Mn-Anti-CTLA4-CREKA-Sericin Nanotheragnostics for Enhanced Magnetic Resonance Imaging and Tumor Immunotherapy.

The integration of magnetic resonance imaging (MRI), cGAS-STING, and anti-CTLA-4 (aCTLA-4) based immunotherapy offers new opportunities for tumor precision therapy. However, the precise delivery of aCTLA-4 and manganese (Mn), an activator of cGAS, to tumors remains a major challenge for enhanced MRI and active immunotherapy. Herein, a theragnostic nanosphere Mn-CREKA-aCTLA-4-SS (MCCS) is prepared by covalently assembling Mn2+, silk sericin (SS), pentapeptide CREKA, and aCTLA-4. MCCS are stable with an average size of 160 nm and is almost negatively charged or neutral at pH 5.5/7.4. T1-weighted images showed MCCS actively targeted tumors to improve the relaxation rate r1 and contrast time of MRI. This studies demonstrated MCCS raises reactive oxygen species levels, activates the cGAS-STING pathway, stimulates effectors CD8+ and CD80+ T cells, reduces regulatory T cell numbers, and increases IFN-γ and granzyme secretion, thereby inducing tumor cells autophagy and apoptosis in vitro and in vivo. Also, MCCS are biocompatible and biosafe. These studies show the great potential of Mn-/SS-based integrative material MCCS for precision and personalized tumor nanotheragnostics.

Rational Design of Abasic Site-Containing DNA Triplexes To Bind Small Molecules with Low Nanomolar Dissociation Constants.

De novo design of functional biomacromolecules is of great interest to a wide range of fundamental science and technological applications, including understanding life evolution and biomacromolecular structures, developing novel catalysts, inventing medicines, and exploring high-performance materials. However, it is an extremely challenging task and its success is very limited. It requires a deep understanding of the relationships among the primary sequences, the 3D structures, and the functions of biomacromolecules. Herein, we report a rational, de novo design of a DNA aptamer that can bind melamine with high specificity and high affinity (dissociation constant Kd = 4.4 nM). The aptamer is essentially a DNA triplex, but contains an abasic site, to which the melamine binds. The aptamer-ligand recognition involves hydrogen-bonding, π-π stacking, and electrostatic interactions. This strategy has been further tested by designing aptamers to bind to guanosine. It is conceivable that such a rational strategy, with further development, would provide a general framework for designing functional DNA molecules.

Solution-Phase Synthesis of DNA Amphiphiles for DNA Micellar Assembly.

Hydrophobic moieties of amphiphilic DNAs can help DNAs penetrate cell membranes, but the conjugation of hydrophobic moieties to DNAs in solution phase remains challenging. Herein we report a solution-phase synthesis method to conjugate hydrophobic molecules to DNAs. This method is simple and efficient. The resulted amphiphilic DNAs can spontaneously assemble into micelles, which may serve as nanocarriers for cellular delivery of nucleic acids and water-insoluble drugs.

Comparison of two different intravitreal treatment regimens combined with systemic antiviral therapy for cytomegalovirus retinitis in patients with AIDS.

PURPOSE: To compare the efficacy and injection frequency of intravitreal low-dose vs. intermediate-dose ganciclovir therapy in acquired immune deficiency syndrome (AIDS) patients exhibiting cytomegalovirus retinitis (CMVR). METHODS: A prospective, single-centre, double-blinded, randomized controlled interventional study was conducted. Fifty patients with a total of 67 included eyes were randomly divided into low-dose (0.4 mg ganciclovir per week) and intermediate-dose (1.0 mg ganciclovir per week) groups. The primary clinical outcomes were the changes in best corrected visual acuity (BCVA) from baseline to the end of treatment and the 12-month follow-up visit as well as the number of intravitreal injections. RESULTS: In both groups, the median BCVA, expressed as the logarithm of the minimum angle of resolution (logMAR), improved significantly from baseline to the end of treatment (both p < 0.001), while vision loss from CMVR continued to occur at the 12-month visit. The mean number of injections was 5.8 in the low-dose group and 5.4 in the intermediate-dose group. No significant differences were detected between the two groups (p > 0.05). Regarding the location of CMVR, we found that Zone I lesions led to a worse visual outcome, more injections and a higher occurrence rate of complications than lesions in other zones (p < 0.05). CONCLUSIONS: The efficacy and frequency of injections to treat CMVR in AIDS patients were not significantly different between low and intermediate doses. Zone I lesions were associated with a worse visual outcome, more injections and a higher occurrence rate of CMVR-related complications than lesions in other zones.

