Parasitism of Mallophaga by Trenomyces histophtorus

Despite current theory that fungi of the order Laboulbeniales are not injurious to their hosts, we found that Trenomyces histophtorus, a parasite of Mallophaga, invaded the body cavity of its host and produced an extensive rhizomycelium that resulted in the loss of fat body and skeletal muscle in these insects.     

Maturation and persistence of the anti-SARS-CoV-2 memory B cell response

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GObar: A Gene Ontology based analysis and visualization tool for gene sets

Background  

Microarray experiments, as well as other genomic analyses, often result in large gene sets containing up to several hundred genes. The biological significance of such sets of genes is, usually, not readily apparent.     

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E₂) are distinct from those in non-E₂-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E₂ proliferative H1793 and non-E₂-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E₂ or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.

     

Widespread Recombination,Reassortment, and Transmission of Unbalanced Compound Viral Genotypes in Natural Arenavirus Infections

Arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. Arenaviruses package a large (L) and small (S) genome segment in their virions. For segmented RNA viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. Although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. Here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. From 48 infected animals, we determined the complete or near complete sequence of 210 genome segments that grouped into 23 L and 11 S genotypes. The majority of snakes were multiply infected, with up to 4 distinct S and 11 distinct L segment genotypes in individual animals. This S/L imbalance was typical: in all cases intrahost L segment genotypes outnumbered S genotypes, and a particular S segment genotype dominated in individual animals and at a population level. We corroborated sequencing results by qRT-PCR and virus isolation, and isolates replicated as ensembles in culture. Numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring 2 intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. Overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. This diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. Thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential.     

Ribonuclear inclusions as biomarker of myotonic dystrophy type 2, even in improperly frozen or defrozen skeletal muscle biopsies

Myotonic dystrophy type 2 (DM2) is a dominantly inherited disorder caused by a CCTG repeat expansion in intron 1 of ZNF9 gene. The size and the somatic instability of DM2 expansion complicate the molecular diagnosis of DM2. In situ hybridization represents a rapid and sensitive method to obtain a definitive diagnosis in few hours, since it allows the direct visualization of the mutant mRNA foci on skeletal muscle sections. This approach makes the muscle biopsy an important tool for definitive diagnosis of DM2. Consequently, a rapid freezing at ultra cold temperature and a good storage of muscle specimens are essential to avoid morphologic alterations and nucleic acids degradation. However incorrect freezing or thawing may accidentally occur. In this work we report that fluorescence in situ hybridization may be applied on improperly frozen or inappropriately stored muscle biopsies since foci of mutant mRNA are well preserved and can still be detected in muscle sections no more useful for histopathological evaluation.Key words: myotonic dystrophy type 2, defrozen muscle biopsy, fluorescence, in situ hybridization, ribonuclear inclusions.Myotonic dystrophy type 2 (DM2) is a neuromuscular disorder due to the unstable (CCTG)n repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene on chromosome 3q21.3 (Liquori et al. 2001). Mutant ZNF9 pre-mRNA is spliced and polyadenylated, and the mRNA is exported to the cytoplasm where normal levels of ZNF9 protein expression occur (Botta et al., 2006; Margolis et al. 2006); however, the expanded repeats remain in cell nuclei as ribonuclear inclusions (Liquori et al. 2001). The DM2 ribonuclear inclusions contain only the CCUG repeat sequence derived from intron 1 but with no detectable flanking intronic RNA (Margolis et al. 2006). CCUG-containing mutant mRNAs form double-stranded hairpin loop structures that bind specific RNA-binding proteins such as muscle-blind-like proteins (MBNLs) that colocalize with ribonuclear inclusions in myonuclei (Mankodi et al., 2001; Fardaei et al., 2002). Sequestration of these proteins which are regulators of alternative splicing, alters the splicing of several pre-mRNA (reviewed by Osborne and Thornton, 2006) such as the insulin receptor (IR) and the chloride channel (ClC1) (Savkur et al., 2004; Charlet et al., 2002; Mankodi et al., 2002). Alterations in IR splicing leads to insulin insensitivity and predisposition to diabetes (Savkur et al. 2004) and alterations in ClC1 splicing results in electrical myotonia (Charlet et al., 2002; Mankodi et al., 2002). Conventional Southern blot analysis is not adequate for a definitive molecular diagnosis in DM2 due to the extremely large size and somatic instability of the expansion mutation (Liquori et al., 2001; Bachinski et al., 2003). The extraordinary somatic instability complicates the analysis of genotype-phenotype correlations including those in the effect of the gender of transmitting parents and anticipation. The copy number of DM2 CCTG is below 30 in phenotypically normal individuals and up 11.000 in patients (Day and Ranum, 2005). A complex genotyping diagnostic procedure is now commonly used consisting of a three-step molecular protocol (Day et al., 2003; Udd et al., 2003). However, a more practical tool to obtain a definitive diagnosis in few hours is represented by in situ hybridization which detects ribonuclear inclusions in cell nuclei of muscle fibers (Cardani et al., 2004; Sallinen et al., 2004). This approach makes muscle biopsy an essential tool for DM2 diagnosis. For this reason muscle specimens should be sent fresh, for rapid freezing, from the operating room to the pathology laboratory.To avoid RNA degradation, biopsies require special precautions with handling of material, such as immediate freezing of fresh tissues, because retrospective genetic analysis is impaired by conventional tissue processing techniques. However, many small hospitals are ill-equipped for snap freezing which requires access to liquid nitrogen or dry ice; thus, frequently outside hospitals provide specimens that are obscured with freeze artefacts because they either were submitted incorrectly or were improperly frozen, at the point of origin prior to shipment. Moreover, an accidental tissue thawing and refreezing may occur (for example power failure of the freezer) causing severe tissue damages and possible RNA degradation.Here we report our experience on DM2 muscle biopsies improperly preserved: these were no more useful for a histopathological analysis since they showed evident morphologic artefacts, but they proved to be still suitable for diagnosis by fluorescence in situ hybridization (FISH) since ribonuclear inclusions were preserved and still detectable on muscle sections.     

