glucose (300 g/L) could effectively stimulate the gene expression of glucose transporters, trehalose synthesis pathways, and xylitol synthesis pathways under a high temperature, especially the expression of the glucose receptor gene RGT2 (up-regulated 193.7 times at 12 h). The corresponding metabolic characteristics showed that the contents of intracellular metabolites, such as glucose (Cmax, 6h = 6.50 ± 0.12 mg/g DCW), trehalose (Cmax, 8h = 369.00 ± 17.82 µg/g DCW), xylitol (Cmax, 8h = 1.79 ± 0.27 mg/g DCW), and glycerol (Cmax, 8h = 268.10 ± 44.49 µg/g DCW), also increased with time. The accumulation of acetic acid, as the main product of overflow metabolism under high temperature stress (intracellular Cmax, 2h = 126.30 ± 10.96 µg/g DCW; extracellular Cmax, 12h = 499.63 ± 27.16 mg/L), indicated that the downstream glycolysis pathway was active. Compared with the normal physiological concentration of glucose, a high glucose concentration can effectively stimulate the gene expression and metabolism of salt-tolerant Z. rouxii under high-temperature conditions to restore growth. This study helps to deepen the current understanding of the thermoadaptive growth mechanism of salt-tolerant Z. rouxii.

Innovative Biomaterials for Bone Tumor Treatment and Regeneration: Tackling Postoperative Challenges and Charting the Path Forward.

Surgical resection of bone tumors is the primary approach employed in the treatment of bone cancer. Simultaneously, perioperative interventions, particularly postoperative adjuvant anticancer strategies, play a crucial role in achieving satisfactory therapeutic outcomes. However, the occurrence of postoperative bone tumor recurrence, metastasis, extensive bone defects, and infection are significant risks that can result in unfavorable prognoses or even treatment failure. In recent years, there has been significant progress in the development of biomaterials, leading to the emergence of new treatment options for bone tumor therapy and bone regeneration. This progress report aims to comprehensively analyze the strategic development of unique therapeutic biomaterials with inherent healing properties and bioactive capabilities for bone tissue regeneration. These composite biomaterials, classified into metallic, inorganic non-metallic, and organic types, are thoroughly investigated for their responses to external stimuli such as light or magnetic fields, internal interventions including chemotherapy or catalytic therapy, and combination therapy, as well as their role in bone regeneration. Additionally, an overview of self-healing materials for osteogenesis is provided and their potential applications in combating osteosarcoma and promoting bone formation are explored. Furthermore, the safety concerns of integrated materials and current limitations are addressed, while also discussing the challenges and future prospects.

Metal nanoparticle hybrid hydrogels: the state-of-the-art of combining hard and soft materials to promote wound healing.

Wounds represent a grave affliction that profoundly impacts human well-being. Establishing barriers, preventing infections, and providing a conducive microenvironment constitute the crux of wound therapy. Hydrogel, a polymer with an intricate three-dimensional lattice, serves as a potent tool in erecting physical barriers and nurturing an environment conducive to wound healing. This enables effective control over exudation, hemostasis, accelerated wound closure, and diminished scar formation. As a result, hydrogels have gained extensive traction in the realm of wound treatment. Metallic nanoparticle carriers, characterized by their multifaceted responses encompassing acoustics, optics, and electronics, have demonstrated efficacy in wound management. Nevertheless, these carriers encounter challenges associated with swift clearance and nonuniform effectiveness. The hybridization of metallic nanoparticle carriers with hydrogels overcomes the shortcomings inherent in metallic nanoparticle-based wound therapy. This amalgamation not only addresses the limitations but also augments the mechanical robustness of hydrogels. It confers upon them attributes such as environmental responsiveness and multifunctionality, thereby synergizing strengths and compensating for weaknesses. This integration culminates in the precise and intelligent management of wounds. This review encapsulates the structural classifications, design strategies, therapeutic applications, and underlying mechanisms of metal nanoparticle hybrid hydrogels in the context of acute and chronic wound treatment. The discourse delves into the generation of novel or enhanced attributes arising from hybridization and how the current paradigm of wound therapy leverages these attributes. Amidst this continually evolving frontier, the potential of metal nanoparticle hybrid hydrogels to revolutionize wound treatment is underscored.

