An Immunohistochemical Analysis of Osteopontin and S100 Calcium-binding Protein P is Useful for Subclassifying Large- and Small-duct Type Intrahepatic Cholangiocarcinomas.

Intrahepatic cholangiocarcinoma (iCCA) has been newly subclassified into two different subtypes: large-duct (LD) type and small-duct (SD) type. However, many cases are difficult to subclassify, and there is no consensus regarding subclassification criteria. LD type expresses the highly sensitive diagnostic marker S100 calcium-binding protein P (S100P), while SD type lacks sensitive markers. We identified osteopontin (OPN) as a highly sensitive marker for SD type. This study aimed to develop new subclassification criteria for LD-type and SD-type iCCA. We retrospectively investigated 74 patients with iCCA and subclassified them based on whole-section immunostaining of S100P and OPN. Of the 74 cases, 41 were subclassified as LD type, 32 as SD type, and one was indeterminate. Notably, all S100P-negative cases had OPN positivity. Seventy-three of the 74 cases (98.6%) were clearly and easily subclassified as LD or SD type using only these 2 markers. We also determined the value of immunohistochemistry in cases that were difficult to diagnose based on hematoxylin-eosin and Alcian blue-periodic acid-Schiff staining. Furthermore, we analyzed the clinicopathological characteristics and prognoses of these 2 subtypes. LD type was a poor prognostic factor on univariate analysis; it had significantly worse overall survival ( P = 0.007) and recurrence-free survival ( P < 0.001) than the SD type. In conclusion, we propose new subclassification criteria for iCCA based on immunostaining of S100P and OPN. These criteria may help pathologists to diagnose subtypes of iCCA, supporting future clinical trials and the development of medications for these 2 subtypes as distinct cancers.

Diagnostic utility and sensitivities of matrix Gla protein (MGP), TRPS1 and GATA3 in breast cancer: focusing on metastatic breast cancer, invasive breast carcinoma with special features, and salivary gland-type tumours.

Matrix Gla protein (MGP) and trichorhinophalangeal syndrome type 1 (TRPS1) have recently emerged as novel breast-specific immunohistochemical (IHC) markers, particularly for triple-negative breast cancer (TNBC) and metaplastic carcinoma. The present study aimed to validate and compare the expression of MGP, TRPS1 and GATA binding protein 3 (GATA3) in metastatic breast carcinoma (MBC), invasive breast carcinoma (IBC) with special features, including special types of invasive breast carcinoma (IBC-STs) and invasive breast carcinoma of no special type with unique features, and mammary and non-mammary salivary gland-type tumours (SGTs). Among all enrolled cases, MGP, TRPS1 and GATA3 had comparable high positivity for ER/PR-positive (p=0.148) and HER2-positive (p=0.310) breast carcinoma (BC), while GATA3 positivity was significantly lower in TNBC (p<0.001). Similarly, the positive rates of MGP and TRPS1 in MBCs (99.4%), were higher than in GATA3 (90.9%, p<0.001). Among the IBC-STs, 98.4% of invasive lobular carcinomas (ILCs) were positive for all three markers. Among neuroendocrine tumours (NTs), all cases were positive for TRPS1 and GATA3, while MGP positivity was relatively low (81.8%, p=0.313). In the neuroendocrine carcinoma (NC) subgroup, all cases were positive for GATA3 and MGP, while one case was negative for TRPS1. All carcinomas with apocrine differentiation (APOs) were positive for GATA3 and MGP, while only 60% of the cases demonstrated moderate staining for TRPS1. Among mammary SGTs, MGP demonstrated the highest positivity (100%), followed by TRPS1 (96.0%) and GATA3 (72.0%). Positive staining for these markers was also frequently observed in non-mammary SGTs. Our findings further validate the high sensitivity of MGP and TRPS1 in MBCs, IBC-STs, and breast SGTs. However, none of these markers are capable of distinguishing between mammary and non-mammary SGTs.

