Comparison of the intestinal flora of wild and artificial breeding green turtles (Chelonia mydas).

Gut microbes are pivotal reference indicators for assessing the health status of animals. Before introducing artificially bred species into the wild, examining their gut microbe composition is crucial to help mitigate potential threats posed to wild populations. However, gut microbiological trait similarities between wild and artificially bred green turtles remain unexplored. Therefore, this study compared the gut microbiological characteristics of wild and artificially bred green turtles (Chelonia mydas) through high-throughput Illumina sequencing technology. The α-diversity of intestinal bacteria in wild green turtles, as determined by Shannon and Chao indices, significantly surpasses that of artificial breeding green turtles (p < 0.01). However, no significant differences were detected in the fungal α-diversity between wild and artificially bred green turtles. Meanwhile, the ß-diversity analysis revealed significant differences between wild and artificially bred green turtles in bacterial and fungal compositions. The community of gut bacteria in artificially bred green turtles had a significantly higher abundance of Fusobacteriota including those belonging to the Paracoccus, Cetobacterium, and Fusobacterium genera than that of the wild green turtle. In contrast, the abundance of bacteria belonging to the phylum Actinobacteriota and genus Nautella significantly decreased. Regarding the fungal community, artificially bred green turtles had a significantly higher abundance of Fusarium, Sterigmatomyces, and Acremonium and a lower abundance of Candida and Rhodotorula than the wild green turtle. The PICRUSt2 analyses demonstrated significant differences in the functions of the gut bacterial flora between groups, particularly in carbohydrate and energy metabolism. Fungal functional guild analysis further revealed that the functions of the intestinal fungal flora of wild and artificially bred green turtles differed significantly in terms of animal pathogens-endophytes-lichen parasites-plant pathogens-soil saprotrophs-wood saprotrophs. BugBase analysis revealed significant potential pathogenicity and stress tolerance variations between wild and artificially bred green turtles. Collectively, this study elucidates the distinctive characteristics of gut microbiota in wild and artificially bred green turtles while evaluating their health status. These findings offer valuable scientific insights for releasing artificially bred green turtles and other artificially bred wildlife into natural habitats.

Enzymes in Synergy: Bacteria Specific Molecular Probe for Locoregional Imaging of Urinary Tract Infection in vivo.

Uropathogenic Escherichia coli (UPECs) is a leading cause for urinary tract infections (UTI), accounting for 70-90% of community or hospital-acquired bacterial infections owing to high recurrence, imprecision in diagnosis and management, and increasing prevalence of antibiotic resistance. Current methods for clinical UPECs detection still rely on labor-intensive urine cultures that impede rapid and accurate diagnosis for timely UTI therapeutic management. Herein, we developed a first-in-class near-infrared (NIR) UPECs fluorescent probe (NO-AH) capable of specifically targeting UPECs through its collaborative response to bacterial enzymes, enabling locoregional imaging of UTIs both in vitro and in vivo. Our NO-AH probe incorporates a dual protease activatable moiety, which first reacts with OmpT, an endopeptidase abundantly present on outer membrane of UPECs, releasing an intermediate amino acid residue conjugated with a NIR hemicyanine fluorophore. Such liberated fragment would be subsequently recognized by aminopeptidase (APN) within periplasm of UPECs, activating localized fluorescence for precise imaging of UTIs in complex living environments. The peculiar specificity and selectivity of NO-AH, facilitated by the collaborative action of bacterial enzymes, features a timely and accurate identification of UPECs-infected UTIs, which could overcome misdiagnosis in conventional urine tests, thus opening new avenues towards reliable UTI diagnosis and personalized antimicrobial therapy management.

An NGS-based assay for accurate detection and quantification of immune gene expression in mouse tumor models.

