Translational modelling to predict human pharmacokinetics and pharmacodynamics of a Bruton's tyrosine kinase-targeted protein degrader BGB-16673.

BACKGROUND AND PURPOSE: Bifunctional small molecule degraders, which link the target protein with E3 ubiquitin ligase, could lead to the efficient degradation of the target protein. BGB-16673 is a Bruton's tyrosine kinase (BTK) degrader. A translational PK/PD modelling approach was used to predict the human BTK degradation of BGB-16673 from preclinical in vitro and in vivo data. EXPERIMENTAL APPROACH: A simplified mechanistic PK/PD model was used to establish the correlation between the in vitro and in vivo BTK degradation by BGB-16673 in a mouse model. Human and mouse species differences were compared using the parameters generated from in vitro human or mouse blood, and human or mouse serum spiked TMD-8 cells. Human PD was then predicted using the simplified mechanistic PK/PD model. KEY RESULTS: BGB-16673 showed potent BTK degradation in mouse whole blood, human whole blood, and TMD-8 tumour cells in vitro. Furthermore, BGB-16673 showed BTK degradation in a murine TMD-8 xenograft model in vivo. The PK/PD model predicted human PD and the observed BTK degradation in clinical studies both showed robust BTK degradation in blood and tumour at clinical dose range. CONCLUSION AND IMPLICATIONS: The presented simplified mechanistic model with reduced number of model parameters is practically easier to be applied to research projects compared with the full mechanistic model. It can be used as a tool to better understand the PK/PD behaviour for targeted protein degraders and increase the confidence when moving to the clinical stage.

Zanubrutinib is effective in non-germinal-center B-cell-like diffuse large B-cell lymphoma with mutated CD79B, high TCL1A expression, or over- expressed MYC/BCL-2.

To evaluate the effects of gene mutations on Bruton tyrosine kinase inhibitor, zanubrutinib's effectiveness in patients with diffuse large B-cell lymphoma (DLBCL), we examined pooled data from four single-arm studies (BGB-3111-AU-003 [NCT02343120], BGB-3111-207 [NCT03145064], BGB-3111_GA101_Study_001 [NCT02569476], BGB-3111-213 [NCT03520920]; n = 121). Objective response rate (ORR) was higher, though not statistically significant, in patients with activated B-cell-like (ABC)- and unclassified DLBCL (42.9% [21/49]) versus those with germinal-center B-cell-like DLBCL (14.3% [1/7]; p = 0.15). Patients with CD79B mutations had better ORR (60%) versus patients with wild-type alleles (25.9%, p < 0.01). Higher TCL1A expression correlated with better zanubrutinib response (p = 0.03), longer progression-free survival (p = 0.01), and longer overall survival (p = 0.12). TCL1A expression was higher in ABC-DLBCL (p < 0.001) and MYD88/CD79B-mutated subtypes (p < 0.0001). Eighteen patients with high MYC/BCL-2 expression responded better to zanubrutinib (ORR = 61 vs. 29%, p = 0.02). Our results support assessing CD79B mutations, co-expressor DLBCL, and TCL1A expression status to identify patients with DLBCL who will benefit from zanubrutinib.

Short- and long-term effects of different forage types supplemented in preweaning dairy calves on performance and milk production into first lactation.

We investigated the short- and long-term effects of different forage types supplemented in preweaning dairy calves on growth performance, blood metabolites, rumen fermentation, bacterial community, and milk production during first lactation. A total of 60 healthy 30-d-old female Holstein calves were blocked by birth date and body weight and randomly assigned to 1 of 3 groups (n = 20): normal milk and pelleted starter feeding (CON), supplemented with chopped oat hay (OAH; 75.0 g/d per calf [DM basis]), or alfalfa hay (ALF; 75.0 g/d per calf [DM basis]). The forage supplementation started when calves were 30 d old (d 1 of the experimental period) and ended when they were 73 d old (d 44 of the experimental period, when calves were weaned). Milk and feed intakes and fecal consistency scores were recorded daily. Growth performance, rumen fluid, and blood samples were collected biweekly. After weaning, all the calves were integrated with the same barn and diets. After calving, the milk production was recorded daily. During the experimental period, the OAH group had greater solid feed and total DM intakes and greater rumen pH than the CON group (P ≤ 0.04), but had lower forage intake and CP digestibility than the ALF group (P ≤ 0.04). The ALF group had higher rumen pH and blood BHB concentration (P ≤ 0.04), lower fecal score (P = 0.02), and greater ether extract digestibility (P = 0.02) than the CON group. The ALF and OAH groups had lower concentrations of ruminal total VFA (P = 0.01). Still, the ALF group had a greater proportion of acetate and a relative abundance of cellulose degradation-related bacteria (Lachnoclostridium_1 and Oribacterium) and a lower relative abundance of inflammation-related bacteria (Erysipelotrichaceae_UCG-009) in the rumen compared with CON. Interestingly, the average milk production from 6 to 200 DIM was greater in the ALF group (P < 0.01), even though no significant effects were found on the rumen fermentation parameters and blood metabolites at 200 DIM. Generally, alfalfa hay supplementation in preweaning dairy calves had positive effects in the short- and long-term for rumen development, health status, and future milk production.

