Fine‐needle aspiration diagnosis of high grade adenoid cystic carcinoma metastatic to the pancreas

Pancreatic tumors are mostly primary tumors, with only rare metastatic tumors described in the literature. Here we report an unusual case of fine‐needle aspiration (FNA) diagnosis of high grade adenoid cystic carcinoma of the parotid gland metastatic to the pancreas. The aspirate smears were moderately cellular and revealed numerous basaloid neoplastic cells. The cytomorphologic differential diagnosis included primary pancreatic tumor with small cell morphology as well as metastatic tumors. By immunocytochemistry, the tumor cells were positive for cytokeratins (AE1/AE3, CAM5.2, and CK7), and CD117 (C‐KIT), and negative for CD45, WT1, synaptophysin, chromogranin, CD56, TTF‐1, and CK20. The cytomorphologic features and immunoprofile in our case were consistent with high‐grade carcinoma metastases from patient's known salivary gland primary. To the best of our knowledge, this case is the first reported encounter of FNA diagnosis of pancreatic metastasis with small cell morphology from a salivary gland neoplasm as primary site. Diagn. Cytopathol. 2015;43:117–120. © 2014 Wiley Periodicals, Inc.     

Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action

Trigger factor (TF), the first chaperone in eubacteria to encounter the emerging nascent chain, binds to the large ribosomal subunit in the vicinity of the protein exit tunnel opening and forms a sheltered folding space. Here, we present the 3.5-A crystal structure of the physiological complex of the large ribosomal subunit from the eubacterium Deinococcus radiodurans with the N-terminal domain of TF (TFa) from the same organism. For anchoring, TFa exploits a small ribosomal surface area in the vicinity of proteins L23 and L29, by using its "signature motif" as well as additional structural elements. The molecular details of TFa interactions reveal that L23 is essential for the association of TF with the ribosome and may serve as a channel of communication with the nascent chain progressing in the tunnel. L29 appears to induce a conformational change in TFa, which results in the exposure of TFa hydrophobic patches to the opening of the ribosomal exit tunnel, thus increasing its affinity for hydrophobic segments of the emerging nascent polypeptide. This observation implies that, in addition to creating a protected folding space for the emerging nascent chain, TF association with the ribosome prevents aggregation by providing a competing hydrophobic environment and may be critical for attaining the functional conformation necessary for chaperone activity.     

Depression and fibromyalgia: treatment and cost when diagnosed separately or concurrently

OBJECTIVE: Depression and fibromyalgia (FM) are often coincident. Both syndromes share common symptoms and impose significant economic burdens. This study compared claims for treatment and costs of FM plus depression with those for FM or depression alone. METHODS: Administrative claims data from a national Fortune 100 manufacturer were used to identify 3 mutually exclusive patient cohorts based on claims with a diagnosis for: FM only, depression only, and FM plus depression. A fourth cohort comprised a random sample of 10% of the employer's overall beneficiary population. Cohorts were compared for demographics, comorbid conditions, and healthcare resources utilization. Mean direct (treatment) costs were calculated and indirect (work loss) costs imputed, and these were assessed using Student's t test and Bonferroni adjustments. RESULTS: Mean annual employer payments (direct plus indirect costs) per patient were 5,163 dollars for FM only, 8,073 dollars for depression only, 11,899 dollars for FM plus depression, and 2,486 dollars for the overall sample. Mean incremental employer payments (i.e., above those for the random sample) per patient with FM plus depression were 9,413 dollars, an amount more than the sum of incremental costs for those with FM or depression alone (8,264 dollars). These costs are consistent with costs of other chronic diseases. CONCLUSION: Patients with FM plus depression are high users of healthcare services. As in studies that established relationships between depression and other medical conditions, incremental costs for patients with FM plus depression were more than additive of costs for each condition alone.     

