Helena, of which only degenerate sequences have been shown to be present in some species (Drosophila melanogaster), whereas putatively active sequences are present in others (D. simulans). Combining experimental and population analyses with the sequence analysis of the 12 Drosophila genomes, we have investigated the evolution of Helena, and propose a possible scenario for the evolution of this element.Several studies have shown that genomes contain a mixture of transposable elements, some of which are still active and others ancient relics that have degenerated. This is true for the non-LTR retrotransposon Drosophila have the Helena transposable element at different stages of its evolution. The copy number is highly variable among these species, but most of them are truncated at the 5' ends and also harbor several internal deletions and insertions suggesting that they are inactive in all species, except in D. mojavensis in which quantitative RT-PCR experiments have identified a putative active copy.We show that six species of Helena was present in the common ancestor of the Drosophila genus, which has been vertically transmitted to the derived lineages, but that it has been lost in some of them. The wide variation in copy number and sequence degeneration in the different species suggest that the evolutionary dynamics of Helena depends on the genomic environment of the host species.Our data suggest that  Drosophila melanogaster genome [Transposable elements (TEs) are ubiquitous components in prokaryotic and eukaryotic genomes. They constitute the largest part of some of them , and plar genome ,4, aboutr genome , and up r genome . Why sper genome ,8, and tr genome  that mayr genome ,11 to epr genome .Several studies have shown that genomes harbor a mixture of TEs, some of which are still active, whereas others are ancient relics that have degenerated -16. DegrHelena, which is a 4,912 bp LINE [D. virilis species [melanogaster subgroup, as well as in D. pseudoobscura [Helena has been shown to be present at different stages of its life-cycle in natural populations of D. melanogaster and D. simulans. Only degenerate copies were found in D. melanogaster [D. simulans several different types of sequences have been identified, ranging from highly degenerate to putatively active ones [Helena in these two closely-related species has shown how important the host genome can be in the evolution of a TE, and how important it is to analyze specific TE families in a wide spectrum of species. This is possible now that the genome sequences of 12 Drosophila species [Helena can be considered to provide an ideal model system for investigating TE evolution across a range of species.One example of this type of TE evolution is the retrotransposon  bp LINE  first re species , and latoobscura . Helena nogaster ,19,21,22ive ones . The ana species -26 are aHelena sequences using a combination of experimental and population analyses with sequence analyses of the 12 Drosophila genomes, and we propose a possible scenario for the evolution of the element in the different host genomic environments that influence the "fate" of TEs.We investigated the evolution of Helena already identified in the draft sequence of the D. simulans genome [melanogaster species group [Helena-like elements in the other 10 Drosophila genomes. We identified Helena reference sequences in all the genomes  with percentage identities to the BS element that were higher than or similar to those to Helena . BS is a previously-described LINE element that belongs to the same "jockey clade" as Helena [BS and Helena have also been reported in D. melanogaster, and it has suggested that they may constitute a new family, named Helena/BS [Helena elements, and performed no further analysis of them. Our results for D. pseudoobscura are not consistent with previous data reported by Petrov et al. [Helena sequences in D. grimshawi, which is consistent with the fact that this a TE-poor genome [Using the full-length copy of s genome , and 23 es group , we perfs Helena . SequencD. sechellia reference sequence . The first ORF is 1,732 bp, and the second 2,870 bp long. Both ORFs are interrupted by three premature stop codons, and the pol-like protein contains only the reverse transcriptase domain.The melanogaster group, the reference copies are smaller than the Helena reference copy in D. simulans. In D. erecta  to 4,888 bp (D. sechellia). The average percentage identity of the copies with the reference within each species ranged from 91.51% (D. ananassae) to 95.95% (D. yakuba). Size-divergence between these copies results from the presence of indels that occur throughout the sequences  to 43.1% (D. melanogaster). These values are closer to the intergenic values for these species (35.3% – 39.9%) than to those for the gene regions (46.8% – 50.4%), suggesting that Helena is more abundant in the non-transcribed genomic environment [D. melanogaster is the only one in which the GC values were intermediate between those of the intergenic and gene regions. This could simply reflect the better annotation available for the D. melanogaster genome.Table ironment ,28-30. THelena-related sequences form a monophyletic clade . The other group contained D. ananassae, D. virilis and D. mojavensis. This phylogeny is typical for a TE with vertical transmission.e Figure  that incin-silico analyses show that only D. simulans and D. mojavensis harbor full-length, putatively active copies, we performed Southern blot analyses on several populations of these two species to infer their TE activity  used to normalize qRT-PCR is equally expressed in both species , in D. mojavensis (repleta