Invivo Assays Research Articles

DUSP4 inhibits autophagic cell death in PTC by inhibiting JNK-BCL2-Beclin1 signaling.

Dual specificity phosphatase 4 (DUSP4) is a prognostic marker and potential target of papillary thyroid carcinoma (PTC); however, the molecular mechanism underlying DUSP4-regulated PTC carcinogenesis is unknown. DUSP4 is a negative regulator of the autophagy promoter, JNK. This study explored the relationship between DUSP4 and JNK-mediated autophagic cell death in PTC, and the roles of DUSP4 in PTC using gain-of-function and loss-of-function assays. In addition, we further identified the significance of the JNK-BCL2-Beclin1-autophagy signaling pathway on DUSP4-regulated PTC carcinogenesis by combining knockdown of DUSP4 with a JNK-specific inhibitor (SP600125). We found that knockdown of DUSP4 promoted the phosphorylation of JNK and BCL2 in PTC cells, and enhanced the release of Beclin1 from the BCL2-Beclin1 complex. Knockdown of DUSP4 promoted autophagy and the death of PTC cells. The death and autophagy enhanced by knockdown of DUSP4 was reversed by the JNK inhibitor. We further extended the in-vitro experiments by subcutaneously injecting nude mice with K1 cells transfected with DUSP4-silencing vector. In-vivo assays showed that knockdown of DUSP4 not only inhibited tumor growth, but also promoted the phosphorylation of JNK and BCL2 and the expression of LC3II. In conclusion, DUSP4 inhibits BCL2-Beclin1-autophagy signaling by negatively regulating JNK activity, thus inhibiting PTC oncogenesis. The data from this study contribute to the prevention and cure of PTC.

Mapping drug-target interactions and synergy in multi-molecular therapeutics for pressure-overload cardiac hypertrophy

Advancements in systems biology have resulted in the development of network pharmacology, leading to a paradigm shift from “one-target, one-drug” to “target-network, multi-component therapeutics”. We employ a chimeric approach involving in-vivo assays, gene expression analysis, cheminformatics, and network biology to deduce the regulatory actions of a multi-constituent Ayurvedic concoction, Amalaki Rasayana (AR) in animal models for its effect in pressure-overload cardiac hypertrophy. The proteomics analysis of in-vivo assays for Aorta Constricted and Biologically Aged rat models identify proteins expressed under each condition. Network analysis mapping protein–protein interactions and synergistic actions of AR using multi-component networks reveal drug targets such as ACADM, COX4I1, COX6B1, HBB, MYH14, and SLC25A4, as potential pharmacological co-targets for cardiac hypertrophy. Further, five out of eighteen AR constituents potentially target these proteins. We propose a distinct prospective strategy for the discovery of network pharmacological therapies and repositioning of existing drug molecules for treating pressure-overload cardiac hypertrophy.

Resistance response enhancement and reduction of Botrytis cinerea infection in strawberry fruit by Morchella conica mycelial extract

Over 200 crops worldwide are susceptible to gray mold infection, caused by the phytopathogenic fungus Botrytis cinerea, including strawberry fruit, both in the field and after harvest. The disease is normally controlled by chemical fungicides. However, increasing public and environmental concern over pesticide applications has prompted the exploration of ecofriendly approaches to combatting pathogens. Morel mushrooms are edible mushrooms with abroad range of bioactive metabolites. We studied the inhibitory effect of Morchella conica Pers. mycelial water extract (MWE) on B. cinerea infection of strawberry fruit for the first time. In-vivo assay showed reduction of gray mold infection, measured by disease index on MWE-treated fruit compared to controls. Enhancement of a range of biochemical and molecular characteristics related to the fruit's defense system was detected by RNA-Seq analysis, quantitative PCR and quantification of total phenols, flavonoids, antioxidant capacity by DPPH, and enzymatic activities in the fruit. In-vitro tests showed no inhibition of B. cinerea compared to a control fungicide fludioxonil. The findings demonstrate the beneficial effect of Morchella conica metabolites in enhancing fruit resistance to pathogenic fungal attack and improving quality of postharvest strawberry fruit.

