Intraoperative cytology (squash smear) in neurosurgical practice – pitfalls in diagnosis experience based on 3057 samples from a single institution

OBJECTIVES: The smear technique is challenging for a neuropathologist where rapid and accurate diagnosis is to be given on small biopsies. The present study, a large retrospective analysis of squash smears in neurosurgical practice, was conducted to assess the usefulness, accuracy and the diagnostic pitfalls of smear diagnosis. METHODS: The authors analysed 3057 central nervous system (CNS) lesions sent for intraoperative cytology (IC) during the years 1988-2005. The stain used was 1% alcoholic toluidine blue. The smear diagnosis was compared with the histological diagnosis to evaluate the diagnostic accuracy. RESULTS: Diagnostic accuracy irrespective of lesion and site ranged from 83.0% to 86.0% per year (mean=85%). The highest rate of correlation among common brain tumours was noted in schwannoma (96.6%) and pituitary adenoma (92.2%), followed by meningiomas (88.9%), astrocytomas (88.4%), chordomas (86.4%) and neurocytomas (86.9%). Infections as a whole contributed 380 cases. The most common infection was tuberculosis. CONCLUSION: This is the largest series reported from India to the best of our knowledge. Squash smear technique is a very reliable and rapid method of intraoperative diagnosis. Knowledge of clinical and neuroimaging details helps the experienced neuropathologist to improve the diagnostic accuracy.     

Immunization with outer-membrane protein(s) of Bacteroides fragilis: an experimental study in mice

A crude outer-membrane protein (OMP) preparation from a strain of Bacteroides fragilis, grown in supplemented brain-heart infusion broth, was tested for its protective effect against subcutaneous infection in mice. Immunization with six doses, each of 100, 150 or 200 g OMP preparation, gave some protection: abscesses completely disappeared 15 to 22 days after immunization. In non-immunized animals and animals immunized with doses of 10, 20, 40 or 80 g each, well demarcated abscesses were seen beyond day 22 post-immunization. Although crude OMP elicited good antibody response, with maximum titres on day 4 post-immunization, high titres could not be associated with healing of the abscesses.     

Association of two novel proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA editing complex

RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre-mRNAs as directed by guide RNAs. This occurs by a series of steps that are catalyzed by endoribonuclease, 3'-terminal uridylyl transferase, 3'-exouridylylase, and RNA ligase activities. A multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched from Trypanosoma brucei mitochondria by sequential ion-exchange and gel filtration chromatography, followed by glycerol gradient sedimentation. The complex size is approximately 1,600 kDa, and the purified fraction contains 20 major polypeptides. A monoclonal antibody that was generated against the enriched complex reacts with an approximately 49-kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity. The protein recognized by the antibody was identified by mass spectrometry, and the corresponding gene, designated TbMP52, was cloned. Recombinant TbMP52 reacts with the monoclonal antibody. Another novel protein, TbMP48, which is similar to TbMP52, and its gene were also identified in the enriched complex. These results suggest that TbMP52 and TbMP48 are components of the RNA editing complex.     

Sanitation,Stress, and Life Stage: A Systematic Data Collection Study among Women in Odisha,India

Emerging evidence demonstrates how inadequate access to water and sanitation is linked to psychosocial stress, especially among women, forcing them to navigate social and physical barriers during their daily sanitation routines. We examine sanitation-related psychosocial stress (SRPS) across women’s reproductive lives in three distinct geographic sites (urban slums, rural villages, and rural tribal villages) in Odisha, India. We explored daily sanitation practices of adolescent, newly married, pregnant, and established adult women (n = 60) and identified stressors encountered during sanitation. Responding to structured data collection methods, women ranked seven sanitation activities (defecation, urination, menstruation, bathing, post-defecation cleaning, carrying water, and changing clothes) based on stress (high to low) and level of freedom (associated with greatest freedom to having the most restrictions). Women then identified common stressors they encountered when practicing sanitation and sorted stressors in constrained piles based on frequency and severity of each issue. The constellation of factors influencing SRPS varies by life stage and location. Overall, sanitation behaviors that were most restricted (i.e., menstruation) were the most stressful. Women in different sites encountered different stressors, and the level of perceived severity varied based on site and life stage. Understanding the influence of place and life stage on SRPS provides a nuanced understanding of sanitation, and may help identify areas for intervention.     

