In vivo three-dimensional brain imaging with chemiluminescence probes in Alzheimer's disease models.

Optical three-dimensional (3D) molecular imaging is highly desirable for providing precise distribution of the target-of-interest in disease models. However, such 3D imaging is still far from wide applications in biomedical research; 3D brain optical molecular imaging, in particular, has rarely been reported. In this report, we designed chemiluminescence probes with high quantum yields, relatively long emission wavelengths, and high signal-to-noise ratios to fulfill the requirements for 3D brain imaging in vivo. With assistance from density-function theory (DFT) computation, we designed ADLumin-Xs by locking up the rotation of the double bond via fusing the furan ring to the phenyl ring. Our results showed that ADLumin-5 had a high quantum yield of chemiluminescence and could bind to amyloid beta (Aß). Remarkably, ADLumin-5's radiance intensity in brain areas could reach 4 × 107 photon/s/cm2/sr, which is probably 100-fold higher than most chemiluminescence probes for in vivo imaging. Because of its strong emission, we demonstrated that ADLumin-5 could be used for in vivo 3D brain imaging in transgenic mouse models of Alzheimer's disease.

Physical- and Chemical-Dually ROS-Responsive Nano-in-Gel Platforms with Sequential Release of OX40 Agonist and PD-1 Inhibitor for Augmented Combination Immunotherapy.

Combination immunotherapy synergizing the PD-1 blockade with OX40 agonism has become a research hotspot, due to its enormous potential to overcome the restricted clinical objective response suffered by monotherapy. Questions of timing and sequence have been important aspects of immunotherapies when considering immunologic mechanisms; however, most of the time the straightforward additive approach was taken. Herein, our work is the first to investigate an alternative timing of aOX40 and aPD-1 treatment in melanoma-bearing mice, and it demonstrates that sequential administration (aOX40 first, then aPD-1 following) provided superior antitumor benefits than concurrent treatment. Based on that, to further avoid the limits suffered by solution forms, we adopted pharmaceutical technologies to construct an in situ-formed physical- and chemical-dually ROS-responsive nano-in-gel platform to implement sequential and prolonged release of aPD-1 and aOX40. Equipped with these advantages, the as-prepared (aPD-1NCs&aOX40)@Gels elicited augmented combination immunity and achieved great eradication of both primary and distant melanoma tumors in vivo.

Catalysts of Healing: A Symphony of Synthesis and Clinical Artistry in Small-Molecule Agents for Breast Cancer Alleviation.

Breast cancer, characterized by its molecular intricacy, has witnessed a surge in targeted therapeutics owing to the rise of small-molecule drugs. These entities, derived from cutting-edge synthetic routes, often encompassing multistage reactions and chiral synthesis, target a spectrum of oncogenic pathways. Their mechanisms of action range from modulating hormone receptor signaling and inhibiting kinase activity, to impeding DNA damage repair mechanisms. Clinical applications of these drugs have resulted in enhanced patient survival rates, reduction in disease recurrence, and improved overall therapeutic indices. Notably, certain molecules have showcased efficacy in drug-resistant breast cancer phenotypes, highlighting their potential in addressing treatment challenges. The evolution and approval of small-molecule drugs have ushered in a new era for breast cancer therapeutics. Their tailored synthetic pathways and defined mechanisms of action have augmented the precision and efficacy of treatment regimens, paving the way for improved patient outcomes in the face of this pervasive malignancy. The present review embarks on a detailed exploration of small-molecule drugs that have secured regulatory approval for breast cancer treatment, emphasizing their clinical applications, synthetic pathways, and distinct mechanisms of action.

Half-curcumin-based chemiluminescence probes and their applications in detecting quasi-stable oxidized protein.