Tumor microenvironment (TME) is a complex dynamic system with many tumor-interacting components including tumor-infiltrating leukocytes (TILs), cancer associated fibroblasts, blood vessels, and other stromal constituents. It intrinsically affects tumor development and pharmacology of oncology therapeutics, particularly immune-oncology (IO) treatments. Accurate measurement of TME is therefore of great importance for understanding the tumor immunity, identifying IO treatment mechanisms, developing predictive biomarkers, and ultimately, improving the treatment of cancer. Here, we introduce a mouse-IO NGS-based (NGSmIO) assay for accurately detecting and quantifying the mRNA expression of 1080 TME related genes in mouse tumor models. The NGSmIO panel was shown to be superior to the commonly used microarray approach by hosting 300 more relevant genes to better characterize various lineage of immune cells, exhibits improved mRNA and protein expression correlation to flow cytometry, shows stronger correlation with mRNA expression than RNAseq with 10x higher sequencing depth, and demonstrates higher sensitivity in measuring low-expressed genes. We describe two studies; firstly, detecting the pharmacodynamic change of interferon-γ expression levels upon anti-PD-1: anti-CD4 combination treatment in MC38 and Hepa 1-6 tumors; and secondly, benchmarking baseline TILs in 14 syngeneic tumors using transcript level expression of lineage specific genes, which demonstrate effective and robust applications of the NGSmIO panel.

Narazaciclib, a novel multi-kinase inhibitor with potent activity against CSF1R, FLT3 and CDK6, shows strong anti-AML activity in defined preclinical models.

CSF1R is a receptor tyrosine kinase responsible for the growth/survival/polarization of macrophages and overexpressed in some AML patients. We hypothesized that a novel multi-kinase inhibitor (TKi), narazaciclib (HX301/ON123300), with high potency against CSF1R (IC50 ~ 0.285 nM), would have anti-AML effects. We tested this by confirming HX301's high potency against CSF1R (IC50 ~ 0.285 nM), as well as other kinases, e.g. FLT3 (IC50 of ~ 19.77 nM) and CDK6 (0.53 nM). An in vitro proliferation assay showed that narazaciclib has a high growth inhibitory effect in cell cultures where CSF1R or mutant FLT3-ITD variants that may be proliferation drivers, including primary macrophages (IC50 of 72.5 nM) and a subset of AML lines (IC50 < 1.5 µM). In vivo pharmacology modeling of narazaciclib using five AML xenografts resulted in: inhibition of MV4-11 (FLT3-ITD) subcutaneous tumor growth and complete suppression of AM7577-PDX (FLT3-ITD/CSF1Rmed) systemic growth, likely due to the suppression of FLT3-ITD activity; complete suppression of AM8096-PDX (CSF1Rhi/wild-type FLT3) growth, likely due to the inhibition of CSF1R ("a putative driver"); and nonresponse of both AM5512-PDX and AM7407-PDX (wild-type FLT3/CSF1Rlo). Significant leukemia load reductions in bone marrow, where disease originated, were also achieved in both responders (AM7577/AM8096), implicating that HX301 might be a potentially more effective therapy than those only affecting peripheral leukemic cells. Altogether, narazaciclib can potentially be a candidate treatment for a subset of AML with CSF1Rhi and/or mutant FLT3-ITD variants, particularly second generation FLT3 inhibitor resistant variants.

A Bioengineered Nanovesicle Vaccine Boosts T-B cell Interaction for Immunotherapy of Echinococcus multilocularis.

Alveolar echinococcosis (AE) is a zoonotic parasitic disease, resulting from being infected with the metacestode larvae of the tapeworm Echinococcus multilocularis (E. multilocularis). Novel prophylactic and therapeutic interventions are urgently needed since the current chemotherapy displays limited efficiency in AE treatment. Bioengineered nano cellular membrane vesicles are widely used for displaying the native conformational epitope peptides because of their unique structure and biocompatibility. In this study, four T-cells and four B-cells dominant epitope peptides of E. multilocularis with high immunogenicity were engineered into the Vero cell surface to construct a membrane vesicle nanovaccine for the treatment of AE. The results showed that the nanovesicle vaccine can efficiently activate dendritic cells, induce specific T/B cells to form a mutually activated circuit, and inhibit E. multilocularis infection. This study presents for the first time a nanovaccine strategy that can completely eliminate the burden of E. multilocularis.

Microplastic Distribution Characteristics and Sources on Beaches That Serve as the Largest Nesting Ground for Green Turtles in China.