group) and in D. virilis (virilis group), as well as in D. melanogaster and D. simulans, which had been studied previously [D. pseudoobscura, D. persimilis and D. willistoni, the small copies that we found displayed similar percentage identities to Helena as to BS, a non-LTR retrotransposon related to Helena. Sequences with the same kind of similarity have been reported in D. melanogaster, and were grouped by the authors as Helena/BS family [D. pseudoobscura, D. persimilis and D. willistoni sequences we found can be included in this family. We agree with the suggestion that there could be members of the Helena/BS family in these species as shown in D. melanogaster [Helena that had been previously described (GenBank AF012036) in a strain  of D. pseudoobscura [Helena is a monophyletic group of sequences patchily distributed in the species of the two subgenera of the genus Drosophila . We would expect to find a similar scenario for D.erecta, which is also a specialized species with a small population size [mariner element, the observed copy number of Helena is not in fact consistent with this hypothesis [Our analysis of the copy numbers reveals a strikingly variable distribution of r of TEs ,33, withr of TEs . This seion size ,36, and ion size ,38. Howepothesis . This meDrosophila subgenus, even though D. mojavensis has a higher copy number than D. virilis. What is more striking is that the only potentially full-length active copies identified were detected in D. mojavensis, together with the other potentially active copy previously described in D. simulans [D. mojavensis and D. simulans suggest that Helena has recently displayed transpositional activity. However, we confirmed that the transcriptional activity is low in D. simulans, as had previously been suggested [Helena is being lost in this species. In D. mojavensis, the situation is completely different. We have observed high levels of Helena transcripts in several populations, confirming its activity. Moreover, expression varied between different populations, indicating that Helena must be present at different stages of its evolutionary cycle, i.e. different stages of activity, in different populations within this species.We did not observe any major differences in copy number between the two species of the simulans . The Souuggested , and thiHelena is influenced by the host species, resulting in differences in copy number, degradation and activity. In all the species analyzed, D. mojavensis is the only one in which the Helena has survived, which gives us a unique opportunity to observe the "fate" of this TE. However, it is crucial to investigate natural populations from this and other species in the repleta group in order to find out how widespread the scenario described here actually is, and to understand the process and speed of the degradation and extinction of Helena.Our analysis has shown that the evolution of Drosophila have Helena transposable elements at different stages of its evolution, and may represent different stages of the TE evolutionary cycle. The copy number is highly variable in different species, but most of them are truncated at the 5' ends and display several internal deletions and insertions. In all the species analyzed, Helena has only survived in D. mojavensis, and this gives us a unique opportunity to track the "fate" of this TE.Here we show that six species of Drosophila Genome Sequencing Projects used in this study are listed in the Table Helena element were carried out using the 4,912 bp full-length sequence of Helena characterized in D. simulans [Helena from the melanogaster group [Helena, BS [X [Drosophila data set were performed using High Scoring Pairs (HSPs) within BLAST algorithms [-10. The sequences correspond to the three best BLAST hits, which were more than 80 bp in length, and were selected and extended to include 3 kb of the flanking regions. These sequences were analyzed using the ORF-FINDER program to identify putative occurrences of coding regions [Helena sequence in each of the 10 Drosophila species to be considered as a reference copy within each genome. To determine the copy number in each species, the previously-determined reference copy was blasted against the full genomes. Significant matches were required to be more than 80 bp long, and to have at least 80% identity [Helena that were separated by less than 200 bp were considered to be a single insertion. For each genome, the copies have been aligned with the reference, using MUSCLE [Helena elements were compared to those of the intergenic regions and genes (exon plus introns) in the other Drosophila genome versions, as noted in the Table The draft sequence data from the 10 related simulans  as the qlena, BS  and X [3a, BS [X  were fougorithms  with an  regions . Conservidentity . Regionsg MUSCLE  and the g MUSCLE . The flag MUSCLE . The GC Jockey (M22874), TART (U14101), Doc (X17551), F (M17214), BS (X77571) and X (AF237761) of D. melanogaster; Jockey of D. funebris (M38437); Amy of Bombyx mori (U07847); JuanA of Aedes aegypti (M95171); JuanC of Culens pipientis (M91082); NCR1th of Chironomus tentans (L79944); Helena of D. mauritiana (AF012043), D. simulans and D. melanogaster [LINEs of the Jockey clade, and the six Helena reference sequences described here , TART U4101, DocJockey M2874, TARand X AF27761 of D, Doc X1751, F . Genomic DNA was prepared from 50 adult flies [Hind III, which has no restriction site within the Helena sequence, so that each hybridized fragment would correspond to a single genomic insertion. The restricted fragments were separated in 1% agarose gels, and transferred to Hybond N+ membranes . Blots were