Numerous methods have been reported for detecting ROS/RNS in vitro and in vivo; however, detecting methods for the secondary products of the ROS/RNS reactions, particularly quasi-stable oxidized products, have been much less explored. In this report, we observed that half-curcumins could generate chemiluminescence. In contrast to other chemiluminescence scaffolds, the distinguishing feature of a half-curcumin is the formation of a carbanion intermediate of its acetylacetone moiety, opening unique avenues for applications. In this study, we designed a series of half-curcumins CRANAD-Xs and found that CRANAD-164 could be used to detect quasi-stable oxidized proteins (QSOP) in vivo and in patient serum samples. We illustrated that CRANAD-164 could be used to monitor the responses of taurine, an amino acid with newly reported anti-aging capacity, in an inflammatory mouse model. Remarkably, we further demonstrated that the QSOP levels were much higher in the disease serum samples, including Alzheimer's disease, compared to the samples from healthy controls. Moreover, our results revealed that the sera chemiluminescence intensities were higher in aged healthy controls compared to young healthy subjects, suggesting that CRANAD-164 can be used to monitor the increase of QSOP during aging.

Bioluminescence Imaging with Functional Amyloid Reservoirs in Alzheimer's Disease Models.

Bioluminescence imaging has changed the daily practice of preclinical research on cancer and other diseases over the last few decades; however, it has rarely been applied in preclinical research on Alzheimer's disease (AD). In this Article, we demonstrated that bioluminescence imaging could be used to report the levels of amyloid beta (Aß) species in vivo. We hypothesized that AkaLumine, a newly discovered substrate for luciferase, could bind to Aß aggregates and plaques. We further speculated that the Aß aggregates/fibrils/plaques could be considered as "functional amyloids", which have a reservoir function to sequester and release AkaLumine to control the bioluminescence intensity, which could be used to report the levels of Aßs. Our hypotheses have been validated via in vitro solution tests, mimic studies with brain tissues and mice, two-photon imaging with AD mice, and in vivo bioluminescence imaging using transgenic AD mice that were virally transduced with AkaLuciferase (AkaLuc), a new luciferase that generates bioluminescence in the near-infrared window. As expected, compared to the control group, we observed that the Aß group showed lower bioluminescence intensity due to AkaLumine sequestering at early time points, while higher intensity was due to AkaLumine releasing at later time points. Lastly, we demonstrated that this method could be used to monitor AD progression and the therapeutic effectiveness of avagacestat, a well-studied gamma-secretase inhibitor. Importantly, a good correlation (R2 = 0.81) was established between in vivo bioluminescence signals and Aß burdens of the tested AD mice. We believe that our approach can be easily implemented into daily imaging experiments and has tremendous potential to change the daily practice of preclinical AD research.

A Photolabile Curcumin-Diazirine Analogue Enables Phototherapy with Physically and Molecularly Produced Light for Alzheimer's Disease Treatment.

The development of Alzheimer's disease (AD) drugs has recently witnessed substantial achievement. To further enhance the pool of drug candidates, it is crucial to explore non-traditional therapeutic avenues. In this study, we present the use of a photolabile curcumin-diazirine analogue, CRANAD-147, to induce changes in properties, structures (sequences), and neurotoxicity of amyloid beta (Aß) species both in cells and in vivo. This manipulation was achieved through irradiation with LED light or molecularly generated light, dubbed as "molecular light", emitted by the chemiluminescence probe ADLumin-4. Next, aided by molecular chemiluminescence imaging, we demonstrated that the combination of CRANAD-147/LED or CRANAD-147/ADLumin-4 (molecular light) could effectively slow down the accumulation of Aßs in transgenic 5xFAD mice in vivo. Leveraging the remarkable tissue penetration capacity of molecular light, phototherapy employing the synergistic effect of a photolabile Aß ligand and molecular light emerges as a promising alternative to conventional AD treatment interventions.

ß-Amyloid Peptides Manipulate Switching Behaviors of Donor-Acceptor Stenhouse Adducts.

