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Background: To evaluate the different patterns of thyroid lesions associated with MNG in surgically resected specimens and biopsies received at department of pathology and correlate the various histological and cytological diagnosis to ensure the usefulness of cytology prior histopathology. Methods: The study period is from 2017 to 2020 January carried out in Department of Pathology, GVPIHCM most common were calcification (85%), cystic change (80%) and Sanderson polsters (75%). 19 nonneoplastic lesions associated with MNG were noted in this study. Most common lesion noted was Hashimoto’s thyroiditis constituting 19 cases seen in the age group of 41-60 yrs. 15 benign lesions associated with nodular goiter were noted in this study. 22 malignant lesions were observed to be associated with nodular goiter with most common malignant lesion being papillary thyroid carcinoma followed by its variant papillary micro-carcinoma. The malignant lesions had lymphnodal enlargement with metastases in 9 cases constituting 7 cases of N1a and 2 cases of N1b.TBSRTC Category – I included 10 cases with inadequate smears for reporting. Most of them i.e. 81 cases constituting 63.28% were benign and were included under Category II. Only 7 cases were indeterminate lesions. Category IV included the next highest reported cases after Category II including 17 cases constituting 13.28%. Only 3 cases were included under suspicious category/category V. 7.8% i.e. 10 cases were reported to be malignant. Conclusions: Taking into consideration histopathology report as a gold standard, correlation of cytological finding with histopathology finding showed 90% sensitivity, 98.8% specificity with 90% positive predictive value.

BACKGROUND A wide variety of non-neoplastic lesions can occur in the posterior cranial fossa. Most of them are asymptomatic and incidentally detected. They may become symptomatic either because of pressure, rupture, or secondary inflammation. We wanted to study the spectrum of non-neoplastic lesions in posterior cranial fossa among intracranial lesions. METHODS This was a combined retrospective and prospective study, done from January 2009 to May 2014. 32 cases of non-neoplastic lesions in posterior cranial fossa were studied for patient demographics and histopathology. RESULTS The patients’ age ranged from one to eighty years and the male to female ratio was 1.2 : 1. Among non-neoplastic lesions, majority were epidermoid cysts i.e. 34.3 % followed by cerebellar abscess 28.1 %, arachnoid cyst 15.6 % and others. Maximum number of cases was in the third and fourth decades. Tuberculous lesions accounted for 12.5 % cases. CONCLUSIONS Non-neoplastic lesions have a wide spectrum of histopathology and histogenesis. Among the non-neoplastic lesions, the most common lesion was epidermoid cyst followed by cerebellar abscess. The peak age incidence was in 3 rd and 4 th decades with male preponderance.

The molecular mechanism of epimorphic regeneration is elusive due to its complexity and limitation in mammals. Epigenetic regulatory mechanisms play a crucial role in development and regeneration. This investigation attempted to reveal the role of epigenetic regulatory mechanisms, such as histone H3 and H4 lysine acetylation and methylation during zebrafish caudal fin regeneration. It was intriguing to observe that H3K9,14 acetylation, H4K20 trimethylation, H3K4 trimethylation and H3K9 dimethylation along with their respective regulatory genes, such as GCN5, SETd8b, SETD7/9, and SUV39h1, were differentially regulated in the regenerating fin at various time points of post-amputation. Annexin genes have been associated with regeneration; this study reveals the significant up-regulation of ANXA2a and ANXA2b transcripts and their protein products during the regeneration process. Chromatin immunoprecipitation and PCR analysis of the regulatory regions of the ANXA2a and ANXA2b genes demonstrated the ability to repress two histone methylations, H3K27me3 and H4K20me3, in transcriptional regulation during regeneration. It is hypothesized that this novel insight into the diverse epigenetic mechanisms that play a critical role during the regeneration process may help to strategize the translational efforts, in addition to identifying the molecules involved in vertebrate regeneration.