Chaperone-Mediated Autophagy Targets IFNAR1 for Lysosomal Degradation in Free Fatty Acid Treated HCV Cell Culture

BackgroundHepatic steatosis is a risk factor for both liver disease progression and an impaired response to interferon alpha (IFN-α)-based combination therapy in chronic hepatitis C virus (HCV) infection. Previously, we reported that free fatty acid (FFA)-treated HCV cell culture induces hepatocellular steatosis and impairs the expression of interferon alpha receptor-1 (IFNAR1), which is why the antiviral activity of IFN-α against HCV is impaired.AimTo investigate the molecular mechanism by which IFNAR1 expression is impaired in HCV cell culture with or without free fatty acid-treatment.MethodHCV-infected Huh 7.5 cells were cultured with or without a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in HCV-infected culture was visualized by oil red staining. Clearance of HCV in FFA cell culture treated with type I IFN (IFN-α) and Type III IFN (IFN-λ) was determined by Renilla luciferase activity, and the expression of HCV core was determined by immunostaining. Activation of Jak-Stat signaling in the FFA-treated HCV culture by IFN-α alone and IFN-λ alone was examined by Western blot analysis and confocal microscopy. Lysosomal degradation of IFNAR1 by chaperone-mediated autophagy (CMA) in the FFA-treated HCV cell culture model was investigated.ResultsFFA treatment induced dose-dependent hepatocellular steatosis and lipid droplet accumulation in HCV-infected Huh-7.5 cells. FFA treatment of infected culture increased HCV replication in a concentration-dependent manner. Intracellular lipid accumulation led to reduced Stat phosphorylation and nuclear translocation, causing an impaired IFN-α antiviral response and HCV clearance. Type III IFN (IFN-λ), which binds to a separate receptor, induces Stat phosphorylation, and nuclear translocation as well as antiviral clearance in FFA-treated HCV cell culture. We show here that the HCV-induced autophagy response is increased in FFA-treated cell culture. Pharmacological inhibitors of lysosomal degradation, such as ammonium chloride and bafilomycin, prevented IFNAR1 degradation in FFA-treated HCV cell culture. Activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased IFNAR1 levels in Huh-7.5 cells. Co-immunoprecipitation, colocalization and siRNA knockdown experiments revealed that IFNAR1 but not IFNLR1 interacts with HSC70 and LAMP2A, which are core components of chaperone-mediated autophagy (CMA).ConclusionOur study presents evidence indicating that chaperone-mediated autophagy targets IFNAR1 degradation in the lysosome in FFA-treated HCV cell culture. These results provide a mechanism for why HCV induced autophagy response selectively degrades type I but not the type III IFNAR1.     

Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk‐secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI‐TOF/TOF MS and 1D‐Gel‐LC‐MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT‐PCR and Western blotting. The 1D‐Gel‐LC‐MS/MS and 2DE‐MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC‐specific proteins that will help the researchers to understand the molecular events taking place during lactation.     

Trypanosoma brucei mitochondrial respiratome: composition and organization in procyclic form

