Parameterization of Physiologically Based Biopharmaceutics Models: Workshop Summary Report.

This Article shares the proceedings from the August 29th, 2023 (day 1) workshop "Physiologically Based Biopharmaceutics Modeling (PBBM) Best Practices for Drug Product Quality: Regulatory and Industry Perspectives". The focus of the day was on model parametrization; regulatory authorities from Canada, the USA, Sweden, Belgium, and Norway presented their views on PBBM case studies submitted by industry members of the IQ consortium. The presentations shared key questions raised by regulators during the mock exercise, regarding the PBBM input parameters and their justification. These presentations also shed light on the regulatory assessment processes, content, and format requirements for future PBBM regulatory submissions. In addition, the day 1 breakout presentations and discussions gave the opportunity to share best practices around key questions faced by scientists when parametrizing PBBMs. Key questions included measurement and integration of drug substance solubility for crystalline vs amorphous drugs; impact of excipients on apparent drug solubility/supersaturation; modeling of acid-base reactions at the surface of the dissolving drug; choice of dissolution methods according to the formulation and drug properties with a view to predict the in vivo performance; mechanistic modeling of in vitro product dissolution data to predict in vivo dissolution for various patient populations/species; best practices for characterization of drug precipitation from simple or complex formulations and integration of the data in PBBM; incorporation of drug permeability into PBBM for various routes of uptake and prediction of permeability along the GI tract.

Isoliquiritigenin Alleviates Diabetic Kidney Disease via Oxidative Stress and the TLR4/NF-κB/NLRP3 Inflammasome Pathway.

Diabetic kidney disease (DKD) is the primary factor that causes chronic kidney disease and causes increasing mortality and morbidity due to its severe consequences. Isoliquiritigenin (ISL) is the primary element of licorice root that is physiologically active and has antifree radical, antioxidation, and antiapoptotic properties. However, the effect of ISL on DKD is still unclear and needs to be further improved. This study aims to evaluate the renoprotective effects of ISL on diabetes-induced renal injury and explores the underlying mechanisms involved. Male C57BL/6 mice are fed a high-fat diet and then injected with streptozotocin for 2 consecutive days to establish a diabetic model, and high-glucose-treated NRK-52E cells are used to investigate the renoprotective effects of ISL in DKD. The results show that ISL significantly preserves renal function and architecture in DKD. ISL suppresses oxidative stress and reduces ROS levels, inhibiting the activation of the NF-κB and the NLRP3 inflammasome and the occurrence of pyroptosis. Moreover, the study finds that ISL can inhibit the mitochondrial apoptotic pathway. In addition, the study confirms the inhibitory effect of ISL on the TLR4/NF-κB/NLRP3 inflammasome pathway. These observations demonstrate that the natural flavonoid compound ISL can be a promising agent for the treatment of DKD.

Dietary patterns and multiple chronic diseases in older adults.

BACKGROUND: The prevalence rate of multiple chronic diseases among the elderly is relatively high, posing a risk to their health and also imposing a financial burden on them. Optimal dietary patterns have positive effects on multiple chronic diseases. This study aimed to identify dietary patterns associated with multiple chronic diseases in older adults. METHODS: Dietary intake was assessed through two non-consecutive 24-hour dietary recalls. The presence of multiple chronic diseases was assessed based on the existence of dyslipidemia, hypertension, chronic kidney disease, sleep disorders, diabetes, moderate or severe depressive symptoms, and cognitive impairment, with two or more of these conditions being considered. Latent class analysis was used to identify types of multiple chronic diseases, and two-step cluster analysis was used to determine individual dietary patterns. Logistic regression analysis with robust standard errors was conducted to determine the associations between dietary patterns and types of multiple chronic diseases. RESULTS: Three dietary patterns and three types of multiple chronic diseases were identified. Individuals following a diet rich in legumes, meat, vegetables and fruits (HLMVF dietary pattern) were 59% less likely to have the cardiometabolic cognitive impairment comorbidity (CCC) than those following a diet rich in milk and eggs but with low grain intake (HME-LG) (OR = 0.41, 95% CI: 0.27-0.64, P < 0.001) and 66% less likely to have the especially sleep disorders comorbidity (ESC) than those following a diet rich in grains but lacking milk and eggs (HG-LME) (OR = 0.34, 95% CI: 0.14-0.87, P < 0.05). DISCUSSION: The HLMVF dietary pattern may serve as a healthy dietary pattern to reduce the incidence of multiple chronic diseases and should be promoted among the older adult population.