The threat of microplastics to marine animals and habitats is increasing, which may affect sea turtle nesting grounds. The Qilianyu Islands are the largest remaining green turtle (Chelonia mydas) nesting grounds in China. Despite being far from the mainland, microplastic pollution cannot be ignored. In this study, the level of microplastic pollution in surface sediments from three different zones, namely, the bottom, intertidal, and supratidal zone, was investigated on North Island, Qilianyu Islands. The results showed that the abundance of microplastics in the supratidal zone was significantly higher than that in the bottom zone and intertidal zone (r = 3.65, p = 0.011), with the highest average abundance of microplastics located on the southwest coast of North Island. In the bottom zone, only plastic blocks (88%) and fibers (12%) were found. The main types of microplastics in the intertidal and supratidal zones were plastic blocks (48%) and foam (42%), with polyethylene (PE) (40%) and polystyrene (PS) (34%) being the predominant components. These types and components of microplastics differed from those in the surrounding seawater, but corresponding types and components were found in the plastic debris on the beach. Meanwhile, it was also observed that there were multiple instances of fragmented plastic on the beach. Thus, we suggest that the microplastics on the beach in North Island were mainly derived from the fragmentation of microplastic debris, indicating secondary microplastics. It is recommended to further strengthen the regular cleaning of plastic debris on the beach, especially the removal of small plastic debris, in order to reduce the pollution from secondary microplastics generated by the fragmentation of beach plastic debris and to better protect China's most important sea turtle nesting site in the South China Sea.

Designing nanodiscs as versatile platforms for on-demand therapy.

Nowadays, there has been an increasing utilization of nanomedicines for disease treatment. Nanodiscs (NDs) have emerged as a novel platform technology that garners significant attention in biomedical research and drug discovery. NDs are nanoscale phospholipid bilayer discs capable of incorporating membrane proteins and lipids within a native-like environment. They are assembled using amphiphilic biomacromolecular materials, such as apolipoprotein A1 or membrane scaffold proteins (MSPs), peptides, and styrene-maleic acid polymers (SMAs). NDs possess well-defined sizes and shapes, offering a stable, homogeneous, and biologically relevant environment for studying membrane proteins and lipids. Their unique properties have made them highly desirable for diverse applications, including cancer immunotherapy, vaccine development, antibacterial and antiviral therapy, and treating Alzheimer's disease (AD) and diabetes-related conditions. This review discusses the classifications, advantages, and applications of NDs in disease therapy.

Nano-sized aggregate Ti3C2-TiO2 supported on the surface of Ag2NCN as a Z-scheme catalyst with enhanced visible light photocatalytic performance.

Exposing the photocatalyst's highly active facets and hybridizing the photocatalyst with suitable cocatalysts in the proper spot have been recognized as strong methods for high-performance photocatalysts. Herein, Ag2NCN/TiO2-Ti3C2 composites were synthesized by applying simple calcination and physically weak interaction deposition processes to obtain an excellent photocatalyst for Rhodamine B (Rh B) degradation when exposed to visible light. The findings from the experiments reveal that the Ag2NCN/TiO2-Ti3C2400 composite exhibited an outstanding photocatalytic rate in 80 min, with the highest Rh B degradation rate (k = 0.03889 min-1), which was 16 times higher than that of pure Ag2NCN (k = 0.00235 min-1) and 2.2 times higher than that of TiO2-Ti3C2400 (k = 0.01761 min-1). The results from the following factors: (i) the powerful interfacial contact created by the in situ formation of TiO2, and the superior electrical conductivity of Ti3C2 that makes carrier separation possible; (ii) TiO2 with electron-rich (101) facets are deposited on the surface of Ag2NCN, significantly reducing charge carrier recombination by trapping photoelectrons; (iii) a Z-type heterojunction is constructed between nanosize aggregate Ti3C2-TiO2 and Ag2NCN with non-metal Ti3C2 as the solid medium, improving the transfer and separation of photogenerated charges and inhibiting the recombination of electrons and holes. Additionally, the redox ability of the composite photocatalyst is enhanced. Furthermore, the analyses of active species showed that photogenerated superoxide radicals and holes were the principal active agents inside the photodegradation of Rh B. Moreover, the composite exhibited outstanding photo-stability.

Bioinformatics analysis to screen the key genes in pediatric Chronic Active Epstein-Barr Virus Infection.