Fam20C overexpression in odontoblasts regulates dentin formation and odontoblast differentiation.

FAM20C phosphorylates secretory proteins at S-x-E/pS motifs, and previous studies of Fam20C-dificient mice revealed that FAM20C played essential roles in bone and tooth formation. Inactivation of FAM20C in mice led to hypophosphatemia that masks direct effect of FAM20C in these tissues, and consequently the direct role of FAM20C remains unknown. Our previous study reported that osteoblast/odontoblast-specific Fam20C transgenic (Fam20C-Tg) mice had normal serum phosphate levels and that osteoblastic FAM20C-mediated phosphorylation regulated bone formation and resorption. Here, we investigated the direct role of FAM20C in dentin using Fam20C-Tg mice. The tooth of Fam20C-Tg mice contained numerous highly phosphorylated proteins, including SIBLINGs, compared to that of wild-type mice. In Fam20C-Tg mice, coronal dentin volume decreased and mineral density unchanged at early age, while the volume unchanged and the mineral density elevated at maturity. In these mice, radicular dentin volume and mineral density decreased at all ages, and histologically, the radicular dentin had wider predentin and abnormal apical-side dentin with embedded cells and argyrophilic canaliculi. Immunohistochemical analyses revealed that abnormal apical-side dentin had bone and dentin matrix properties accompanied with osteoblast-lineage cells. Further, in Fam20C-Tg mice, DSPP content which is important for dentin formation, was reduced in dentin, especially radicular dentin, which might lead to defects mainly in radicular dentin. Renal subcapsular transplantations of tooth germ revealed that newly formed radicular dentin replicated apical abnormal dentin of Fam20C-Tg mice, corroborating that FAM20C overexpression indeed caused the abnormal dentin. Our findings indicate that odontoblastic FAM20C-mediated phosphorylation in the tooth regulates dentin formation and odontoblast differentiation.

Protein atlas of fibroblast specific protein 1 (FSP1)/S100A4.

Fibroblast specific protein 1 (FSP1)/S100A4 is a calcium binding protein which has been linked to epithelial-mesenchymal transition, tissue fibrosis, pulmonary vascular disease, metastatic tumour development, increased tumour cell motility and invasiveness. This protein is reported to be also expressed in newly formed and differentiated fibroblasts and has been used in various studies to demonstrate epithelial-mesenchymal transition (EMT). We aimed to characterize S100A4 positive cells in different human tissue compartments, with the focus on fibroblasts/myofibroblast. We found S100A4 expression in a wide range of cells. Fibroblasts/myofibroblasts showed a broad spectrum of staining intensity, ranging from negative to strong expression of S100A4, with the strongest expression in smooth muscle actin positive myofibroblasts. Cells of haematopoietic lineage, namely CD4 and CD8 positive T-lymphocytes, but not B-lymphocytes expressed S100A4. All investigated monocytes, macrophages and specialised histiocytes were positive for S100A4. Even some epithelial cells of the kidney and bladder were positive for S100A4. Expression was also found in the vasculature. Here, cells of the subendothelial space, tunica adventitia and some smooth muscle cells of the tunica media were positive for S100A4. In summary, S100A4 is expressed in various cell types of different lineage and is not, as originally believed, specific for fibroblasts (FSP). Results attained under the premise of specificity of FSP1/S100A4 for fibroblasts, like the founding research on EMT type 2 in kidney and liver, therefore need to be reinterpreted.

Highly sensitive fluorescent detection of EDIL3 overexpressed exosomes for the diagnosis of triple-negative breast cancer.

