Vivo Screening Research Articles

Bioinformatic Prediction and High Throughput In Vivo Screening to Identify Cis-Regulatory Elements for the Development of Algal Synthetic Promoters.

Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, their development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME, and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2, or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high levels of GFP expression. Further, we individually tested 14 identified promoters, revealing substantial increases in GFP expression─up to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome data set.

High-Throughput In Vivo Screening Identifies Differential Influences on mRNA Lipid Nanoparticle Immune Cell Delivery by Administration Route.

Immune modulation through the intracellular delivery of nucleoside-modified mRNA to immune cells is an attractive approach for in vivo immunoengineering, with applications in infectious disease, cancer immunotherapy, and beyond. Lipid nanoparticles (LNPs) have come to the fore as a promising nucleic acid delivery platform, but LNP design criteria remain poorly defined, making the rate-limiting step for LNP discovery the screening process. In this study, we employed high-throughput in vivo LNP screening based on molecular barcoding to investigate the influence of LNP composition on immune tropism with applications in vaccines and systemic immunotherapies. Screening a large LNP library under both intramuscular (i.m.) and intravenous (i.v.) injection, we observed differential influences on LNP uptake by immune populations across the two administration routes, gleaning insight into LNP design criteria for in vivo immunoengineering. In validation studies, the lead LNP formulation for i.m. administration demonstrated substantial mRNA translation in the spleen and draining lymph nodes with a more favorable biodistribution profile than LNPs formulated with the clinical standard ionizable lipid DLin-MC3-DMA (MC3). The lead LNP formulations for i.v. administration displayed potent immune transfection in the spleen and peripheral blood, with one lead LNP demonstrating substantial transfection of splenic dendritic cells and another inducing substantial transfection of circulating monocytes. Altogether, the immunotropic LNPs identified by high-throughput in vivo screening demonstrated significant promise for both locally- and systemically-delivered mRNA and confirmed the value of the LNP design criteria gleaned from our screening process, which could potentially inform future endeavors in mRNA vaccine and immunotherapy applications.

Multiplexed In Vivo Screening Using Barcoded Aptamer Technology to Identify Oligonucleotide-Based Targeting Reagents.

Recent FDA approvals of mRNA vaccines, short-interfering RNAs, and antisense oligonucleotides highlight the success of oligonucleotides as therapeutics. Aptamers are excellent affinity reagents that can selectively label protein biomarkers, but their clinical application has lagged. When formulating a given aptamer for invivo use, molecular design details can determine biostability and biodistribution; therefore, extensive postselection manipulation is often required for each new design to identify clinically useful reagents harboring improved pharmacokinetic properties. Few methods are available to comprehensively screen such aptamers, especially invivo, constituting a significant bottleneck in the field. In this study, we introduce barcoded aptamer technology (BApT) for multiplexed screening of predefined aptamer formulations in vitro and invivo. We demonstrate this technology by simultaneously investigating 20 aptamer formulations, each harboring different molecular designs, for targeting Non-Small Cell Lung Cancer cells and tumors. Screening in vitro identified a 45 kDa bispecific formulation as the best cancer cell targeting reagent, whereas screening invivo identified a 30 kDa monomeric formulation as the best tumor-specific targeting reagent. The multiplexed analysis pipeline also identified biodistribution phenotypes shared among formulations with similar molecular architectures. The BApT approach we describe here has the potential for broad application to fields where oligonucleotide-based targeting reagents are desired.

Analgesic and Anti-Inflammatory Activity: A Comprehensive Review on Invitro and In Vivo Screening Methods