A poly(thymine)-melamine duplex for the assembly of DNA nanomaterials.

The diversity of DNA duplex structures is limited by a binary pair of hydrogen-bonded motifs. Here we show that poly(thymine) self-associates into antiparallel, right-handed duplexes in the presence of melamine, a small molecule that presents a triplicate set of the hydrogen-bonding face of adenine. X-ray crystallography shows that in the complex two poly(thymine) strands wrap around a helical column of melamine, which hydrogen bonds to thymine residues on two of its three faces. The mechanical strength of the thymine-melamine-thymine triplet surpasses that of adenine-thymine base pairs, which enables a sensitive detection of melamine at 3 pM. The poly(thymine)-melamine duplex is orthogonal to native DNA base pairing and can undergo strand displacement without the need for overhangs. Its incorporation into two-dimensional grids and hybrid DNA-small-molecule polymers highlights the poly(thymine)-melamine duplex as an additional tool for DNA nanotechnology.

Rational Design of Ag/ZnO Hybrid Nanoparticles on Sericin/Agarose Composite Film for Enhanced Antimicrobial Applications.

Silver-based hybrid nanomaterials are receiving increasing attention as potential alternatives for traditional antimicrobial agents. Here, we proposed a simple and eco-friendly strategy to efficiently assemble zinc oxide nanoparticles (ZnO) and silver nanoparticles (AgNPs) on sericin-agarose composite film to impart superior antimicrobial activity. Based on a layer-by-layer self-assembly strategy, AgNPs and ZnO were immobilized on sericin-agarose films using the adhesion property of polydopamine. Scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray powder diffraction spectroscopy were used to show the morphology of AgNPs and ZnO on the surface of the composite film and analyze the composition and structure of AgNPs and ZnO, respectively. Water contact angle, swelling ratio, and mechanical property were determined to characterize the hydrophilicity, water absorption ability, and mechanical properties of the composite films. In addition, the antibacterial activity of the composite film was evaluated against Gram-positive and Gram-negative bacteria. The results showed that the composite film not only has desirable hydrophilicity, high water absorption ability, and favorable mechanical properties but also exhibits excellent antimicrobial activity against both Gram-positive and Gram-negative bacteria. It has shown great potential as a novel antimicrobial biomaterial for wound dressing, artificial skin, and tissue engineering.

Stimulator of interferon genes (STING) provides insect antiviral immunity by promoting Dredd caspase-mediated NF-κB activation.

The antiviral cGMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway is well characterized in mammalian cells. However, whether this pathway also plays a role in insect antiviral immunity is unknown. In this study, we found that cGAMP is produced in silkworm (Bombyx mori) cells infected with nucleopolyhedrovirus (NPV). In searches for STING-related sequences, we identified BmSTING, a potential cGAMP sensor in B. mori We observed that BmSTING overexpression effectively inhibits NPV replication in silkworm larvae, whereas dsRNA-mediated BmSTING knockdown resulted in higher viral load. Cleavage and nuclear translocation of BmRelish, a NF-κB-related transcription factor, was also observed when BmSTING was overexpressed and was enhanced by cGAMP stimulation or viral infection of B. mori larvae. Moreover, we identified a caspase-8-like protein (BmCasp8L) as a BmSTING-interacting molecule and as a suppressor of BmSTING-mediated BmRelish activation. Interestingly, cGAMP stimulation decreased BmCasp8L binding to BmSTING and increased BmRelish activity. Of note, an interaction between death-related ced-3/Nedd2-like caspase (BmDredd) and BmSTING promoted BmRelish cleavage for efficient antiviral signaling and protection of insect cells from viral infection. Our findings have uncovered BmSTING as a critical mediator of antiviral immunity in the model insect B. mori and have identified several BmSTING-interacting proteins that control antiviral defenses.

