WEE1 promotes endometriosis via the Wnt/ß-catenin signaling pathway.

BACKGROUND: Endometriosis, the presence of active endometrial tissue outside the lining membrane of the uterine cavity, is a common disease in women of childbearing age. The ectopic endometrium has some characteristics of tumor tissue, including invasive and migratory abilities. In addition, endometriosis is associated with inflammation and reduced cellular apoptosis. METHODS: Western blot analysis, qPCR, immunohistochemistry, immunofluorescence microscopy, Transwell assay, wound healing assay, and TUNEL staining. RESULTS: Interleukin-1ß (IL-1ß) induced WEE1 expression in endometrial stromal cells (ESCs), suggesting that WEE1 may be upregulated during the endometriosis-induced inflammatory response. Overexpression of WEE1 in cultured ESCs promoted ESC migration while inhibiting apoptosis, whereas WEE1 knockdown reduced ESC migration while promoting apoptosis. Inhibition of WEE1 attenuates fibrosis in ESCs and female C57BL/6 J mice. This pro-fibrotic effect of WEE1 was significantly decreased by treatment with the Wnt/ß-catenin inhibitor XAV939, suggesting that WEE1 acts via the Wnt/ß-catenin signaling pathway. CONCLUSION: Our study demonstrates that WEE1 promotes ESC migration and fibrosis via the Wnt/ß-catenin signaling pathway. Thus, WEE1 may serve as a potential therapeutic target for the treatment of endometriosis.

Overexpressed MPS-1 contributes to endometrioma development through the NF-κB signaling pathway.

BACKGROUND: Endometriosis is a benign gynecological disease that shares some characteristics with malignant tumors and affects approximately 10% of women of reproductive age. Endometrioma refers to endometriosis that appears in the ovary. Metallopanstimulin-1 (MPS-1) is a component of the 40S subunit of ribosomes that has extra-ribosomal functions that contribute to the development of diseases. This study aimed to explore the expression pattern and role of MPS-1 in endometrioma development. METHODS: Quantitative real time polymerase chain reaction, western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay were used to determine the expression of MPS-1 in patients with endometrioma. Following the successful knockdown of MPS-1 by siRNA, CCK-8 assays, flow cytometry, and transwell assays were performed to detect ectopic endometrial stromal cells (EcESCs) proliferation, the rate of apoptosis, and cell cycle, migration, and invasion, respectively. Western blotting was used to explore the effect of MPS-1 knockdown on protein levels in the NF-κB signaling pathway. RESULTS: The expression of MPS-1 was significantly higher in endometrioma and the serum of endometrioma patients than in the patients without endometriosis. In addition, the downregulation of MPS-1 expression inhibited EcESCs proliferation, migration, and invasion. This downregulation led to the arrest of the EcESCs cycle in the G0/G1 phase and apoptosis and depressed the NF-κB signaling pathway. CONCLUSION: MPS-1 can regulate EcESCs proliferation, motility, invasion, apoptosis, and cell cycle via the NF-κB signaling pathway in endometrioma. This may contribute to the formation or development of endometriotic foci. This study suggests the potential role of MPS-1 in the pathogenesis of endometriosis and enabled further research into the use of MPS-1 in the clinical diagnosis of endometrioma.

Antidepressant-like effect of guanosine involves activation of AMPA receptor and BDNF/TrkB signaling.

Guanosine is a purine nucleoside that has been shown to exhibit antidepressant effects, but the mechanisms underlying its effect are not well established. We investigated if the antidepressant-like effect induced by guanosine in the tail suspension test (TST) in mice involves the modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, voltage-dependent calcium channel (VDCC), and brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) pathway. We also evaluated if the antidepressant-like effect of guanosine is accompanied by an acute increase in hippocampal and prefrontocortical BDNF levels. Additionally, we investigated if the ability of guanosine to elicit a fast behavioral response in the novelty suppressed feeding (NSF) test is associated with morphological changes related to hippocampal synaptogenesis. The antidepressant-like effect of guanosine (0.05 mg/kg, p.o.) in the TST was prevented by DNQX (AMPA receptor antagonist), verapamil (VDCC blocker), K-252a (TrkBantagonist), or BDNF antibody. Increased P70S6K phosphorylation and higher synapsin I immunocontent in the hippocampus, but not in the prefrontal cortex, were observed 1 h after guanosine administration. Guanosine exerted an antidepressant-like effect 1, 6, and 24 h after its administration, an effect accompanied by increased hippocampal BDNF level. In the prefrontal cortex, BDNF level was increased only 1 h after guanosine treatment. Finally, guanosine was effective in the NSF test (after 1 h) but caused no alterations in dendritic spine density and remodeling in the ventral dentate gyrus (DG). Altogether, the results indicate that guanosine modulates targets known to be implicated in fast antidepressant behavioral responses (AMPA receptor, VDCC, and TrkB/BDNF pathway).

