MYC-Targeting Inhibitors Generated from a Stereodiversified Bicyclic Peptide Library.

Here, we present the second generation of our bicyclic peptide library (NTB), featuring a stereodiversified structure and a simplified construction strategy. We utilized a tandem ring-opening metathesis and ring-closing metathesis reaction (ROM-RCM) to cyclize the linear peptide library in a single step, representing the first reported instance of this reaction being applied to the preparation of macrocyclic peptides. Moreover, the resulting bicyclic peptide can be easily linearized for MS/MS sequencing with a one-step deallylation process. We employed this library to screen against the E363-R378 epitope of MYC and identified several MYC-targeting bicyclic peptides. Subsequent in vitro cell studies demonstrated that one candidate, NT-B2R, effectively suppressed MYC transcription activities and cell proliferation.

Preparation of High-Performance Mn-Doped SnTe Materials at High Pressure and High Temperature.

SnTe is an environmentally friendly medium-temperature thermoelectric material, but its inherent low power factor (PF) and high lattice thermal conductivity severely limit its application. In this study, based on the fact that Mn doping can induce band convergence, the high-pressure and high-temperature (HPHT) synthesis method was used to optimize the sample preparation and shorten the synthesis cycle to 30 min. The results show that the Sn0.93Mn0.10Te sample achieves the maximum PF value of 34.00 µW cm-1 K-2 at 775 K and PFave value of 21.36 µW cm-1 K-2 between 300-875 K. Microstructure analysis shows that the high-pressure synthesis method introduces abundant grain boundaries, various grain sizes, multiple defects, and pore structures into the sample. These microscopic crystal structures can effectively scatter phonons and lower the lattice thermal conductivity. The modification of these micromorphologies results in the Sn0.92Mn0.11Te sample attaining a minimum lattice thermal conductivity of 0.45 W m-1 K-1 at 625 K. The thermoelectric figure of merit (zT) of sample Sn0.92Mn0.11Te reaches a maximum value of 1.1 at 775 K, and the zTave reaches 0.63 in the range of 300-875 K. This study indicates that the synergistic effect of Mn element doping and microstructure modification can effectively optimize the thermoelectric transport performance of SnTe materials.

Non-Mass Spectrometric Targeted Single-Cell Metabolomics.

Metabolic assays serve as pivotal tools in biomedical research, offering keen insights into cellular physiological and pathological states. While mass spectrometry (MS)-based metabolomics remains the gold standard for comprehensive, multiplexed analyses of cellular metabolites, innovative technologies are now emerging for the targeted, quantitative scrutiny of metabolites and metabolic pathways at the single-cell level. In this review, we elucidate an array of these advanced methodologies, spanning synthetic and surface chemistry techniques, imaging-based methods, and electrochemical approaches. We summarize the rationale, design principles, and practical applications for each method, and underscore the synergistic benefits of integrating single-cell metabolomics (scMet) with other single-cell omics technologies. Concluding, we identify prevailing challenges in the targeted scMet arena and offer a forward-looking commentary on future avenues and opportunities in this rapidly evolving field.

Hydroxyl-Rich Hydrophilic Endocytosis-Promoting Peptide with No Positive Charge.

Delivering cargo molecules across the plasma membrane is critical for biomedical research, and the need to develop molecularly well-defined tags that enable cargo transportation is ever-increasing. We report here a hydrophilic endocytosis-promoting peptide (EPP6) rich in hydroxyl groups with no positive charge. EPP6 can transport a wide array of small-molecule cargos into a diverse panel of animal cells. Mechanistic studies revealed that it entered the cells through a caveolin- and dynamin-dependent endocytosis pathway, mediated by the surface receptor fibrinogen C domain-containing protein 1. After endocytosis, EPP6 trafficked through early and late endosomes within 30 min. Over time, EPP6 partitioned among cytosol, lysosomes, and some long-lived compartments. It also demonstrated prominent transcytosis abilities in both in vitro and in vivo models. Our study proves that positive charge is not an indispensable feature for hydrophilic cell-penetrating peptides and provides a new category of molecularly well-defined delivery tags for biomedical applications.