Synthesis of urea-modified magnetic nanocomposites iron oxide/carbon as a potential biomaterial produced by arc discharge in liquid medium and its in-vivo toxicity assessment

Carbon-encapsulated magnetic nanoparticles are promising candidate materials for drug-delivery applications. However, due to their hydrophobic and aggregation properties, which indicate lower biocompatibility, proper surface modification of the carbon-based material is required. In the present study, we present the facile route to producing biocompatible magnetic nanocomposite iron oxide/carbon using the liquid medium arc-discharge method. The medium used was ethanol 50% with urea added in various concentrations. Using x-ray diffraction (XRD), the nanocomposite produced was confirmed to have a crystalline structure with distinctive peaks representing iron oxide, graphite, and urea. Fourier transform infrared spectroscopy (FTIR) analysis of the nanocomposite produced in ethanol/acetic acid or ethanol/urea medium shows several vibrations, including Fe–O, C–H, C–O, C=C, C–H, O–H, and C–N, which are intended to be the attached aromatic oxygen- and amine-containing functional groups. The nanocomposite particle was observed to have a core–shell structure that had an iron-compound core coated in a carbon shell possibly modified by polymeric urea groups. The presence of these groups suggested that the nanocomposite would be biocompatible with biological entities in the living body. Lastly, the prepared nanocomposite Fe3O4/C-urea underwent an in-vivo acute toxicity assay to confirm its toxicity. The highest dose of 2000 mg kg−1 BW in this study caused no deaths in the test animals even though cell damages were observed, especially in the liver. This highest dose is considered a maximum tolerable dose and is defined as practically non-toxic.

Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems

Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, invivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13acrRNA in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with invitro biochemical and invivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.

Anti-inflammatory, Antioxidant, Lung and Liver Protective Activity of Galaxaura oblongata as Antagonistic Efficacy against LPS using Hematological Parameters and Immunohistochemistry as Biomarkers

This study aimed to investigate the potential bioactivity and the ameliorative role of Galaxaura oblongata (G. oblongata) against LPS-induced toxicity using hematological parameters. The objective of this study is to examine its protective effect using the immunohistochemistry of the liver and lungs as biomarkers in male BALB/C albino mice. The current study was carried out using different in-vitro and in-vivo assays, such as phytochemicals, antioxidants and anti-inflammatory for in-vitro where the hematological and immunohistochemistry for lung and liver were investigated in vivo. No previous studies were performed to investigate the in vivo and in vitro effects of the G. oblongata extracts as antioxidant and anti-inflammatory due to their rareness compared to other red algae. LPS treated mice revealed a significant decrease in the total number of WBCs, RBCs, platelets, and HGB%, MPV, MCV and MCHC compared to the control group. In contrast, the HCT and MCHC were increased in the induction group, which was treated with LPS compared to the control group. Furthermore, the immunohistochemistry results of the present study revealed the protective effect of G. oblongata compared to the induction group. G. oblongata can be used as protective marine natural products against the toxicity induced by LPS. It exhibited a significant ameliorative role against the alterations in the hematological parameters and immunohistochemistry of the liver and lungs, and reduced as well as coordinated the acute inflammations caused by TNF.

A bispecific antibody targeting GPC3 and CD47 induced enhanced antitumor efficacy against dual antigen-expressing HCC

Glypican-3 (GPC3) is a well-characterized hepatocellular carcinoma (HCC)-associated antigen, yet anti-GPC3 therapies have achieved only minimal clinical progress. CD47 is a ubiquitously expressed innate immune checkpoint that promotes evasion of tumors from immune surveillance. Given both the specific expression of GPC3 in HCC and the known phagocytosis inhibitory effect of CD47 in liver cancer, we hypothesized that a bispecific antibody (BsAb) that co-engages with GPC3 and CD47 may offer excellent antitumor efficacy with minimal toxicity. Here, we generated a novel BsAb: GPC3/CD47 biAb. With the use of both invitro and invivo assays, we found that GPC3/CD47 biAb exerts strong antitumor activity preferentially against dual antigen-expressing tumor cells. In hCD47/human signal regulatory protein alpha (hCD47/hSIRPα) humanized mice, GPC3/CD47 biAb had an extended serum half-life without causing systemic toxicity. Importantly, GPC3/CD47 biAb induced enhanced Fc-mediated effector functions to dual antigen-expressing HCC cells invitro, and both macrophages and neutrophils are required for its strong efficacy against xenograft HCC tumors. Notably, GPC3/CD47 biAb outperformed monotherapies and a combination therapy with anti-CD47 and anti-GPC3 monoclonal antibodies (mAbs) in a xenograft HCC model. Our study illustrates a strategy for improving HCC treatment by boosting innate immune responses and presents new insights to inform antibody design for the future development of innovative immune therapies.