Lyme borreliosis, caused by the spirochete Borrelia burgdorferi sensu lato, has grown into a major public health problem. We recently identified a novel morphological form of B. burgdorferi, called biofilm, a structure that is well known to be highly resistant to antibiotics. However, there is no evidence of the existence of Borrelia biofilm in vivo; therefore, the main goal of this study was to determine the presence of Borrelia biofilm in infected human skin tissues. Archived skin biopsy tissues from borrelial lymphocytomas (BL) were reexamined for the presence of B. burgdorferi sensu lato using Borrelia-specific immunohistochemical staining (IHC), fluorescent in situ hybridization, combined fluorescent in situ hybridization (FISH)–IHC, polymerase chain reaction (PCR), and fluorescent and atomic force microscopy methods. Our morphological and histological analyses showed that significant amounts of Borrelia-positive spirochetes and aggregates exist in the BL tissues. Analyzing structures positive for Borrelia showed that aggregates, but not spirochetes, expressed biofilm markers such as protective layers of different mucopolysaccharides, especially alginate. Atomic force microscopy revealed additional hallmark biofilm features of the Borrelia/alginate-positive aggregates such as inside channels and surface protrusions. In summary, this is the first study that demonstrates the presence of Borrelia biofilm in human infected skin tissues.

Background Plasmodium knowlesi is primarily responsible for zoonotic malaria in several Southeast Asian countries. Precise identification of the parasite in the blood of patients presently relies on an expensive and elaborate PCR procedure because microscopic examination of blood and other available field identification techniques lack adequate specificity. Therefore, the use of a simple and inexpensive dual-colour fluorescence in situ hybridization (FISH) assay, analogous to FISH assays recently described for Plasmodium falciparum and Plasmodium vivax, was investigated as a potential tool for identifying P. knowlesi. Results A P. knowlesi 18S rDNA sequence-based DNA probe was used to test thin blood smears of P. knowlesi by FISH, and fluorescence viewed in a light microscope fitted with a light emitting diode light source and appropriate emission and barrier filters. The limit of detection in the P. knowlesi FISH assay was 84 parasites per μl in infected monkey blood and 61 parasites per μl for P. knowlesi cultured in human blood. The P. knowlesi-specific FISH probe detected only P. knowlesi and not P. falciparum, Plasmodium malariae, Plasmodium ovale, P. vivax or a panel of other human blood-borne pathogens. A previously described Plasmodium genus-specific probe used simultaneously in the P. knowlesi FISH assay reacted with all tested Plasmodium species. Conclusions To our knowledge, this is the first description of a FISH assay for P. knowlesi that is potentially useful for diagnosing infections in remote laboratories in endemic countries. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2273-7) contains supplementary material, which is available to authorized users.

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.

The aminoacyl-tRNA synthetases are prominently known for their classic function in the first step of protein synthesis, where they bear the responsibility of setting the genetic code. Each enzyme is exquisitely adapted to covalently link a single standard amino acid to its cognate set of tRNA isoacceptors. These ancient enzymes have evolved idiosyncratically to host alternate activities that go far beyond their aminoacylation role and impact a wide range of other metabolic pathways and cell signaling processes. The family of aminoacyl-tRNA synthetases have also been suggested as a remarkable scaffold to incorporate new domains that would drive evolution and the emergence of new organisms with more complex function. Because they are essential, the tRNA synthetases have served as pharmaceutical targets for drug and antibiotic development. The recent unfolding of novel important functions for this family of proteins offers new and promising pathways for therapeutic development to treat diverse human diseases.

Tick-borne relapsing fever (TBRF) is caused by spirochete bacteria of the genus Borrelia termed relapsing fever Borreliae (RFB). TBRF shares symptoms with Lyme disease (LD) caused by related Lyme disease Borreliae (LDB). TBRF and LD are transmitted by ticks and occur in overlapping localities worldwide. Serological detection of antibodies used for laboratory confirmation of LD is not established for TBRF. A line immunoblot assay using recombinant proteins from different RFB species, termed TBRF IB, was developed and its diagnostic utility investigated. The TBRF IBs were able to differentiate between antibodies to RFB and LDB and had estimated sensitivity, specificity, and positive and negative predictive values of 70.5%, 99.5%, 97.3%, and 93.4%, respectively, based on results with reference sera from patients known to be positive and negative for TBRF. The use of TBRF IBs and analogous immunoblots for LD to test sera of patients from Australia, Ukraine, and the USA with LD symptoms revealed infection with TBRF alone, LD alone, and both TBRF and LD. Diagnosis by clinical criteria alone can, therefore, underestimate the incidence of TBRF. TBRF IBs will be useful for laboratory confirmation of TBRF and understanding its epidemiology worldwide.