EDIL3 is a strong and highly accurate diagnostic marker for breast cancer, meanwhile, EDIL3 overexpressed exosomes are novel biomarkers for the early diagnosis of triple-negative breast cancer (TNBC). Here, we proposed a fluorescent detection method for EDIL3 overexpressed exosomes, which is simple and sensitive. Basically, we utilized a magnetic nanospheres (MNS) based liquid sandwich immunoassay strategy. MNS were modified with CD63 aptamers, which can immunologically bound to the CD63 protein on the surface of exosomes. Alexa Fluor 647 labeled anti-EDIL3 antibodies (Anti-EDIL3/AF647) were used as the fluorescent probes to recognize the EDIL3 on exosomes derived from a TNBC cell line (MDA-MB-231). With the target TNBC exosomes present, sandwich structures containing MNS, exosomes and fluorescent probes were formed. After magnetic purification, optical super resolution imaging of the products was conducted to check the specificity of the assay. In addition, fluorescence signals of the products were detected to quantitatively analyze the EDIL3 overexpressed exosomes. The linear range was found to be 7.78 × 101to 7.78× 106particlesµl-1. The detection limit was approximately 10 particlesµl-1. The feasibility of the method for the detection of exosomes in complex biological samples was also demonstrated. Such a simple and sensitive detection method for EDIL3 overexpressed exosomes holds a great potential in clinical diagnosis of TNBC.

S100A1 expression characterizes terminally differentiated superficial cells in the urothelium of the murine bladder and ureter.

The urothelium is a stratified epithelium that lines the inner surface of the components of the urinary drainage system. It is composed of a layer of basal cells, one or several layers of intermediate cells, and a layer of large luminal superficial or umbrella cells. In the mouse, only a small set of markers is available that allows easy molecular distinction of these urothelial cell types. Here, we analyzed expression of S100A1, a member of the S100 family of calcium-binding proteins, in the urothelium of the two major organs of the murine urinary tract, the ureter and the bladder. Using RNA in situ hybridization analysis, we found exclusive expression of S100a1 mRNA in luminal cells of the ureter from embryonic day (E)17.5 onwards and of the bladder from E15.5 to adulthood. Immunofluorescence analysis showed that expression of S100A1 protein is confined to terminally differentiated superficial cells of both the ureter and bladder where it localized to the nucleus and cytoplasm. We conclude that S100A1 is a suitable marker for mature superficial cells in the urothelial lining of the drainage system of the developing and mature mouse.

Mycobacterium vaccae immunization in rats ameliorates features of age-associated microglia activation in the amygdala and hippocampus.

Aging and reduced exposure to environmental microbes can both potentiate neuroinflammatory responses. Prior studies indicate that immunization with the immunoregulatory and anti-inflammatory bacterium, Mycobacterium vaccae (M. vaccae), in aged rats limits neuroimmune activation and cognitive impairments. However, the mechanisms by which M. vaccae immunization ameliorates age-associated neuroinflammatory "priming" and whether microglia are a primary target remain unclear. Here, we investigated whether M. vaccae immunization protects against microglia morphological changes in response to aging. Adult (3 mos) and aged (24 mos) Fisher 344 × Brown Norway rats were immunized with either M. vaccae or vehicle once every week for 3 weeks. Aging led to elevated Iba1 immunoreactivity, microglial density, and deramification of microglia processes in the hippocampus and amygdala but not other brain regions. Additionally, aged rats exhibited larger microglial somas in the dorsal hippocampus, suggestive of a more activated phenotype. Notably, M. vaccae treatment ameliorated indicators of microglia activation in both the amygdala and hippocampus. While changes in morphology appeared to be region-specific, gene markers indicative of microglia activation were upregulated by age and lowered in response to M. vaccae in all brain regions evaluated. Taken together, these data suggest that peripheral immunization with M. vaccae quells markers of age-associated microglia activation.

Terminal 6q deletions cause brain malformations, a phenotype mimicking heterozygous DLL1 pathogenic variants: A multicenter retrospective case series.