Single-Cell Imaging of mRNA by Target RNA-Initiated RCA.

We present a powerful method for direct mRNA detection based on ligation-based recognition and in situ amplification, capable of single-cell imaging mRNA at single-nucleotide and single-molecule resolution. Attributed to the use of Splint R ligase that can ligate padlock probe with RNA as target template, this method can efficiently detect mRNA in the absence of reverse transcription. This method enables spatial localization and correlation analysis of gene expression in single cells, which helps us to elucidate gene function and regulatory mechanisms.

A probe for NIR-II imaging and multimodal analysis of early Alzheimer's disease by targeting CTGF.

To date, earlier diagnosis of Alzheimer's disease (AD) is still challenging. Recent studies revealed the elevated expression of connective tissue growth factor (CTGF) in AD brain is an upstream regulator of amyloid-beta (Aß) plaque, thus CTGF could be an earlier diagnostic biomarker of AD than Aß plaque. Herein, we develop a peptide-coated gold nanocluster that specifically targets CTGF with high affinity (KD ~ 21.9 nM). The probe can well penetrate the blood-brain-barrier (BBB) of APP/PS1 transgenic mice at early-stage (earlier than 3-month-old) in vivo, allowing non-invasive NIR-II imaging of CTGF when there is no appearance of Aß plaque deposition. Notably, this probe can also be applied to measuring CTGF on postmortem brain sections by multimodal analysis, including fluorescence imaging, peroxidase-like chromogenic imaging, and ICP-MS quantitation, which enables distinguishment between the brains of AD patients and healthy people. This probe possesses great potential for precise diagnosis of earlier AD before Aß plaque formation.

Study on the Impact of Dietary Patterns on Cardiovascular Metabolic Comorbidities among Adults.

Background: The prevalence of cardiovascular metabolic comorbidities (CMM) among adults is relatively high, imposing a heavy burden on individuals, families, and society. Dietary patterns play a significant role in the occurrence and development of CMM. This study aimed to identify the combined types of CMM in adult populations and investigate the impact of dietary patterns on CMM. Methods: Participants in this study were from the sixth wave of the China Health and Nutrition Survey (CHNS). Dietary intake was assessed using a three-day 24-hour dietary recall method among 4,963 participants. Latent profile analysis was used to determine dietary pattern types. Two-step cluster analysis was performed to identify the combined types of CMM based on the participants' conditions of hyperuricemia, dyslipidemia, diabetes, renal dysfunction, hypertension, and stroke. Logistic regression analysis with robust standard errors was used to determine the impact of dietary patterns on CMM. Results: Participants were clustered into three dietary patterns (Pattern 1 to 3) and five CMM types (Class I to V). Class I combined six diseases, with a low proportion of diabetes. Class II also combined six diseases but with a high proportion of diabetes. Class III combined four diseases, with a high proportion of hypertension. Class IV combined three diseases, with the highest proportions of hyperuricemia, diabetes, and renal dysfunction. Class V combined two diseases, with high proportions of dyslipidemia and renal dysfunction. Patients with Class III CMM had a significantly higher average age than the other four classes (P ≤ 0.05). Compared to those with isolated dyslipidemia, individuals with a low-grain, high-fruit, milk, and egg (LCHFM) dietary pattern had a higher risk of developing dyslipidemia combined with renal dysfunction (Class V CMM) with an odds ratio of 2.001 (95% CI 1.011-3.960, P≤ 0.05). Conclusion: For individuals with isolated dyslipidemia, avoiding a low-grain, high-fruit, milk, and egg (LCHFM) dietary pattern may help reduce the risk of developing dyslipidemia combined with renal dysfunction (Class V CMM).

Reactive Oxygen Species Amplifier for Apoptosis-Ferroptosis Mediated High-Efficiency Radiosensitization of Tumors.