A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughput screening.

Bone Marrow Tyrosine kinase in the chromosome X (BMX) is a TEC family kinase associated with numerous pathological pathways in cancer cells. Covalent inhibition of BMX activity holds promise as a therapeutic approach against cancer. To screen for potent and selective covalent BMX inhibitors, large quantities of highly pure BMX are normally required which is challenging with the currently available production and purification processes. Here, we developed a scalable production process for the human recombinant BMX (hrBMX) using the insect cell-baculovirus expression vector system. Comparable expression levels were obtained in small-scale shake flasks (13 mL) and in stirred-tank bioreactors (STB, 5 L). A two-step chromatographic-based process was implemented, reducing purification times by 75% when compared to traditional processes, while maintaining hrBMX stability. The final production yield was 24 mg of purified hrBMX per litter of cell culture, with a purity of > 99%. Product quality was assessed and confirmed through a series of biochemical and biophysical assays, including circular dichroism and dynamic light scattering. Overall, the platform herein developed was capable of generating 100 mg purified hrBMX from 5 L STB in just 34 days, thus having the potential to assist in-vitro covalent ligand high-throughput screening for BMX activity inhibition.

Kinome Array Profiling of Patient-Derived Pancreatic Ductal Adenocarcinoma Identifies Differentially Active Protein Tyrosine Kinases.

Pancreatic cancer remains one of the most difficult malignancies to treat. Minimal improvements in patient outcomes and persistently abysmal patient survival rates underscore the great need for new treatment strategies. Currently, there is intense interest in therapeutic strategies that target tyrosine protein kinases. Here, we employed kinome arrays and bioinformatic pipelines capable of identifying differentially active protein tyrosine kinases in different patient-derived pancreatic ductal adenocarcinoma (PDAC) cell lines and wild-type pancreatic tissue to investigate the unique kinomic networks of PDAC samples and posit novel target kinases for pancreatic cancer therapy. Consistent with previously described reports, the resultant peptide-based kinome array profiles identified increased protein tyrosine kinase activity in pancreatic cancer for the following kinases: epidermal growth factor receptor (EGFR), fms related receptor tyrosine kinase 4/vascular endothelial growth factor receptor 3 (FLT4/VEGFR-3), insulin receptor (INSR), ephrin receptor A2 (EPHA2), platelet derived growth factor receptor alpha (PDGFRA), SRC proto-oncogene kinase (SRC), and tyrosine kinase non receptor 2 (TNK2). Furthermore, this study identified increased activity for protein tyrosine kinases with limited prior evidence of differential activity in pancreatic cancer. These protein tyrosine kinases include B lymphoid kinase (BLK), Fyn-related kinase (FRK), Lck/Yes-related novel kinase (LYN), FYN proto-oncogene kinase (FYN), lymphocyte cell-specific kinase (LCK), tec protein kinase (TEC), hemopoietic cell kinase (HCK), ABL proto-oncogene 2 kinase (ABL2), discoidin domain receptor 1 kinase (DDR1), and ephrin receptor A8 kinase (EPHA8). Together, these results support the utility of peptide array kinomic analyses in the generation of potential candidate kinases for future pancreatic cancer therapeutic development.

Overexpression of PKMYT1 Facilitates Tumor Development and Is Correlated with Poor Prognosis in Clear Cell Renal Cell Carcinoma.