OBJECTIVE: Terminal 6q deletion is a rare genetic condition associated with a neurodevelopmental disorder characterized by intellectual disability and structural brain anomalies. Interestingly, a similar phenotype is observed in patients harboring pathogenic variants in the DLL1 gene. Our study aimed to further characterize the prenatal phenotype of this syndrome as well as to attempt to establish phenotype-genotype correlations. METHOD: We collected ultrasound findings from 22 fetuses diagnosed with a pure 6qter deletion. We reviewed the literature and compared our 22 cases with 14 fetuses previously reported as well as with patients with heterozygous DLL1 pathogenic variants. RESULTS: Brain structural alterations were observed in all fetuses. The most common findings (>70%) were cerebellar hypoplasia, ventriculomegaly, and corpus callosum abnormalities. Gyration abnormalities were observed in 46% of cases. Occasional findings included cerebral heterotopia, aqueductal stenosis, vertebral malformations, dysmorphic features, and kidney abnormalities. CONCLUSION: This is the first series of fetuses diagnosed with pure terminal 6q deletion. Based on our findings, we emphasize the prenatal sonographic anomalies, which may suggest the syndrome. Furthermore, this study highlights the importance of chromosomal microarray analysis to search for submicroscopic deletions of the 6q27 region involving the DLL1 gene in fetuses with these malformations.

Effects of Light Isoflurane Anesthesia on Organization of Direction and Orientation Selectivity in the Superficial Layer of the Mouse Superior Colliculus.

The superior colliculus (SC) is the midbrain center for integrating visual and multimodal sensory information. Neurons in the SC exhibit direction and orientation selectivity. Recent studies reported that neurons with similar preferences formed clusters in the mouse SC (Ahmadlou and Heimel, 2015; Feinberg and Meister, 2015; de Malmazet et al., 2018; Li et al., 2020). However, it remains controversial as to how these clusters are organized within the SC (Inayat et al., 2015; Chen et al., 2021). Here, we found that different brain states (i.e., awake or anesthetized with isoflurane) changed the selectivity of individual SC neurons and organizations of the neuronal population in both male and female mice. Using two-photon Ca2+ imaging, we examined both individual neuronal responses and the spatial patterns of their population responses. Under isoflurane anesthesia, orientation selectivity increased and a larger number of orientation-selective cells were observed when compared with the awake condition, whereas the proportions of direction-selective cells were similar in both conditions. Furthermore, direction- and orientation-selective cells located at closer positions showed more similar preferences, and cluster-like spatial patterns were enhanced. Inhibitory responses of direction-selective neurons were also reduced under isoflurane anesthesia. Thus, the changes in the spatial organization of response patterns were considered to be because of changes in the balance of excitation and inhibition, with excitation dominance, in the local circuits. These results provide new insights into the possibility that the functional organization of feature selectivity in the brain is affected by brain state.SIGNIFICANCE STATEMENT Recent large-scale recording studies are changing our view of visual maps in the superior colliculus (SC), including findings of cluster-like localizations of direction- and orientation-selective neurons. However, results from several laboratories are conflicting regarding the presence of cluster-like organization. Here, we demonstrated that light isoflurane anesthesia affected the direction- and orientation-tuning properties in the mouse superficial SC and that their cluster-like localization pattern was enhanced by the anesthesia. Furthermore, the effect of anesthesia on direction selectivity appeared to be different in the excitatory and inhibitory populations in the SC. Our results suggest that the functional organization of direction and orientation selectivity might be regulated by the excitation-inhibition balance that depends on the brain state.

Identification and Clinical Validation of Key Extracellular Proteins as the Potential Biomarkers in Relapsing-Remitting Multiple Sclerosis.