Self-Assembly of Microparticles by Supramolecular Homopolymerization of One Component DNA Molecule.

DNA is a superb molecule for self-assembly of nanostructures. Often many DNA strands are required for the assembly of one DNA nanostructure. For lowering the cost of synthesizing DNA strands and facilitating the assembly process, it is highly desirable to use a minimal number of unique strands for potential technological applications. Herein, a strategy is reported to assemble a series of DNA microparticles (DNAµPs) from one component DNA strand. As a demonstration of the application of the resulting DNAµPs, the design and assembled DNAµPs are modified to carry additional single-stranded tails on their surfaces. The modified DNAµPs can either capture other nucleic acids or display CpG motifs to stimulate immune responses.

A Novel Adenosine Kinase from Bombyx mori: Enzymatic Activity, Structure, and Biological Function.

Adenosine kinase (ADK) is the first enzyme in the adenosine remediation pathway that catalyzes adenosine phosphorylation into adenosine monophosphate, thus regulating adenosine homeostasis in cells. To obtain new insights into ADK from Bombyx mori (BmADK), we obtained recombinant BmADK, and analyzed its activity, structure, and function. Gel-filtration showed BmADK was a monomer with molecular weight of approximately 38 kDa. Circular dichroism spectra indicated BmADK had 36.8% α-helix and 29.9% ß-strand structures, respectively. The structure of BmADK was stable in pH 5.0-11.0, and not affected under 30 °C. The melting temperature and the enthalpy and entropy changes in the thermal transition of BmADK were 46.51 ± 0.50 °C, 253.43 ± 0.20 KJ/mol, and 0.79 ± 0.01 KJ/(mol·K), respectively. Site-directed mutagenesis demonstrated G68, S201, E229, and D303 were key amino acids for BmADK structure and activity. In particular, S201A mutation significantly increased the α-helix content of BmADK and its activity. BmADK was located in the cytoplasm and highly expressed in the silk gland during the pre-pupal stage. RNA interference revealed the downregulation of BmADK decreased ATG-8, Caspase-9, Ec-R, E74A, and Br-C expression, indicating it was likely involved in 20E signaling, apoptosis, and autophagy to regulate silk gland degeneration and silkworm metamorphosis. Our study greatly expanded the knowledge on the activity, structure, and role of ADK.

Rational Design and Fabrication of ZnONPs Functionalized Sericin/PVA Antimicrobial Sponge.

The interests of developing antimicrobial biomaterials based on silk sericin from Bombyx mori cocoon, have been shooting up in the last decades. Sericin is a valuable natural protein owing to its hydrophilicity, biodegradability, and biocompatibility. Here, we fabricated a sponge with antibacterial capacities for potential wound dressing application. By co-blending of sericin, polyvinyl alcohol (PVA) and zinc oxide nanoparticles (ZnONPs), the ZnONPs-sericin/PVA composite sponge (ZnONPs-SP) was successfully prepared after freeze-drying. Scanning electron microscopy showed the porous structure of ZnONPs-SP. Energy dispersive spectroscopy indicated the existence of Zn in the sponge. X-ray diffractometry revealed the hexagonal wurtzite structure of ZnONPs. Fourier transform infrared spectroscopy showed the biologic coupling of ZnONPs and sericin resulted in a decrease of α-helix and random coil contents, and an increase of ß-sheet structure in the sponge. The swelling experiment suggested ZnONPs-SP has high porosity, good hydrophilicity, and water absorption capability. The plate bacterial colony counting coupled with growth curve assays demonstrated that the composite sponge has an efficiently bacteriostatic effect against Staphylococcus aureus and Escherichia coli, respectively. Furthermore, the cell compatibility analysis suggested the composite sponge has excellent cytocompatibility on NIH3T3 cells. In all, ZnONPs-SP composite sponge has significant potentials in biomaterials such as wound dressing and tissue engineering.

