Previous studies have investigated a potential method for targeted drug delivery in the central nervous system that uses focused ultrasound bursts combined with an ultrasound contrast agent to temporarily disrupt the blood-brain barrier (BBB). The purpose of this work was to investigate the integrity of the tight junctions (TJs) in rat brain microvessels after this BBB disruption. 1.5-MHz ultrasound bursts in combination with a gas contrast agent (Optison) was applied at two locations in the brain in 25 rats to induce BBB disruption. Using immunoelectron microscopy, the distributions of the TJspecific transmembrane proteins occludin, claudin-1, claudin-5, and of submembranous ZO-1 were examined at 1, 2, 4, 6 and 24 h after sonication. A quantitative evaluation of the protein expression was made by counting the number of immunosignals per micrometer in the junctional clefts. BBB disruption at the sonicated locations was confirmed by the leakage of intravenously administered horseradish peroxidase (HRP, m.w. 40,000 Da) and lanthanum chloride (La 3+ , m.w. ~ 139 Da). Leakage of these agents was observed at 1 and 2 h and in a few vessels at 4 h after ultrasound application. These changes were paralleled by the apparent disintegration of the TJ complexes, as evidenced by the redistribution and loss of the immunosignals for occludin, claudin-5 and ZO-1. Claudin-1 seemed less involved. At 6 and 24 h after sonication, no HRP or lanthanum leakage was observed, and the barrier function of the TJs, as indicated by the localization and density of immunosignals, appeared to be completely restored. This study provides the first direct evidence that ultrasound bursts combined with a gas contrast agent cause disassembling of the TJ molecular structure, leading to loss of the junctional barrier functions in brain microvessels. The BBB disruption appears to last up to 4 h after sonication and permits the paracellular passage of agents with molecular weights up to at least 40 kDa. These promising features can be exploited in the future development of this method that could enable the delivery of drugs, antibodies or genes to targeted locations in the brain.

At the time of implantation, the trophoblast cells of the embryo adhere and then invade into the maternal endometrium and eventually establish placentation. The endometrium at the same time undergoes decidualization, which is essential for successful pregnancy. While the NK cells of the decidua have been implicated to play a key role in trophoblast invasion, few evidence are now available to demonstrate a pro-invasive property of decidual stromal cells. Secretions from decidualized endometrial stromal cells promote invasion of primary trophoblasts and model cell lines by activating proteases and altering expression of adhesion-related molecules. The decidual secretions contain high amounts of pro-invasive factors that include IL-1b, IL-5, IL-6, IL-7, IL-8, IL-9, IL-13, IL-15, Eotaxin CCL11, IP-10 and RANTES, and anti-invasive factors IL-10, IL-12 and VEGF. It appears that these decidual factors promote invasion by regulating the protease pathways and integrin expression utilizing the STAT pathways in the trophoblast cells. At the same time the decidua also seem to secrete some anti-invasive factors that are antagonist to the matrix metalloproteinases and/or are activators of tissue inhibitors of metalloproteinases. This might be essential to neutralize the effects of the invasionpromoting factors and restrain overinvasion. It is tempting to propose that during the course of pregnancy, the decidua must balance the production of these pro and anti-invasive molecules and such harmonizing production would allow a timely and regulated invasion.

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The interplay between influenza virus and host factors to support the viral life cycle is well documented. Influenza A virus (IAV) proteins interact with an array of cellular proteins and hijack host pathways which are at the helm of cellular responses to facilitate virus invasion. The multifaceted nature of the ubiquitination pathway for protein regulation makes it a vulnerable target of many viruses including IAV. To this end we conducted a yeast two-hybrid screen to search for cellular ubiquitin ligases important for influenza virus replication. We identified host protein, RING finger protein 43 (RNF43), a RING-type E3 ubiquitin ligase, as a novel interactor of nucleoprotein (NP) of IAV and an essential partner to induce NP-driven p53-mediated apoptosis in IAV-infected cells. In this study, we demonstrate that IAV leads to attenuation of RNF43 transcripts and hence its respective protein levels in the cellular milieu whereas in RNF43 depleted cells, viral replication was escalated several folds. Moreover, RNF43 polyubiquitinates p53 which further leads to its destabilization resulting in a decrease in induction of the p53 apoptotic pathway, a hitherto unknown process targeted by NP for p53 stabilization and accumulation. Collectively, these results conclude that NP targets RNF43 to modulate p53 ubiquitination levels and hence causes p53 stabilization which is conducive to an enhanced apoptosis level in the host cells. In conclusion, our study unravels a novel strategy adopted by IAV for utilizing the much conserved ubiquitin proteasomal pathway.

Background:The NA protein is required for the release of progeny virions. Results: The NA/C6 interaction leads to increased tyrosyl phosphorylation of Src, FAK, Akt, GSK3␤, and Bcl-2, which affects cell survival. Conclusion: A novel role exists for NA in enhancing host cell survival. Significance: NA not only aids in the release of progeny virions, but also cell survival during viral replication.