Structural investigation of pathogenic RFC1 AAGGG pentanucleotide repeats reveals a role of G-quadruplex in dysregulated gene expression in CANVAS.

An expansion of AAGGG pentanucleotide repeats in the replication factor C subunit 1 (RFC1) gene is the genetic cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), and it also links to several other neurodegenerative diseases including the Parkinson's disease. However, the pathogenic mechanism of RFC1 AAGGG repeat expansion remains enigmatic. Here, we report that the pathogenic RFC1 AAGGG repeats form DNA and RNA parallel G-quadruplex (G4) structures that play a role in impairing biological processes. We determine the first high-resolution nuclear magnetic resonance (NMR) structure of a bimolecular parallel G4 formed by d(AAGGG)2AA and reveal how AAGGG repeats fold into a higher-order structure composed of three G-tetrad layers, and further demonstrate the formation of intramolecular G4s in longer DNA and RNA repeats. The pathogenic AAGGG repeats, but not the nonpathogenic AAAAG repeats, form G4 structures to stall DNA replication and reduce gene expression via impairing the translation process in a repeat-length-dependent manner. Our results provide an unprecedented structural basis for understanding the pathogenic mechanism of AAGGG repeat expansion associated with CANVAS. In addition, the high-resolution structures resolved in this study will facilitate rational design of small-molecule ligands and helicases targeting G4s formed by AAGGG repeats for therapeutic interventions.

ARTC1-mediated VAPB ADP-ribosylation regulates calcium homeostasis.

Mono-ADP-ribosylation (MARylation) is a post-translational modification that regulates a variety of biological processes, including DNA damage repair, cell proliferation, metabolism, and stress and immune responses. In mammals, MARylation is mainly catalyzed by ADP-ribosyltransferases (ARTs), which consist of two groups: ART cholera toxin-like (ARTCs) and ART diphtheria toxin-like (ARTDs, also known as PARPs). The human ARTC (hARTC) family is composed of four members: two active mono-ADP-ARTs (hARTC1 and hARTC5) and two enzymatically inactive enzymes (hARTC3 and hARTC4). In this study, we systematically examined the homology, expression, and localization pattern of the hARTC family, with a particular focus on hARTC1. Our results showed that hARTC3 interacted with hARTC1 and promoted the enzymatic activity of hARTC1 by stabilizing hARTC1. We also identified vesicle-associated membrane protein-associated protein B (VAPB) as a new target of hARTC1 and pinpointed Arg50 of VAPB as the ADP-ribosylation site. Furthermore, we demonstrated that knockdown of hARTC1 impaired intracellular calcium homeostasis, highlighting the functional importance of hARTC1-mediated VAPB Arg50 ADP-ribosylation in regulating calcium homeostasis. In summary, our study identified a new target of hARTC1 in the endoplasmic reticulum and suggested that ARTC1 plays a role in regulating calcium signaling.

Molecular mechanism of human ISG20L2 for the ITS1 cleavage in the processing of 18S precursor ribosomal RNA.

The exonuclease ISG20L2 has been initially characterized for its role in the mammalian 5.8S rRNA 3' end maturation, specifically in the cleavage of ITS2 of 12S precursor ribosomal RNA (pre-rRNA). Here, we show that human ISG20L2 is also involved in 18S pre-rRNA maturation through removing the ITS1 region, and contributes to ribosomal biogenesis and cell proliferation. Furthermore, we determined the crystal structure of the ISG20L2 nuclease domain at 2.9 Å resolution. It exhibits the typical αßα fold of the DEDD 3'-5' exonuclease with a catalytic pocket located in the hollow near the center. The catalytic residues Asp183, Glu185, Asp267, His322 and Asp327 constitute the DEDDh motif in ISG20L2. The active pocket represents conformational flexibility in the absence of an RNA substrate. Using structural superposition and mutagenesis assay, we mapped RNA substrate binding residues in ISG20L2. Finally, cellular assays revealed that ISG20L2 is aberrantly up-regulated in colon adenocarcinoma and promotes colon cancer cell proliferation through regulating ribosome biogenesis. Together, these results reveal that ISG20L2 is a new enzymatic member for 18S pre-rRNA maturation, provide insights into the mechanism of ISG20L2 underlying pre-rRNA processing, and suggest that ISG20L2 is a potential therapeutic target for colon adenocarcinoma.