Chronic active EBV infection (CAEBV) is associated with poor prognosis and high mortality. We performed bioinformatics analysis to screen out key genes associated with CAEBV. Weighted gene co-expression network analysis (WGCNA) was used to identify the gene module which was most correlated with pediatric CAEBV. Furthermore, the differentially expressed genes (DEGs) between pediatric acute infectious mononucleosis (AIM) and pediatric CAEBV were investigated. Least absolute shrinkage and selection operator (LASSO) and random forest then were performed to identify the key variables associated with pediatric CAEBV. We also explored the correlation between these hub genes with EBV infection related pathway and immune cell abundance. Compared with pediatric AIM, 1561 DEGs were up-regulated in pediatric CAEBV, and these genes were mainly enriched in inflammatory response and inflammation-related pathways. WGCNA analysis showed that genes in blue module were mostly related to pediatric CAEBV. Genes in the blue module and DEGs are intersected to get 174 genes and these genes are also enriched in inflammatory response-related pathways. The key CAEBV-related genes were selected from these 174 genes by applying the random Forest and LASSO algorithm, resulting in TPST1, TNFSF8 and RAB3GAP1. These three genes showed good diagnostic performance in distinguishing pediatric CAEBV from pediatric AIM. Furthermore, Cibersort and GSEA analysis indicated that these three genes were positively correlated with myeloid cell enrichment and persistent EBV infection pathway, respectively. Our finding systematically analyzed the difference between AIM and CAEBV and identified TPST1, TNFSF8 and RAB3GAP1 were the key genes in the development of CAEBV.

HX009, a novel BsAb dual targeting PD1 x CD47, demonstrates potent anti-lymphoma activity in preclinical models.

Both PD1/PD-L1 and CD47 blockades have demonstrated limited activity in most subtypes of NHL save NK/T-cell lymphoma. The hemotoxicity with anti-CD47 agents in the clinic has been speculated to account for their limitations. Herein we describe a first-in-class and rationally designed bispecific antibody (BsAb), HX009, targeting PD1 and CD47 but with weakened CD47 binding, which selectively hones the BsAb for tumor microenvironment through PD1 interaction, potentially reducing toxicity. In vitro characterization confirmed: (1) Both receptor binding/ligand blockade, with lowered CD47 affinity; (2) functional PD1/CD47 blockades by reporter assays; (3) T-cell activation in Staphylococcal-enterotoxin-B-pretreated PBMC and mixed-lymphocyte-reaction. In vivo modeling demonstrated antitumor activity in Raji-B and Karpass-229-T xenograft lymphomas. In the humanized mouse syngeneic A20 B-lymphoma (huCD47-A20) HuGEMM model, which has quadruple knocked-in hPD1xhPD-L1xhCD47xhSIRPα genes and an intact autologous immune-system, a contribution of effect is demonstrated for each targeted biologic (HX008 targeting PD1 and SIRPα-Fc targeting CD47), which is clearly augmented by the dual targeting with HX009. Lastly, the expression of the immune-checkpoints PD-L1/L2 and CD47 seemed co-regulated among a panel of lymphoma-derived-xenografts, where HX009 maybe more effective in those with upregulated CD47. Our data warrants HX009's further clinical development for treating NHLs.

A living biobank of matched pairs of patient-derived xenografts and organoids for cancer pharmacology.

Patient-derived tumor xenograft (PDX)/organoid (PDO), driven by cancer stem cells (CSC), are considered the most predictive models for translational oncology. Large PDX collections reflective of patient populations have been created and used extensively to test various investigational therapies, including population-trials as surrogate subjects in vivo. PDOs are recognized as in vitro surrogates for patients amenable for high-throughput screening (HTS). We have built a biobank of carcinoma PDX-derived organoids (PDXOs) by converting an existing PDX library and confirmed high degree of similarities between PDXOs and parental PDXs in genomics, histopathology and pharmacology, suggesting "biological equivalence or interchangeability" between the two. Here we demonstrate the applications of PDXO biobank for HTS "matrix" screening for both lead compounds and indications, immune cell co-cultures for immune-therapies and engineering enables in vitro/in vivo imaging. This large biobank of >550 matched pairs of PDXs/PDXOs across different cancers could become powerful tools for the future cancer drug discovery.

Cellular-Membrane-Derived Vesicles for Cancer Immunotherapy.

The medical community is constantly searching for new and innovative ways to treat cancer, and cellular-membrane-derived artificial vesicles are emerging as a promising avenue for cancer immunotherapy. These vesicles, which are derived from mammal and bacteria cell membranes, offer a range of benefits, including compatibility with living organisms, minimal immune response, and prolonged circulation. By modifying their surface, manipulating their genes, combining them with other substances, stimulating them externally, and even enclosing drugs within them, cellular vesicles have the potential to be a powerful tool in fighting cancer. The ability to merge drugs with diverse compositions and functionalities in a localized area is particularly exciting, as it offers a way to combine different immunotherapy treatments for maximum impact. This review contains information on the various sources of these vesicles and discusses some recent developments in cancer immunotherapy using this promising technology. While there are still obstacles to overcome, the possibilities for cellular vesicles in cancer treatment are truly exciting.