Fruits of <i>Vitex Doniana</i> Sweet: Toxicity Profile, Anti-Inflammatory and Antioxidant Activities, and Quantification of One of its Bioactive Constituents Oleanolic Acid

Background: Vitex doniana Sweet fruit, an under-utilised crop specie of Ghana, has not been validated for its ethnomedical use in managing inflammatory conditions. Therefore, the study sought to investigate its anti-inflammatory and antioxidant activities as well as isolate and quantify one of its active constituents. Methods: In-vivo anti-inflammatory assay was done using the carrageenan- induced oedema model in chicks. The in-vitro antioxidant property was also investigated using the 2, 2-diphenyl-1-picrylhydrayl radical scavenging assay. The acute and subacute toxicity studies of the fruit extract were evaluated in rodent models. Results: No signs of autonomic and central nervous system stimulation/depression were recorded. The median lethal dose (LD50 ), by oral route, was estimated to be beyond 3000 mg/kg body weight. Subacute studies revealed an increase in red blood cell and lymphocyte counts. Liver enzymes, serum proteins and bilirubin levels did not significantly increase. The crude extracts at doses of 10, 30 and 100 mg/kg inhibited paw oedema considerably. The ethyl acetate fraction showed the highest antioxidant activity (half-maximal inhibitory concentration, IC50 = 99.35 ± 0.77 µg/mL). 3β-hydroxyolean-12-en-28-oic acid (oleanolic acid), isolated from the ethyl acetate extract, showed significant anti-inflammatory and antioxidant activities. A sensitive high-performance liquid chromatography method for the detection and estimation of oleanolic acid, as a biomarker compound for V. doniana fruit, was developed and validated for quality assurance purposes. Conclusion: The extract of V. doniana possesses considerable anti-inflammatory and antioxidant properties and was non-toxic under laboratory conditions.

A simple theoretical approach to converging of Myoglobin-Assay with different pH values

AbstractMany metal carbonyl complexes have been synthesized and analyzed as CO-releasing agents. As in many bioactivity assays, differences between in-vitro and in-vivo studies in Myoglobin Assay have been observed. Adjustment of in-vitro conditions to in-vivo conditions is one way to overcoming this problem. Changing the conditions of each in-vivo assay is not possible considering the available grant, material, and labor facilities. In-silico methods are suitable as they provide better in-vitro conditions before experimental procedures. A method which is easy to employ on a basic computer could be more suitable to observe the assay convergence. In this study, global reactivity descriptors were used as an approach to investigate pH differences in myoglobin assay. Global reactivity descriptors of the molecules were compared with myoglobin assay results at different pH values and molecular docking results performed with optimized molecules in different solvents. The following complexes were studied: [Mn(CO)3(bpy)(L)]PF6(bpy: 2,2-bipyridyl, L: benzylbenzimidazole, 4-chlorobenzylbenzimidazole).

Leaf extract of ethnomedicinally important Bharangi (Clerodendrum serratum) may improve neuromodulatory activity in mice model

Ancient civilization to present-day cultural and technological development, human over the world has been using plants as a source of natural medicine for their basic needs. Clerodendrum serratum (Linn.) Moonis one such traditionally well-accepted plant used vigorously in Indian ayurvedic therapeutic purposes. Despite its ethnomedicinal significance, the neuroprotective prospects and relevance of Clerodendrum serratum (CSL) remain unaddressed. Therefore, in the present study, we explored several in-vivo antioxidant enzymatic assays followed by acetylcholinesterase enzyme (AChE) inhibitory activity against neurodegenerative disorders (NDs) in mice model and explore the beneficial impact of the plant extract. Furthermore, the in-silico molecular docking approach was employed to evaluate the inhibitory potential of the plant-derived bioactive compounds against the imperative proteins associated with neurodegenerative disorders. The plant extract showed promising results in all in-vivo enzymatic assays indicating its positive role over several neurodegenerative disseases namely, Alzheimer’s and Parkinson’s disease. The histological and in-silico investigation also reveled this plant extract is promising in elevating neurodegenerative disorders. Therefore, it seems likely that this plant can be used as an anti-cognitive agent for future drug development.