Modern pharmacotherapy includes analgesic and anti-inflammatory medicines as essential components to relieve pain and inflammation brought on by a variety of medical diseases. Robust screening techniques are essential for the identification of possible candidates with appropriate safety and effectiveness profiles in the search and development of new analgesic and anti-inflammatory medications. This study looks at the many screening methods used in preclinical studies to assess new drugs' analgesic and anti-inflammatory quality. Conventional techniques like the tail flick, hot plate, and writhing’s tests measure analgesic activity by having animals respond to unpleasant stimuli. Comparably, anti-inflammatory activity is frequently assessed using assays like the cotton pellet granuloma test, which gauges tissue granuloma formation, and the carrageenan-induced paw edema model, which measures inflammation. These traditional techniques offer insightful information about the pharmacological effects of test substances. Despite the wide range of screening techniques available, each strategy has advantages and disadvantages. Preclinical studies are more reliable and have higher predictive value when various assays and techniques are integrated into a tiered screening strategy. Furthermore, the successful translation of preclinical findings to human applications depends on taking into account translational variables including species differences and clinical relevancies. As a result, choosing the right screening techniques is critical to the effective identification and characterization of new analgesic and anti-inflammatory drugs.

Unified Methodology for the Primary Preclinical In Vivo Screening of New Anticoagulant Pharmaceutical Agents from Hematophagous Organisms.

The development of novel anticoagulants requires a comprehensive investigational approach that is capable of characterizing different aspects of antithrombotic activity. The necessary experiments include both in vitro assays and studies on animal models. The required in vivo approaches include the assessment of pharmacokinetic and pharmacodynamic profiles and studies of hemorrhagic and antithrombotic effects. Comparison of anticoagulants with different mechanisms of action and administration types requires unification of the experiment scheme and its adaptation to existing laboratory conditions. The rodent thrombosis models in combination with the assessment of hemostasis parameters and hematological analysis are the classic methods for conducting preclinical studies. We report an approach for the comparative study of the activity of different anticoagulants in vivo, including the investigation of pharmacodynamics and the assessment of hemorrhagic effects (tail-cut bleeding model) and pathological thrombus formation (inferior vena cava stenosis model of venous thrombosis). The reproducibility and uniformity of our set of experiments were illustrated on unfractionated heparin and dabigatran etexilate (the most common pharmaceuticals in antithrombic therapy) as comparator drugs and an experimental drug variegin from the tick Amblyomma variegatum. Variegin is notorious since it is a potential analogue of bivalirudin (Angiomax, Novartis AG, Basel, Switzerland), which is now being actively introduced into antithrombotic therapy.

Biotransformation of Organophosphate Diesters Characterized via In Vitro Metabolism and In Vivo Screening.

Organophosphate diesters (di-OPEs), as additives in industrial applications and/or transformation products of emerging environmental pollutants, such as organophosphate triesters (tri-OPEs), have been found in the environment and biological matrices. The metabolic fate of di-OPEs in biological media is of great significance for tracing the inherent and precursor toxicity variations. This is the first study to investigate the metabolism of a suite of di-OPEs by liver microsomes and to identify any metabolite of metabolizable di-OPEs in in vitro and in vivo samples. Of the 14 di-OPEs, 5 are significantly metabolizable, and their abundant metabolites with hydroxyl, carboxyl, dealkylated, carbonyl, and/or epoxide groups are tentatively identified. More than half of the di-OPEs are detectable in human serum and/or wild fish tissues, and dibenzyl phosphate (DBzP), bis(2,3-dibromopropyl) phosphate (BDBPP), and isopropyl diphenyl phosphate (ip-DPHP) are first reported at a detectable level in humans and wildlife. Using an in vitro assay and a known biotransformation rule-based integrated screening strategy, 2 and 10 suspected metabolite peaks of DEHP are found in human serum and wild fish samples, respectively, and are then identified as phase I and phase II metabolites of DEHP. This study provides a novel insight into fate and persistence of di-OPE and confirms the presence of di-OPE metabolites in humans and wildlife.

Functional In Vivo Screening Identifies microRNAs Regulating Metastatic Dissemination of Prostate Cancer Cells to Bone Marrow.