Structural characterization, acute toxicity assessment and protective effects of selenylated apple pectin on dextran sulfate sodium-induced ulcerative colitis.

This study was aimed at investigating the structural characterization, acute toxicity and protective effect of selenylated apple pectin on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. Selenylated apple pectin was characterized by ion chromatography, NMR and SEC-RI-MALLS. The acute toxicity and protective effect of selenylated apple pectin against UC were investigated by gavage administration in mice. The organ state and coefficients, inflammatory cytokine (IL-6, IL-10 and TNF-α) contents in serum, GSH-Px activity and MPO content in colon tissues were also evaluated. The results indicated that selenylated apple pectin was non-toxic and contained 244.28 µgselenium per g. The monosaccharide composition with different molar ratios, different relative molecular weights and a weakened signal peak (CH2-O group) at 3-4 ppm were observed after selenylation. The selenylated apple pectin showed the protective effect against UC by down-regulating IL-6 and TNF-α contents and up-regulating the IL-10 content in serum, as well as increasing the GSH-Px activity and decreasing the MPO content in colon tissues. Moreover, DSS-induced alterations were effectively recovered by a high-dose sample. These findings provide evidence in support of selenylated apple pectin as a novel dietary selenium supplement for UC protection.

Different syngeneic tumors show distinctive intrinsic tumor-immunity and mechanisms of actions (MOA) of anti-PD-1 treatment.

Cancers are immunologically heterogeneous. A range of immunotherapies target abnormal tumor immunity via different mechanisms of actions (MOAs), particularly various tumor-infiltrate leukocytes (TILs). We modeled loss of function (LOF) in four common anti-PD-1 antibody-responsive syngeneic tumors, MC38, Hepa1-6, CT-26 and EMT-6, by systematical depleting a series of TIL lineages to explore the mechanisms of tumor immunity and treatment. CD8+-T-cells, CD4+-T-cells, Treg, NK cells and macrophages were individually depleted through either direct administration of anti-marker antibodies/reagents or using DTR (diphtheria toxin receptor) knock-in mice, for some syngeneic tumors, where specific subsets were depleted following diphtheria toxin (DT) administration. These LOF experiments revealed distinctive intrinsic tumor immunity and thus different MOAs in their responses to anti-PD-1 antibody among different syngeneic tumors. Specifically, the intrinsic tumor immunity and the associated anti-PD-1 MOA were predominately driven by CD8+ cytotoxic TILs (CTL) in all syngeneic tumors, excluding Hepa1-6 where CD4+ Teff TILs played a key role. TIL-Treg also played a critical role in supporting tumor growth in all four syngeneic models as well as M2-macrophages. Pathway analysis using pharmacodynamic readouts of immuno-genomics and proteomics on MC38 and Hepa1-6 also revealed defined, but distinctive, immune pathways of activation and suppression between the two, closely associated with the efficacy and consistent with TIL-pharmacodynamic readouts. Understanding tumor immune-pathogenesis and treatment MOAs in the different syngeneic animal models, not only assists the selection of the right model for evaluating new immunotherapy of a given MOA, but also can potentially help to understand the potential disease mechanisms and strategize optimal immune-therapies in patients.

Design of a Single-Channel Chaotic Secure Communication System Implemented by DNA Strand Displacement.

DNA strand displacement (DSD) is regarded as a foundation for the construction of biological computing systems because of the predictability of DNA molecular behaviors. Some complex system dynamics can be approximated by cascading DSD reaction modules with different functions. In this paper, four DSD reaction modules are used to realize chaotic secure communication based on drive-response synchronization of four-dimensional chaotic systems. The system adopts the communication technology of chaos masking and uses a single-channel synchronization scheme to achieve high accuracy. The simulation results demonstrate that encryption and decryption of the signal are achieved by the design. Moreover, the system is robust to noise signals and interference during the DNA reactions. This work provides a method for the application of DNA molecular computation in the communication field.

Preclinical pharmacology modeling of chimeric antigen receptor T therapies.