The timing of activity rhythms in patients with dementia is related to survival

BACKGROUND: Older adults with dementia often have disruptions in circadian rhythms, including disruptions of the rest-activity rhythm. These disruptions are a product of internal neuronal activity and external environmental influences, both of which are deficient in dementia. However, the consequences of disturbed rhythms are unknown. This study examined the relationship between rest-activity rhythms and death in patients with dementia. METHODS: The authors recruited 149 older adults with dementia (104 women; mean age, 84.1 years) from nursing homes. Activity was recorded with wrist actigraphs from each participant for 3 days. Survival was determined by examining public death records. Cox proportional hazards models were used to determine which aspects of rest-activity rhythms were related to survival. RESULTS: The timing of each participant's rest-activity rhythm compared with a sample of persons without dementia was related to survival, such that those who more closely resembled the persons without dementia lived longer. CONCLUSIONS: Although rest-activity rhythms as a whole were not related to survival, the timing of the rhythm was. Patients with dementia appear to develop an abnormal timing of their rhythms, which is predictive of shorter survival. It may be possible to intervene with these patients to correct the timing of their rhythms and possibly prolong their lives.     

Tyrosine phosphorylation-dependent localization of TmaR that controls activity of a major bacterial sugar regulator by polar sequestration

The poles of Escherichia coli cells are emerging as hubs for major sensory systems, but the polar determinants that allocate their components to the pole are largely unknown. Here, we describe the discovery of a previously unannotated protein, TmaR, which localizes to the E. coli cell pole when phosphorylated on a tyrosine residue. TmaR is shown here to control the subcellular localization and activity of the general PTS protein Enzyme I (EI) by binding and polar sequestration of EI, thus regulating sugar uptake and metabolism. Depletion or overexpression of TmaR results in EI release from the pole or enhanced recruitment to the pole, which leads to increasing or decreasing the rate of sugar consumption, respectively. Notably, phosphorylation of TmaR is required to release EI and enable its activity. Like TmaR, the ability of EI to be recruited to the pole depends on phosphorylation of one of its tyrosines. In addition to hyperactivity in sugar consumption, the absence of TmaR also leads to detrimental effects on the ability of cells to survive in mild acidic conditions. Our results suggest that this survival defect, which is sugar- and EI-dependent, reflects the difficulty of cells lacking TmaR to enter stationary phase. Our study identifies TmaR as the first, to our knowledge, E. coli protein reported to localize in a tyrosine-dependent manner and to control the activity of other proteins by their polar sequestration and release.