Insufficient reactive oxygen species (ROS) production and radioresistance have consistently contributed to the failure of radiotherapy (RT). The development of a biomaterial capable of activating ROS-induced apoptosis and ferroptosis is a potential strategy to enhance RT sensitivity. To achieve precision and high-efficiency RT, the theranostic nanoplatform Au/Cu nanodots (Au/CuNDs) were designed for dual-mode imaging, amplifying ROS generation, and inducing apoptosis-ferroptosis to sensitize RT. A large amount of ROS is derived from three aspects: (1) When exposed to ionizing radiation, Au/CuNDs effectively absorb photons and emit various electrons, which can interact with water to produce ROS. (2) Au/CuNDs act as a catalase-like to produce abundant ROS through Fenton reaction with hydrogen peroxide overexpressed of tumor cells. (3) Au/CuNDs deplete overexpressed glutathione, which causes the accumulation of ROS. Large amounts of ROS and ionizing radiation further lead to apoptosis by increasing DNA damage, and ferroptosis by enhancing lipid peroxidation, significantly improving the therapeutic efficiency of RT. Furthermore, Au/CuNDs serve as an excellent nanoprobe for high-resolution near-infrared fluorescence imaging and computed tomography of tumors. The promising dual-mode imaging performance shows their potential application in clinical cancer detection and imaging-guided precision RT, minimizing damage to adjacent normal tissues during RT. In summary, our developed theranostic nanoplatform integrates dual-mode imaging and sensitizes RT via ROS-activated apoptosis-ferroptosis, offering a promising prospect for clinical cancer diagnosis and treatment.

METTL3-mediated m6A modification increases Hspa1a stability to inhibit osteoblast aging.

Senile osteoporosis is mainly caused by osteoblasts attenuation, which results in reduced bone mass and disrupted bone remodeling. Numerous studies have focused on the regulatory role of m6A modification in osteoporosis; however, most of the studies have investigated the differentiation of bone marrow mesenchymal stem cells (BMSCs), while the direct regulatory mechanism of m6A on osteoblasts remains unknown. This study revealed that the progression of senile osteoporosis is closely related to the downregulation of m6A modification and methyltransferase-like 3 (METTL3). Overexpression of METTL3 inhibits osteoblast aging. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that METTL3 upregulates the stability of Hspa1a mRNA, thereby inhibiting osteoblast aging. Moreover, the results demonstrated that METTL3 enhances the stability of Hspa1a mRNA via m6A modification to regulate osteoblast aging. Notably, YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) participates in stabilizing Hspa1a mRNA in the METTL3-mediated m6A modification process, rather than the well-known degradation function. Mechanistically, METTL3 increases the stability of Hspa1a mRNA in a YTHDF2-dependent manner to inhibit osteoblast aging. Our results confirmed the significant role of METTL3 in osteoblast aging and suggested that METTL3 could be a potential therapeutic target for senile osteoporosis.

Sparse CBX2 nucleates many Polycomb proteins to promote facultative heterochromatinization of Polycomb target genes.

Facultative heterochromatinization of genomic regulators by Polycomb repressive complex (PRC) 1 and 2 is essential in development and differentiation; however, the underlying molecular mechanisms remain obscure. Using genetic engineering, molecular approaches, and live-cell single-molecule imaging, we quantify the number of proteins within condensates formed through liquid-liquid phase separation (LLPS) and find that in mouse embryonic stem cells (mESCs), approximately 3 CBX2 proteins nucleate many PRC1 and PRC2 subunits to form one non-stoichiometric condensate. We demonstrate that sparse CBX2 prevents Polycomb proteins from migrating to constitutive heterochromatin, demarcates the spatial boundaries of facultative heterochromatin, controls the deposition of H3K27me3, regulates transcription, and impacts cellular differentiation. Furthermore, we show that LLPS of CBX2 is required for the demarcation and deposition of H3K27me3 and is essential for cellular differentiation. Our findings uncover new functional roles of LLPS in the formation of facultative heterochromatin and unravel a new mechanism by which low-abundant proteins nucleate many other proteins to form compartments that enable them to execute their functions.

METTL3-mediated m6A modification of SOX4 regulates osteoblast proliferation and differentiation via YTHDF3 recognition.