Single-Cell Profiling of Fatty Acid Uptake Using Surface-Immobilized Dendrimers.

We present a chemical approach to profile fatty acid uptake in single cells. We use azide-modified analogues to probe the fatty acid influx and surface-immobilized dendrimers with dibenzocyclooctyne (DBCO) groups for detection. A competition between the fatty acid probes and BHQ2-azide quencher molecules generates fluorescence signals in a concentration-dependent manner. By integrating this method onto a microfluidics-based multiplex protein analysis platform, we resolved the relationships between fatty acid influx, oncogenic signaling activities, and cell proliferation in single glioblastoma cells. We found that p70S6K and 4EBP1 differentially correlated with fatty acid uptake. We validated that cotargeting p70S6K and fatty acid metabolism synergistically inhibited cell proliferation. Our work provided the first example of studying fatty acid metabolism in the context of protein signaling at single-cell resolution and generated new insights into cancer biology.

Digitonin-facilitated delivery of imaging probes enables single-cell analysis of AKT signalling activities in suspension cells.

Analyzing intracellular signalling protein activities in living cells promises a better understanding of the signalling cascade and related biological processes. We have previously developed cyclic peptide-based probes for analyzing intracellular AKT signalling activities, but these peptide probes were not cell-permeable. Implementing fusogenic liposomes as delivery vehicles could circumvent the problem when analyzing adherent cells, but it remained challenging to study suspension cells using similar approaches. Here, we present a method for delivering these imaging probes into suspension cells using digitonin, which could transiently perforate the cell membrane. Using U87, THP-1, and Jurkat cells as model systems representing suspended adherent cells, myeloid cells, and lymphoid cells, we demonstrated that low concentrations of digitonin enabled a sufficient amount of probes to enter the cytosol without affecting cell viability. We further combined this delivery method with a microwell single-cell chip and interrogated the AKT signalling dynamics in THP-1 and Jurkat cells, followed by immunofluorescence-based quantitation of AKT expression levels. We resolved the cellular heterogeneity in AKT signalling activities and showed that the kinetic patterns of AKT signalling and the AKT expression levels were related in THP-1 cells, but decoupled in Jurkat cells. We expect that our approach can be adapted to study other suspension cells.

A cyclic peptide antenna ligand for enhancing terbium luminescence.

We present here a cyclic peptide ligand, cy(WQETR), that binds to the terbium ion (Tb3+) and enhances Tb3+ luminescence intensity through the antenna effect. This peptide was identified through screening a cyclic peptide library against Tb3+ with an apparent EC50 of 540 µM. The tryptophan residue from the peptide directly interacts with the Tb3+ ion, which provides access to a low-lying triplet excited state of the tryptophan. Direct excitation of this triplet state enables energy transfer to the Tb3+ ion and enhances Tb3+ luminescence intensity by 150 fold. We further showcase the application of this cy(WQETR)-Tb3+ system by demonstrating the detection of tromethamine with a detection limit of 0.5 mM.

Construction of a Sequenceable Protein Mimetic Peptide Library with a True 3D Diversifiable Chemical Space.

We present here a library of protein mimetic bicyclic peptides. These nanosized structures exhibit rigid backbones and spatially diversifiable side chains. They present modular amino acids on all three linkages, providing access to a true 3D diversifiable chemical space. These peptides are synthesized through a Cu-catalyzed click reaction and a Ru-catalyzed ring-closing metathesis reaction. Their bicyclic topology can be reduced to a linear one, using Edman degradation and Pd-catalyzed deallylation reactions. The linearization approaches allow de novo sequencing through mass spectrometry methods. We demonstrate the function of a particular peptide that was identified through a high throughput screening against the E363-R378 epitope on the intrinsically disordered c-Myc oncoprotein. Intracellular delivery of this peptide could interfere with the c-Myc-mediated transcription and inhibit proliferation in a human glioblastoma cell line.