Pharmacophore hybridization approach to discover novel pyrazoline-based hydantoin analogs with anti-tumor efficacy

In search for new and safer anti-cancer agents, a structurally guided pharmacophore hybridization strategy of two privileged scaffolds, namely diaryl pyrazolines and imidazolidine-2,4-dione (hydantoin), was adopted resulting in a newfangled series of compounds (H1-H22). Herein, a bio-isosteric replacement of “pyrrolidine-2,5-dione” moiety of our recently reported antitumor hybrid incorporating diaryl pyrazoline and pyrrolidine-2,5-dione scaffolds with “imidazoline-2,4-dione” moiety has been incorporated. Complete biological studies revealed the most potent analog among all i.e. compound H13, which was at-least 10-fold more potent compared to the corresponding pyrrolidine-2,5-dione, in colon and breast cancer cells. In-vitro studies showed activation of caspases, arrest of G0/G1 phase of cell cycle, decrease in the expression of anti-apoptotic protein (Bcl-2) and increased DNA damage. In-vivo assay on HT-29 (human colorectal adenocarcinoma) animal xenograft model unveiled the significant anti-tumor efficacy along with oral bioavailability with maximum TGI 36% (i.p.) and 44% (per os) at 50 mg/kg dose. These findings confirm the suitability of hybridized pyrazoline and imidazolidine-2,4-dione analog H13 for its anti-cancer potential and starting-point for the development of more efficacious analogs.

TFAP2A inhibits microRNA-126 expression at the transcriptional level and aggravates ischemic neuronal injury

Neuronal injury induced by cerebral ischemia poses a serious health risk globally, and there is no effective clinical therapy. This study was performed to investigate the role of transcription factor AP-2 alpha (TFAP2A) in cerebral ischemia, and the underlying mechanisms, using an in-vitro model (PC-12 cells) of oxygen–glucose deprivation (OGD), and an in-vivo model (rat) of transient global cerebral ischemia (tGCI). The results for CCK-8 and Hoechst staining showed that silencing of TFAP2A enhanced the viability and decreased the rate of apoptosis of PC12 cells subjected to OGD. ChIP assays were performed to evaluate the binding of TFAP2A to the promoter region of microRNA (miR)-126, and we found that TFAP2A inhibits the expression of miR-126. Further mechanistic investigation revealed that miR-126 targets polo like kinase 2 (PLK2), and that overexpression of PLK2 activates the IκBα–NF-κB signaling pathway and suppresses the growth of PC12 cells subjected to OGD. For our in-vivo assay, we used TTC staining to analyze the infarction area in the brain tissues of rats, and Nissl staining to evaluate the number of surviving brain neurons. The pathological conditions associated with neuronal injury in rat brain tissues were assessed by staining the tissues with hematoxylin–eosin. Our results indicate that TFAP2A downregulates miR-126, and thereby upregulates PLK2 and activates the IκBα–NF-κB pathway, which increased neuronal injury following cerebral ischemia.

Function and Regulation of the Pyruvate Transporter CstA in Escherichia coli.

Pyruvate is a central metabolite that connects many metabolic pathways in living organisms. To meet the cellular pyruvate requirements, the enterobacterium Escherichia coli has at least three pyruvate uptake systems—the H+/pyruvate symporter BtsT, and two thus far less well-characterized transporters, YhjX and CstA. BtsT and CstA belong to the putative carbon starvation (CstA) family (transporter classification TC# 2.A.114). We have created an E. coli mutant that cannot grow on pyruvate as the sole carbon source and used it to characterize CstA as a pyruvate transporter. Transport studies in intact cells confirmed that CstA is a highly specific pyruvate transporter with moderate affinity and is energized by a proton gradient. When cells of a reporter strain were cultured in complex medium, cstA expression was maximal only in stationary phase. A DNA affinity-capture assay combined with mass spectrometry and an in-vivo reporter assay identified Fis as a repressor of cstA expression, in addition to the known activator cAMP-CRP. The functional characterization and regulation of this second pyruvate uptake system provides valuable information for understanding the complexity of pyruvate sensing and uptake in E. coli.

Treating Hyperglycemia From Eryngium caeruleum M. Bieb: In-vitro α-Glucosidase, Antioxidant, in-vivo Antidiabetic and Molecular Docking-Based Approaches.