Distant metastasis is the major cause of cancer-related deaths in men with prostate cancer (PCa). An in vivo functional screen was used to identify microRNAs (miRNAs) regulating metastatic dissemination of PCa cells. PC3 cells transduced with pooled miRZiP™ lentivirus library (anti-miRNAs) were injected intraprostatic to 13 NSG mice followed by targeted barcode/anti-miR sequencing. PCa cells in the primary tumours showed a homogenous pattern of anti-miRNAs, but different anti-miRNAs were enriched in liver, lung, and bone marrow, with anti-miR-379 highly enriched in the latter. The bone metastasis-promoting phenotype induced by decreased miR-379 levels was also confirmed in a less metastatic PCa cell line, 22Rv1, where all mice injected intracardially with anti-miR-379-22Rv1 cells developed bone metastases. The levels of miR-379 were found to be lower in bone metastases compared to primary tumours and non-cancerous prostatic tissue in a patient cohort. In vitro functional studies suggested that the mechanism of action was that reduced levels of miR-379 gave an increased colony formation capacity in conditions mimicking the bone microenvironment. In conclusion, our data suggest that specific miRNAs affect the establishment of primary tumours and metastatic dissemination, with a loss of miR-379 promoting metastases in bone.

In Vivo Screening of Immunotherapy Resistant Head and Neck Squamous Cell Carcinoma Identifies B7-H3 Inhibition as a Potential Radiosensitizer

<h3>Purpose/Objective(s)</h3> Patients with locally advanced head and neck squamous cell carcinoma (HNSCC) have poor outcomes following conventionally fractionated radiation (conRT) alone or in combination with chemotherapy. Unfortunately, the addition of anti-PD-1/L1 therapy to conRT has not been found to improve patient outcome. To identify other, more active conRT and immunotherapy combinations, we performed an <i>in vivo</i> shRNA screen of immune-related and other "druggable" genes in an immunotherapy and comparative conRT resistant murine HNSCC model (MOC2) to identify potential radiosensitizers. <h3>Materials/Methods</h3> The <i>in vivo</i> screen utilized shRNA libraries focused on immune-related and other "druggable" targets (n=242) in murine immunoevasive HNSCC tumor cells (MOC2), which were implanted into C57BL/6 flanks and treated with conRT (2Gy x 5) or sham. For validation, the same MOC2 tumor model was treated with murine isotype IgG or anti-B7H3 antibody with conRT. Flow cytometry was performed on single tumor cells approximately 10 days following the last conRT treatment using a murine specific adaptive immunotherapy panel. Data from the HNSCC Cancer Genome Atlas (TCGA) cohort was used for expression of B7-H3 in HPV-negative (-) and HPV-positive (+) HNSCC tumors. A subset of these patients with HPV (-) tumors (n=94) treated uniformly with surgery and post-operative radiation were analyzed for locoregional recurrence (LRR). Statistical analysis was performed in a scientific 2-D graphing and statistics software. <h3>Results</h3> Our <i>in vivo</i> shRNA screen identified several immune-related genes associated with conRT sensitization, including the under-studied CD276 (gene encoding immune checkpoint molecule B7-H3) (RSA log p-value -2.8). Notably, PD-L1 was predicted to have a relatively poor response in combination with conRT (RSA log p-value -0.38). Although ongoing at the time of writing, the validation study showed that conRT + anti-B7-H3 treatment led to a median survival of at least 28.3 days, significantly greater than RT + IgG of 22.3 days (p=3.5e⁻³), IgG (p=4.4e⁻⁶) or anti-B7-H3 alone (p=7e⁻⁶) (both 15.5 days). On initial flow cytometry, RT + anti-B7H3 had a potential increase in NK cells (1.55) compared to no RT + anti-B7H3 (0.22) or no RT + IgG (1.00). B7-H3 was expressed at a significantly higher level in HPV (-) versus HPV (+) HNSCC tumors from the TCGA (p=1.37e-13). In a subset of HPV (-) tumors from the HNSCC TCGA cohort treated with surgery and conRT, B7-H3 expression was significantly associated with increased LRR (p=0.035). <h3>Conclusion</h3> Our immunocompetent <i>in vivo</i> screen identified B7H3 inhibition as a potential novel radiosensitizer in HNSCC. In an immunocompetent syngeneic model, conRT + anti-B7H3 treatment demonstrated improved survival. As B7-H3 is targetable clinically, it provides an exciting and rational target for combinational therapy with RT.