BACKGROUND Protein kinase membrane-associated tyrosine/threonine (PKMYT1) has been found in many tumors, but its association with clear cell renal cell carcinoma (ccRCC) remains unclear. MATERIAL AND METHODS PKMYT1 expression in ccRCC was examined in the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Tumor Immune Estimation Resource databases. The correlation between PKMYT1 expression and clinicopathological parameters was explored via the chi-square test. Receiver operating characteristic curves were used to estimate the diagnostic performance of PKMYT1. Kaplan-Meier curves, a Cox model, nomogram, time-dependent receiver operating characteristic curves, and decision curve analysis (DCA) were used to evaluate the prognostic value and clinical utility of PKMYT1. Genes coexpressed with PKMYT1 in ccRCC were identified based on TCGA, the gene expression profiling interactive, and cBioPortal. Gene Set Enrichment Analysis revealed biological pathways associated with PKMYT1 in ccRCC. RESULTS Weighted gene coexpression network analysis identified PKMYT1 as one of the genes most significantly correlated with progression of histological grade. PKMYT1 was significantly upregulated in ccRCC compared with normal tissue (P<0.001), with a trend toward differentiating between individuals with ccRCC and those who were healthy (area under the curve=0.942). High PKMYT1 expression was correlated with unsatisfactory survival (hazard ratio=1.67, P=0.001), indicating that it is a risk factor for ccRCC. A nomogram incorporating PKMYT1 level was created and showed a clinical net benefit. PKMYT1 was strongly positively correlated with the anti-silencing function of 1B histone chaperone (ASF1B) gene in ccRCC. CONCLUSIONS PKMYT1 is upregulated in ccRCC and its presence indicates poor prognosis, making it a potential therapeutic target for ccRCC.

Tropomyosin Receptor Kinase B Expressed in Oligodendrocyte Lineage Cells Functions to Promote Myelin Following a Demyelinating Lesion.

The levels of brain-derived neurotrophic factor (BDNF) in the corpus callosum have previously been shown to have a critical impact on oligodendrocyte (OLG) lineage cells during cuprizone-elicited demyelination. In particular, BDNF+/- mice exhibit greater losses in myelin protein levels compared to wild-type mice after cuprizone. To investigate whether OLGs may directly mediate these effects of BDNF during a lesion in vivo, we used the cuprizone model of demyelination with inducible conditional male knockout mice to specifically delete the high-affinity tropomyosin receptor kinase B (TrkB) receptor from proteolipid protein + OLGs during cuprizone-elicited demyelination and subsequent remyelination. The loss of TrkB during cuprizone-elicited demyelination results in an increased sensitivity to demyelination as demonstrated by greater deficits in myelin protein levels, greater decreases in numbers of mature OLGs, increased numbers of demyelinated axons, and decreased myelin thickness. When mice are removed from cuprizone, they exhibit a delayed recovery in myelin proteins and myelin. Our data indicate that following a demyelinating lesion, TrkB in OLGs positively regulates myelin protein expression, myelin itself, and remyelination.

Expression, purification and crystallization of CLK1 kinase - A potential target for antiviral therapy.

Cdc-like kinase 1 (CLK1) is a dual-specificity kinase capable of autophosphorylation on tyrosine residues and Ser/Thr phosphorylation of its substrates. CLK1 belongs to the CLK kinase family that regulates alternative splicing through phosphorylation of serine-arginine rich (SR) proteins. Recent studies have demonstrated that CLK1 has an important role in the replication of influenza A and chikungunya viruses. Furthermore, CLK1 was found to be relevant for the replication of HIV-1 and the West Nile virus, making CLK1 an interesting cellular candidate for the development of a host-directed antiviral therapy that might be efficient for treatment of newly emerging viruses. We describe here our attempts and detailed procedures to obtain the recombinant kinase domain of CLK1 in suitable amounts for crystallization in complex with specific inhibitors. The key solution for the reproducibility of crystals resides in devising and refining expression and purification protocols leading to homogeneous protein. Co-expression of CLK1 with λ-phosphatase and careful purification has yielded crystals of CLK1 complexed with the KH-CB19 inhibitor that diffracted to 1.65 Å. These results paved the path to the screening of more structures of CLK1 complexed compounds, leading to further optimization of their inhibitory activity. Moreover, since kinases are desired targets in numerous pathologies, the approach we report here, the co-expression of kinases with λ-phosphatase, previously used in other kinases, can be adopted as a general protocol in numerous kinase targets for obtaining reproducible and homogenic non-phosphorylated (inactive) forms suitable for biochemical and structural studies thus facilitating the development of novel inhibitors.

Negative Regulation of Tec Kinase Alleviates LPS-Induced Acute Kidney Injury in Mice via theTLR4/NF-κB Signaling Pathway.