Establishment of dermal sheath cell line from Cashmere goat and characterizing cytokeratin 13 as its novel biomarker.

OBJECTIVE: To establish a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from Cashmere goat and clarify the similarities and differences among them. RESULTS: We established a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from the pelage skin hair follicles of Cashmere goat. The growth rate of dermal sheath cells was intermediate between that of dermal papilla cells and outer root sheath cells. Immunofluorescence experiments and reverse transcription-polymerase chain reaction analysis showed that at both the transcriptional and translational levels, the dermal sheath cells were alpha-smooth muscle actin (α-SMA)+/cytokeratin 13+, while the dermal papilla cells were α-SMA+/cytokeratin 13- and the outer root sheath cells were α-SMA-/cytokeratin 13+. Patterns of cytokeratin 13 expression could distinguish the dermal sheath cells from the dermal papilla cells. CONCLUSIONS: These results suggest that cytokeratin 13 could serve as a novel biomarker for dermal sheath cells of Cashmere goat, and should prove useful for researchers investigating dermal stem cells or interaction of different types of cells during hair cycle.

Surface Immobilization of Redox-Labile Fluorescent Probes: Enabling Single-Cell Co-Profiling of Aerobic Glycolysis and Oncogenic Protein Signaling Activities.

An analytical method is described for profiling lactate production in single cells via the use of coupled enzyme reactions on surface-grafted resazurin molecules. The immobilization of the redox-labile probes was achieved through chemical modifications on resazurin, followed by bio-orthogonal click reactions. The lactate detection was demonstrated to be sensitive and specific. The method was incorporated into a single-cell barcode chip for simultaneous quantification of aerobic glycolysis activities and oncogenic signaling phosphoproteins in cancer. The interplay between glycolysis and oncogenic signaling activities was interrogated on a glioblastoma cell line. Results revealed a drug-induced oncogenic signaling reliance accompanying shifted metabolic paradigms. A drug combination that exploits this induced reliance exhibited synergistic effects in growth inhibition.

Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury.

Peripheral nerve injury (PNI) is a common disease, which results in a partial or total loss of motor, sensory and autonomic functions, leading to a decrease in quality of life. Schwann cells play a vital role in maintaining the peripheral nervous system and in injury and repair. Using immunohistochemistry, Western blot, calcium assay and bromodeoxyuridine (BrdU) proliferation assay, the present study clearly demonstrated that P2X7 receptors (R) were expressed in myelinating and non-myelinating Schwann cells in longitudinal sections of sciatic nerves. After sciatic nerve injury (SNI), P2X7R expression in Schwann cells of injured sciatic nerves was significantly up-regulated during the early days of SNI. Double immunofluorescence of proliferating cell nuclear antigen (PCNA) and P2X7R implied that P2X7R may be involved in proliferation of Schwann cells. Further experiments on primary cultures of Schwann cells showed that P2X7R are functionally expressed in Schwann cells of rat sciatic nerves; ATP via P2X7R can promote Schwann cell proliferation, possibly via the MAPK/ERK intracellular signalling pathway. Other possible roles of P2X7R on Schwann cells are discussed.

DanHong injection dose-dependently varies amino acid metabolites and metabolic pathways in the treatment of rats with cerebral ischemia.