Natural-based drugs are believed to be safe, effective and economical. Based on the medicinal importance of the genus Eryngium and unexplored nature of Eryngium caeruleum, we have evaluated its antidiabetic and antioxidant potentials. Both in-vitro and in-vivo assays have been carried out for antidiabetic assays. The antioxidant activity was determined by using different free radicals [i.e., 1,1-diphenyl,2-picrylhydrazyl (DPPH), 2,2-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS), and hydrogen peroxide (H2O2)]. Moreover, different phytoconstituents were identified in the most active solvent fraction by GC-MS analysis. Furthermore, comparative fingerprints of methanolic extract and chloroform fraction were also analyzed via High Performance Liquid Chromatography coupled with Diode Array Detector (HPLC-DAD). The crude methanolic extract of E. caeruleum (Ec.Cr) and its sub-fractions [i.e., n-hexane (Ec.Hex), chloroform (Ec.Chf), ethyl acetate (Ec.EtAc), and aqueous (Ec.Aq) were employed in this study]. In the α-glucosidase inhibition assay, a concentration-dependent inhibitory response was observed against the enzyme. The most active sample was Ec.Chf which revealed an IC50 of 437 μg/ml in comparison to the standard acarbose (IC50 25 μg/ml). The rest of the samples showed moderate inhibition of α-glucosidase. In antioxidant assays, Ec.Chf and Ec.Cr exhibited a considerable scavenging effect against all the free radicals. The IC50 values recorded for Ec.Chf were 112, 109, and 150 μg/ml against DPPH, ABTS, and H2O2 respectively. Based on the in-vitro potential of Ec.Chf, this was subjected to the in-vivo model experiment. The Ec.Chf lowered the blood glucose level up to 10.3 mmol/L at 500 μg/Kg. The Ec.Chf was also subjected to GC-MS analysis. The GC-MS analysis confirmed the presence of 60 compounds. The identified phytoconstituents consist of some essential compounds previously reported with antidiabetic and antioxidant studies, which include thymol, tocopherol, phytol, nerolidol, (I)-neophytadiene, linolenic acid, and falcarinol. Similarly, the HPLC-DAD chromatograms of Ec.Cr and Ec.Chf exhibited a variety of peaks, which further demonstrates the possibility of important phytochemicals. In a nutshell, we can conclude that Eryngium caeruleum is a potential source of bioactive compounds which may be beneficial for the management of ailments like diabetes and free radicals mediated disorders. Molecular docking was performed to explore the possible role of all the identified bioactive compounds in the chloroform fraction of Eryngium caeruleum into active sites of the homology model of α-glucosidase.

Erythroid Specificity and Safety of Globin-Encoding Lentiviral Vectors

The beta-thalassemias and sickle cell disease are the most common monogenic inherited blood disorders, both arising from mutations affecting the beta-globin locus. Recent clinical trials utilizing lentiviral-mediated beta-globin gene transfer in autologous CD34+ cells have shown encouraging results in patients with non-beta-zero thalassemias and sickle cell anemia. A case report of insertional mutagenesis in a beta-thalassemia patient resulted in a prolonged clonal expansion that eventually regressed without progressing to leukemic transformation. Nonetheless, this occurrence and the need to insert multiple vector copies per cell when using globin vectors that provide insufficient globin expression to achieve curative responses from a single integrated copy per cell, raise some questions about the erythroid specificity and safety of globin vectors. We thus found it imperative to investigate globin vector expression in hematopoietic progenitors and in non-erythroid cells. To this end, we investigated what regulatory elements, including locus control region (LCR) hypersensitive sites (HS) and others, achieve the highest beta-globin expression per vector copy while at the same time minimizing non-erythroid transcriptional activity. We developed an invivo assay to track the enhancer/promoter activity of different HS elements in hematopoietic progenitor and differentiated cell subsets. We designed lentiviral vectors expressing hrGFP under the control of a short β-globin promoter (137bp) controlled by a set of LCR HS elements and/or a novel biliverdin (BLV) enhancer. Analysis of hrGFP expression in bone marrow cells derived from C57BL6 mice transplanted with transduced syngeneic bone marrow cells revealed that many of the vectors encompassing elements thought to encode erythroid-specific elements were in fact expressed in long term-HSC (LT-HSC), short term-HSC (ST-HSC), multipotent progenitors (MPP), pre-GM, Granulocyte/macrophage progenitors (GMP) and megakaryocyte progenitors (MKP). These findings confirmed that ectopically integrated erythroid regulatory elements can serve as transcriptional enhancers in non-erythroid cells. This transcriptional leakiness was confirmed in therapeutic globin vectors harboring a combination of LCR elements. To avoid these effects, we flanked the globin transcription unit with a small human enhancer-blocking element called A1, kindly provided by the late George Stamatoyannopoulos (Liu, M. et al. 2015). The insulated vector showed markedly reduced non-erythroid expression in non-erythroid cells including ST-HSC, MPP, Pre-GM and GMP. Expression in mature erythroid cells was unchanged. Our findings underscore the benefits of selecting lineage-specific regulatory elements with the least lineage promiscuity for therapeutic vectors, with the eventual addition of enhancer-blocking elements. Based on these results, we are now investigating new globin expression vectors with optimal combination of HS elements. Such novel globin vectors should minimize the risk of oncogene trans-activation in hematopoietic progenitor cells and therefore improve the safety of globin gene therapy while providing lineage-specific high-level expression. Disclosures Sadelain: Mnemo: Patents & Royalties; Fate Therapeutics: Patents & Royalties, Research Funding; Atara: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents & Royalties.