Tec kinase is an important mediator in inflammatory immune response that enhances the activity of neutrophils and macrophages. However, information on its function in lipopolysaccharide- (LPS-) induced acute kidney injury (AKI) is limited. This study is aimed at determining whether Tec kinase was a regulator in AKI. An AKI model in mice was successfully established using intraperitoneal LPS. Results showed that the serum levels of creatinine (Cr), blood urea nitrogen (BUN), and cystatin-C (Cys-C) increased after intraperitoneal LPS injection. Renal tissue sustained significantly severe injury as measured by pathological scores. Pretreatment with LFM-A13 improved the function of the kidney in mice and decreased the renal injury score. Enzyme-linked immunosorbent assay showed that LFM-A13 significantly reduced the release of IL-1ß and TNF-α in mice exposed to LPS. LFM-A13 can evidently abrogate the expression of Tec protein, MyD88, TLR4, NF-κB p65, and Tec's phosphorylated protein as determined by Western blot. Immunohistochemistry analysis revealed that LFM-A13 markedly downregulated the expression of Tec kinase in renal tubular epithelial cells. In vitro, Tec kinase protein was expressed highly in NRK-52E cells after LPS exposure. Tec-siRNA also decreased IL-1ß and TNF-α production and obviously abolished phospho-p65 and phospho-IκBα expression in NRK-52E cell stimulated by LPS; however, Tec-siRNA increased the IκBα level. Altogether, these data suggested that Tec kinase can be a modulating protein in AKI through TLR4/NF-κB activation.

Targeting CLK3 inhibits the progression of cholangiocarcinoma by reprogramming nucleotide metabolism.

CDC-like kinase 3 (CLK3) is a dual specificity kinase that functions on substrates containing serine/threonine and tyrosine. But its role in human cancer remains unknown. Herein, we demonstrated that CLK3 was significantly up-regulated in cholangiocarcinoma (CCA) and identified a recurrent Q607R somatic substitution that represented a gain-of-function mutation in the CLK3 kinase domain. Gene ontology term enrichment suggested that high CLK3 expression in CCA patients mainly was associated with nucleotide metabolism reprogramming, which was further confirmed by comparing metabolic profiling of CCA cells. CLK3 directly phosphorylated USP13 at Y708, which promoted its binding to c-Myc, thereby preventing Fbxl14-mediated c-Myc ubiquitination and activating the transcription of purine metabolic genes. Notably, the CCA-associated CLK3-Q607R mutant induced USP13-Y708 phosphorylation and enhanced the activity of c-Myc. In turn, c-Myc transcriptionally up-regulated CLK3. Finally, we identified tacrine hydrochloride as a potential drug to inhibit aberrant CLK3-induced CCA. These findings demonstrate that CLK3 plays a crucial role in CCA purine metabolism, suggesting a potential therapeutic utility.

Overexpression of miR-26a-5p Suppresses Tau Phosphorylation and Aß Accumulation in the Alzheimer's Disease Mice by Targeting DYRK1A.

OBJECTIVE: It is reported that miR-26a-5p could regulate neuronal development, but its underlying mechanisms in Alzheimer's disease (AD) progression is unclear. METHODS: APP (swe)/PS1 (ΔE9) transgenic mice served as AD mice. Morris water maze test was used to measure the spatial learning and memory ability of mice. The expressions of miR-26a-5p, DYRK1A, phosphorylated-Tau, Aß40, and Aß42 were detected. The relationship between miR- 26a-5p and DYRK1A was explored using dual luciferase reporter assay. The effects of miR-26a- 5p on AD mice was determined. RESULTS: AD mice walked a lot of wrong ways to find the platform area and the latency time to reach the platform was longer. There was low expression of MiR-26a-5p in AD mice. Overexpression of miR-26a-5p inhibited Tau phosphorylation and Aß accumulation. MiR-26a-5p negatively regulated DYRK1A via targeting its 3'UTR. In vivo, increased miR-26a-5p down-regulated Aß40, Aß42, p-APP and p-Tau levels in AD mice through decreasing DYRK1A. Meanwhile, the swimming path and the latency time, to reach the platform, was shorten after enhancing miR-26a-5p expression. CONCLUSION: Overexpression of miR-26a-5p could repress Tau phosphorylation and Aß accumulation via down-regulating DYRK1A level in AD mice.

TTK inhibition radiosensitizes basal-like breast cancer through impaired homologous recombination.