Patient satisfaction with humanistic nursing in Chinese secondary and tertiary public hospitals: a cross-sectional survey.

Background: Humanistic care pertains to the abilities, attitudes, and behaviors central to patient-centered care, contributing to patients' sense of safety and wellbeing. This study aimed to assess the satisfaction of patients with humanistic nursing care in Chinese secondary and tertiary public hospitals. Methods: A national cross-sectional survey was conducted across 30 provinces and 83 hospitals in China. Patient satisfaction with humanistic care was assessed using the Methodist Health Care System Nurse Caring Instrument (NCI), which encompasses 20 items across 12 dimensions. Each item was rated on a 7-point Likert scale, yielding a total score of 140. Multiple linear regression analysis was employed to identify factors associated with patients' satisfaction. Results: Moderate satisfaction (mean score 91.26 ± 13.14) with humanistic nursing care was observed among the 17,593 participants. Factors significantly associated with patient satisfaction included age, hospital type, presence of children, educational attainment, place of residence, family monthly income, and medical insurance type. Conclusion: The study findings highlight the importance of tailored interventions, evidence-based practice guidelines, and patient-centered care in improving patients' satisfaction with humanistic nursing care. Continuous emphasis on nursing education and professional development is crucial for enhancing humanistic care and patient satisfaction.

Novel Abs targeting the N-terminus of fibroblast growth factor 19 inhibit hepatocellular carcinoma growth without bile-acid-related side-effects.

Hepatocellular carcinoma (HCC) is a common and particularly fatal form of cancer for which very few drugs are effective. The fibroblast growth factor 19 (FGF19) has been viewed as a driver of HCC development and a potential Ab target for developing novel HCC therapy. However, a previously developed anti-FGF19 Ab disrupted FGF19's normal regulatory function and caused severe bile-acid-related side-effects despite of having potent antitumor effects in preclinical models. Here, we developed novel human Abs (G1A8 and HS29) that specifically target the N-terminus of FGF19. Both Abs inhibited FGF19-induced HCC cell proliferation in vitro and significantly suppressed HCC tumor growth in mouse models. Importantly, no bile-acid-related side effects were observed in preclinical cynomolgus monkeys. Fundamentally, our study demonstrates that it is possible to target FGF19 for anti-HCC therapies without adversely affecting its normal bile acid regulatory function, and highlights the exciting promise of G1A8 or HS29 as potential therapy for HCC.

Increased sulfation of bile acids in mice and human subjects with sodium taurocholate cotransporting polypeptide deficiency.

Sodium taurocholate cotransporting polypeptide (NTCP, encoded by Slc10a1/SLC10A1) deficiency can result in hypercholanemia but no obvious symptoms in both mice and humans. However, the consequence of and response to long-term hypercholanemia caused by NTCP deficiency remain largely unexplored. Here, we analyzed lifelong dynamics of serum total bile acid (TBA) levels in Slc10a1-/- mice, and we also assessed changes of TBA levels in 33 young individuals with SLC10A1 loss-of-function variant p.Ser267Phe. We found that overall serum TBA levels tended to decrease gradually with age in both Slc10a1-/- mice and p.Ser267Phe individuals. Liver mRNA profiling revealed notable transcription alterations in hypercholanemic Slc10a1-/- mice, including inhibition of bile acid (BA) synthesis, enhancement of BA detoxification, and altered BA transport. Members of the sulfotransferase (SULT) family showed the most dramatic increases in livers of hypercholanemic Slc10a1-/- mice, and one of their BA sulfates, taurolithocholic acid 3-sulfate, significantly increased. Importantly, consistent with the mouse studies, comprehensive profiling of 58 BA species in sera of p.Ser267Phe individuals revealed a markedly increased level of BA sulfates. Together, our findings indicate that the enhanced BA sulfation is a major mechanism for BA detoxification and elimination in both mice and humans with Slc10a1/SLC10A1 deficiency.

Integrated analysis of hematopoietic differentiation outcomes and molecular characterization reveals unbiased differentiation capacity and minor transcriptional memory in HPC/HSC-iPSCs.