prehybridized for 1 h at 60°C in 5× SSC, in 5% dextran sulfate, subjected to 20-fold dilution of the liquid block, and hybridized overnight with the probes. Blots were washed twice with 0.2× SSC, 0.5% SDS, and then exposed to autoradiographic film for 20 minutes at room temperature. A 644 bp Helena sequence amplified from plasmid AF012044 (DsechF: 5' AGGATTTGTCATGCCACGCT 3' e DsechR R 5' TGTTTGGTGCTGCCATGTGT 3'), and a 674 bp sequence, corresponding to the RTase of D. mojavensis Helena (DmojF: 5' TAAGAGGCCATAGTACGGAGCAGGTA3' and DmojR: 5' GCGAATTGGAACAGGCTAACGCAT 3'), were used as probes for the D. simulans and D. mojavensis populations, respectively.The occurrence of lt flies  and digeHelena RTase in different populations of D. simulans and D. mojavensis  that amplify 193 bp and 204 bp respectively. Quantitative PCR cycling conditions were 5 min at 95°C (1 cycle), 15 s at 95°C, followed by 10 s at 62°C and 20 s at 72°C (50 cycles). A negative control for DNA contamination of cDNA of each population (without Thermoscript enzyme) was tested (data not shown). Reactions were done in duplicate, and standard curves were calculated from serial dilutions of specific amplified PCR fragments. The quantity of the transcripts was estimated relative to the RP49 expression (qPCR fragments of 182 bp and 167 bp for D. simulans and D. mojavensis respectively). Primers were: RP49_DsimF: 5' CGGATCGATATGCTAAGCTGT 3', RP49_DsimR: 5' GCGCTTGTTCGATCCGTA 3', RP49_DmojF: 5' GTCGTCGCTTCAAGGGCCAAT 3', RP49_DmojR: 5' ATGGGCGATCTCACCGCAGTA 3'. In both species, RP49 expression is equivalent  allowing relative expression of Helena to be comparable between D. mojavensis and D. simulans. Hence, Helena transcripts plotted in Figure Helena transcripts normalized by the quantification of RP49 transcripts for each strain.The expression profiles of DOA: dead on arrival; LINE: long interspersed nuclear element; LTR: long terminal repeat; ORF: open reading frame; TE: transposable element; RTase: reverse transcriptase.AG carried out the molecular and genetic studies, AG, FRL and EL did the bioinformatic analyses, CV and CMAC designed and coordinated the study. All the authors contributed to data analyses and the writing of the paper. All the authors have read and approved the final manuscript.Helena reference copiesSequences of the . Helena sequences used to determine the structure of the element, for each species.Click here for fileHelena copies in the Drosophila sechellia sequenced genome. The data provided is a list of D. sechellia copies.Click here for fileHelena copies in the Drosophila erecta sequenced genome. The data provided is a list of D. erecta copies.Click here for fileHelena copies in the Drosophila ananassae sequenced genome. The data provided is a list of D. ananassae copies.Click here for fileHelena copies in the Drosophila yakuba sequenced genome. The data provided is a list of D. yakuba copies.Click here for fileHelena copies in the Drosophila virilis sequenced genome. The data provided is a list of D. virilis copies.Click here for fileHelena copies in the Drosophila mojavensis sequenced genome. The data provided is a list of D. mojavensis.Click here for fileHelenaSchematic representation of . Schematic representation of Helena copies in D. sechellia, D. yakuba, D. erecta, D. ananassae, D. mojavensis and D. virilis. The sequences represented have at least 90% identity and 50% of the length of the reference copy, and with e-values of less than 10e-10. Spaces = indels. The first schematic representation is the reference copy in each species. White = gag. Gray = RTase.Click here for fileSpecies and strains, geographic origin and year of collection. The data provided is a list of D. simulans and D. mojavensis used in this study for analysis of Helena activity in natural populations.Click here for filerp49 control gene and HelenaMean CT of the . Ct comparison between rp49 (reference) and Helena real time PCR assays of D. simulans and D. mojavensis. Gray = mean CT of D. simulans. Black = mean CT of D. mojavensis. Std = standard deviation.Click here for file


Enterobacteriaceae carrying extended spectrum β-lactamases(ESBLs), Vancomycin resistant Enterococcus (VRE), methicillin resistant Staphylococcus aureus (MRSA) have emerged as significant pathogens. Many of the health care workers, who come in contact with patients regularly, are carriers of many of these organisms. They are potential source of transmission of these organisms.Antimicrobial resistance among nosocomial pathogens is a significant problem in most clinical settings adding to the cost of medical care and the morbidity and mortality of patients. This study was undertaken to determine the extent of prevalence of faecal carriers of multi-drug resistant organisms among health care workers.A total of 211 faecal samples were processed on MacConkey agar with Ceftazidime, bile esculin azide agar, Salmonella – Shigella agar and blood agar as per standard microbiological methods. Identification of the organisms and ESBL production was done as per CLSI guidelines.Of the 211 faecal samples received, 66(31.2 %) had growth of VRE’s , 99(46.9 %) had ESBLs and 13(6.6 %) had Salmonella spp.Although several studies have been reported on faecal carriage of ESBLs and VRE’s in patients, there has been no report on health care workers. Since we have high incidence of ESBLs and VRE’s in our hospital, this study was undertaken. Health care workers have a high risk of transmitting as many of our patients are neutropenic. Many of our patients are VRE and ESBLs carriers who have got the infection from health care workers. This needs to be further investigated.None declared.