Background: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) mediated by autoimmunity. No objective clinical indicators are available for the diagnosis and prognosis of MS. Extracellular proteins are most glycosylated and likely to enter into the body fluid to serve as potential biomarkers. Our work will contribute to the in-depth study of the functions of extracellular proteins and the discovery of disease biomarkers. Methods: MS expression profiling data of the human brain was downloaded from the Gene Expression Omnibus (GEO). Extracellular protein-differentially expressed genes (EP-DEGs) were screened by protein annotation databases. GO and KEGG were used to analyze the function and pathway of EP-DEGs. STRING, Cytoscape, MCODE and Cytohubba were used to construct a protein-protein interaction (PPI) network and screen key EP-DEGs. Key EP-DEGs levels were detected in the CSF of MS patients. ROC curve and survival analysis were used to evaluate the diagnostic and prognostic ability of key EP-DEGs. Results: We screened 133 EP-DEGs from DEGs. EP-DEGs were enriched in the collagen-containing extracellular matrix, signaling receptor activator activity, immune-related pathways, and PI3K-Akt signaling pathway. The PPI network of EP-DEGs had 85 nodes and 185 edges. We identified 4 key extracellular proteins IL17A, IL2, CD44, IGF1, and 16 extracellular proteins that interacted with IL17A. We clinically verified that IL17A levels decreased, but Del-1 and resolvinD1 levels increased. The diagnostic accuracy of Del-1 (AUC: 0.947) was superior to that of IgG (AUC: 0.740) with a sensitivity of 82.4% and a specificity of 100%. High Del-1 levels were significantly associated with better relapse-free and progression-free survival. Conclusion: IL17A, IL2, CD44, and IGF1 may be key extracellular proteins in the pathogenesis of MS. IL17A, Del-1, and resolvinD1 may co-regulate the development of MS and Del-1 is a potential biomarker of MS. We used bioinformatics methods to explore the biomarkers of MS and validated the results in clinical samples. The study provides a theoretical and experimental basis for revealing the pathogenesis of MS and improving the diagnosis and prognosis of MS.

Protocol for measuring NLRC4 inflammasome activation and pyroptosis in murine bone-marrow-derived macrophages.

NLR family CARD domain containing protein 4 (NLRC4) inflammasome activation and the associated pyroptosis are critical for protection against infection by bacterial pathogens. This protocol presents a detailed procedure to activate and measure NLRC4 inflammasome activation and pyroptosis upon Salmonella Typhimurium infection. The techniques can be adapted to monitoring the activation of other types of inflammasomes and pathogenic stimuli. For comprehensive details on the use and execution of this protocol, please refer to Dong et al. (2021).

The Health Hazards of Volcanoes: First Evidence of Neuroinflammation in the Hippocampus of Mice Exposed to Active Volcanic Surroundings.

Neuroinflammation is a process related to the onset of neurodegenerative diseases; one of the hallmarks of this process is microglial reactivation and the secretion by these cells of proinflammatory cytokines such as TNFα. Numerous studies report the relationship between neuroinflammatory processes and exposure to anthropogenic air pollutants, but few refer to natural pollutants. Volcanoes are highly inhabited natural sources of environmental pollution that induce changes in the nervous system, such as reactive astrogliosis or the blood-brain barrier breakdown in exposed individuals; however, no neuroinflammatory event has been yet defined. To this purpose, we studied resting microglia, reactive microglia, and TNFα production in the brains of mice chronically exposed to an active volcanic environment on the island of São Miguel (Azores, Portugal). For the first time, we demonstrate a proliferation of microglial cells and an increase in reactive microglia, as well an increase in TNFα secretion, in the central nervous system of individuals exposed to volcanogenic pollutants.

Regulatory role of local tissue signal Del-1 in cancer and inflammation: a review.

Developmental endothelial locus-1 (Del-1) is a secretory, multifunctional domain protein. It can bind to integrins and phosphatidylserine. As a local tissue signal, it plays a regulatory role in the cancer microenvironment and inflammation. Del-1 has destructive effects in most cancers and is associated with the progression and invasion of some cancers. In contrast, Del-1 also plays a protective role in inflammation. Del-1 regulates inflammation by regulating the generation of neutrophils in bone marrow, inhibiting the recruitment and migration of neutrophils and accelerating the clearance of neutrophils by macrophages. Del-1 and IL-17 are reciprocally regulated, and their balance maintains immune system homeostasis. Del-1 is expected to become a new therapeutic target for inflammatory disorders such as multiple sclerosis.