Chimeric antigen receptor (CAR) T cells have largely been successful in treating hematological malignancies in the clinic but have not been as effective in treating solid tumors, in part, owing to poor access and the immunosuppressive tumor microenvironment. In addition, CAR-T therapy can cause potentially life-threatening side effects, including cytokine release syndrome and neurotoxicity. Current preclinical testing of CAR-T therapy efficacy is typically performed in mouse tumor models, which often fails to predict toxicity. Recent developments in humanized models and transgenic mice as well as in vitro three-dimensional organoids in early development and nonhuman primate models are being adopted for CAR-T cell efficacy and toxicity assessment. However, because no single model perfectly recapitulates the human immune system and tumor microenvironment, careful model selection based on their respective pros and cons is crucial for adequate evaluation of different CAR-T treatments, so that their clinical development can be better supported.

Isolation of Thalassobius mangrovi sp. nov., a novel bacterium in the family Rhodobacteraceae, from marine mangrove sediment.

A Gram-stain-negative, strictly aerobic and oval-shaped bacterial strain with a flagellum, designated GS-10T, was isolated from mangrove wetland sediment. GS-10T grew at 20-40 °C (optimum, 37 °C), in the pH range of 5.0-11.0 (optimum, 6.0-8.0) and under various NaCl concentrations from 1 to 11 % (w/v) (optimum, 5-6 %). The respiratory quinone was ubiquinone-10, and the predominant polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids (>10 % of the total fatty acids) were summed feature 4 (C17 : 1iso I/anteiso B) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The G+C content of the genomic DNA was 63.71 %. On the basis of the results from comparative analysis of the 16S rRNA gene sequence, GS-10T represents a member of the family Rhodobacteraceae and had the highest sequence similarity to Thalassobius gelatinovorus CECT 4357T (97.47 %), followed by Lutimaribacter pacificus W11-2BT (97.03 %), Marivita cryptomonadis CL-SK44T (96.83 %), Thalassobius autumnalis CECT 5118T (96.75 %) and Thalassobius mediterraneus CECT 5383T (96.68 %). Phylogenetic trees based on 16S rRNA gene sequences, multilocus sequence analysis (MLSA) and whole genome sequences revealed that GS-10T clustered with species within the genus Thalassobius. The average nucleotide identity (ANI) and the average amino acid identity (AAI) values were calculated from complete genome sequences and indicated that GS-10T represented a novel species of the genus Thalassobius, and the name Thalassobius mangrovi sp. nov. is proposed for this species. The type strain of Thalassobius mangrovi is GS-10T (=MCCC 1K03624T=KCTC 82131T).

Research Advances of Purple Sweet Potato Anthocyanins: Extraction, Identification, Stability, Bioactivity, Application, and Biotransformation.

Purple sweet potato anthocyanins are kinds of natural anthocyanin red pigments extracted from the root or stem of purple sweet potato. They are stable and have the functions of anti-oxidation, anti-mutation, anti-tumor, liver protection, hypoglycemia, and anti-inflammation, which confer them a good application prospect. Nevertheless, there is not a comprehensive review of purple sweet potato anthocyanins so far. The extraction, structural characterization, stability, functional activity, application in the food, cosmetics, medicine, and other industries of anthocyanins from purple sweet potato, together with their biotransformation in vitro or by gut microorganism are reviewed in this paper, which provides a reference for further development and utilization of anthocyanins.

In Vivo Pharmacology Models for Cancer Target Research.

Experimental animal tumor models have been broadly used to evaluate anticancer drugs in the preclinical setting. They have also been widely applied for drug target discovery and validation, which usually follows four experimental strategies: first, assess the roles of putative drug targets using in vivo tumorigenicity and tumor growth kinetics assays of transplanted tumors, engineered through gain-of-function (GOF) by overexpressing transgene or knock-in (KI) or loss-of-function by gene silencing using knockdown (KD) or knockout (KO) or mutation via mutagenesis procedures; second, similarly genetically engineered mouse models (GEMM), through either germline or somatic cell procedures, are used to test the roles of potential targets in spontaneous tumorigenicity assays; third, patient-derived xenografts (PDXs), which most closely resemble patient genetics and histopathology, are used in tumor inhibition assays for evaluating target-/pathway-specific inhibitors, including large and small molecules, thus assessing the drug target; and fourth, the targets can be assessed in population-based trials, mouse clinical trials (MCT), so that the validation can be generally meaningful as performed in human clinical trials. This chapter outlines the commonly used protocols in cancer drug target research: the first four sections describe four sets of different, specific pharmacology protocols used in the respective cancer modeling stages, with the last section summarizing the common protocols applicable to all four pharmacology modeling steps.