Background: Kaposi’s sarcoma (KS) is a rare neoplasm of lymphatic endothelial cells. Human herpes virus 8 (HHV-8) is considered to be a necessary, but not sufficient causal agent of KS and additional cofactors remain unknown. In this study we evaluated the expression of human β defensin (HBD)-3 and LL-37 in cutaneous lesions of KS in comparison to the healthy skin of normal subjects.Methods: We performed a quantitative immunohistochemical study of HBD-3 and LL-37 on skin lesions from 18 patients having KS, and on healthy skin from 12 normal controls.Results: HBD-3 and LL-37 were significantly upregulated in epidermal and dermal specimens of all KS patients in comparison to normal skin of healthy controls. The immunostaining score of dermal HBD-3 was significantly higher in nodular lesions (9.6 ± 2.4) versus plaque lesions (4.1 ± 2.2), P = 0.001. Also the immunostaining score of dermal LL-37 was significantly higher in nodular lesions versus plaque lesions (P = 0.001).Conclusions: We have demonstrated for the first time that HBD-3 and LL-37 are significantly upregulated in lesional skin of KS in comparison to the skin of healthy controls. The obtained data suggest a possible involvement of these antimicrobial peptides in the pathogenesis of KS. However, the biological significance of HBD-3 and LL-37 in KS lesions needs further research. The four clinico-epidemiological forms of KS are: a classic form typically affecting elderly men of the Mediterranean, the endemic presence in Southern Africa, the epidemic form in patients infected with human immunodeficiency virus (HIV) and the iatrogenic KS complicating iatrogenic immunosuppression2.Kaposi’s sarcoma (KS) is a rare disease of lymphatic endothelial cells frequently evident as multiple vascular cutaneous and mucosal nodules. Lymph node and visceral manifestation is seen in cases of strong immunosuppression or aggressive disease3. HHV-8 is present in the vast majority (> 90%) of spindle cells and in the neoangiogenic vessels6. It is considered to be a necessary, but not sufficient causal agent of KS. Besides immunosuppression and AIDS, additional cofactors remain unknown7.KS is strongly associated with human herpes virus 8 (HHV-8), which is implicated in the pathogenesis of all forms of KSet al.8 speculated that early stage KS is a reactive inflammatory angiogenic process that may be triggered or enhanced by infection with HHV-8 with many lymphocytes and monocytes infiltrating the lesion. These cells produce inflammatory cytokines including interferon-y (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-2, IL-6 and others9. The inflammatory cytokines induce the recruitment of circulating cells into tissues, induce the production of angiogenic factors that mediate angiogenesis and edema and activate endothelial cells to acquire the phenotype of KS spindle cells10.Ensoli11. This diverse group of peptides is separated into several classes. Defensins and cathelicidin  are considered the two main groups of AMPs in human skin12. It was confirmed that human β defensin (HBD)-3 and LL-37 demonstrated the ability to inhibit viral infection, affecting both enveloped RNA and DNA viruses and non-enveloped viruses21. Moreover, beside their role in protecting the host from the invasion of pathogens, HBD-3 and LL-37 have immunomodulatory properties in inflammatory skin diseases as psoriasis23 and rosacea24.Antimicrobial peptides (AMPs) are an evolutionarily conserved component of the innate immune system that defend against invading bacteria, viruses and fungi through membrane or metabolic disruption25, this study was carried out to assess the expression of the two main antimicrobial peptides (HBD-3 and LL-37) in skin lesions of KS in comparison to normal skin from healthy subjects.Based on the antiviral and immunomodulatory effect of HBD-3 and LL-37, as well as the angiogenic role of LL-37This is a case control study including a convenient sample of 18 Egyptian patients with KS recruited sequentially from the outpatient clinic of the Dermatology unit of Mansoura University Hospital from 2006 to 2011. The diagnosis was based on classic clinical, histopathological features of KS and immunohistochemical staining using the endothelial marker CD34. The controls were 12 healthy subjects that underwent surgical removal of benign lesions.26, gastrointestinal tract endoscopies were done only to patients with widespread skin lesions (four patients) to evaluate the presence of gut lesions. Inclusion criteria were newly diagnosed patients without previous treatment of KS. Only patients with fully developed lesions such as plaques and nodules were included in this study. Patch (macular) stage lesions were not included. Staging of KS was assigned using the classification proposed by Schwartzet al.27 and modified by Schwartzet al.28 as follows:Stage I: Localized nodular KS, with ≤ 15 cutaneous lesions or involvement restricted to one bilateral anatomic site, and few, if any gut nodules.Stage II: Includes both exophytic destructive KS and locally