Extracellular vesicle biopotentiated hydrogels for diabetic wound healing: The art of living nanomaterials combined with soft scaffolds.

Diabetic wounds (DWs) pose a major challenge for the public health system owing to their high incidence, complex pathogenesis, and long recovery time; thus, there is an urgent need to develop innovative therapies to accelerate the healing process of diabetic wounds. As natural nanovesicles, extracellular vesicles (EVs) are rich in sources with low immunogenicity and abundant nutritive molecules and exert potent therapeutic effects on diabetic wound healing. To avoid the rapid removal of EVs, a suitable delivery system is required for their controlled release. Owing to the advantages of high porosity, good biocompatibility, and adjustable physical and chemical properties of hydrogels, EV biopotentiated hydrogels can aid in achieving precise and favorable therapy against diabetic wounds. This review highlights the different design strategies, therapeutic effects, and mechanisms of EV biopotentiated hydrogels. We also discussed the future challenges and opportunities of using EV biopotentiated hydrogels for diabetic wound healing.

RNF167-mediated ubiquitination of Tollip inhibits TNF-α-triggered NF-κB and MAPK activation.

Toll-interacting protein (Tollip) is a multifunctional regulator in cellular activities. However, whether its functions are subjected to post-translational modifications remains elusive. Here, we identified ubiquitination as a post-translational modification on Tollip. We found that Tollip interacted with ring finger protein 167 (RNF167) through its C-terminal coupling of ubiquitin to ER degradation (CUE) domain, and RNF167 functioned as the potential E3 ligase to attach K33-linked poly-ubiquitin chains to the Lys235 (K235) site of Tollip. Furthermore, we discovered Tollip could inhibit TNF-α-induced nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) activation, and substitution of Lys235 on Tollip to arginine failed to suppress TNF-α-NF-κB/MAPK (JNK) cascades, revealing the role of Tollip and its ubiquitination in NF-κB/MAPK pathways. Thus, our study reveals the novel biological function of Tollip and RNF167-dependent ubiquitination of Tollip in TNF-α signaling.

FBL promotes cancer cell resistance to DNA damage and BRCA1 transcription via YBX1.

Fibrillarin (FBL) is a highly conserved nucleolar methyltransferase responsible for methylation of ribosomal RNA and proteins. Here, we reveal a role for FBL in DNA damage response and its impact on cancer proliferation and sensitivity to DNA-damaging agents. FBL is highly expressed in various cancers and correlates with poor survival outcomes in cancer patients. Knockdown of FBL sensitizes tumor cells and xenografts to DNA crosslinking agents, and leads to homologous recombination-mediated DNA repair defects. We identify Y-box-binding protein-1 (YBX1) as a key interacting partner of FBL, and FBL increases the nuclear accumulation of YBX1 in response to DNA damage. We show that FBL promotes the expression of BRCA1 by increasing the binding of YBX1 to the BRCA1 promoter. Our study sheds light on the regulatory mechanism of FBL in tumorigenesis and DNA damage response, providing potential therapeutic targets to overcome chemoresistance in cancer.

Structural and biochemical insights into the molecular mechanism of TRPT1 for nucleic acid ADP-ribosylation.