Dysfunction in Sertoli cells participates in glucocorticoid-induced impairment of spermatogenesis.

The effect of stress on male fertility is a widespread public health issue, but less is known about the related signaling pathway. To investigate this, we established a hypercortisolism mouse model by supplementing the drinking water with corticosterone for four weeks. In the hypercortisolism mice, the serum corticosterone was much higher than in the control, and serum testosterone was significantly decreased. Moreover, corticosterone treatment induced decrease of sperm counts and increase of teratozoospermia. Increased numbers of multinucleated giant cells and apoptotic germ cells as well as downregulated meiotic markers suggested that corticosterone induced impaired spermatogenesis. Further, upregulation of macrophage-specific marker antigen F4/80 as well as inflammation-related genes suggested that corticosterone induced inflammation in the testis. Lactate content was found to be decreased in the testis and Sertoli cells after corticosterone treatment, and lactate metabolism-related genes were downregulated. In vitro phagocytosis assays showed that the phagocytic activity in corticosterone-treated Sertoli cells was downregulated and accompanied by decreased mitochondrial membrane potential, while pyruvate dehydrogenase kinase-4 inhibitor supplementation restored this process. Taken together, our results demonstrated that dysfunctional phagocytosis capacity and lactate metabolism in Sertoli cells participates in corticosterone-induced impairment of spermatogenesis.

Senescence Marker Protein 30 (SMP30): A Novel Pan-Species Diagnostic Marker for the Histopathological Diagnosis of Breast Cancer in Humans and Animals.

Senescence marker protein 30 (SMP30) is a cell survival factor playing an important role in vitamin C synthesis and antiapoptosis. Moreover, its cytoprotective role suggests a possibility to be related to cancer cell survival. Mammary carcinoma is a common cancer in both humans and animals. Because of its histopathological diversity, especially in the early stage, histopathological diagnosis may be complicated; therefore, a diagnostic marker is helpful for confirmation. The present study analyzed the expression pattern of SMP30 in mammary carcinoma in humans, dogs, and cats. Immunohistochemistry, immunofluorescence, and western blot analysis were used to investigate SMP30 expression patterns. The expression was specifically observed in neoplastic glandular epithelial cells. The expression increased with the malignancy of glandular epithelial cells with a highly proliferative status. However, SMP30 expression was low in normal mammary gland tissues or well-differentiated adenoma tissues. The patterns were consistently reproduced in canine primary mammary carcinoma cells and MCF-7 and MDA-MB-231 human carcinoma cell lines. This study provides useful information to understand SMP30 expression in various stages of mammary carcinoma and to suggest its utility as a pan-species diagnostic marker, thereby helping to establish strategies for diagnosing mammary carcinoma in several species.

Ionized Calcium Binding Adaptor Molecule 1 (IBA1).

OBJECTIVES: Ionized calcium binding adaptor molecule 1 (IBA1), a marker of microglia/macrophages, has not been investigated in human hematopathologic contexts. We evaluated its expression in mature and immature neoplasms of monocytic/histiocytic and dendritic cell (DC) origin. METHODS: Immunohistochemistry for IBA1, CD14, CD68, and CD163 was performed on a total of 114 cases, including a spectrum of monocytic/histiocytic and DC neoplasms (20 tissue based and 59 bone marrow based) and several nonhistiocytic/monocytic/DC neoplasms as control groups (15 tissue based and 20 bone marrow based). RESULTS: IBA1 expression was observed in all types of mature tissue-based histiocytic/DC neoplasms (20/20) but not in the corresponding control group (0/15). In bone marrow-based cases, IBA1 was expressed in most acute myeloid leukemias (AMLs) with monocytic differentiation (48/53), both blastic plasmacytoid dendritic cell neoplasms (2/2), and all chronic myelomonocytic leukemias (4/4), while it was positive in only one nonmonocytic AML (1/15) and none of the acute lymphoblastic leukemias (0/5). Collectively, IBA1 showed much higher sensitivity and specificity (93.7%, 97.1%) compared with CD14 (65.4%, 88.2%), CD68 (74.4%, 74.2%), and CD163 (52.6%, 90.6%). CONCLUSIONS: IBA1 is a novel, highly sensitive, and specific marker for diagnosing neoplasms of monocytic/histiocytic and DC origin.