The central dogma describes the flow of genetic information from DNA to RNA to protein. However, for this process to be successful, the final product—the protein—needs to be in the right place and at the right time. The consequences of mislocalization can be harmful to any cell type, let alone to the unicellular bacterial cell, whose survival depends on fast and efficient response to environmental changes. Hence, protein localization is an important posttranslational regulatory step. Thus far, most examples of protein targeting were reported in eukaryotic cells, usually in the context of transport from one organelle to another (1). In recent years, it became evident that localization of proteins and RNAs to specific subcellular domains occurs also in prokaryotic cells and is vital for many cellular processes (2–5). However, the mechanisms underlying macromolecules targeting to specific subcellular domains in bacterial cells, with the exception of membrane and cell division proteins, remain largely unknown.The bacterial cell poles are emerging as important domains that accommodate protein and RNA assemblies (5, 6). Pole-localized proteins are involved in a wide range of cellular functions, including motility, regulation of cell cycle, metabolism, differentiation, pathogenesis, and secretion (7). Several proteins were reported to be kept as inactive at the Escherichia coli cell pole until needed, e.g., MurG (8), and FtsZ (9). The phosphotransferase system (PTS), which controls sugar utilization and metabolism in most bacteria, provides an example for regulation via polar cluster formation. Execution of the PTS functions depends on a phosphorylation cascade that initiates with EI and HPr—the general PTS proteins—that deliver the phosphate to the PTS sugar permeases, which import and phosphorylate the incoming sugars (10). The PTS proteins also exert different effects on non-PTS proteins depending on their phosphorylation on histidine residues, thus modulating the hierarchy in sugar utilization (10). The PTS-imported sugars enter glycolysis, whose product, phosphoenolpyruvate (PEP), phosphorylates EI, making EI an important link between glycolysis and sugar uptake (11). We have previously shown that the general PTS proteins localize to the E. coli cell poles (12), although their localization depends on yet-unknown factors (13), that during growth EI polar clusters form stochastically from preexisting dispersed molecules, and that EI clustering negatively correlates with EI function (14). Still, conclusive proof for polar localization as an inhibitory mechanism of EI function is lacking and the identity of the factor that captures EI at the pole remained unknown.Polar clusters offer additional benefits, such as communication between signal transduction systems in order to generate an optimal response, e.g., the chemotaxis and the PTS system (15), or the establishment of cellular asymmetry to coordinate developmental programs with cell cycle progression (7). Polar proteins that recruit other proteins to the poles, thus regulating cell cycle progression, were discovered in some bacteria, e.g., DivIVA in Bacillus subtilis, PopZ and TipN in Caulobacter crescentus, and HubP in Vibrio cholerae (7). In E. coli, the three Min proteins, MinCDE, cooperate to position the cell division site through pole-to-pole oscillation (16).In some bacteria, e.g., C. crescentus, specific localization of proteins is linked to their phosphorylation or to the phosphorylation of factors regulating them (17, 18). In most cases, these proteins are members of the two-component systems, which mediate sensing and regulation by phosphorylation on histidine and aspartic acid residues (19), events considered most prevalent in bacteria. Only in recent years, improved methodologies revealed numerous previously unknown Ser/Thr/Tyr phosphorylation sites, once thought to be hallmarks of eukaryotes, in bacterial and archaeal proteins. The degree to which these putative sites are phosphorylated is still unclear and proofs for their importance in vivo are just beginning to emerge. Also, the linkage between phosphorylation on Ser/Thr/TyR and localization of the phosphorylated bacterial proteins remained unknown.In this study, we show that a previously uncharacterized E. coli protein, YeeX, which is prevalent among Gram-negative bacteria, clusters at the pole in a tyrosine phosphorylation-dependent manner and recruits the major sugar utilization regulator EI. We, therefore, renamed this protein TmaR for targeting of sugar metabolism-associated regulator. TmaR and EI are shown to physically interact and to colocalize. TmaR is necessary for EI polar clustering, but the opposite is not true. Only phosphorylated, TmaR can release EI from the poles, since the diffuse nonphosphorylated TmaR binds to EI quite irreversibly. Notably, tyrosine phosphorylation of EI is also required for its polar localization. We further show that TmaR-mediated EI clustering inversely correlates with EI-mediated sugar uptake, implying that the polar clusters serve as a reservoir for ready-to-act EI molecules. Cells lacking TmaR have detrimental effects on cell survival, which is affected by EI and sugar concentration, when challenged with mildly acidic conditions that are typical to various E. coli habitats. Taken together, our study identifies TmaR as a spatial regulator of sugar metabolism and bacterial survival.     

HIV type 1-specific IgE in serum of long-term surviving children inhibits HIV type 1 production in vitro

We previously identified a group of long-term pediatric survivors who had acquired HIV-1 through maternal transmission; had not received antiretroviral therapy; are now >8 years old, in good health, and with no opportunistic infections; and have not failed to thrive, although they have greatly decreased numbers of blood CD4+ T cells (<500/mm(3)). All the children have elevated total serum IgE levels (210-2475 IU/ml) and make anti-HIV-1 IgE or IgE directed against non-HIV-1 specificities (radioimmunoassay, Western blot assay); they have no detectable antigenemia. We have now studied the ability of anti-HIV-1 IgE in serum obtained from these children to regulate (1) production of HIV-1 by interleukin 2/phytohemagglutinin (IL-2/PHA)-stimulated peripheral blood mononuclear cells (PBMCs) taken from HIV-1-seronegative donors and infected with a T cell-tropic clone of HIV-1, and (2) transmission of a primary HIV-1 strain from adult AIDS patients to uninfected IL-2/PHA-stimulated PBMCs (p24 core antigen production). High levels of HIV-1 production were observed when PBMCs were cultured for 5 days in the presence of HIV-1-seronegative donor serum that was either IgE positive or IgE negative (IgE, >100 or <100 IU/ml, respectively). HIV-1 production also was observed when PBMCs were cultured with HIV-1-infected donor serum that either contained IgE directed against non-HIV-1 specificities or was IgE negative; these levels were 40% less than those seen with sera from the HIV-1-seronegative donors. Far greater inhibition of virus production was observed if the serum in culture contained anti-HIV-1 IgE (>95%). Virus neutralization did not appear to account for the inhibition obtained with anti-HIV-1 IgE-containing serum because virus production was not suppressed in cultures to which serum was added immediately preinfection (<10%), but was strongly suppressed when serum was added 1.5 hr postinfection (>95%). The inhibition of virus production obtained with serum containing anti-HIV-1 IgE was reversed when (1) serum was depleted of IgE (immunoaffinity), but not when it was depleted of IgG (protein G-Sepharose) before inclusion in culture postinfection, (2) anti-IgE, but not anti-IgG, was included in culture, or (3) serum was heat treated before culture. The results indicate that serum from certain HIV-1-infected pediatric long-term survivors contains agents that inhibit HIV-1 production in vitro, and that these agents include anti-HIV-1 IgE. They suggest that a cytotoxic event, rather than virus neutralization, plays an important role in anti-HIV-1 IgE-mediated inhibition of virus production.     

Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells

Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue.     

Combined local blood–brain barrier opening and systemic methotrexate for the treatment of brain tumors

Despite aggressive therapy, existing treatments offer poor prognosis for glioblastoma multiforme patients, in part due to poor penetration of most drugs across the blood–brain barrier (BBB). We propose a minimal-invasive combined treatment approach consisting of local BBB disruption in the tumor in parallel to systemic drug administration. Local BBB disruption is obtained by convection-enhanced delivery of a novel BBB disruption agent, enabling efficient/targeted delivery of the systemically administered drug by the tumors own vasculature. Various human serum albumin (HSA) analogs were synthesized and screened for BBB disruption efficacy in custom in vitro systems. The candidate analogs were then delivered into naïve rat brains by convection-enhanced delivery and screened for maximal BBB disruption and minimal brain toxicity. These studies found a noncationized/neutralized analog, ethylamine (EA)–HSA, to be the optimal BBB-opening agent. Immunocytochemical studies suggested that BBB disruption by EA–HSA may be explained by alterations in occludin expression. Finally, an efficacy study in rats bearing intracranial gliomas was performed. The rats were treated by convection-enhanced delivery of EA–HSA in parallel to systemic administration of Methotrexate, showing significant antineoplastic effects of the combined approached reflected in suppressed tumor growth and significantly (~x3) prolonged survival.     

Noncanonical Wnt Signaling Promotes Obesity-Induced Adipose Tissue Inflammation and Metabolic Dysfunction Independent of Adipose Tissue Expansion

Adipose tissue dysfunction plays a pivotal role in the development of insulin resistance in obese individuals. Cell culture studies and gain-of-function mouse models suggest that canonical Wnt proteins modulate adipose tissue expansion. However, no genetic evidence supports a role for endogenous Wnt proteins in adipose tissue dysfunction, and the role of noncanonical Wnt signaling remains largely unexplored. Here we provide evidence from human, mouse, and cell culture studies showing that Wnt5a-mediated, noncanonical Wnt signaling contributes to obesity-associated metabolic dysfunction by increasing adipose tissue inflammation. Wnt5a expression is significantly upregulated in human visceral fat compared with subcutaneous fat in obese individuals. In obese mice, Wnt5a ablation ameliorates insulin resistance, in parallel with reductions in adipose tissue inflammation. Conversely, Wnt5a overexpression in myeloid cells augments adipose tissue inflammation and leads to greater impairments in glucose homeostasis. Wnt5a ablation or overexpression did not affect fat mass or adipocyte size. Mechanistically, Wnt5a promotes the expression of proinflammatory cytokines by macrophages in a Jun NH₂-terminal kinase–dependent manner, leading to defective insulin signaling in adipocytes. Exogenous interleukin-6 administration restores insulin resistance in obese Wnt5a-deficient mice, suggesting a central role for this cytokine in Wnt5a-mediated metabolic dysfunction. Taken together, these results demonstrate that noncanonical Wnt signaling contributes to obesity-induced insulin resistance independent of adipose tissue expansion.