Polydopamine-Based Surface Modification of ZnO Nanoparticles on Sericin/Polyvinyl Alcohol Composite Film for Antibacterial Application.

Silk sericin (SS) is a type of natural macromolecular protein with excellent hydrophilicity, biocompatibility and biodegradability, but also has very poor mechanical properties. To develop sericin-based wound dressings, we utilized polyvinyl alcohol (PVA) to reinforce the mechanical property of sericin by blending PVA and sericin, then modified zinc oxide nanoparticles (ZnO NPs) on SS/PVA film with the assistance of polydopamine (PDA) to endow SS/PVA film with antibacterial activity. Scanning electron microscopy, energy dispersive spectroscopy and X-ray powder diffraction demonstrated ZnO NPs were well grafted on PDA-SS/PVA film. Fourier transform infrared spectra suggested PDA coating and ZnONPs modification did not alter the structure of sericin and PVA. Water contact angle and swelling tests indicated the excellent hydrophilicity and swellability of ZnO NPs-PDA-SS/PVA composite film. Mass loss analysis showed ZnO NPs-PDA-SS/PVA film had excellent stability. The mechanical performance test suggested the improved tensile strength and elongation at break could meet the requirement of ZnO NPs-PDA-SS/PVA film in biomaterial applications. The antibacterial assay suggested the prepared ZnO NPs-PDA-SS/PVA composite film had a degree of antimicrobial activity against Escherichia coli and Staphylococcus aureus. The excellent hydrophilicity, swellability, stability, mechanical property and antibacterial activity greatly promote the possibility of ZnO NPs-PDA-SS/PVA composite film in antibacterial biomaterials application.

A Convenient, Rapid, Sensitive, and Reliable Spectrophotometric Assay for Adenylate Kinase Activity.

Enzymatic activity assays are essential and critical for the study of enzyme kinetics. Adenylate kinase (Adk) plays a fundamental role in cellular energy and nucleotide homeostasis. To date, assays based on different principles have been used for the determination of Adk activity. Here, we show a spectrophotometric analysis technique to determine Adk activity with bromothymol blue as a pH indicator. We analyzed the effects of substrates and the pH indicator on the assay using orthogonal design and then established the most optimal assay for Adk activity. Subsequently, we evaluated the thermostability of Adk and the inhibitory effect of KCl on Adk activity with this assay. Our results show that this assay is simple, rapid, and precise. It shows great potential as an alternative to the conventional Adk activity assay. Our results also suggest that orthogonal design is an effective approach, which is very suitable for the optimization of complex enzyme reaction conditions.

A rapid and sensitive colorimetric assay for the determination of adenosine kinase activity.

Adenosine kinase (ADK) plays an important role in the growth and development of organisms. A convenient, quick, reliable, sensitive and low-cost assay for ADK activity is of great significance. Here, we found the reaction system with bromothymol blue as the pH indicator had a maximum absorption peak at 614 nm. The absorbance change in 614 nm was positively correlated with the generated hydrogen ions in the reaction catalyzed by ADK. Then, we demonstrated this assay was feasible for ADK activity. Further, we analyzed the effects of buffer, bromothymol blue concentrations on the sensitivity of the assay, and investigated the sensitivity of ADK contents and adenosine concentration on the assay. Finally, we calculated the Km and Vmax of ADK from Bombyx mori with this assay. Our results suggested this assay was quick, convenient, reliable, sensitive and economic for the activity of ADK. It is an excellent alternative for the conventional ADK assays.

Polydopamine-Assisted Silver Nanoparticle Self-Assembly on Sericin/Agar Film for Potential Wound Dressing Application.