Acrylamide treatment alters the level of Ca2+ and Ca2+-related protein kinase in spinal cords of rats.

This study aimed to analyze the neurological changes induced by acrylamide (ACR) poisoning and their underlying mechanisms within the spinal cords of male adult Wistar rats. The rats were randomly divided into three groups (n = 9 rats per group). ACR was intraperitoneally injected to produce axonopathy according to the daily dosing schedules of 20 or 40 mg/kg/day of ACR for eight continuous weeks (three times per week). During the exposure period, body weights and gait scores were assessed, and the concentration of Ca2+ was calculated in 27 mice. Protein kinase A (PKA), protein kinase C (PKC), cyclin-dependent protein kinase 5 (CDK5), and P35 were assessed by electrophoretic resolution and Western blotting. The contents of 3'-cyclic adenosine monophosphate (cAMP) and calmodulin (CaM) were determined using ELISA kits, and the activities of calcium/calmodulin-dependent protein kinase II (CaMKII), PKA, and PKC were determined using the commercial Signa TECTPKAassay kits. Compared with control rats, treatment with 20 and 40 mg/kg of ACR decreased body weight and increased gait scores at 8 weeks. Intracellular Ca2+ levels increased significantly in treated rats; CaM, PKC, CDK5, and P35 levels were significantly decreased; and PKA and cAMP levels remained unchanged. CaMKII, PKA, and PKC activities increased significantly. The results indicated that ACR can damage neurofilaments by affecting the contents and activities of CaM, CaMKII, PKA, cAMP, PKC, CDK5, and P35, which could result in ACR toxic neuropathy.

Jasplakinolide Attenuates Cell Migration by Impeding Alpha-1-syntrophin Protein Phosphorylation in Breast Cancer Cells.

BACKGROUND: Alpha-1-syntrophin (SNTA1) is emerging as a novel modulator of the actin cytoskeleton. SNTA1 binds to F-actin and regulates intracellular localization and activity of various actin organizing signaling molecules. Aberration in syntrophin signaling has been closely linked with deregulated growth connected to tumor development/metastasis and its abnormal over expression has been observed in breast cancer. In the present work the effect of jasplakinolide, an actin-binding cyclodepsipeptide, on the SNTA1 protein activity and SNTA1 mediated downstream cellular events was studied in MDA-MB-231 breast cancer cell line. METHODS: SNTA1 protein levels and phosphorylation status were determined in MDA-MB-231 cells post jasplakinolide exposure using western blotting and immunoprecipitation techniques respectively. MDA-MB-231 cells were transfected with WT SNTA1 and DM SNTA1 (Y215/229 phospho mutant) and simultaneously treated with jasplakinolide. The effect of jasplakinolide and SNTA1 protein on cell migration was determined using the boyden chamber assay. RESULTS: Jasplakinolide treatment decreases proliferation of MDA-MB-231 cells in both dose and time dependent manner. Results suggest that subtoxic doses of jasplakinolide induce morphological changes in MDA-MB-231 cells from flat spindle shape adherent cells to round weakly adherent forms. Mechanistically, jasplakinolide treatment was found to decrease SNTA1 protein levels and its tyrosine phosphorylation status. Moreover, migratory potential of jasplakinolide treated cells was significantly inhibited in comparison to control cells. CONCLUSION: Our results demonstrate that jasplakinolide inhibits cell migration by impairing SNTA1 functioning in breast cancer cells.

Time-restricted feeding prevents depressive-like and anxiety-like behaviors in male rats exposed to an experimental model of shift-work.