High‐throughput Assay and In Vivo Screen Identify α‐2,3‐sialylation of CD98 by ST3GAL1 and ST3GAL2 as Essential to Melanoma Survival

Melanoma is an aggressive type of skin cancer that accounts for most skin cancer deaths, and the incidence of melanoma has increased rapidly over the past decades. Early‐staged disease can be cured by surgery, however, a lack of curative treatments for patients with established melanoma metastasis results in significantly low survival rates. Thus, identification of key drivers in melanoma progression is essential to our understanding of melanoma biology. Glycosylation is a hallmark of cancer biology and altered glycosylation influences multiple facets of both tumor growth and progression. Herein, we utilized lectin microarrays to compare the glycomes of early transformation and melanoma progression. We found common glycan signatures, including an increase of α‐2,3‐sialosides, in both biological processes, and revealed glycans associated with site‐specific metastasis. Tandem analysis of using an innovative functional in vivo growth screening of essential glycogenes identified the underlying sialyltransferases ST3GAL1 and ST3GAL2 as essential for melanoma growth. We confirmed upregulation of ST3GAL1 and ST3GAL2 in melanoma via examination of transcriptomic datasets and human tissue microarrays. Proteomic analysis identified CD98 as a candidate glycoprotein responsible for promoting melanoma proliferation. Our studies reveal glycans may act as molecular drivers that functionally contribute to melanoma biology and opens up novel paths to develop glycan‐based therapeutics to treat melanoma patients.

Zebrafish: An In Vivo Screening Model to Study Ocular Phenotypes.

PurposeTo establish a set of assays that allow the in vivo screening of candidate genes for ocular diseases in zebrafish, with an emphasis on refractive error.MethodsOur pipeline includes the most relevant ocular screening measurements to assess (1) ocular biometry using spectral domain optical coherence tomography, (2) refractive status using an eccentric photorefractor, (3) intraocular pressure by tonometry, and (4) optokinetic response to study visual capability in zebrafish. To validate our pipeline and to demonstrate the potential of zebrafish as a valid animal model, we chose two well-characterized genes with an ocular phenotype (PRSS56 and FBN1) and generated two mutant zebrafish lines (prss56 and fbn1). Mutant fish were assessed at 2, 4, and 6 months after fertilization.ResultsWith the proposed phenotyping pipeline, we showed that ocular biometry, refractive status, intraocular pressure, and visual function can be studied in zebrafish. In the prss56 mutant, the pipeline revealed a dramatic decrease in axial length, mainly owing to a decreased vitreous chamber depth, whereas in the fbn1 mutant, ectopia lentis was the most distinctive ocular phenotype observed. Tonometry in both mutant lines showed an increase in intraocular pressure.ConclusionsThe proposed pipeline was applied successfully in zebrafish and can be used for future genetic screenings of candidate genes. While validating our pipeline, we found a close resemblance between the ocular manifestations in the zebrafish mutants and patients harboring mutations in PRSS56 and FBN1. Our results support the validity of our pipeline and highlight the potential of zebrafish as an animal model for in vivo screening of candidate genes for ocular diseases.

Construction and Application of a High-Throughput In Vivo Screening Platform for the Evolution of Nitrile Metabolism-Related Enzymes Based on a Desensitized Repressive Biosensor.

Transcription factor (TF)-based biosensors are expected to serve as powerful tools for the high-throughput screening of biocatalytic systems; however, most of them respond to ligands in a narrow concentration range, which limits their application. In this study, we constructed a heterogenous niacin biosensor using the repressive TF BsNadR and its target promoters from Bacillus subtilis. The fine-tunable output of the niacin biosensor was expanded to a wide range of niacin concentrations (0-50 mM) through desensitization engineering, which was suitable for the accurate identification of differences in enzyme activity. Structural mechanism analysis indicated that weakening the affinity of BsNadR with the ligand niacin and with DNA alters its regulatory properties. Based on the desensitized niacin biosensor, a high-throughput in vivo screening platform was developed for evolving nitrile metabolism-related enzymes. The evolved nitrilase, amidase, and nitrile hydratase with 6.6-, 2.1-, and 21.3-fold improvements in activity were achieved, respectively. In addition, these mutants also exhibited elevated activity toward other cognate substrates, indicating the broad applicability of the screening platform. This study not only provided a universal high-throughput screening platform for different nitrile metabolism-related enzymes but also demonstrated the advantages of repressive biosensors and the vital role of desensitization engineering of the TF in the development of high-throughput screening platforms for enzymes.