N6-methyladenosine (m6A), the most prevalent internal modification in mRNA, is related to the pathogenesis of osteoporosis (OP). Although methyltransferase Like-3 (METTL3), an m6A transferase, has been shown to mitigate OP progression, the mechanisms of METTL3-mediated m6A modification in osteoblast function remain unclear. Here, fluid shear stress (FSS) induced osteoblast proliferation and differentiation, resulting in elevated levels of METTL3 expression and m6A modification. Through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and Transcriptomic RNA Sequencing (RNA-seq), SRY (Sex Determining Region Y)-box 4 (SOX4) was screened as a target of METTL3, whose m6A-modified coding sequence (CDS) regions exhibited binding affinity towards METTL3. Further functional experiments demonstrated that knockdown of METTL3 and SOX4 hampered osteogenesis, and METTL3 knockdown compromised SOX4 mRNA stability. Via RNA immunoprecipitation (RIP) assays, we further confirmed the direct interaction between METTL3 and SOX4. YTH N6-Methyladenosine RNA Binding Protein 3 (YTHDF3) was identified as the m6A reader responsible for modulating SOX4 mRNA and protein levels by affecting its degradation. Furthermore, in vivo experiments demonstrated that bone loss in an ovariectomized (OVX) mouse model was reversed through the overexpression of SOX4 mediated by adeno-associated virus serotype 2 (AAV2). In conclusion, our research demonstrates that METTL3-mediated m6A modification of SOX4 plays a crucial role in regulating osteoblast proliferation and differentiation through its recognition by YTHDF3. Our research confirms METTL3-m6A-SOX4-YTHDF3 as an essential axis and potential mechanism in OP.

Visual genetic typing of glioma using proximity-anchored in situ spectral coding amplification.

Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity-anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single-cell and tissue levels. The ligation-based padlock probe can discriminate one-nucleotide variations, and the design of proximity primers enables the anchoring of amplicons on target RNA, thus improving localization accuracy. The DNA module-based spectral coding strategy can dramatically improve the multiplexing capacity for imaging RNA mutations through one-time labelling, with low cost and simple operation. One-target-one-amplicon amplification confers ProxISCA the ability to quantify RNA mutation copy number with single-molecule resolution. Based on this approach, it is found that gliomas with higher malignant grades express more genes with high correlation at the cellular and tissue levels and show greater cellular heterogeneity. ProxISCA provides a tool for glioma research and precise diagnosis, which can reveal the relationship between cellular heterogeneity and glioma occurrence or development and assist in pathological prognosis.

One-Base-Gap Circular Probe-Mediated Dual Amplification for Isothermal Detection of N6-Methyladenosine Modifications.

N6-Methyladenosine (m6A) stands out as the predominant internal modification in mammalian RNA, exerting crucial regulatory functions in the metabolism of mRNA. Currently available methods have been limited by an inability to quantify m6A modification at precise sites. In this work, we screened a Bst 2.0 warm start DNA polymerase with the capability of discriminating m6A from adenosine (A) and developed a robust m6A RNA detection method that enables isothermal and ultrasensitive quantification of m6A RNA at single-base resolution. The detection limit of the assay could reach about 0.02 amol, and the quantitative accuracy of the assay was verified in real cell samples. Furthermore, we applied this assay to single-cell analysis and found that the coefficients of variation of the MALAT1 m6A 2611 site in glioblastoma U251 cells showed over 20% higher than in oligodendrocytes MO3.13 cells. This method provides a highly sensitive analytical tool for site-specific m6A detection and quantification, which is expected to provide a basis for precise disease diagnosis and epigenetic transcriptional regulation.

One Stone, Three Birds: Multifunctional Nanodots as "Pilot Light" for Guiding Surgery, Enhanced Radiotherapy, and Brachytherapy of Tumors.