infiltrative cutaneous lesions and locally aggressive KS or nodular KS, or > 15 cutaneous lesions or involvement of more than one bilateral anatomic site, and few or many gut nodules.Stage III: Widespread lymph node involvement, with or without cutaneous KS, but with limited if any visceral involvement.Stage IV: Widespread KS, usually progressing from stage II or III, with involvement of multiple visceral organs with or without cutaneous KS.A thorough general and skin examination of patients was done, focusing on morphology, localization and number of skin lesions, as well as oral mucosa and lymph nodes examination. A blood cell count, blood chemistry, ELISA test for HIV serology, chest X-ray and abdominal ultrasound were conducted. As classic KS rarely affects other organsA lesional skin biopsy (intact lesion without evidence of secondary infection) was obtained from each patient. Biopsy from 12 normal subjects served as controls. They were obtained from normal skin beside benign neoplasms such as melanocytic naevus, infundibular cyst and lipoma of age, sex and localization-related subjects. Informed consent was taken from all participants. The study protocol was approved by the ethical committee of the College of Medicine of Mansoura University.2O2. After blocking, sections were incubated at room temperature for 60 minutes with antibodies to HBD-3 using rabbit polyclonal antibody  at a dilution of 1:2 and with human LL-37 monoclonal antibody  at a dilution of 1:500. Diaminobenzidine (DAB) reaction was used for visualization, followed by a hematoxylin counterstain. Negative controls for all studies were obtained by omission of the primary antibodies of an adjacent section to assess the degree of non-specific staining. All slides were examined by Olympus light microscope.All specimens were fixed in formalin 10% and sections from paraffin blocks (3–4 um) were cut on glass slides for routine hematoxylin and eosin stains as well as immunohistochemical staining using an bindirect avidin-biotin-peroxidase method. Endogenous peroxidase activity was blocked with 0.6% Het al.20 as follows: 0, none; 1, stratum corneum only; 2, stratum corneum and stratum granulosum; 3, stratum corneum, stratum granulosum and stratum spinosum; 4, whole epidermis. Next an intensity score was assigned, which represented the average intensity of positive epidermal cells as follows: 0, none; 1, weak; 2, moderate; 3, intense staining. Both scores were then added to obtain a total epidermal score which ranged from 0 to 7. Skin samples of psoriasis that are known to exhibit high expression of HBD-3 and LL-37 in the epidermis were used as positive controls. Immunostaining results of dermal lesions of KS were scored as previously described29. Immunoreactivity of HBD-3 and LL-37 were evaluated in the two basic component of this disease . The percentage of positive cells was graded from 0 to 4 as follows: 0, zero to 10%; 1, 11 to 33%; 2, 33 to 66%; 3, 67 to 90%; and 4, 91 to 100%. Specimens were considered immunopositive when more than 10% of cells showed clear evidence of immunostaining. The intensity of immunostaining was rated as follows: 0, none; 1, weak; 2, moderate; and 3, intense. Because KS lesions frequently showed significant intra-specimen heterogeneity, a score was calculated in which the percentage positive rating was multiplied by the intensity rating. Each component of the lesion was scored independently and the results were added up. The score was calculated on 6 to 10 representative high–power fields after examination of the totality of lesion present in one section for each case. Sweat glands served as internal positive controls for LL-37. Inflammatory cells (monocytes and macrophages) served as internal positive controls for both studied AMPs. However, specimens from healthy subjects served as negative controls. Slides were scored by two independent and trained researchers (the authors).Immunostaining results of HBD-3 and LL-37 were evaluated in four layers of the epidermis and the proposed score was modified from Meyer-HofferData were analyzed using SPSS version 16. Qualitative variables were presented as numbers and percentages. Quantitative variables were presented as mean ± SD (median) and a Mann-Whitney test was used for group comparison. Spearman’s correlation coefficient was used to calculate correlation between variables in patients with KS. P ≤ 0.05 was considered statistically significant.The mean age of the patients  was 63.9 ± 7.4 years (ranged from 55 to 82 years). The age and sex-matched control group were eight males and four females with a mean age of 63.2 ± 3.7 years (ranged from 45 to 67 years). All investigated patients had classic KS without clinical evidence of immunodeficiency and no history of immunosuppressive drug intake. They had normal total and differential leukocytic count, normal blood chemistry and negative HIV serology. Fourteen KS patients were classified as stage I (≤ 15 cutaneous lesions) and 4 patients as stage II (> 15 cutaneous lesions) . No patiImmunohistochemical