Sandwich panels were fabricated with nanoclay-filled polyurethane foams and glass fiber-reinforced polyamide and polypropylenes face sheets. Nanoclay-filled foam cores, with organophilic montmorillonite loadings of 0–10 wt%, were synthesized through polyaddition of the polyol premix with 4,4'-diphenylmethane diisocyanate, and bound to the injected molded face sheets. Produced sandwich structures were then subjected to low energy impact (15 J) tests under localized point and surface loads, in an instrumented impact test setup. Additionally, quasi-static compressive behavior of the sandwiches panels was studied. The results showed that the addition of nanoclay in the polyurethane foam core improved both energy absorption and maximal deflection during impact. The improvement in energy absorption was between 66% and 92% for polypropylenes face sheet sandwiches and 23% and 34% for the polyamide face sheet sandwiches during point load. Furthermore, an increase in the compression modulus of 20–37% was recorded for the sandwiches with polyamide face sheets.

Photograph showing a laboratory microscope fitted with a LED and filter attachment for viewing fluorescence in FISH assays (reproduced with permission from [18]. (PPTX 70Â kb)

Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.

The molecular mechanism of epimorphic regeneration is elusive due to its complexity and limitation in mammals. Epigenetic regulatory mechanisms play a crucial role in development and regeneration. This investigation attempted to reveal the role of epigenetic regulatory mechanisms, such as histone H3 and H4 lysine acetylation and methylation during zebrafish caudal fin regeneration. It was intriguing to observe that H3K9,14 acetylation, H4K20 trimethylation, H3K4 trimethylation and H3K9 dimethylation along with their respective regulatory genes, such as GCN5, SETd8b, SETD7/9, and SUV39h1, were differentially regulated in the regenerating fin at various time points of post-amputation. Annexin genes have been associated with regeneration; this study reveals the significant up-regulation of ANXA2a and ANXA2b transcripts and their protein products during the regeneration process. Chromatin immunoprecipitation and PCR analysis of the regulatory regions of the ANXA2a and ANXA2b genes demonstrated the ability to repress two histone methylations, H3K27me3 and H4K20me3, in transcriptional regulation during regeneration. It is hypothesized that this novel insight into the diverse epigenetic mechanisms that play a critical role during the regeneration process may help to strategize the translational efforts, in addition to identifying the molecules involved in vertebrate regeneration.

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.

Aminoacyl-tRNA synthetases (AARSs) are a group of essential and ubiquitous "house-keeping" enzymes responsible for charging corresponding amino acids to their cognate transfer RNAs (tRNAs) and providing the correct substrates for high-fidelity protein synthesis. During the last three decades, wide-ranging biochemical and genetic studies have revealed non-catalytic regulatory functions of multiple AARSs in biological processes including gene transcription, mRNA translation, and mitochondrial RNA splicing, and in diverse species from bacteria through yeasts to vertebrates. Remarkably, ongoing exploration of non-canonical functions of AARSs has shown that they contribute importantly to control of inflammation, angiogenesis, immune response, and tumorigenesis, among other critical physiopathological processes. In this chapter we consider the non-canonical functions of AARSs in regulating gene expression by mechanisms not directly related to their enzymatic activities, namely, at the levels of mRNA production, processing, and translation. The scope of AARS-mediated gene regulation ranges from negative autoregulation of single AARS genes to gene-selective control, and ultimately to global gene regulation. Clearly, AARSs have evolved these auxiliary regulatory functions that optimize the survival and well-being of the organism, possibly with more complex regulatory mechanisms associated with more complex organisms. In the first section on transcriptional control, we introduce the roles of autoregulation by Escherichia coli AlaRS, transcriptional activation by human LysRS, and transcriptional inhibition by vertebrate SerRS. In the second section on translational control, we recapitulate the roles of GluProRS in translation repression at the initiation step, auto-inhibition of E. coli thrS mRNA translation by ThrRS, and global translational arrest by phosphorylated human MetRS. Finally, in the third section, we describe the RNA splicing activities of mitochondrial TyrRS and LeuRS in Neurospora and yeasts, respectively.