Molecular switching plays a critical role in biological and displaying systems. Donor-acceptor Stenhouse adducts (DASAs) is a newly re-discovered series of switchable photochromes, and light is the most used approach to control its switching behavior. In this report, we speculated that hydrophobic binding pockets of biologically relevant peptides/proteins could be harnessed to alter its switching behavior without the assistance of light. We designed and synthesized a DASA compound SHA-2, and we demonstrated that the Aß40 species could stabilize SHA-2 in the linear conformation and decrease the rate of molecular switching via fluorescence spectral studies. Moreover, molecular dynamics simulation revealed that SHA-2 could bind to the hydrophobic fragment of the peptide and resulted in substantial changes in the tertiary structure of Aß40 monomer. This structural change is likely to impede the aggregation of Aß40, as evidenced by the results from thioflavin T fluorescence and ProteoStat aggregation detection experiments. We believe that our study opens a new window to alter the switching behavior of DASA via DASA-peptide/protein interactions.

CRANAD-28: A Robust Fluorescent Compound for Visualization of Amyloid Beta Plaques.

CRANAD-28, a difluoroboron curcumin analogue, has been demonstrated in earlier reports to successfully label amyloid beta (Aß) plaques for imaging both ex vivo and in vivo. CRANAD-28's imaging brightness, ability to penetrate the blood brain barrier, and low toxicity make the compound a potentially potent imaging tool in Alzheimer's research. In this study, the Aß-labeling ability of CRANAD-28 was investigated in further detail using histological staining to assess different criteria, including stained Aß plaque brightness, Aß plaque size, and Aß plaque number count. The results of this study demonstrated CRANAD-28 to be superior across all criteria assessed. Furthermore, CRANAD-28 and IBA-1 antibody were used to label Aß-plaques and microglia respectively. Statistical analysis with Spearman regression revealed a statistically significant negative correlation between the size of labeled Aß plaques and surrounding microglia density. This finding provides interesting insight into Aß plaque and microglia dynamism in AD pathology and corroborates the findings of previous studies. In addition, we found that CRANAD-28 provided distinct spectral signatures for Aßs in the core and periphery of the plaques. Based on the study's results, CRANAD-28 could be considered as an alternative standard for imaging Aß-plaques in future research studies.

Synthesis and evaluation of styrylpyran fluorophores for noninvasive detection of cerebral ß-amyloid deposits.

The development of amyloid-specific fluorophores allows the visualization of cerebral ß-amyloid deposits using optical imaging technology. In the present study, a series of smart styrylpyran fluorophores with compact donor-acceptor architecture were designed and evaluated for noninvasive detection of cerebral ß-amyloid deposits. Spectral behavior of the fluorophores changed significantly (optical turn-on) upon binding to ß-amyloid aggregates. Computational studies were conducted to correlate the experimental Kd values with calculated binding energies, speculating the relationship between fluorophore structure and ß-amyloid affinity. In vivo studies demonstrated that PAD-2 could discriminate APP/PS1 transgenic mice from wild type controls, with specific labeling of cerebral ß-amyloid deposits confirmed by ex vivo observation. Collectively, these styrylpyran fluorophores could provide a new scaffold for the development of optical imaging probes targeting cerebral ß-amyloid deposits.

In vivo detection of cerebral amyloid fibrils with smart dicynomethylene-4H-pyran-based fluorescence probe.

In vivo detection of cerebral ß-amyloid fibrils may facilitate the monitoring of ß-amyloidosis in the brain and effectiveness of antiamyloid therapies. Thioflavin T (ThT) is a widely used dye for the spectroscopic determination of ß-amyloid fibrils, but its ability to detect cerebral ß-amyloid fibrils in vivo is limited due to the charged molecule. To this end, a smart dicynomethylene-4H-pyran (DCM) fluorophore, namely, (E)-2-(2-(4-(dimethylamino)styryl)-6-methyl-4H-pyran-4-ylidene) malononitrile (PAD-1), was evaluated for in vivo fluorescence imaging of cerebral ß-amyloid fibrils. PAD-1 rapidly entered the brain with high initial brain uptake after intravenous injection, which is highly desirable for in vivo detection of ß-amyloid fibrils. PAD-1 displayed a turn-on effect, showing significant enhancement in fluorescence when bound to the aggregated ß-amyloid fibrils. It also showed specific labeling of ß-amyloid deposits in APP/PS1 transgenic mouse brains. Thus, PAD-1 proved to be a valuable alternative to ThT for cerebral ß-amyloid detection and may enable quantitative imaging in vivo.