staining of HBD-3 and LL-37 were generally cytoplasmic, and membranous staining was also seen. There was significant upregulation of HBD-3 and LL-37 in both epidermal and dermal specimens of all studied patients in comparison to normal skin of healthy controls. The expressions of HBD-3 and LL-37 were seen in the epidermis as well as in the dermis   with difThe immunostaining scores of HBD-3 and LL-37 are detailed inAge in years Sex: M=male, F=female Duration of lesion in monthsClick here for additional data file.13, vaccinia virus14, and HIV15. Similarly LL-37 is found to inhibit replication of vaccinia virus16, kill HSV17, is effective against adenovirus18 and inhibits HIV-1 replication in peripheral blood mononuclear cells19. Furthermore, HBD-3 expression was shown to be upregulated in human papillomavirus induced lesions21.Recently many reports about the antiviral effect of defensins and cathelicidin have been published. The antiviral activity of HBD-3 has been reported against herpes simplex virus (HSV)In our study we investigated for the first time the expression of HBD-3 and LL-37 in KS. They were significantly upregulated in epidermal and dermal  regions of all studied KS lesions in comparison to normal skin of healthy subjects. These findings suggest a potential role of HBD-3 and LL-37 in the pathogenesis of KS. Furthermore, we found that the expressions of these AMPs were increased with progression of KS lesions from plaque stage to nodules. The stage-related differences that we found in HBD-3 and LL-37 expression add more support to the possible role of these AMPs in the progression of KS.3. HBD-3 and LL-37 might exhibit antiviral activity against HHV-8 as shown for the related HSV17.The induction of HBD-3 and LL-37 in KS may be in response to infection with HHV-8 that is implicated in the pathogenesis of all forms of KS30. It was found that LL-37 increases natural killer cell proliferation by activating the Toll like receptor 9. LL-37 increases proinflammatory cytokines at the dendritic cell level, promoting CD4+ TH1 cell responses31. LL-37 can synergize with IL-1β to increase the production of cytokines, such as IL-6, IL-8 and IL-10, and chemokines, such as the cc-chemokine ligand 232. Similarly to cathelicidin, HBD-3 has chemoattractant properties on different cell types such as T lymphocytes and dendritic cells33. Furthermore, HBD-3 and cathelicidin induce the production of diverse chemokines and cytokines such as monocyte chemotactic protein-1, macrophage inflammatory protein-3, interferon-inducible protein-10, IL-1, IL-6, IL-8, IL-10 and TNF-α mainly in keratinocytes35.In addition to functioning as direct antimicrobial compounds, AMPs can function as chemokines36.Taken together, it is possible that HBD-3 and LL-37 can function in KS by promoting Th1 cell responses or by increasing the production of cytokines such as IL-1, IL-6, IL-8 and TNF-α. Several cytokines have been shown to support the growth of cultured KS spindle cells: these include IL-1β, IL-6, the soluble IL-6 receptor 2 and TNF-α+ T cells were present in KS lesions. Also, monocytes-macrophages and dendritic cells were present in lesions8. Furthermore, Sirianni and coworkers37 had shown that NK cell function is important for the control of latent HHV-8 infection and abrogation of this important immune response can lead to a more aggressive KS disease. Moreover, besides LL 37’s function as chemoattractant to T-cells and monocytes, it can stimulate angiogenesis through an increase in endothelial cell proliferation and vessel formation25. AMPs can indirectly sustain angiogenic signals by production of TNF-α and IL-1. These cytokines are powerful inducers of vascular endothelial growth factors38. So the implicated role of HBD-3 and LL-37 in KS may be inhibition of HHV-8 replication, production of several inflammatory cytokines and stimulation of angiogenesis. Ensoliet al.8 speculated that KS is a multistep process including not only HHV-8 infection, but also genetic and angiogenic factors, as well as the production of several inflammatory cytokines. Furthermore, Kawsaret al.39 reported that HBD-2 is expressed in vascular endothelial cells associated with KS. The authors suggested that HBD-2 may play a role in tumor angiogenesis and metastasis.HHV-8-specific cytotoxic T-lymphocyte and T helper responses are found in KS patients, and CD4 and CD828. Whatever the nature of KS, HBD-3 and IL-37 may serve as a growth factor for KS. This is supported by the finding that the expression of these AMPs were increased with progression of KS lesions from plaque stage to nodules in our study. Recent evidence suggests cathelicidin LL-37 to be a putative growth factor for various human cancers43. Similarly, HBD-3 may play an important role in the development and progression of oral cancer. HBD-3 stimulated the expression of tumor-promoting cytokines, including IL-1α, Il-6, Il-8 and TNF-α in macrophages44. These cytokines are also important in the progression of KS36. Further study to elucidate the hypothesis that