Silver nanoparticles (AgNPs) are extensively applied for their broad-spectrum and excellent antibacterial ability in recent years. Polydopamine (PDA) has great advantages for synthesizing large amounts of AgNPs, as it has multiple sites for silver ion binding and phenolic hydroxyl structure to reduce silver ions to AgNPs. Here, we mixed sericin and agar solution and dried at 65 °C to prepare a sericin (SS)/Agar composite film, and then coated polydopamine (PDA) on the surface of SS/Agar film by soaking SS/Agar film into polydopamine solution, subsequently synthesizing high-density AgNPs with the assistance of PDA to yield antibacterial AgNPs-PDA- SS/Agar film. Scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) spectra indicated the successful synthesis of high-density AgNPs on the surface of PDA-SS/Agar film. PDA coating and AgNPs modification did not affect the structure of sericin and agar. Furthermore, water contact angle, water absorption and mechanical property analysis showed that AgNPs-PDA-SS/Agar film had excellent hydrophilicity and proper mechanical properties. Inhibition zone and growth curve assays suggested the prepared film had excellent and long-lasting antibacterial ability. In addition, it had excellent cytocompatibility on the fibroblast NIH/3T3 cells. The film shows great potential as a novel kind of wound dressing.

A Novel AgNPs/Sericin/Agar Film with Enhanced Mechanical Property and Antibacterial Capability.

Silk sericin is a protein from a silkworm's cocoon. It has good biocompatibility, hydrophilicity, bioactivity, and biodegradability. However, sericin could not be used in biomedical materials directly because of its frangible characteristic. To develop multifunctional sericin-based materials for biomedical purposes, we prepared a sericin/agar (SS/agar) composite film through the blending of sericin and agar and repetitive freeze-thawing. Then, we synthesized silver nanoparticles (AgNPs) in situ on the surface of the composite film to endow it with antibacterial activity. Water contact angle, swelling and losing ratio, and mechanical properties analysis indicated that the composite film had excellent mechanical property, hydrophilicity, hygroscopicity, and stability. Scanning electron microscopy and X-ray photoelectron spectroscopy analysis confirmed the successful modification of AgNPs on the composite film. X-ray powder diffraction showed the face-centered cubic structures of the AgNPs. This AgNPs modified composite film exhibited an excellent antibacterial capability against Escherichia coli and Staphylococcus aureus. Our study develops a novel AgNPs/sericin/agar composite film with enhanced mechanical performance and an antimicrobial property for potential biomedical applications.

One-Step Synthesis of Silver Nanoparticles on Polydopamine-Coated Sericin/Polyvinyl Alcohol Composite Films for Potential Antimicrobial Applications.

Silk sericin has great potential as a biomaterial for biomedical applications due to its good hydrophilicity, reactivity, and biodegradability. To develop multifunctional sericin materials for potential antibacterial application, a one-step synthesis method for preparing silver nanoparticles (AgNPs) modified on polydopamine-coated sericin/polyvinyl alcohol (PVA) composite films was developed. Polydopamine (PDA) acted as both metal ion chelating and reducing agent to synthesize AgNPs in situ on the sericin/PVA composite film. Scanning electron microscopy and energy dispersive spectroscopy analysis revealed that polydopamine could effectively facilitate the high-density growth of AgNPs as a 3-D matrix. X-ray diffractometry studies suggested the synthesized AgNPs formed good face-centered cubic crystalline structures. Contact angle measurement and mechanical test indicated AgNPs modified PDA-sericin/PVA composite film had good hydrophilicity and mechanical property. The bacterial growth curve and inhibition zone assays showed the AgNPs modified PDA-sericin/PVA composite film had long-term antibacterial activities. This work develops a new method for the preparation of AgNPs modified PDA-sericin/PVA film with good hydrophilicity, mechanical performance and antibacterial activities for the potential antimicrobial application in biomedicine.

Sensitive detection of strong acidic condition by a novel rhodamine-based fluorescent pH chemosensor.

A novel rhodamine-based fluorescent pH probe responding to extremely low pH values has been synthesized and characterized. This probe showed an excellent photophysical response to pH on the basis that the colorless spirocyclic structure under basic conditions opened to a colored and highly fluorescent form under extreme acidity. The quantitative relationship between fluorescence intensity and pH value (1.75-2.62) was consistent with the equilibrium equation pH = pKa + log[(Imax - I)/(I - Imin)]. This sensitive pH probe was also characterized with good reversibility and no interaction with interfering metal ions, and was successfully applied to image Escherichia coli under strong acidity.