AIM: To determine how the relative amino acid contents and metabolic pathways regulate the pharmacological phenotypes in rats with cerebral ischemia after treatment with varying doses of DanHong injection (DHI). METHODS: Adult male rats underwent middle cerebral artery occlusion (MCAO), and were injected with DHI (DH-1: 1 mL/kg; DH-2: 2.5 mL/kg; DH-3: 5 mL/kg, and DH-4: 10 mL/kg, iv) daily for 3 d. The neurological deficit score, body weights and infarct volume were assessed. Serum levels of 20 free amino acids were determined using HPLC, and the values were transformed through the quantitative analysis of the amino acids in the serum metabolic spectrum. Multivariate statistical analysis methods (PCA and PLS-DA) and web-based metabolomics tools (MetPa and MetaboAnalyst) were used to analyze the biological data sets for the amino acids. RESULTS: Administration of DHI dose-dependently decreased cerebral infarct volume, and ameliorated neurological deficits. A total of 5, 6, 7 and 7 non-overlapping metabolites were identified in the DH-1, DH-2, DH-3, and DH-4 groups, respectively. Eight metabolites were shared between the DHI groups and the vehicle group. In addition, the serum levels of glutamic acid, aspartic acid and serine increased with increasing DHI dose. A total of 3, 2, 2 and 5 non-overlapping metabolic pathways were identified in the DH-1, DH-2, DH-3 and DH-4 groups, respectively, and glycine, serine, threonine and histidine metabolism were identified as overlapping pathways among the 4 dose groups. CONCLUSION: Overlapping and non-overlapping amino acid metabolites and metabolic pathways are associated with the dose-dependent neuroprotective effect of DHI.

Phenotype-dependent alteration of pathways and networks reveals a pure synergistic mechanism for compounds treating mouse cerebral ischemia.

AIM: Our previous studies have showed that ursodeoxycholic acid (UA) and jasminoidin (JA) effectively reduce cerebral infarct volume in mice. In this study we explored the pure synergistic mechanism of these compounds in treatment of mouse cerebral ischemia, which was defined as synergistic actions specific for phenotype variations after excluding interference from ineffective compounds. METHODS: Mice with focal cerebral ischemia were treated with UA, JA or a combination JA and UA (JU). Concha margaritifera (CM) was taken as ineffective compound. Cerebral infarct volume of the mice was determined, and the hippocampi were taken for microarray analysis. Particular signaling pathways and biological functions were enriched based on differentially expressed genes, and corresponding networks were constructed through Ingenuity Pathway Analysis. RESULTS: In phenotype analysis, UA, JA, and JU significantly reduced the ischemic infarct volume with JU being superior to UA or JA alone, while CM was ineffective. As a result, 4 pathways enriched in CM were excluded. Core pathways in the phenotype-positive groups (UA or JA) were involved in neuronal homeostasis and neuropathology. JU-contributing pathways included all UA-contributing and the majority (71.7%) of JA-contributing pathways, and 10 new core pathways whose effects included inflammatory immunity, apoptosis and nervous system development. The functions of JU group included all functions of JA group, the majority (93.1%) of UA-contributing functions, and 3 new core functions, which focused on physiological system development and function. CONCLUSION: The pure synergism between UA and JA underlies 10 new core pathways and 3 new core functions, which are involved in inflammation, immune responses, apoptosis and nervous system development.

The potential of vascular normalization for sensitization to radiotherapy.

Radiotherapy causes apoptosis mainly through direct or indirect damage to DNA via ionizing radiation, leading to DNA strand breaks. However, the efficacy of radiotherapy is attenuated in malignant tumor microenvironment (TME), such as hypoxia. Tumor vasculature, due to the imbalance of various angiogenic and anti-angiogenic factors, leads to irregular morphology of tumor neovasculature, disordered arrangement of endothelial cells, and too little peripheral coverage. This ultimately leads to a TME characterized by hypoxia, low pH and high interstitial pressure. This deleterious TME further exacerbates the adverse effects of tumor neovascularization and weakens the efficacy of conventional radiotherapy. Whereas normalization of blood vessels improves TME and thus the efficacy of radiotherapy. In addition to describing the research progress of radiotherapy sensitization and vascular normalization, this review focuses on the strategy and application prospect of modulating vascular normalization to improve the efficacy of radiotherapy sensitization.

Block of P2X7 receptors could partly reverse the delayed neuronal death in area CA1 of the hippocampus after transient global cerebral ischemia.