Nucleic acid ADP-ribosylation has been established as a novel modification found in a wide diversity of prokaryotic and eukaryotic organisms. tRNA 2'-phosphotransferase 1 (TRPT1/TPT1/KptA) possesses ADP-ribosyltransferase (ART) activity and is able to ADP-ribosylate nucleic acids. However, the underlying molecular mechanism remains elusive. Here, we determined crystal structures of TRPT1s in complex with NAD+ from Homo sapiens, Mus musculus and Saccharomyces cerevisiae. Our results revealed that the eukaryotic TRPT1s adopt common mechanisms for both NAD+ and nucleic acid substrate binding. The conserved SGR motif induces a significant conformational change in the donor loop upon NAD+ binding to facilitate the catalytic reaction of ART. Moreover, the nucleic acid-binding residue redundancy provides structural flexibility to accommodate different nucleic acid substrates. Mutational assays revealed that TRPT1s employ different catalytic and nucleic acid-binding residues to perform nucleic acid ADP-ribosylation and RNA 2'-phosphotransferase activities. Finally, cellular assays revealed that the mammalian TRPT1 is able to promote endocervical HeLa cell survival and proliferation. Together, our results provide structural and biochemical insights into the molecular mechanism of TRPT1 for nucleic acid ADP-ribosylation.

Chinese herbal medicine for the treatment of cardiovascular diseases ─ Targeting cardiac ion channels.

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality, imposing an increasing global health burden. Cardiac ion channels (voltage-gated NaV, CaV, KVs, and others) synergistically shape the cardiac action potential (AP) and control the heartbeat. Dysfunction of these channels, due to genetic mutations, transcriptional or post-translational modifications, may disturb the AP and lead to arrhythmia, a major risk for CVD patients. Although there are five classes of anti-arrhythmic drugs available, they can have varying levels of efficacies and side effects on patients, possibly due to the complex pathogenesis of arrhythmias. As an alternative treatment option, Chinese herbal remedies have shown promise in regulating cardiac ion channels and providing anti-arrhythmic effects. In this review, we first discuss the role of cardiac ion channels in maintaining normal heart function and the pathogenesis of CVD, then summarize the classification of Chinese herbal compounds, and elaborate detailed mechanisms of their efficacy in regulating cardiac ion channels and in alleviating arrhythmia and CVD. We also address current limitations and opportunities for developing new anti-CVD drugs based on Chinese herbal medicines.

A Novel Minidumbbell DNA-Based Sensor for Silver Ion Detection.

Silver ion (Ag+) is one of the most common heavy metal ions that cause environmental pollution and affect human health, and therefore, its detection is of great importance in the field of analytical chemistry. Here, we report an 8-nucleotide (nt) minidumbbell DNA-based sensor (M-DNA) for Ag+ detection. The minidumbbell contained a unique reverse wobble C·C mispair in the minor groove, which served as the binding site for Ag+. The M-DNA sensor could achieve a detection limit of 2.1 nM and sense Ag+ in real environmental samples with high accuracy. More importantly, the M-DNA sensor exhibited advantages of fast kinetics and easy operation owing to the usage of an ultrashort oligonucleotide. The minidumbbell represents a new and minimal non-B DNA structural motif for Ag+ sensing, allowing for the further development of on-site environmental Ag+ detection devices.

A global perspective on compositions, risks, and ecological genesis of antibiotic resistance genes in biofilters of drinking water treatment plants.

Antibiotic resistance genes (ARGs) in biofilters of drinking water treatment plants (DWTPs) are regarded to be a remarkable potential health risk to human. A global survey on ARGs in biofilters may help evaluate their risk features as a whole. This study aims to explore the compositions, risks, and ecological genesis of ARGs in the biofilters of DWTPs. In total, 98 metagenomes of DWTP biofilters were collected from Sequence Read Archive  (SRA)  of National center for Biotechnology Information (NCBI), and the main ARG types were recognized, with multidrug, bacitracin, and beta-lactam as the first three types. Source water types (surface water vs. groundwater) were found to significantly influence antibiotic resistome, overpassing biofilter media and locations. Although ARG abundances of surface water biofilters were approximately five times higher than that of groundwater biofilters, the risk pattern of ARGs was highly similar between surface water biofilters and groundwater biofilters, and up to 99.61% of the ARGs on average belong to the least risk and unassessed ranks, and only 0.23% the highest risk rank. Monobactam biosynthesis pathway and prodigiosin biosynthesis pathway, two antibiotics biosynthesis pathways, were observed to be positively correlated with several ARG types and total ARG abundance in samples of surface water and groundwater biofilters, respectively, suggesting their potential roles in ecological genesis of ARGs. Overall, the results of this study would deepen our understanding of the ARG risks in biofilters of DWTPs and shed light on their ecological genesis inside.