In Vivo Screening Method for the Identification and Characterization of Prokaryotic, Metabolite-Responsive Transcription Factors.

In prokaryotes, transcription factors (TFs) are of uttermost importance for the regulation of gene expression. However, the majority of TFs are not characterized today, which hampers both the understanding of fundamental processes and the development of TF-based applications, such as biosensors, used in metabolic engineering, synthetic biology, diagnostics, etc. One way of analyzing TFs is through in vivo screening, enabling the study of TF-promoter interactions, ligand inducibility, and ligand specificity in a high-throughput fashion. Here, an approach is described for the selection and cloning of TF-promoter pairs, the development of a reporter system, and the measurement and analysis of fluorescent reporter assays. Furthermore, the importance of a suitable inducible plasmid system is illustrated together with prospective adaptations to modify a reporter system's output signal. The given approach can be used for the investigation of native, heterologous, or even artificially created TFs in Escherichia coli, and can be extended toward use in other microorganisms.

SEMMs: Somatically Engineered Mouse Models. A New Tool for In Vivo Disease Modeling for Basic and Translational Research.

Most experimental oncology therapies fail during clinical development despite years of preclinical testing rationalizing their use. This begs the question of whether the current preclinical models used for evaluating oncology therapies adequately capture patient heterogeneity and response to therapy. Most of the preclinical work is based on xenograft models where tumor mis-location and the lack of the immune system represent a major limitation for the translatability of many observations from preclinical models to patients. Genetically engineered mouse models (GEMMs) hold great potential to recapitulate more accurately disease models but their cost and complexity have stymied their widespread adoption in discovery, early or late drug screening programs. Recent advancements in genome editing technology made possible by the discovery and development of the CRISPR/Cas9 system has opened the opportunity of generating disease-relevant animal models by direct mutation of somatic cell genomes in an organ or tissue compartment of interest. The advent of CRISPR/Cas9 has not only aided in the production of conventional GEMMs but has also enabled the bypassing of the construction of these costly strains. In this review, we describe the Somatically Engineered Mouse Models (SEMMs) as a new category of models where a specific oncogenic signature is introduced in somatic cells of an intended organ in a post-natal animal. In addition, SEMMs represent a novel platform to perform in vivo functional genomics studies, here defined as DIVoS (Direct In Vivo Screening).

Optimization of 1,4-Naphthoquinone Hit Compound: A Computational, Phenotypic, and In Vivo Screening against Trypanosoma cruzi.

Chagas disease (CD) still represents a serious public health problem in Latin America, even after more than 100 years of its discovery. Clinical treatments (nifurtimox and benznidazole) are considered inadequate, especially because of undesirable side effects and low efficacy in the chronic stages of the disease, highlighting the urgency for discovering new effective and safe drugs. A small library of compounds (1a–i and 2a–j) was designed based on the structural optimization of a Hit compound derived from 1,4-naphthoquinones (C2) previously identified. The biological activity, structure-activity relationship (SAR), and the in silico physicochemical profiles of the naphthoquinone derivatives were analyzed. Most modifications resulted in increased trypanocidal activity but some substitutions also increased toxicity. The data reinforce the importance of the chlorine atom in the thiophenol benzene ring for trypanocidal activity, highlighting 1g, which exhibit a drug-likeness profile, as a promising compound against Trypanosoma cruzi. SAR analysis also revealed 1g as cliff generator in the structure-activity similarity map (SAS maps). However, compounds C2 and 1g were unable to reduce parasite load, and did not prevent mouse mortality in T. cruzi acute infection. Phenotypic screening and computational analysis have provided relevant information to advance the optimization and design of new 1,4-naphthoquinone derivatives with a better pharmacological profile.