A pyrane based fluorescence probe for noninvasive prediction of cerebral ß-amyloid fibrils.

A potential fluorescence probe for in vivo detection of cerebral ß-amyloid fibrils, (E)-2-(2-(2-(5-(dimethylamino)thiophen-2-yl)vinyl)-6-methyl-4H-pyran-4-ylidene)malononitrile (PT-1), was synthesized and evaluated. In experiments in vitro, PT-1 exhibited clear labeling of ß-amyloid fibrils and significant fluorescence changes upon binding to aggregated ß-amyloid fibrils. It also showed favorite kinetics in the brain, which is critical for cerebral imaging. In vivo fluorescence imaging with PT-1 and semi-quantitative analysis of the images further confirmed noninvasive visualization of cerebral ß-amyloid fibrils in vivo and obvious distinction between APP/PS1 transgenic mice and wild-type controls. The results demonstrate the potential of PT-1 as a novel fluorescence probe for noninvasive prediction of cerebral ß-amyloid fibrils.

Azithromycin cationic non-lecithoid nano/microparticles improve bioavailability and targeting efficiency.

PURPOSE: The purpose of this study was to develop and evaluate the azithromycin cationic non-lecithoid nano/microparticles with high bioavailability and lung targeting efficiency. METHODS: The cationic niosomes with different sizes (AMCNS-S and AMCNS-L) along with varied built-in characteristics were produced to achieve high bioavailability and lung targeting efficiency of azithromycin (AM) via two administration routes widely used in clinical practice, i.e., oral and intravenous routes, instead of transdermal route (by which the only marketed niosome-based drug delivery dermatologic products were given). The possible explanations for improved bioavailability and lung targeting efficacy were put forward here. RESULTS: AMCNS-S (or AMCNS-L) had high bioavailability, for example, the oral (or intravenous) relative bioavailability of AMCNS-S (or AMCNS-L) to free AM increased to 273.19% (or 163.50%). After intravenous administration, AMCNS-S (or AMCNS-L) had obvious lung targeting efficiency, for example, the lung AM concentration of AMCNS-S (or AMCNS-L) increased 16 (or 28) times that of free AM at 12 h; the AM concentration of AMCNS-S (or AMCNS-L) in lung was higher than that in heart and kidney all the time. CONCLUSIONS: The development of niosome-based AM nanocarriers provides valuable tactics in antibacterial therapy and in non-lecithoid niosomal application.

Federated attention consistent learning models for prostate cancer diagnosis and Gleason grading.

Artificial intelligence (AI) holds significant promise in transforming medical imaging, enhancing diagnostics, and refining treatment strategies. However, the reliance on extensive multicenter datasets for training AI models poses challenges due to privacy concerns. Federated learning provides a solution by facilitating collaborative model training across multiple centers without sharing raw data. This study introduces a federated attention-consistent learning (FACL) framework to address challenges associated with large-scale pathological images and data heterogeneity. FACL enhances model generalization by maximizing attention consistency between local clients and the server model. To ensure privacy and validate robustness, we incorporated differential privacy by introducing noise during parameter transfer. We assessed the effectiveness of FACL in cancer diagnosis and Gleason grading tasks using 19,461 whole-slide images of prostate cancer from multiple centers. In the diagnosis task, FACL achieved an area under the curve (AUC) of 0.9718, outperforming seven centers with an average AUC of 0.9499 when categories are relatively balanced. For the Gleason grading task, FACL attained a Kappa score of 0.8463, surpassing the average Kappa score of 0.7379 from six centers. In conclusion, FACL offers a robust, accurate, and cost-effective AI training model for prostate cancer pathology while maintaining effective data safeguards.