Transient global ischemia (which closely resembles clinical situations such as cardiac arrest, near drowning or severe systemic hypotension during surgical procedures), often induces delayed neuronal death in the brain, especially in the hippocampal CA1 region. The mechanism of ischemia/reperfusion (I/R) injury is not fully understood. In this study, we have shown that the P2X7 receptor antagonist, BBG, reduced delayed neuronal death in the hippocampal CA1 region after I/R injury; P2X7 receptor expression levels increased before delayed neuronal death after I/R injury; inhibition of the P2X7 receptor reduced I/R-induced microglial microvesicle-like components, IL-1ß expression, P38 phosphorylation, and glial activation in hippocampal CA1 region after I/R injury. These results indicate that antagonism of the P2X7 receptor and signaling pathways of microglial MV shedding, such as src-protein tyrosine kinase, P38 MAP kinase and A-SMase, might be a promising therapeutic strategy for clinical treatment of transient global cerebral I/R injury.

Hepatitis B DNA vaccine-polycation nano-complexes enhancing immune response by percutaneous administration with microneedle.

Percutaneous immune method is becoming an attractive alternative for DNA vaccine as a lot of antigen presenting cells are existed in the viable epidermis. However, due to the barrier function of stratum corneum, it would be hard for DNA vaccine to reach the viable epidermis of the skin. In order to deliver the DNA vaccine successfully cross the stratum corneum, pentagram silicon microneedle array was prepared in this study, and fluorescently labeled nanoparticle was taken as the model to observe the situation inside the skin processed by microneedle. Via microneedle nanoparticles could enter the skin through the micro-channel (diameter about 20-30 µm) and its amount is greatly larger than that enter though the hair follicle of intact skin. A new type of gene vector Pluronic P123-modified polyethyleneimine (P123-PEI) was synthesized by high molecular weight polyethylenimine and Pluronic P123 with the molar ratio of 1 : 1 to take the advantage of P123-PEI as low cytotoxicity and high transfection efficiency. Mice were immunized percutaneously with Hepatitis B DNA vaccine/P123-PEI nano-complexes by microneedle. The humoral and cellular immunity generated in percutaneously immunized mice through microneedle array by Hepatitis B DNA vaccine/P123-PEI nano-complex was significantly higher than that of DNA vaccine intramuscular administration.

Upregulation of chicken TLR4, TLR15 and MyD88 in heterophils and monocyte-derived macrophages stimulated with Eimeria tenella in vitro.

Coccidiosis, caused by Eimeria parasites, is a major parasitic disease responsible for great economic losses in the poultry industry. Toll-like receptor (TLR) family is one of the most important innate immune receptors, which involved in pathogen detection by initiating host responses, and it plays important roles in the reduction and clearance of pathogens. Very little information is available about the roles of chicken TLRs (ChTLRs) during Eimeria tenella infection. In the current study, mRNA expression of ChTLRs and associated signal adaptors in heterophils and monocyte-derived macrophages stimulated with E. tenella in vitro were measured by real-time quantitative polymerase chain reaction. The results showed that ChTLR4 and ChTLR15 expression were increased significantly in heterophils and monocyte-derived macrophages following live E. tenella sporozoites stimulation. The heat-killed E. tenella sporozoites stimulated higher expression of ChTLRs and signal adaptors than live sporozoites, the expression of ChTLR4, ChTLR15 and MyD88 in heterophils and monocyte-derived macrophages stimulated with heat-killed E. tenella sporozoites were up-regulated significantly than unstimulated cells. The results suggest that ChTLR4 and ChTLR15 are involved in response to E. tenella infection, and may operate in a MyD88-dependent manner for host defense.

Molecular features and antimicrobial susceptibility of Mycoplasma pneumoniae isolates from paediatric inpatients in Weihai, China: Characteristics of M. pneumoniae In Weihai.