Surgery, radiotherapy (RT), and brachytherapy are crucial treatments for localized deep tumors. However, imprecise tumor location often leads to issues such as positive surgical margins, extended radiotherapy target volumes, and radiation damage to healthy tissues. Reducing side effects in healthy tissue and enhancing RT efficacy are critical challenges. To address these issues, we developed a multifunctional theranostic platform using Au/Ag nanodots (Au/AgNDs) that act as a "pilot light" for real-time guided surgery, high-efficiency RT, and brachytherapy, achieving a strategy of killing three birds with one stone. First, dual-mode imaging of Au/AgNDs enabled precision RT, minimizing damage to adjacent normal tissue during X-ray irradiation. Au/AgNDs enhanced ionizing radiation energy deposition, increased intracellular reactive oxygen species (ROS) generation, regulated the cell cycle, promoted DNA damage formation, and inhibited DNA repair in tumor cells, significantly improving RT efficacy. Second, in brachytherapy, precise guidance provided by dual-mode imaging addressed challenges related to non-visualization of existing interstitial brachytherapy and multiple adjustments of insertion needle positions. Meanwhile, the effect of brachytherapy was improved. Third, the excellent fluorescence imaging of Au/AgNDs accurately distinguished tumors from normal tissue, facilitating their use as a powerful tool for assisting surgeons during tumor resection. Taken together, our multifunctional theranostic platform offers real-time guidance for surgery and high-efficiency RT, and improves brachytherapy precision, providing a novel strategy and vision for the clinical diagnosis and treatment of cancer.

Exploration of eMSCs with HA-GEL system in repairing damaged endometrium after endometrial cancer with fertility-sparing treatment.

Despite the high complete response rate of fertility-sparing treatment in early-stage endometrial cancer (EC), the low pregnancy rate is a clinical challenge. Whether endometrium-derived mesenchymal stem cells (eMSCs) can repair damaged endometrium after EC reversal remains unclear. This study explored the potential therapeutic effects of eMSCs with suitable scaffold materials on endometrial damage caused by EC. Here, appropriate engineering scaffold materials were compared to identify the most suitable materials to carry eMSCs. Then, safety and efficacy evaluations of eMSCs with a suitable hyaluronic acid hydrogel (eMSCs/HA-GEL) were investigated in in vivo experiments with subcutaneous xenotransplantation in Balb/C nude mice and a model of endometrial mechanical injury in rats. HA-GEL has minimal cytotoxicity to eMSCs compared to other materials. Then, in vitro experiments demonstrate that eMSCs/HA-GEL enhance the inhibitory effects of progestins on EC cell biological behaviors. eMSCs/HA-GEL significantly inhibit EC cell growth and have no potential safety hazards of spontaneous tumorigenesis in Balb/C nude mouse subcutaneous xenotransplantation assays. eMSCs/HA-GEL intrauterine transplantation effectively increases endometrial thickness and glandular number, improves endometrial blood supply, reduces fibrotic areas, and improves pregnancy rates in a rat endometrial mechanical injury model. GFP-eMSCs/HA-GEL intrauterine transplantation in rats shows more GFP-eMSCs in the endometrium than GFP-eMSCs transplantation alone, and no tumor formation or suspicious cell nodules are found in the liver, kidney, or lung tissues. Our results reveal the safety and efficacy of eMSCs/HA-GEL in animal models and provide preliminary evidence for the use of eMSCs/HA-GEL as a treatment for EC-related endometrial damage.

Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation.

Polycomb repressive complex 1 (PRC1) undergoes phase separation to form Polycomb condensates that are multi-component hubs for silencing Polycomb target genes. In this study, we demonstrate that formation and regulation of PRC1 condensates are consistent with the scaffold-client model, where the Chromobox 2 (CBX2) protein behaves as the scaffold while the other PRC1 proteins are clients. Such clients induce a re-entrant phase transition of CBX2 condensates. The composition of the multi-component PRC1 condensates (1) determines the dynamic properties of the scaffold protein; (2) selectively promotes the formation of CBX4-PRC1 condensates while dissolving condensates of CBX6-, CBX7-, and CBX8-PRC1; and (3) controls the enrichment of CBX4-, CBX7-, and CBX8-PRC1 in CBX2-PRC1 condensates and the exclusion of CBX6-PRC1 from CBX2-PRC1 condensates. Our findings uncover how multi-component PRC1 condensates are assembled via an intricate scaffold-client mechanism whereby the properties of the PRC1 condensates are sensitively regulated by its composition and stoichiometry.