BACKGROUND: Transcription factor-mediated reprogramming can reset the epigenetics of somatic cells into a pluripotency compatible state. Recent studies show that induced pluripotent stem cells (iPSCs) always inherit starting cell-specific characteristics, called epigenetic memory, which may be advantageous, as directed differentiation into specific cell types is still challenging; however, it also may be unpredictable when uncontrollable differentiation occurs. In consideration of biosafety in disease modeling and personalized medicine, the availability of high-quality iPSCs which lack a biased differentiation capacity and somatic memory could be indispensable. METHODS: Herein, we evaluate the hematopoietic differentiation capacity and somatic memory state of hematopoietic progenitor and stem cell (HPC/HSC)-derived-iPSCs (HPC/HSC-iPSCs) using a previously established sequential reprogramming system. RESULTS: We found that HPC/HSCs are amenable to being reprogrammed into iPSCs with unbiased differentiation capacity to hematopoietic progenitors and mature hematopoietic cells. Genome-wide analyses revealed that no global epigenetic memory was detectable in HPC/HSC-iPSCs, but only a minor transcriptional memory of HPC/HSCs existed in a specific tetraploid complementation (4 N)-incompetent HPC/HSC-iPSC line. However, the observed minor transcriptional memory had no influence on the hematopoietic differentiation capacity, indicating the reprogramming of the HPC/HSCs was nearly complete. Further analysis revealed the correlation of minor transcriptional memory with the aberrant distribution of H3K27me3. CONCLUSIONS: This work provides a comprehensive framework for obtaining high-quality iPSCs from HPC/HSCs with unbiased hematopoietic differentiation capacity and minor transcriptional memory.

Genome-wide gene expression analyses reveal unique cellular characteristics related to the amenability of HPC/HSCs into high-quality induced pluripotent stem cells.

BACKGROUND: Transcription factor-mediated reprogramming can efficiently convert differentiated cells into induced pluripotent stem cells (iPSCs). Furthermore, many cell types have been shown to be amenable to reprogramming into iPSCs, such as neural stem cells, hematopoietic progenitor and stem cells (HPC/HSCs). However, the mechanisms related to the amenability of these cell types to be reprogrammed are still unknown. METHODS: Herein, we attempt to elucidate the mechanisms of HPC/HSC reprogramming using the sequential reprogramming system that we have previously established. RESULTS: We found that HPC/HSCs were amenable to transcription factor-mediated reprogramming, which yielded a high frequency of fully reprogrammed HPC/HSC-iPSCs. Genome-wide gene expression analyses revealed select down-regulated tumor suppressor and mesenchymal genes as well as up-regulated oncogenes in HPC/HSCs compared with mouse embryonic fibroblasts (MEFs), indicating that these genes may play important roles during the reprogramming of HPC/HSCs. Additional studies provided insights into the contribution of select tumor suppressor genes (p21, Ink4a and Arf) and an epithelial-to-mesenchymal transition (EMT) factor (Snail1) to the reprogramming process of HPC/HSCs. CONCLUSIONS: Our findings demonstrate that HPC/HSCs carry unique cellular characteristics, which determine the amenability of HPC/HSCs to be reprogrammed into high-quality iPSCs.

Nucleosome organizations in induced pluripotent stem cells reprogrammed from somatic cells belonging to three different germ layers.

BACKGROUND: Nucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells. RESULTS: We generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs' tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs. CONCLUSIONS: The re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.

High-throughput sequencing reveals the disruption of methylation of imprinted gene in induced pluripotent stem cells.

It remains controversial whether the abnormal epigenetic modifications accumulated in the induced pluripotent stem cells (iPSCs) can ultimately affect iPSC pluripotency. To probe this question, iPSC lines with the same genetic background and proviral integration sites were established, and the pluripotency state of each iPSC line was characterized using tetraploid (4N) complementation assay. Subsequently, gene expression and global epigenetic modifications of "4N-ON" and the corresponding "4N-OFF" iPSC lines were compared through deep sequencing analyses of mRNA expression, small RNA profile, histone modifications (H3K27me3, H3K4me3, and H3K4me2), and DNA methylation. We found that methylation of an imprinted gene, Zrsr1, was consistently disrupted in the iPSC lines with reduced pluripotency. Furthermore, the disrupted methylation could not be rescued by improving culture conditions or subcloning of iPSCs. Moreover, the relationship between hypomethylation of Zrsr1 and pluripotency state of iPSCs was further validated in independent iPSC lines derived from other reprogramming systems.

MiR-335-5p promotes chondrogenesis in mouse mesenchymal stem cells and is regulated through two positive feedback loops.

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is regulated by many factors and signal pathways, including transcription factors such as Sox9 and microRNAs. MiR-335-5p has been previously reported to regulate osteogenic and adipogenic differentiations of MSCs, but its role in chondrogenic differentiation of MSC remains unknown. In this study, we found that miR-335-5p and its host gene Mest are co-expressed and greatly upregulated during mouse MSCs (mMSCs) chondrogenesis. Overexpression of miR-335-5p in mMSCs increased expression of chondrogenic marker genes. Molecular mechanism explorations revealed that miR-335-5p targets Daam1 and ROCK1, a set of negative regulators of Sox9; Sox9 downregulates the expression of miR-29a and 29b, both negative regulators of Mest expression, thus forming a positive loop from miR-335-5p to Sox9 to Mest/miR-335-5p. In addition, miR-335-5p targets DKK1 during mMSC chondrogenic differentiation to increase ß-catenin/TCF activity, which leads to increased level of Mest transcription. These data showed miR-335-5p positively regulates MSC chondrogenesis, and two positive feedback loops are identified for the expression of miR-335-5p and its host gene Mest during the early phase of mMSC chondrogenic differentiation.