Virulence potential of Aeromonas hydrophila isolated from apparently healthy freshwater food fish

Aeromonas hydrophila is considered an important pathogen of fish causing substantial economic loss in aquaculture worldwide. The objective of the current study was to assess the virulence potential of A. hydrophila strains isolated from gills of apparently healthy fish using phenotypic assays, PCR based genotypic tests, and in-vivo pathogenicity assays in Indian catfish, Clarias magur. For this, motile aeromonads (n = 50) were isolated from gills of apparently healthy freshwater food fish (n = 50) collected from domestic fish market. Gill swabs were incubated for primary enrichment in alkaline peptone water supplemented with cephalothin (10 mg/mL) for 12 hours followed by plating on Starch Ampicillin Agar. A single big yellow colony was selected from each sample, purified, and presumptively identified as Aeromonas with the help of Aerokey-II. Out of the fifty isolates of Aeromonas, five isolates of diverse origin (9C, 10G1, 7C, 10C and 4P) were confirmed to be A. hydrophila by automated bacterial identification system VITEK 2 with confidence interval of 90–98%. In the phenotypic assays, strains 9C and 10G1 showed high serum resistance, swimming and swarming activity and low biofilm producing capabilities, which were indicative of putative virulence. Both the strains belonged to genotype aah+act+ alt+ascV+eno+lip+ast+ on the basis of detection of virulence genes by PCR, i.e. extracellular haemolysin (aah), cytolytic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), type-III secretion system (ascV), enolase (eno), lipase (lip) and heat-stable cytotonic enterotoxin (ast). In-vivo pathogenicity assays confirmed that both the strains were pathogenic to magur fish. The LD50 for 9C strain was 6.81 × 104 CFU/mL and 7.62 × 105 CFU/mL for 10G1strain. In conclusion, our phenotypic and genotypic findings showed that A. hydrophila isolated from apparently healthy fish harbour number of important virulence genes/factors and could have important implications in triggering disease in farmed fish under stress.

Cell Biology: Deciphering the ABCs of SLiMs in G1-CDK Signaling

A new study uses an elegant invivo assay to comprehensively characterize the LP docking motif, which determines G1-CDK substrate specificity in fungi. The authors show that LP-cyclin docking strength determines the timing of Sic1 degradation, a key cell cycle event.

Long noncoding RNA KCNQ1OT1 contributes to tumor growth and activates Wnt/β‑catenin signaling inosteosarcoma by targeting the miR‑3666/KLF7 axis.

Osteosarcoma (OS) is a type of primary malignant cancer occurring in the bone and poses a threat to the lives of children and young adults. Long non‑coding RNAs (lncRNAs) have been certified to play important roles in various human malignant tumors, including OS. lncRNA KCNQ1OT1 has been investigated in certain types of cancer; however, its role and molecular mechanisms in OS remain to be determined. In the present study, a high KCNQ1OT1 expression was detected in human OS tissues and cell lines. Moreover, patients with OS with a high expression of KCNQ1OT1 presented a worse prognosis. Loss‑of‑function assays demonstrated that KCNQ1OT1 silencing suppressed cell proliferative, migratory and invasive abilities in OS. Importantly, the knockdown of KCNQ1OT1 suppressed the Wnt/β‑catenin signaling pathway in OS. Invivo assays displayed the inhibitory role of the silencing of KCNQ1OT1 in OS tumor growth. As regards the underlying mechanisms, KCNQ1OT1 could sponge miR‑3666, and its expression was negatively associated with that of miR‑3666 in OS tissues. Thereafter, Kruppel‑like factor 7 (KLF7), upregulated in OS tissues and cells, was discerned as a target gene of miR‑3666. Furthermore, KLF7 expression negatively correlated with miR‑3666 expression, whereas it positively correlated with KCNQ1OT1 expression. A rescue assay delineated that the overexpression of KLF7 counteracted the KCNQ1OT1 knockdown‑induced suppression of OS cell proliferation, migration, invasion and Wnt/β‑catenin signaling. Collectively, the present study demonstrates that KCNQ1OT1 facilitates OS progression and activates Wnt/β‑catenin signaling by targeting the miR‑3666/KLF7 axis.