Background:The diagnosis of retroperitoneal lesions is one of the most difficult areas in surgical pathology. The retroperitoneal space allows both primary and metastatic tumors to grow silently before the appearance of clinical signs and symptoms. Fine needle aspiration cytology has shown promising role in establishing the diagnosis in this region.Objectives:This study was undertaken to evaluate the reliability of ultrasonography (USG)-guided fine needle aspiration cytology (FNAC) in distinguishing between benign and malignant lesions in the retroperitoneum, and to correlate the diagnosis by cytology of retroperitoneal masses with the results obtained by histology.Materials and Methods:The study was carried out on 85 patients presenting over the last five years with retroperitoneal masses on ultrasound.Results:Out of 85 cases, 32 were of kidney, 27 of lymph nodes, 24 of retroperitoneal soft tissues, and two were of the adrenals. Malignant lesions (47) were more common than nonmalignant lesions (38). In the kidney, the maximum number of cases were of renal cell carcinoma (12-38%), followed by Wilm's tumor (6-19%), pyonephrosis (5-16%), renal cyst (4), angiomyolipoma (2), cortical pseudotumor (2), and tuberculosis (1). Out of 27 cases of retroperitoneal lymphadenopathy, 12 cases (44%) were of metastatic carcinoma followed by non-Hodgkin's lymphoma (8-30%), tuberculosis (6-22%), and Hodgkin's lymphoma (1). The two cases of the adrenals were of angiomyolipoma and metastatic carcinoma. Among the 24 soft tissue tumors in the study, seven (29%) were malignant and 17 (71%) were benign (lipoma being the most common benign neoplasm). Results from histopathological investigations were available in 47 cases, out of which 45 were consistent with the FNAC-based diagnoses. Two cases for which the histopathological results were inconsistent with the FNAC diagnoses, were of renal cell carcinoma, which had been diagnosed as renal cysts on cytology.Conclusions:USG-guided FNAC is an inexpensive, rapid, safe, and accurate procedure for the diagnosis of retroperitoneal masses.

Influenza A virus (IAV), similar to other viruses, exploits the machinery of human host cells for its survival and replication. We identified a-actinin-4, a host cytoskeletal protein, as an interacting partner of IAV nucleoprotein (NP). We confirmed this interaction using co-immunoprecipitation studies, first in a coupled in vitro transcription-translation assay and then in cells either transiently co-expressing the two proteins or infected with whole IAV. Importantly, the NP-actinin-4 interaction was observed in several IAV subtypes, including the 2009 H1N1 pandemic virus. Moreover, immunofluorescence studies revealed that both NP and actinin-4 co-localized largely around the nucleus and also in the cytoplasmic region of virus-infected A549 cells. Silencing of actinin-4 expression resulted in not only a significant decrease in NP, M2 and NS1 viral protein expression, but also a reduction of both NP mRNA and viral RNA levels, as well as viral titers, 24 h post-infection with IAV, suggesting that actinin-4 was critical for viral replication. Furthermore, actinin-4 depletion reduced the amount of NP localized in the nucleus. Treatment of infected cells with wortmannin, a known inhibitor of actinin-4, led to a decrease in NP mRNA levels and also caused the nuclear retention of NP, further strengthening our previous observations. Taken together, the results of the present study indicate that actinin-4, a novel interacting partner of IAV NP, plays a crucial role in viral replication and this interaction may participate in nuclear localization of NP and/or viral ribonucleoproteins.

Apoptosis of host cells profoundly influences virus propagation and dissemination, events that are integral to influenza A virus (IAV) pathogenesis. The trigger for activation of apoptosis is regulated by an intricate interplay between cellular and viral proteins, with a strong bearing on IAV replication. Though the knowledge of viral proteins and mechanisms employed by IAV to induce apoptosis has advanced considerably of late, we know relatively little about the repertoire of host factors targeted by viral proteins. Thus, identification of cellular proteins that are hijacked by the virus will help us not only to understand the molecular underpinnings of IAV-induced apoptosis, but also to design future antiviral therapies. Here we show that the nucleoprotein (NP) of IAV directly interacts with and suppresses the expression of API5, a host antiapoptotic protein that antagonizes E2F1-dependent apoptosis. siRNA-mediated depletion of API5, in NP-overexpressed as well as IAV-infected cells, leads to upregulation of apoptotic protease activating factor 1 (APAF1), a downstream modulator of E2F1-mediated apoptosis, and cleavage of caspases 9 and 3, although a reciprocal pattern of these events was observed on ectopic overexpression of API5. In concordance with these observations, annexin V and 7AAD staining assays exhibit downregulation of early and late apoptosis in IAV-infected or NP-transfected cells on overexpression of API5. Most significantly, while overexpression of API5 decreases viral titers, cellular NP protein as well as mRNA levels in IAV-infected A549 cells, silencing of API5 expression causes a steep rise in the same parameters. From the data reported in this manuscript, we propose a proapoptotic role for NP in IAV pathogenesis, whereby it suppresses expression of antiapoptotic factor API5, thus potentiating the E2F1-dependent apoptotic pathway and ensuring viral replication.