Increased rates of locoregional recurrence are observed in patients with basal-like breast cancer (BC) despite the use of radiation therapy (RT); therefore, approaches that result in radiosensitization of basal-like BC are critically needed. Using patients' tumor gene expression data from 4 independent data sets, we correlated gene expression with recurrence to find genes significantly correlated with early recurrence after RT. The highest-ranked gene, TTK, was most highly expressed in basal-like BC across multiple data sets. Inhibition of TTK by both genetic and pharmacologic methods enhanced radiosensitivity in multiple basal-like cell lines. Radiosensitivity was mediated, at least in part, through persistent DNA damage after treatment with TTK inhibition and RT. Inhibition of TTK impaired homologous recombination (HR) and repair efficiency, but not nonhomologous end-joining, and decreased the formation of Rad51 foci. Reintroduction of wild-type TTK rescued both radioresistance and HR repair efficiency after TTK knockdown; however, reintroduction of kinase-dead TTK did not. In vivo, TTK inhibition combined with RT led to a significant decrease in tumor growth in both heterotopic and orthotopic, including patient-derived xenograft, BC models. These data support the rationale for clinical development of TTK inhibition as a radiosensitizing strategy for patients with basal-like BC, and efforts toward this end are currently underway.

Antidepressant Drugs Correct the Imbalance Between proBDNF/p75NTR/Sortilin and Mature BDNF/TrkB in the Brain of Mice with Chronic Stress.

Depression is a worldwide problem with a great social and economic burden in many countries. In our previous research, we found that the expression of proBDNF/p75NTR/sortilin is upregulated in patients with major depressive disorder. In addition, the treatment of proBDNF antibodies reversed both the depressive behaviors and the reduced BDNF mRNA detected in our rodent chronic stress models. Antidepressant drugs are usually only effective in a subpopulation of patients with major depression with a delayed time window of 2-4 weeks to exert their efficacy. The mechanism underlying such delayed response is not known. In this study, we hypothesize that antidepressant drugs exert their therapeutic effect by modulating proBDNF/p75NTR and mature BDNF/TrkB signaling pathways. To test the hypothesis, C57 mice were randomly divided into normal control, chronic unpredictable mild stress (CUMS), vehicle (VEH), fluoxetine (FLU), and clozapine (CLO) groups. Behavioral tests (sucrose preference, open field, and tail suspension tests) were performed before and after 4 weeks of CUMS. The gene and protein expression of proBDNF, the neurotrophin receptor (p75NTR), sortilin, and TrkB in the cortex and hippocampus were examined. At the protein level, CUMS induced a significant increase in proBDNF, p75NTR, and sortilin production while the TrkB protein level was found to be lower in the cortex and hippocampus compared with the control group. Consistently, at the mRNA level, p75NTR expression increased with reduced BDNF/TrkB mRNA in both cortex and hippocampus, while sortilin increased only in the hippocampus after CUMS. FLU and CLO treatments of CUMS mice reversed all protein and mRNA expression of the biomarkers in both cortex and hippocampus, except for sortilin mRNA in the cortex and proBDNF in the hippocampus, respectively. This study further confirms that the imbalance between proBDNF/p75NTR/sortilin and mBDNF/TrkB production is important in the pathogenesis of depression. It is likely that antidepressant FLU and antipsychotic CLO exert their antidepressant-like effect correcting the imbalance between proBDNF/p75NTR/sortilin and mBDNF/TrkB.

microRNAs expression correlates with levels of APP, DYRK1A, hyperphosphorylated Tau and BDNF in the hippocampus of a mouse model for Down syndrome during ageing.

Down syndrome (DS) patients are more susceptible to Alzheimer's disease (AD) due to the presence of three copies of genes on chromosome 21 such as DYRK1A, which encodes a broad acting kinase, and APP (amyloid precursor protein), leading to formation of amyloid beta (Aß) peptide and hyperphosphorylation of Tau. In this study, we investigated the association among miRNAs miR-17, -20a, -101, -106b, -199b, -26a, 26b and some of their target mRNAs such as APP, DYRK1A and BDNF, as well as the levels of hyperphosphorylated Tau in the hippocampus of a 2 and 5 months old mice model of trisomy 21 (Ts65Dn). Results indicated that increased APP expression in the hippocampus of 5 months old DS mice might be correlated with decrease in miR-17, -20a, -101 and -106b. Whereas at 2 months of age normal levels of APP expression in the hippocampus was correlated with increased levels of miR-17, -101 and -106b in DS mice. DYRK1A mRNA also increased in the hippocampus of 5 months old DS mice and it is associated with decreased levels of miR-199b. Increased levels of DYRK1A in 5-month old mice are associated with increased phosphorylation of Tau at Thr212 residue but not at Ser199-202. Tau pathology is accompanied by decreased expression of BDNF and increased miR-26a/b in mice of 5 months of age. Taken together, data indicate that miR-17, -20a, -26a/b, -101, -106b and -199b might be interesting targets to mitigate Tau and Aß pathology in DS.

Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels.

Brain-derived neurotrophic factor (BDNF) is a potent modulator of brain synaptic plasticity. Signaling defects caused by dysregulation of its Ntrk2 (TrkB) kinase (TrkB.FL) and truncated receptors (TrkB.T1) have been linked to the pathophysiology of several neurological and neurodegenerative disorders. We found that upregulation of Rbfox1, an RNA binding protein associated with intellectual disability, epilepsy and autism, increases selectively hippocampal TrkB.T1 isoform expression. Physiologically, increased Rbfox1 impairs BDNF-dependent LTP which can be rescued by genetically restoring TrkB.T1 levels. RNA-seq analysis of hippocampi with upregulation of Rbfox1 in conjunction with the specific increase of TrkB.T1 isoform expression also shows that the genes affected by Rbfox1 gain of function are surprisingly different from those influenced by Rbfox1 deletion. These findings not only identify TrkB as a major target of Rbfox1 pathophysiology but also suggest that gain or loss of function of Rbfox1 regulate different genetic landscapes.

Effects of prenatal cigarette smoke exposure on BDNF, PACAP, microglia and gliosis expression in the young male mouse brainstem.

Cigarette smoke exposure during pregnancy into infancy affects brain growth and development in both short and long term (into adulthood). Using a mouse model of pre- into post- natal cigarette smoke exposure (SE), we aimed to determine the effects on brain derived neurotrophic factor (BDNF) and its receptor TrkB, neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor PAC1, and astrocyte (GFAP) and microglia (Iba-1) immunohistochemical expression, in seven nuclei of the medulla and the facial (FAC) nucleus of the pons. Male pups of dams exposed to two cigarettes (nicotine <1.2 mg, CO <15 mg) twice daily for six weeks prior to mating, during gestation and lactation (n = 5; SE), were compared to pups exposed to air under the same condition (n = 5; SHAM) at postnatal day 20. Expression changes were only evident for BDNF, TrkB and PAC1 and included decreased BDNF in the hypoglossal (XII) nucleus and nucleus of the solitary tract (NTS), increased TrkB in XII but decreased TrkB in the FAC, and increased PAC1 in 4 nuclei of the medulla including the NTS. These results suggest that the effect of SE on the brainstem are region and marker selective, affecting regions of respiratory control (XII and NTS), and restricted to the BDNF system and PAC1, with no effect on activation states of astrocytes or microglia.

Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model.

Chronic liver inflammation (CLI) is a risk factor for development of hepatocellular carcinoma (HCC). Galectin-1 (Gal1) is involved in the regulation of inflammation, angiogenesis, and tumorigenesis, exhibiting multiple anti-inflammatory and protumorigenic activities. We aimed to explore its regulatory role in CLI and HCC progression using an established model of CLI-mediated HCC development, Abcb4 [multidrug-resistance 2 (Mdr2)]-knockout (KO) mice, which express high levels of Gal1 in the liver. We generated double-KO (dKO) Gal1-KO/Mdr2-KO mice on C57BL/6 and FVB/N genetic backgrounds and compared HCC development in the generated strains with their parental Mdr2-KO strains. Loss of Gal1 increased liver injury, inflammation, fibrosis, and ductular reaction in dKO mice of both strains starting from an early age. Aged dKO mutants displayed earlier hepatocarcinogenesis and increased tumor size compared with control Mdr2-KO mice. We found that osteopontin, a well-known modulator of HCC development, and oncogenic proteins Ntrk2 (TrkB) and S100A4 were overexpressed in dKO compared with Mdr2-KO livers. Our results demonstrate that in Mdr2-KO mice, a model of CLI-mediated HCC, Gal1-mediated protection from hepatitis, liver fibrosis, and HCC initiation dominates over its known procarcinogenic activities at later stages of HCC development. These findings suggest that anti-Gal1 treatments may not be applicable at all stages of CLI-mediated HCC.-Potikha, T., Pappo, O., Mizrahi, L., Olam, D., Maller, S. M., Rabinovich, G. A., Galun, E., Goldenberg, D. S. Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model.