Formation of the ternary complex between GTP-bound form of elongation factor Tu (EF-Tu) and aminoacylated transfer RNA (aa-tRNA) is a key event in protein biosynthesis. Here we show that fluorescently modified Escherichia coli EF-Tu carrying three mutations, C137A, C255V and E348C, and fluorescently modified Phe-tRNA(Phe) form functionally active ternary complex that has properties similar to those of the naturally occurring (unmodified) complex. Similarities include the binding and binding rate constants, behavior in gel retardation assay, as well as activities in tRNA protection and in vitro translation assays. Proper labeling of EF-Tu was demonstrated in MALDI mass spectroscopy experiments. To generate the mutant EF-Tu, a series of genetic constructions were performed. Two native cysteine residues in the wild-type EF-Tu at positions 137 and 255 were replaced by Ala and Val, respectively, and an additional cysteine was introduced either in position 324 or 348. The assembly FRET assay showed a 5- to 7-fold increase of Cy5-labeled EF-Tu E348C mutant fluorescence upon formation of ternary complex with charged tRNA(Phe)(Cy3-labeled) when the complex was excited at 532 nm and monitored at 665 nm. In a control experiment, we did not observe FRET using uncharged tRNA(Phe)(Cy3), nor with wild-type EF-Tu preparation that was allowed to react with Cy5 maleimide, nor in the absence of GTP. The results obtained demonstrate that the EF-Tu:tRNA FRET system described can be used for investigations of ribosomal translation in many types of experiments.

The QSY9 derivative of E348C-EF-Tu (EF-TuQ348) is fully functional and is an efficient quencher of Cy3 fluorescence within the ternary complex made with Phe-tRNAPhe(Cy3). Incubation of this ternary...

Elongation factor Tu (EF-Tu) ensures fidelity in protein synthesis by mediating the entry of cognate aminoacyl-tRNA (aa-tRNA) into the A-site of the ribosome via formation of an EF-Tu.GTP.aa-tRNA ternary complex (TC). In order to probe the kinetic details of EF-Tu interactions with both aminoacyl tRNA and the ribosome during the tRNA selection process, we have constructed, purified, and labeled an E. coli EF-Tu mutant at position 348 (E348C) with either a fluorescence quencher (QSY9) or a fluorescent dye (Cy3 or Cy5). This position of labeling allows monitoring of EF-Tu interactions with fluorescent derivatives of ribosomal protein L11 (labeled at position 87) and aa-tRNA (labeled in the dihydroU loop). These three positions form an almost equilateral triangle within the ribosome, at distances that are appropriate for sensitive monitoring by single molecule fluorescence resonance energy transfer (smFRET). Two kinetic steps, denoted as TC association (4.7±0.3×107 M−1s−1) and EF-Tu dissociation (12±1 s−1), are found with Cy3-Cy5 FRET pairs or Cy3-QSY9 pairs placed on L11/EF-Tu, tRNA/EF-Tu (A-site) or tRNA/tRNA (A-site/P-site) pairs. The reaction rates are almost independent of labeling strategy and agree with ensemble measurements. At 10 ms time resolution, the FRET between L11/EF-Tu and tRNA/EF-Tu (A-site) pairs showed only one EF-Tu bound conformational state that is detectable during the tRNA selection process, providing strong evidence that, at this time resolution, EF-Tu loses proximity essentially simultaneously with both L11 and aa-tRNA. Alternating-laser excitation experiments demonstrate that, following EF-Tu dissociation, a fraction of aa-tRNA remains stably bound to the ribosome, corresponding to aa-tRNA that has successfully accommodated into the A-site, while the remainder dissociates rapidly, presumably due to rejection via proofreading.Supported by NIH (GM080376, GM071014 and HG004364) and NIST (70NANB7H0711).