OBJECTIVES: We analysed the molecular features and antimicrobial susceptibility of Mycoplasma pneumoniae isolates from Weihai, China, in 2019. METHODS: Pharyngeal swabs of 160 paediatric patients with pneumonia-related symptoms were collected and were subjected to culture and subsequent characteristic analysis. The characteristics of M. pneumoniae isolates were analysed by real-time PCR and genotyping. Antimicrobial susceptibility testing was performed against four antibiotics. All isolates were amplified for analysis of macrolide (ML) resistance mutations in domain V of the 23S rRNA gene and were genotyped using multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and 'AGT' VNTR detection in the p1 gene. RESULTS: The M. pneumoniae nucleic acid and culture-positive rate of 160 specimens were 88.1% (141/160) and 51.3% (82/160), respectively. Almost all isolates were ML-resistant (81/82). Point mutation at position 2063 in 23S rRNA was identified in all ML-resistant isolates. The ML resistance rate of M. pneumoniae genotype 2 isolates was 97.6% (41/42). MLVA types 4/5/7/2 and 4/5/7/3 belonged to genotype 1, while type 3/5/6/2 belonged to genotype 2. The numbers of 'AGT' VNTR in the p1 gene from all isolates was in the range of 5-15. CONCLUSION: This is the first report that the two genotypes of M. pneumoniae were present in a relatively equivalent ratio, with genotype 2 slightly predominant, in paediatric patients in Weihai in 2019, and the overall ML resistance rate was close to 100%. The minimum inhibitory concentration (MIC) of erythromycin in M. pneumoniae with ML resistance mutation A2063T in Weihai was higher than previously reported.

Treatment of psoriasis with recombinant human LFA3-antibody fusion protein: a multi-center, randomized, double-blind trial in a Chinese population.

To evaluate clinical efficacy and safety of injectable recombinant human LFA3-antibody fusion protein (rhLFA3-IgFP), a multi-center, randomized, double-blind, double-dummy, parallel-controlled clinical trial was performed in 212 cases of moderate to severe psoriasis. Intramuscular injection of rhLFA3-IgFP (15 mg/week) and oral administration of blank dummy methotrexate at the dose of 7.5 mg/week was performed in the patients in the experimental group, and control patients were orally administered with methotrexate at the dose of 7.5 mg/week and intramuscularly injected with the blank dummy rhLFA3-IgFP (15 mg/week). PASI was determined prior to and at 2, 4, 6, 8, 12, 16, 20 weeks after the treatment. The efficacy evaluation was carried out on 192 patients, and no significant differences were found in PASI50, PASI75 & PASI90 between the two groups after twelve weeks' treatment (p>0.05). After discontinuation, PASI scores continued to decrease drastically in the experiment group, whereas they increased in the control group. At 8 weeks after discontinuation, PASI scores were decreased by 62.32% (p<0.05) and 52.67% (p<0.05) in the experimental and control groups, respectively. No serious adverse reactions were observed. In conclusion, the results of our investigation demonstrated that rhLFA3-IgFP was an effective therapy for chronic plaque psoriasis with lasting action and low incidence of adverse reactions.

Single-cell profiling of D-2-hydroxyglutarate using surface-immobilized resazurin analogs.

D-2-hydroxyglutarate (D2HG) is over-produced as an oncometabolite due to mutations in isocitrate dehydrogenases (IDHs). Accumulation of D2HG can cause the dysfunction of many enzymes and genome-wide epigenetic alterations, which can promote oncogenesis. Quantification of D2HG at single-cell resolution can help understand the phenotypic signatures of IDH-mutant cancers and identify effective therapeutics. In this study, we developed an analytical method to detect D2HG levels in single cancer cells by adapting cascade enzymatic reactions on a resazurin-based fluorescence reporter. The resazurin probe was immobilized to the sensing surface via biotin-streptavidin interaction. This surface chemistry was rationally optimized to translate the D2HG levels to sensitive fluorescence readouts efficiently. This D2HG assay demonstrated good selectivity and high sensitivity toward D2HG, and it was compatible with the previously developed single-cell barcode chip (SCBC) technology. Using the SCBC platform, we performed simultaneous single-cell profiling of D2HG, glucose uptake, and critical oncogenic signaling proteins in single IDH-mutant glioma cells. The results unveiled the complex interplays between metabolic and oncogenic signaling and led to the identification of effective combination targeted therapy against these IDH-mutant glioma cells.