The Kinome of Human Alveolar Type II and Basal Cells, and Its Reprogramming in Lung Cancer.

The discovery of mutant tyrosine kinases as oncogenic drivers of lung adenocarcinomas has changed the basic understanding of lung cancer development and therapy. Yet, expressed kinases (kinome) in lung cancer progenitor cells, as well as whether kinase expression and the overall kinome changes or is reprogrammed upon transformation, is incompletely understood. We hypothesized that the kinome differs between lung cancer progenitor cells, alveolar type II cells (ATII), and basal cells (BC) and that their respective kinomes undergo distinct lineage-specific reprogramming to adenocarcinomas and squamous cell carcinomas upon transformation. We performed RNA sequencing on freshly isolated human ATII, BC, and lung cancer cell lines to define the kinome in nontransformed cells and transformed cells. Our studies identified a unique kinome for ATII and BC and changes in their kinome upon transformation to their respective carcinomas.

Antibiotic treatment-induced dysbiosis differently affects BDNF and TrkB expression in the brain and in the gut of juvenile mice.

Antibiotic use during adolescence may result in dysbiosis-induced neuronal vulnerability both in the enteric nervous system (ENS) and central nervous system (CNS) contributing to the onset of chronic gastrointestinal disorders, such as irritable bowel syndrome (IBS), showing significant psychiatric comorbidity. Intestinal microbiota alterations during adolescence influence the expression of molecular factors involved in neuronal development in both the ENS and CNS. In this study, we have evaluated the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TrkB) in juvenile mice ENS and CNS, after a 2-week antibiotic (ABX) treatment. In both mucosa and mucosa-deprived whole-wall small intestine segments of ABX-treated animals, BDNF and TrKB mRNA and protein levels significantly increased. In longitudinal muscle-myenteric plexus preparations of ABX-treated mice the percentage of myenteric neurons staining for BDNF and TrkB was significantly higher than in controls. After ABX treatment, a consistent population of BDNF- and TrkB-immunoreactive neurons costained with SP and CGRP, suggesting up-regulation of BDNF signaling in both motor and sensory myenteric neurons. BDNF and TrkB protein levels were downregulated in the hippocampus and remained unchanged in the prefrontal cortex of ABX-treated animals. Immunostaining for BDNF and TrkB decreased in the hippocampus CA3 and dentate gyrus subregions, respectively, and remained unchanged in the prefrontal cortex. These data suggest that dysbiosis differentially influences the expression of BDNF-TrkB in the juvenile mice ENS and CNS. Such changes may potentially contribute later to the development of functional gut disorders, such as IBS, showing psychiatric comorbidity.

A study detection of the ROS1 gene fusion by FISH and ROS1 protein expression by IHC methods in patients with ovarian malignant or borderline serous tumors.

OBJECTIVE: ROS1 is an orphan receptor protein tyrosine kinase which is supposed to undergo genetic rearrangement in carcinogenesis. In the current study, we aimed to investigate the frequency and clinicopathologic features associated with ROS1 gene fusion and ROS1 protein expression in patients with ovarian serous carcinoma or serous borderline tumors. MATERIALS AND METHODS: Tissue samples of 102 patients with high or low grade serous carcinoma and borderline serous tumors were selected randomly from the archives of Department of Gyneco-pathology, and analyzed for ROS1 gene expression. (Fluorescence in situ hybridization (FISH) method was used to assess ROS1 gene rearrangement, while ROS1 protein expression was analyzed using immunohistochemistry. RESULTS: The study consisted of 94 cases of high-grade serous carcinoma (92.1%), 2 cases of low-grade serous carcinoma (%2) and 6 cases of serous borderline tumor (5.9%). ROS1 gene rearrangement analysis revealed that 4 patients (3.9%) were FISH-positive; whereas the immunohistochemical analysis yielded only 1 patient (0.9%) exhibiting faint positive expression of ROS1 protein. Given the low incidences of ROS1 gene rearrangement and protein expression, their relationships with clinicopathologic parameters could not be statistically analyzed. CONCLUSION: Although rare, patients with ovarian serous carcinoma or serous borderline tumor may exhibit ROS1 gene rearrangement and ROS1 protein expression. Further large-scale studies are necessary to explore the clinicopathologic significance of ROS1 gene expression in ovarian serous carcinoma.