Molecular Docking and In Vivo Screening of Some Bioactive Phenoxyacetanilide Derivatives as Potent Non-Steroidal Anti-Inflammatory Drugs

Molecular Docking and In Vivo Screening of Some Bioactive Phenoxyacetanilide Derivatives as Potent Non-Steroidal Anti-Inflammatory Drugs

In Vivo Screening of Xanthones from Garcinia oligantha Identified Oliganthin H as a Novel Natural Inhibitor of Convulsions.

Epilepsy is a chronic neurological disorder, characterized by recurrent, spontaneous, and transient seizures, and affects more than 70 million people worldwide. Although two dozen antiepileptic drugs (AEDs) are approved and available in the market, seizures remain poorly controlled in one-third of epileptic patients who are suffering from drug resistance or various adverse effects. Recently, the xanthone skeleton has been regarded as an attractive scaffold for the discovery and development of emerging anticonvulsants. We had isolated several dihydroxanthone derivatives previously, including oliganthin H, oliganthin I, and oliganthin N, whose structures were similar and delicately elucidated by spectrum analysis or X-ray crystallographic data, from extracts of leaves of Garcinia oligantha. These xanthone analogues were evaluated for anticonvulsant activity, and a novel xanthone, oliganthin H, has been identified as a sound and effective natural inhibitor of convulsions in zebrafish in vivo. A preliminary structure-activity relationship analysis on the relationship between structures of the xanthone analogues and their activities was also conducted. Oliganthin H significantly suppressed convulsant behavior and reduced to about 25% and 50% of PTZ-induced activity, in 12.5 and 25 μM treatment groups (P < 0.01 and 0.001), respectively. Meanwhile, it reduced seizure activity, velocity, seizure duration, and number of bursts in zebrafish larvae (P < 0.05). Pretreatment of oliganthin H significantly restored aberrant induction of gene expressions including npas4a, c-fos, pyya, and bdnf, as well as gabra1, gad1, glsa, and glula, upon PTZ treatment. In addition, in silico analysis revealed the stability of the oliganthin H-GABAA receptor complex and their detailed binding pattern. Therefore, direct interactions with the GABAA receptor and involvement of downstream GABA-glutamate pathways were possible mechanisms of the anticonvulsant action of oliganthin H. Our findings present the anticonvulsant activity of oliganthin H, provide a novel scaffold for further modifications, and highlight the xanthone skeleton as an attractive and reliable resource for the development of emerging AEDs.

In Vivo Screening and Antidiabetic Potential of Polyphenol Extracts from Guava Pulp, Seeds and Leaves.

Simple SummaryThe incidence of diabetes has risen from 151 million people in 2000 to 463 million in 2019, with 4.2 million estimated deaths in 2019, and over 700 million people will be affected with diabetes worldwide by 2045. Thus, the screening of anti-diabetic plants is inevitable in order to overcome diabetes. This study has investigated the anti-diabetic potential of polyphenol extracts from guava leaves, seeds and pulp using an albino rat model. The in vivo study has reported that polyphenols from leaves and pulp have an ability to improve diabetic parameters, such as insulin, blood glucose and triglycerides levels. The improvement in blood parameters is also an indication of these extracts that are valuable for diabetes management. The findings of this study reveal that polyphenols from guava leaves, pulp and seeds can be used for drug development.The present study investigates the antidiabetic potential of polyphenol extracts purified from guava pulp, seeds and leaves using an in vivo experiment on albino rats. The polyphenols from guava pulp, seeds and leaves were extracted using methanol solvent and the sonication method while being evaluated by total phenolic contents and radical scavenging activity assay. The proximate composition of powders revealed that ash, protein and total sugars were significantly (p < 0.05) higher in leaves and seeds, while vitamin C was highest in pulp. Total phenolic and antioxidant activities were highest in pulp followed by leaves and seeds. The findings of feed intake and body gain revealed that the supplementation of polyphenols, especially from pulp, significantly (p < 0.05) increased the feed intake, which resulted in increased body weight. Moreover, total cholesterol (TC) and low-density lipoprotein (LDL) levels were significantly (p < 0.05) decreased, while the level of high-density lipoprotein (HDL) was increased in groups fed with polyphenols from guava pulp compared to both (+ive and –ive) control groups. Furthermore, blood glucose and triglycerides were significantly (p < 0.05) decreased in supplemented groups compared to the control group of diabetes mice, which resulted in the inhibition of α-amylase and glucose transport. Besides this, packed cell volume (PCV), mean corpuscular volume (MCV), hemoglobin, red blood cells (RBCs), white blood cells (WBCs) and platelet levels were increased significantly (p < 0.05) in pulp’s extract followed by leaves and seeds compared to both control groups. Overall, the antidiabetic potential of different extracts was in the following order: pulp > leaves > seeds. The findings suggest the feasibility of adding 200–250 mg/kg.bw of polyphenol extracts of pulp as an alternative to diabetic drugs.