[Simultaneous determination of six ingredients in Huoxiang Zhengqi oral liquid by UPLC].

OBJECTIVE: To develop a UPLC method for the simultaneous determination of liquiritin, narirutin, hesperidin, ammonium glycyrrhetate, honokiol and magnolol in Huoxiang Zhengqi oral liquid. METHOD: A Zorbax Eclipse C18 column was used with the mobile phase of acetonitrile and 0. 05% phosphate acid by gradient elution at the detection wavelength of 220 nm. The flow rate was 0.42 mL x min(-1) and the column temperature was 30 degrees C. RESULT: The calibration curves were linear in the ranges of 0.001 7-0.034, 0.003 4-0.068, 0.006 4-0.128, 0.012 8-0.256, 0.003 2-0.064, 0.006 4-0.128 microg, respectively. The average recoveries were 103.3%, 98.39%, 98.29%, 102.1%, 98.45%, 102.2% with RSDs of 2.1%,1.0%, 0.50%, 2.3%, 0.9%, 2.0%, respectively. CONCLUSION: The UPLC method was simple, rapid and accurate, it could be used for quality control of Huoxiang Zhengqi oral liquid.

The Application of Bio-orthogonality for In Vivo Animal Imaging.

The application of bio-orthogonality has greatly facilitated numerous aspects of biological studies in recent years. In particular, bio-orthogonal chemistry has transformed biological research, including in vitro conjugate chemistry, target identification, and biomedical imaging. In this review, we highlighted examples of bio-orthogonal in vivo imaging published in recent years. We grouped the references into two major categories: bio-orthogonal chemistry-related imaging and in vivo imaging with bio-orthogonal nonconjugated pairing. Lastly, we discussed the challenges and opportunities of bio-orthogonality for in vivo imaging.

Imaging Brown Adipose Tissue with TSPO PET Tracers in Preclinical Animal Studies.

Brown adipose tissue (BAT) is closely associated with thermogenesis and related to numerous diseases, including type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), and obesity. Using molecular imaging technologies to monitor BAT could facilitate etiology elucidation, disease diagnosis, and therapeutics development. Translocator protein (TSPO), an 18 kDa protein that mainly locates on the outer mitochondrial membrane, has been proven as a promising biomarker for monitoring BAT mass. Here, we lay out the steps for imaging BAT with TSPO PET tracer [18F]-DPA in mouse studies.

In Vivo Three-dimensional Brain Imaging with Chemiluminescence Probes in Alzheimer's Disease Models.

Optical three-dimensional (3D) molecular imaging is highly desirable for providing precise distribution of the target-of-interest in disease models. However, such 3D imaging is still far from wide applications in biomedical research; 3D brain optical molecular imaging, in particular, has rarely been reported. In this report, we designed chemiluminescence probes with high quantum yields (QY), relatively long emission wavelengths, and high signal-to-noise ratios (SNRs) to fulfill the requirements for 3D brain imaging in vivo. With assistance from density-function theory (DFT) computation, we designed ADLumin-Xs by locking up the rotation of the double-bond via fusing the furan ring to the phenyl ring. Our results showed that ADLumin-5 had a high quantum yield of chemiluminescence and could bind to amyloid beta (Aß). Remarkably, ADLumin-5's radiance intensity in brain areas could reach 4×107 photon/s/cm2/sr, which is probably 100-fold higher than most chemiluminescence probes for in vivo imaging. Because of its strong emission, we demonstrated that ADLumin-5 could be used for in vivo 3D brain imaging in transgenic mouse models of Alzheimer's disease (AD).

Epitope alteration by small molecules and applications in drug discovery.