Silk gland-specific proteinase inhibitor serpin16 from the Bombyx mori shows cysteine proteinase inhibitory activity.

Serpins (serine proteinase inhibitors) are widely distributed in different species and are well known for their inhibitory activities towards serine proteinases. Here, we report the functional characterization of Bombyx mori serpin16. Expression analysis showed that serpin16 was specifically expressed at high levels in the silk gland at both the transcriptional and translational levels. Moreover, homology modeling and multi-sequence alignment suggested that serpin16 had a canonical serpin fold, but it contained a unique reactive center loop, which was obviously shorter than that of typical serpins. Inhibitory activity analyses revealed that the target proteinase of serpin18 is a cysteine proteinase, rather than a serine proteinase. Furthermore, a Michaelis complex model of serpin16 with its target proteinase was constructed to explain the structural basis of how serpin16 recognizes the cysteine proteinase and its target specificity.

A connectome-wide association study of altered functional connectivity in schizophrenia based on resting-state fMRI.

BACKGROUND: Aberrant resting-state functional connectivity is a neuropathological feature of schizophrenia (SCZ). Prior investigations into functional connectivity abnormalities have primarily employed seed-based connectivity analysis, necessitating predefined seed locations. To address this limitation, a data-driven multivariate method known as connectome-wide association study (CWAS) has been proposed for exploring whole-brain functional connectivity. METHODS: We conducted a CWAS analysis involving 46 patients with SCZ and 40 age- and sex-matched healthy controls. Multivariate distance matrix regression (MDMR) was utilized to identify key nodes in the brain. Subsequently, we conducted a follow-up seed-based connectivity analysis to elucidate specific connectivity patterns between regions of interest (ROIs). Additionally, we explored the spatial correlation between changes in functional connectivity and underlying molecular architectures by examining correlations between neurotransmitter/transporter distribution densities and functional connectivity. RESULTS: MDMR revealed the right medial frontal gyrus and the left calcarine sulcus as two key nodes. Follow-up analysis unveiled hypoconnectivity between the right medial frontal superior gyrus and the right fusiform gyrus, as well as hypoconnectivity between the left calcarine sulcus and the right lingual gyrus in SCZ. Notably, a significant association between functional connectivity strength and positive symptom severity was identified. Furthermore, altered functional connectivity patterns suggested potential dysfunctions in the dopamine, serotonin, and gamma-aminobutyric acid systems. CONCLUSIONS: This study elucidated reduced functional connectivity both within and between the medial frontal regions and the occipital cortex in patients with SCZ. Moreover, it indicated potential alterations in molecular architecture, thereby expanding current knowledge regarding neurobiological changes associated with SCZ.

Decoding silkworm spinning programmed by pH and metal ions.

Silk is one of the toughest fibrous materials known despite spun at ambient temperature and pressure with water as a solvent. It is a great challenge to reproduce high-performance artificial fibers comparable to natural silk by bionic for the incomplete understanding of silkworm spinning in vivo. Here, we found that amphipol and digitonin stabilized the structure of natural silk fibroin (NSF) by a large-scale screening in vitro, and then studied the close-to-native ultrastructure and hierarchical assembly of NSF in the silk gland lumen. Our study showed that NSF formed reversible flexible nanofibrils mainly composed of random coils with a sedimentation coefficient of 5.8 S and a diameter of about 4 nm, rather than a micellar or rod-like structure assembled by the aggregation of globular NSF molecules. Metal ions were required for NSF nanofibril formation. The successive pH decrease from posterior silk gland (PSG) to anterior silk gland (ASG) resulted in a gradual increase in NSF hydrophobicity, thus inducing the sol-gelation transition of NSF nanofibrils. NSF nanofibrils were randomly dispersed from PSG to ASG-1, and self-assembled into anisotropic herringbone patterns at ASG-2 near the spinneret ready for silkworm spinning. Our findings reveal the controlled self-assembly mechanism of the multi-scale hierarchical architecture of NSF from nanofibrils to herringbone patterns programmed by metal ions and pH gradient, which provides novel insights into the spinning mechanism of silk-secreting animals and bioinspired design of high-performance fibers.