Assessment of pharmacological activities of two medicinal plant of Bangladesh: Launaea sarmentosa and Aegialitis rotundifolia roxb in the management of pain,pyrexia and inflammation

Background

The current study aims at evaluating the analgesic, anti-pyretic and anti-inflammatory properties of methanolic extract of the stem, bark and leaves of Launaea sarmentosa and Aegialitis rotundifolia roxb.

Results

The AELS and AEAR extract presented a significant (***p < 0.001) dose dependent increase in reaction time in writhing method and showed inhibition of 63.1% and 57.1% respectively at the doses of 400 mg/kg body weight while standard drug showed (P < 0.001) inhibition of 69.23%. In tail immersion method, AELS and AEAR showed maximum time of tail retention at 30 min in hot water i.e. 6.93 sec and 6.54 sec respectively at highest doses of 400 mg/kg body weight than lower dose while standard pentazocine showed reaction time of 7.62 sec. The AELS and AEAR extract also exhibited promising anti-inflammatory effect as demonstrated by statistically significant inhibition of paw volume by 32.48% and 26.75% respectively at the dose of 400 mg/kg body weight while the value at the dose of 200 mg/kg body weight were linear to higher dose at the 3^rd hour of study. On the other hand, Standard indomethacin inhibited 40.13% of inflammation (***P < 0.001). In Cotton-pellet granuloma method, AELS and AEAR extract at the dose of 400 mg/kg body weight exhibited inhibition of inflammation of 34.7% and 29.1% respectively while standard drug showed (P < 0.001) inhibition of 63.22%. Intraperitoneal administration of AELS and AEAR showed dose dependent decrease in body temperature in brewer’s yeast induced hyperthermia in rats at both doses. However, AELS significantly decreased body temperature (***p < 0.001) at 400 mg/kg compared to control.

Conclusions

Present work propose that the methanolic extract of Launaea sarmentosa and Aegialitis rotundifolia roxb possesses dose dependent pharmacological action which supports its therapeutic use in folk medicine possibly mediated through the inhibition or blocking of release of prostaglandin and/or actions of vasoactive substances such as histamine, serotonin and kinins.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Advanced microscopic and histochemical techniques: diagnostic tools in the molecular era of myology

Over the past two centuries, myology (i.e. the basic and clinical science of muscle and muscle disease) has passed through 3 stages of development: the classical period, the modern stage and the molecular era. The classical period spans the last part of nineteenth century and the earlier part of the twentieth century. During this time, several major muscle diseases were clinically and pathologically characterized, including Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM) and facioscapulohumeral dystrophy (FSHD). The modern stage in the second half of the twentieth century is characterized by the adaptation of histo and cytochemical techniques to the study of muscle biopsies. These tools improved the diagnostic accuracy and made possible the identification of new changes and structures (Engel and Cunningham, 1963; Scarlato, 1975).