Lightweight forest smoke and fire detection algorithm based on improved YOLOv5.

Smoke and fire detection technology is a key technology for automatically realizing forest monitoring and forest fire warning. One of the most popular algorithms for object detection tasks is YOLOv5. However, it suffers from some challenges, such as high computational load and limited detection performance. This paper proposes a high-performance lightweight network model for detecting forest smoke and fire based on YOLOv5 to overcome these problems. C3Ghost and Ghost modules are introduced into the Backbone and Neck network to achieve the purpose of reducing network parameters and improving the feature's expressing performance. Coordinate Attention (CA) module is introduced into the Backbone network to highlight the object's important information about smoke and fire and to suppress irrelevant background information. In Neck network part, in order to distinguish the importance of different features in feature fusing process, the weight parameter of feature fusion is added which is based on PAN (path aggregation network) structure, which is named PAN-weight. Multiple sets of controlled experiments were conducted to confirm the proposed method's performance. Compared with YOLOv5s, the proposed method reduced the model size and FLOPs by 44.75% and 47.46% respectively, while increased precision and mAP(mean average precision)@0.5 by 2.53% and 1.16% respectively. The experimental results demonstrated the usefulness and superiority of the proposed method. The core code and dataset required for the experiment are saved in this article at https://github.com/vinchole/zzzccc.git.

Combination of RCA and DNAzyme for Dual-Signal Isothermal Amplification of Exosome RNA.

The RNA contained in exosomes plays a crucial role in information transfer between cells in various life activities. The accurate detection of low-abundance exosome RNA (exRNA) is of great significance for cell function studies and the early diagnosis of diseases. However, their intrinsic properties, such as their short length and high sequence homology, represent great challenges for exRNA detection. In this paper, we developed a dual-signal isothermal amplification method based on rolling circle amplification (RCA) coupled with DNAzyme (RCA-DNAzyme). The sensitive detection of low-abundance exRNA, the specific recognition of their targets and the amplification of the detection signal were studied and explored. By designing padlock probes to specifically bind to the target exRNA, while relying on the ligation reaction to enhance recognition, the precise targeting of exosome RNA was realized. The combination of RCA and DNAzyme could achieve a twice-as-large isothermal amplification of the signal compared to RCA alone. This RCA-DNAzyme assay could sensitively detect a target exRNA at a concentration as low as 527 fM and could effectively distinguish the target from other miRNA sequences. In addition, this technology was successfully proven to be effective for the quantitative detection of miR-21 by spike recovery, providing a new research approach for the accurate detection of low-abundance exRNA and the exploration of unknown exRNA functions.

The Fe-S cluster assembly protein IscU2 increases α-ketoglutarate catabolism and DNA 5mC to promote tumor growth.

IscU2 is a scaffold protein that is critical for the assembly of iron-sulfur (Fe-S) clusters and the functions of Fe-S-containing mitochondrial proteins. However, the role of IscU2 in tumor development remains unclear. Here, we demonstrated that IscU2 expression is much higher in human pancreatic ductal adenocarcinoma (PDAC) tissues than in adjacent normal pancreatic tissues. In PDAC cells, activated KRAS enhances the c-Myc-mediated IscU2 transcription. The upregulated IscU2 stabilizes Fe-S cluster and regulates the activity of tricarboxylic acid (TCA) cycle enzymes α-ketoglutarate (α-KG) dehydrogenase and aconitase 2, which promote α-KG catabolism through oxidative and reductive TCA cycling, respectively. In addition to promoting mitochondrial functions, activated KRAS-induced and IscU2-dependent acceleration of α-KG catabolism results in reduced α-KG levels in the cytosol and nucleus, leading to an increase in DNA 5mC due to Tet methylcytosine dioxygenase 3 (TET3) inhibition and subsequent expression of genes including DNA polymerase alpha 1 catalytic subunit for PDAC cell proliferation and tumor growth in mice. These findings underscore a critical role of IscU2 in KRAS-promoted α-KG catabolism, 5mC-dependent gene expression, and PDAC growth and highlight the instrumental and integrated regulation of mitochondrial functions and gene expression by IscU2 in PDAC cells.