Evaluation of Enterococcus hirae LAB3 as potential probiont against Vibrio harveyi in Artemia nauplii and Asian seabass larvae (Lates calcarifer) cultures

Aim: To evaluate the ability of to inhibit the growth of V. harveyi in in-vitro co-culture assay and confer protection towards Artemia nauplii and seabass larvae against V. harveyi in in-vivo assay. Methodology: Enterococcus hirae was co-cultured with V. harveyi for 96 hrs and the samples were taken every 6 hr interval to determine the growth of the pathogen in in-vitro assay. In the in-vivo assay, E. hirae at different concentrations were introduced to the Artemia nauplii and seabass larvae cultures. After 24-hr incubation period, V. harveyi 6 -1 at concentration 10 CFU ml were added into the respective experimental tanks. Mortality was observed and recorded daily, until 50% mortality was observed in negative control group which challenged with V. harveyi only. Results: E. hirae was able to reduce the numbers of V. harveyi after 12 hr of co-culture incubation. In addition, E. hirae was administered into the Artemia nauplii and conferred protection 6 against V. harveyi with the best survival rate at concentration of 10 -1 CFUmL (70±3.1%). The untreated group seabass larvae challenged with V. harveyi presented low survival of (16.7±3.3%), 6 -1 while fish treated with probiotic E. hirae at 10 CFUml showed significantly increased survival rates (68.3±0.9%) after challenged. The survival of healthy unchallenged fish treated with probiotic was not significantly different with the control group. The pathogen loaded also reduced in groups treated with E. hirae respectively Interpretation: E. hirae strain LAB3, a potential probiont was able to reduce the number of Vibrios and conferred protection to Artemia and seabass larvae.

Constitutively active androgen receptor supports the metastatic phenotype of endocrine-resistant hormone receptor-positive breast cancer

BackgroundHormone receptor positive (HR+) breast cancer (BCa) is the most frequently diagnosed subtype. Acquired and intrinsic resistance to conventional endocrine therapy (ET) commonly occurs and prompts incurable metastatic disease. Hence, ET-resistant (ET-R) HR+ BCa presents a therapeutic challenge. Previous studies show elevated androgen receptor (AR) that supports resistance to ET tamoxifen and correlates with HR+ BCa metastasis. Yet surprisingly, studies with AR-blocker enzalutamide (Enz) in ET-R HR+ BCa present conflicting results. We now report that a constitutively active, unique from canonical Enz-targeted, AR accumulates in endocrine resistant HR+ BCa cells.MethodsAR protein profiles in acquired and intrinsic ET-R HR + -BCa were defined with cell-free modification tests, in-house in-vivo SUMOylation assays, and PLA imaging. Genomic activity of native AR and modified-AR mimetic was tested with reporter assays and limited transcriptome analysis. Spheroid growth and migration studies were used to evaluate inhibitory actions of Enz and combinatorial therapy.ResultsSustained higher molecular weight SUMO-modified AR (SUMO-AR) persists in acquired and intrinsic ET-R BCa cell lines. Concurrently, SUMO isoforms and global SUMO-modified proteome also accumulates in the same cell lines. We identified AR as a novel substrate for the SUMO-E3 ligase HSPB1/Hsp27. Independent of ligand, SUMO-AR is resilient to ubiquitin-mediated proteasomal degradation, enriched in the nucleus, readily chromatin-bound, and transcriptionally active. Constitutive SUMO-AR initiates a gene-expression profile that favors epithelial-mesenchymal transition. Enz combined with a SUMO inhibitor attenuates migration and metastatic phenotype of ET-R HR+ BCa.ConclusionTargeting both unmodified and SUMO-modified AR prevents the metastatic progression of HR+ BCa with ET-R.1cz6dozgZCQes7_eQqKRNVVideo abstract