Trophic interrelationships of bacteria are important for shaping soil protist communities.

Protists occupy multiple trophic positions in soil food webs and significantly contribute to organic matter decomposition and biogeochemical cycling. Protists can ingest bacteria and fungi as main food sources while being subjected to predation of invertebrates, but our understanding of how bottom-up and top-down regulations structure protists in natural soil habitats is limited. Here, we disentangle the effects of trophic regulations to the diversity and structure of soil protists in natural settings across northern and eastern Australia. Bacterial and invertebrate diversity were identified as important drivers of the diversity of functional groups of protists. Moreover, the compositions of protistan taxonomic and functional groups were better predicted by bacteria and fungi, than by soil invertebrates. There were strong trophic interconnections between protists and bacteria in multiple organismic network analysis. Altogether, the study provided new evidence that, bottom-up control of bacteria played an important role in shaping the soil protist community structure, which can be derived from feeding preferences of protists on microbial prey, and their intimate relationships in soil functioning or environmental adaptation. Our findings advance our knowledge about the impacts of different trophic groups on key soil organismic communities, with implications for ecosystem functions and services.

CD-NTase family member MB21D2 promotes cGAS-mediated antiviral and antitumor immunity.

cGAS/DncV-like nucleotidyltransferase (CD-NTase) family members are immune sensors that synthesize diverse nucleotide signals to initiate antiviral response in bacteria and animals. As a founding member of CD-NTase enzyme, cGAS has been identified as a key sensor for cytoplasmic DNA and type I interferons (IFNs) signaling in metazoan. However, the functions of other metazoan CD-NTases remain enigmatic. Here, we showed that Mab-21 domain-containing protein 2 (MB21D2), another member of the CD-NTase family, plays a positive role in modulating the cGAS-STING signaling in myeloid cells. Deficiency of MB21D2 in THP-1 cells or mice macrophages led to impaired production of type I interferon upon DNA stimulation. Consistently, Mb21d2-/- mice showed more susceptible to infection with DNA virus and faster growth of melanoma, compared to its counterparts. Mechanistically, MB21D2 specially bound with the N-terminal of cGAS, facilitated its liquid phase condensation and DNA-binding activity, leading to the enhanced production of cGAMP and subsequent IFN-ß production. Thus, our findings unveiled that the CD-NTase family member MB21D2 contributes to host antiviral and antitumor responses by enhancing cGAS activation.

Identification and Characterization of a Novel Carboxylesterase Belonging to Family VIII with Promiscuous Acyltransferase Activity Toward Cyanidin-3-O-Glucoside from a Soil Metagenomic Library.

An alkaline esterase, designated as EstXT1, was identified through functional screening from a metagenomic library. Sequence analysis revealed that EstXT1 belonged to the family VIII carboxylesterases and contained a characteristic conserved S-x-x-K motif and a deduced catalytic triad Ser56-Lys59-Tyr165. EstXT1 exhibited the strongest activity toward methyl ferulate at pH 8.0 and temperature 55°C and retained over 80% of its original activity after incubation in the pH range of 7.0-10.6 buffers. Biochemical characterization of the recombinant enzyme showed that it was activated by Zn2+ and Co2+ metal ion, while inhibited by Cu2+ and CTAB. EstXT1 exhibited significant promiscuous acyltransferase activity preferred to the acylation of benzyl alcohol acceptor using short-chain pNP-esters (C2-C8) as acyl-donors. A structure-function analysis indicated that a WAG motif is essential to acyltransferase activity. This is the first report example that WAG motif plays a pivotal role in acyltransferase activity in family VIII carboxylesterases beside WGG motif. Further experiment indicated that EstXT1 successfully acylated cyanidin-3-O-glucoside in aqueous solution. The results from the current investigation provided new insights for the family VIII carboxylesterase and lay a foundation for the potential applications of EstXT1 in food and biotechnology fields.