Efficient In Vivo Screening Method for the Identification of C4 Photosynthesis Inhibitors Based on Cell Suspensions of the Single-Cell C4 Plant Bienertia sinuspersici.

The identification of novel herbicides is of crucial importance to modern agriculture. We developed an efficient in vivo assay based on oxygen evolution measurements using suspensions of chlorenchyma cells isolated from the single-cell C4 plant Bienertia sinuspersici to identify and characterize inhibitors of C4 photosynthesis. This novel approach fills the gap between conventional in vitro assays for inhibitors targeting C4 key enzymes and in vivo experiments on whole plants. The assay addresses inhibition of the target enzymes in a plant context thereby taking care of any reduced target inhibition due to metabolization or inadequate uptake of small molecule inhibitors across plant cell walls and membranes. Known small molecule inhibitors targeting C4 photosynthesis were used to validate the approach. To this end, we tested pyruvate phosphate dikinase inhibitor bisindolylmaleimide IV and phosphoenolpyruvate carboxylase inhibitor okanin. Both inhibitors show inhibition of plant photosynthesis at half-maximal inhibitory concentrations in the sub-mM range and confirm their potential to act as a new class of C4 selective inhibitors.

(OA30) In Vivo Screening Identifies ESR1 as a Clinically Relevant Target for Radiosensitization In HNSCC

(OA30) In Vivo Screening Identifies ESR1 as a Clinically Relevant Target for Radiosensitization In HNSCC

Discovery of Trypanocidal Bioactive Leads by Docking Study, Molecular Dynamic Simulation and In Vivo Screening

AbstractNine selected potential antitrypanosomal hits from plants obtained by docking calculation according to the docking protocols implemented in Molecular Operating Environment were further screened by evaluating their binding affinities toward an ‘all important’ antitrypanosomal drug target – trypanothione reductase (TryR), which is absent in humans, using a more stringent scoring function obtainable in AutoDock vina. All the compounds showed binding affinity for TryR at the range of −4.90±1.78 to −8.70±0.22 Kcal/mol. Also, the compounds demonstrated affinity toward an analogous enzyme to TryR, glutathione reductase (GR) that is found in vertebrates which suggests that they could pose a selectivity challenge. However, on comparison, β‐sitosterol; the candidate that scored topmost against three out of six earlier studied antitrypanosomal drug targets (uridylyl transferase ‐ UTF, farnesyl diphosphate synthase ‐ FDS and adenosine kinase ‐ ADK), showed a significant (P&lt;0.05) binding affinity for TryR (‐8.70±0.22 Kcal/mol) over GR (‐7.50±0.36 Kcal/mol). The complexes formed between β‐sitosterol and the proteins: UTF, FDS and ADK, remained stable over 100 ps molecular dynamic simulation. Moreover, 200 mg/kg b.w. of the candidate significantly (P&lt;0.05) killed the pathogen in T. b. brucei infected mice from third day post treatment. This study presents β‐sitosterol as an interesting antitrypanosomal candidate.