The mitochondrial respiratory chain is comprised of four different protein complexes (I–IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F_oF₁-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen.Mitochondria are dynamic organelles essential for cellular life, death, and differentiation of virtually every eukaryotic cell. They house systems for energy production through oxidative phosphorylation, synthesis of key metabolites, and iron-sulfur cluster assembly. The oxidative phoshorylation system of eukaryotic mitochondria comprises five major complexes located in the mitochondrial (mt)¹ inner membrane, and often abbreviated as mt complexes I–V. The redox energy of the substrates NADH and succinate is first converted into an electrochemical proton potential across the inner mt membrane by respiratory complexes I (NADH:ubiquinone reductase), II (SDH, succinate:ubiquinone reductase), III (bc₁, ubiquinone:cytochrome c reductase), and IV (cytochrome c oxidase). The electrochemical proton potential is then used by complex V (F_oF₁-ATP synthase) to synthesize ATP from ADP and inorganic phosphate, a mechanism that has essentially remained unchanged from bacteria to human (1). However, parasitic organisms have exploited unique energy metabolic pathways by adapting to their natural host habitats (2). Indeed, the respiratory systems of parasites typically show greater diversity in electron transfer pathways than those of their host, and Trypanosoma brucei is no exception to this rule (3).T. brucei, the causative agent of human African trypanosomiasis (HAT), or sleeping sickness, is a blood-borne pathogenic parasite transmitted by tsetse flies. It has a complex life cycle that alternates between the bloodstream forms (BF) in the mammalian host and several stages in the insect vector starting with the procyclic form (PF) in the midgut. During T. brucei differentiation between the distinct life-cycle stages, the mitochondrion undergoes morphological and functional changes, and the parasite switches its energy metabolism from amino acid to glucose oxidation (4). BF cells, which live in sugar-rich environment, use energy metabolism predominantly through the glycolytic pathway (5). They contain no cytochrome-mediated respiratory chain and they possess a unique electron transport chain in the mitochondria, the glycerol-3-phosphate dehydrogenase and the salicyl hydroxamic acid (SHAM)-sensitive alternative oxidase, which is known as the trypanosome alternative oxidase (TAO) (6). Despite the absence of complete cytochrome-containing complexes III and IV in BF trypanosomes, a mt membrane potential is maintained and involves the hydrolytic activity of the F_oF₁-ATP synthase complex (7). Conversely, PF cells are dependent on the cytochrome-containing respiratory chain and ATP generated by conventional function of the F_oF₁-ATP synthase complex for their energy production (8, 9). The branched electron-transport chain contains four complexes that donate electrons to the ubiquinone pool, two NADH:ubiquinone oxidoreductases (complex I and a rotenone-insensitive enzyme), complex II, and glycerol-3-phosphate dehydrogenase. Reduced ubiquinol can be reoxidized by the transfer of electron to either the TAO, which does not translocate protons, or to the cytochrome-containing complexes III and IV that produce a proton motive force by translocation of protons and thus create essential membrane potential (10).Although the T. brucei genome has been sequenced (11), little information is available on the subunit composition of mt complexes I–V based on similarity searches. However, some respiratory complexes have been partially characterized in other trypanosomatids such as Crithidia fasciculata, T. cruzi, and Leishmania tarentolae (12–15). In recent studies, we have determined the protein composition of complexes IV and V, and part of complex I purified from mitochondria of T. brucei PF cells (8, 16, 17, 25). These analyses revealed the uniqueness of respiratory complexes in trypanosomes, where large numbers of component proteins have no homologs outside of the Kinetoplastida.In this study, we focus on the comprehensive characterization of all respiratory complexes in T. brucei, collectively termed the respiratome. We report the composition of complexes II and III from PF cells, and extend the characterization of complex I by identifying additional protein constituents. This included the identification of two subunits of the respiratory complex IV, both encoded by mt edited RNAs. We also present a predicted protein-protein interaction network of the respiratome, which was generated using proteomics data collected from numerous tagged proteins in each of the complexes I–V. Our results provide a comprehensive insight into the unique composition of the respiratory complexes in one of the life-cycle stages of T. brucei.     

Bacterial and archaea community present in the Pine Barrens Forest of Long Island, NY: unusually high percentage of ammonia oxidizing bacteria

Of the few preserved areas in the northeast of United States, the soil in the Pine Barrens Forests presents a harsh environment for the microorganisms to grow and survive. In the current study we report the use of clustering methods to scientifically select the sampling locations that would represent the entire forest and also report the microbial diversity present in various horizons of the soil. Sixty six sampling locations were selected across the forest and soils were collected from three horizons (sampling depths). The three horizons were 0-10 cm (Horizon O); 11-25 cm (Horizon A) and 26-40 cm (Horizon B). Based on the total microbial substrate utilization pattern and K-means clustering analysis, the soil in the Pine Barrens Forest can be classified into four distinct clusters at each of the three horizons. One soil sample from each of the four clusters were selected and archaeal and bacterial populations within the soil studied using pyrosequencing method. The results show the microbial communities present in each of these clusters are different. Within the microbial communities present, microorganisms involved in nitrogen cycle occupy a major fraction of microbial community in the soil. High level of diversity was observed for nitrogen fixing bacteria. In contrast, Nitrosovibrio and Nitrosocaldus spp are the single bacterial and archaeal population respectively carrying out ammonia oxidation in the soil.