Individuals who regularly shift their sleep timing, like night and/or shift-workers suffer from circadian desynchrony and are at risk of developing cardiometabolic diseases and cancer. Also, shift-work is are suggested to be a risk factor for the development of mood disorders such as the burn out syndrome, anxiety, and depression. Experimental and clinical studies provide evidence that food intake restricted to the normal activity phase is a potent synchronizer for the circadian system and can prevent the detrimental health effects associated with circadian disruption. Here, we explored whether adult male Wistar rats exposed to an experimental model of shift-work (W-AL) developed depressive and/or anxiety-like behaviors and whether this was associated with neuroinflammation in brain areas involved with mood regulation. We also tested whether time-restricted feeding (TRF) to the active phase could ameliorate circadian disruption and therefore would prevent depressive and anxiety-like behaviors as well as neuroinflammation. In male Wistar rats, W-AL induced depressive-like behavior characterized by hypoactivity and anhedonia and induced increased anxiety-like behavior in the open field test. This was associated with increased number of glial fibrillary acidic protein and IBA-1-positive cells in the prefrontal cortex and basolateral amygdala. Moreover W-AL caused morphological changes in the microglia in the CA3 area of the hippocampus indicating microglial activation. Importantly, TRF prevented behavioral changes and decreased neuroinflammation markers in the brain. Present results add up evidence about the importance that TRF in synchrony with the light-dark cycle can prevent neuroinflammation leading to healthy mood states in spite of circadian disruptive conditions.

The Myoepithelial Cells of Salivary Intercalated Duct-type Intraductal Carcinoma Are Neoplastic: A Study Using Combined Whole-slide Imaging, Immunofluorescence, and RET Fluorescence In Situ Hybridization.

Intraductal carcinoma (IDC) is a salivary gland tumor currently believed to be analogous to breast ductal carcinoma in situ, consisting of a complex neoplastic epithelial proliferation surrounded by a continuous layer of myoepithelial cells presumed to be native and non-neoplastic. Recent molecular insights have shown that there are at least 3 different types of IDC: (1) intercalated duct-like, with frequent NCOA4-RET fusions; (2) apocrine, with multiple mutations similar to salivary duct carcinoma; and (3) mixed intercalated duct-like and apocrine with frequent RET fusions, especially TRIM27-RET. Recent observations (eg, IDC occurring in lymph nodes) have challenged the notion that the myoepithelial cells of IDC are non-neoplastic. Five IDCs with known RET fusions by RNA sequencing were retrieved from the authors' archives, including 4 intercalated duct-like IDCs with NCOA4-RET, and 1 mixed intercalated duct-like/apocrine IDC with TRIM27-RET. A panel of immunohistochemistry antibodies (S100 protein, p63 or p40, mammaglobin, smooth muscle actin, calponin, androgen receptor) was tested. To precisely localize RET split-positive cells, each case was subjected to sequential retrieval of whole-slide imaging data of hematoxylin and eosin (HE) staining, immunofluorescence staining for calponin, and fluorescence in situ hybridization (FISH) for RET. Because NCOA4-RET is an inversion difficult to visualize on conventional RET FISH, a novel 3-color FISH technique was utilized to demonstrate it clearly. In all 5 cases, the proliferative ducts were completely surrounded by a layer of myoepithelial cells that were positive for p63 or p40, smooth muscle actin, and calponin. Using combined HE, calponin immunofluorescence, and RET FISH imaging, the positive signals were unmistakably identified in both calponin-negative ductal cells and peripheral, calponin-positive myoepithelial cells in all 5 cases. Utilizing combined HE, calponin immunofluorescence, and RET FISH imaging, we demonstrated that IDCs with RET fusions harbored this alteration in both the ductal and myoepithelial cells. This is compelling evidence that the myoepithelial cells of IDC are not mere bystanders, but are rather a component of the neoplasm itself, similar to other biphasic salivary gland neoplasms like pleomorphic adenoma and epithelial-myoepithelial carcinoma. This finding raises questions about the appropriate terminology, classification, and staging of IDC.