Small molecules and antibodies are normally considered separately in drug discovery, except in the case of covalent conjugates. We unexpectedly discovered several small molecules that could inhibit or enhance antibody-epitope interactions which opens new possibilities in drug discovery and therapeutic modulation of auto-antibodies. We first discovered a small molecule, CRANAD-17, that enhanced the binding of an antibody to amyloid beta (Aß), one of the major hallmarks of Alzheimer's disease, by stable triplex formation. Next, we found several small molecules that altered antibody-epitope interactions of tau and PD-L1 proteins, demonstrating the generality of this phenomenon. We report a new screening technology for ligand discovery, screening platform based on epitope alteration for drug discovery (SPEED), which is label-free for both the antibody and small molecule. SPEED, applied to an Aß antibody, led to the discovery of a small molecule, GNF5837, that inhibits Aß aggregation and another, obatoclax, that binds Aß plaques and can serve as a fluorescent reporter in brain slices of AD mice. We also found a small molecule that altered the binding between Aß and auto-antibodies from AD patient serum. SPEED reveals the sensitivity of antibody-epitope interactions to perturbation by small molecules and will have multiple applications in biotechnology and drug discovery.

Natural medicine HLXL targets multiple pathways of amyloid-mediated neuroinflammation and immune response in treating alzheimer's disease.

BACKGROUND: Based on the complex pathology of AD, a single chemical approach may not be sufficient to deal simultaneously with multiple pathways of amyloid-tau neuroinflammation. A polydrug approach which contains multiple bioactive components targeting multiple pathways in AD would be more appropriate. Here we focused on a Chinese medicine (HLXL), which contains 56 bioactive natural products identified in 11 medicinal plants and displays potent anti-inflammatory and immuno-modulatory activity. HYPOTHESIS/PURPOSE: We investigated the neuroimmune and neuroinflammation mechanisms by which HLXL may attenuate AD neuropathology. Specifically, we investigated the effects of HLXL on the neuropathology of AD using both transgenic mouse models as well as microglial cell-based models. STUDY DESIGN: The 5XFAD transgenic animals and microglial cell models were respectively treated with HLXL and Aß42, and/or lipopolysaccharide (LPS), and then analyzed focusing on microglia mediated Aß uptake and clearance, as well as pathway changes. METHODS: We showed that HLXL significantly reduced amyloid neuropathology by upregulation of microglia-mediated phagocytosis of Aß both in vivo and in vitro. HLXL displayed multi-modal mechanisms regulating pathways of phagocytosis and energy metabolism. RESULTS: Our results may not only open a new avenue to support pharmacologic modulation of neuroinflammation and the neuroimmune system for AD intervention, but also identify HLXL as a promising natural medicine for AD. CONCLUSION: It is conceivable that the traditional wisdom of natural medicine in combination with modern science and technology would be the best strategy in developing effective therapeutics for AD.

Differentiating Aß40 and Aß42 in amyloid plaques with a small molecule fluorescence probe.

Differentiating amyloid beta (Aß) subspecies Aß40 and Aß42 has long been considered an impossible mission with small-molecule probes. In this report, based on recently published structures of Aß fibrils, we designed iminocoumarin-thiazole (ICT) fluorescence probes to differentiate Aß40 and Aß42, among which Aß42 has much higher neurotoxicity. We demonstrated that ICTAD-1 robustly responds to Aß fibrils, evidenced by turn-on fluorescence intensity and red-shifting of emission peaks. Remarkably, ICTAD-1 showed different spectra towards Aß40 and Aß42 fibrils. In vitro results demonstrated that ICTAD-1 could be used to differentiate Aß40/42 in solutions. Moreover, our data revealed that ICTAD-1 could be used to separate Aß40/42 components in plaques of AD mouse brain slides. In addition, two-photon imaging suggested that ICTAD-1 was able to cross the BBB and label plaques in vivo. Interestingly, we observed that ICTAD-1 was specific toward plaques, but not cerebral amyloid angiopathy (CAA) on brain blood vessels. Given Aß40 and Aß42 species have significant differences of neurotoxicity, we believe that ICTAD-1 can be used as an important tool for basic studies and has the potential to provide a better diagnosis in the future.