HBD-3 and LL-37 serve as progression factor for KS is therefore recommended.It remains unclear whether KS itself is a true malignancy, a reactive proliferation or both22. However IL-6 is a potent inducer of LL-3745. Interestingly, these inflammatory cytokines are abundant in KS lesions9. Furthermore, the possibility of induction of these AMPs by HHV-8 protein can be supported by the finding of increased expression of HBD-3 and LL-37 with progression of KS lesions from plaque into nodular stage in our study. Similarly, increased load of HHV-8 has been found with progression of KS lesions5. Further study is recommended to verify this point.It is not known whether upregulation of HBD-3 and LL-37 in KS lesions is signaled indirectly by locally produced proinflammatory cytokines or directly by HHV8 molecules. HBD-3 is upregulated by proinflammatory cytokines, TNF-α, IFN-γ and IL-1βHBD-3 and LL-37 are for the first time shown to be significantly upregulated in KS skin lesions as compared with skin of healthy controls. The obtained data suggest a possible contribution of HBD-3 and LL-37 in the innate and adaptive immune response target against HHV-8. Another possibility is the potential involvement of these antimicrobial peptides in the pathogenesis of KS. However, the biological significance of HBD-3 and LL-37 in KS lesion needs further research.Written informed consent for publication of clinical details was obtained from the patients. In this study the authors have evaluated for the first time the expression of human β defensin (HBD)-3 and LL-37 in cutaneous lesions of KS and showed that these antimicrobial peptides (AMPs) are significantly up-regulated in KS skin lesions as compared with skin of healthy controls. The results are of potential interest to the understanding of KS pathogenesis, however there are several points that should be addressed which can improve the work.1. The authors should explain why they excluded from the immunohistochemical evaluation macular lesions considering that these lesions are the one with the highest degree of inflammatory infiltrate, and given the role of the peptides in inflammatory skin diseases.2. It is not clear how the scoring for the immunohistochemical analysis was calculated . In particular it seems that a total epidermal score (ranging from 0 to 7) was obtained by the addition of a score representing the extension of the expression detected in the four epidermal layers with an intensity score, which represented the average intensity of positive epidermal cells. Then the percentage of positive cells is calculated, and a grading from 0 to 4 was assigned. Finally, the authors stated that, because KS lesions frequently showed significant intraspecimen heterogeneity, a score was calculated in which the percentage positive rating was multiplied by the intensity rating. Each component of the lesion was scored independently and the results were added up, so how were all these scorings were combined? Which are the scores indicated in Table 1-3? Finally, it seem that the scoring was done in the open and this may represent a bias.3. In the Table summarizing the “Demographic, clinical and immunohistochemical scoring of HBD-3 and LL-37 of studied population” it would be important to indicate also the patient clinical stage.4. Figure 1: arrows should be included in panels to point out positive cells, particularly in panels E and I. In addition, in order to correctly compare the expression of LL37 in plaque and nodules, panel I and J should have the same magnification.5. The authors stated in Discussion that the expression of AMPs was increased with progression of KS lesions from plaque stage to nodules. However, to support of the possible role of these AMPs in progression of KS lesions from stage III and IV patients should be studied.Kawsar et al. (2010). This citation should be included in the manuscript and results discussed.6. The manuscript does not cite a paper reporting that human beta defensin-2 (HBD-2) is expressed by endothelial cells present in Kaposi’s sarcoma lesions , and that inflammatory mediators induce HBD-2 expression by normal endothelial cellsI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Firstly I thank Professor Barbara Ensoli for her valued comments. Below, I have some answers to the points she has made:1. Patch lesions were not included in this study because it is very difficult to discriminate between tumor cells and activated endothelial cells in this stage of KS (Reference number 29 in our manuscript).2. In this study we used two different independent (i.e not added to each other) scores, one epidermal score and the other is dermal score as detailed in material and method section.3.Tumor stage is presented in table 1. It is based on clinical examination and radiological study as mentioned in material and method section.4. The panel i is replaced by another one with higher magnification (x400) and arrows are included.5. Unfortunately none of the studied patients had stage III or IV.6. We have included the findings ofKawsar et al. 2010 in the discussion.Many Thanks. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Although I approve this paper, I have a few comments:- The authors stated that “we performed a quantitative immunohistochemistry” however, it seems that they have done a qualitative assessment as quantitative immunohistochemistry is done by image analysis program which is reproducible, avoids interpersonal variations and potential bias. Furthermore, it provides quantitative data. If the authors wished to conduct quantitative immunohistochemistry then they would have to revise the statistical tests used.- It seems that all authors evaluating the immunohistochemistry results were aware of the study design. This may be a potential source of bias.- The authors used specimens of psoriasis as a positive control. Psoriasis is an inflammatory but not infective dermatosis which raises an argument against the impact of the expression of AMPs in KS.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Firstly I thank Professor Naser for his valued comments. Below, I have some answers to the points he has made:1. We performed a quantitative immunohistochemical analysis because the scoring of epidermal and dermal immunostain of the studied AMPs is a quantitative in nature and not a qualitative measure. We do agree that the image analysis program could be more accurate, but unfortunately this was not available to us. Furthermore, false positive results by positive staining of sweat glands (which is not a component of the tumor) with LL-37 can be obtained by the image analysis program used, so there is no need for reanalysis the data.2. The specimens prepared hematoxylin, eosin and the immunohistochemical analysis were coded numerically. So the first two authors evaluating the immunohistochemistry results were unaware about the clinical data of the patients at the time of pathological evaluation. Even so, the stage of the disease (plaques versus nodules) can be determined by hematoxylin and eosin stained sections; Fernández-Figueraset al. (Reference 29 in our manuscript).3. Up until now the pathogenesis of KS has not been clear. HHV8 is considered to be a necessary, but not sufficient causal agent of KS. Apart from immunosuppression and AIDS, additional cofactors still remain unknown (Reference number 7 in our manuscript). This means that the etiology of KS is not just a viral infection. LL-37 was found to be highly expressed in malignant melanoma and squamous cell carcinoma . Similarly, HBD-3 was found to be highly expressed in oral squamous cell carcinoma . These AMPs have been found to be expressed in some inflammatory, infective and malignant diseases, so further research is needed to elucidate the biological function of these AMPs in KS.


Bovine tuberculosis (bTB) is a chronic debilitating disease and is a cause of morbidity and mortality in livestock, wildlife and humans. This study estimated the prevalence and risk factors associated with bovine tuberculosis transmission in indigenous cattle at the human-animal interface in the Serengeti ecosystem of Tanzania.A total of 1,103 indigenous cattle from 32 herds were investigated for the presence of bTB using the Single Intradermal Comparative Tuberculin Test. Epidemiological data on herd structure, management and grazing system were also collected.The apparent individual animal prevalence of tuberculin reactors was 2.4% , 1.7 – 3.5%), whereas the true prevalence was 0.6% CI, 0.6 – 0.7% as indicated by a reaction to avian tuberculin purified protein derivatives (PPD) which is more than 4 mm greater than the reaction to avian tuberculin PPD. The results showed that 10.6%  showed non-specific reactions . The herd prevalence of 50% (16/32) was found. Tuberculin skin test results were found to be significantly associated with age, location, size of the household and animal tested. Of 108 respondents, 70 (64.8%) individuals had not heard about bovine tuberculosis at all. Thirty five percent (38/108) of respondents at least were aware of bTB. About 60% (23/38) of respondents who were aware of bTB had some knowledge on how bTB is spread. Eighty one percent (87/108) of respondents were not aware of the presence of bTB in wildlife. There is regular contact between cattle and wild animals due to sharing of grazing land and water sources, with 99% (107/108) of households grazing cattle in communal pastures.The study has demonstrated a high reported interaction of livestock with wildlife and poor knowledge of most cattle owners concerning bTB and its transmission pathways among people, livestock and wildlife. Although the overall proportion of animals with bTB is relatively low, herd prevalence is 50% and prevalence within herds varied considerably. Thus there is a possibility of cross transmission of bTB at wildlife-livestock interface areas that necessitates use of genetic strain typing methods to characterize them accurately. Mycobacterium bovis, is a chronic debilitating disease of livestock, wildlife and humans [M. bovis worldwide [Bovine tuberculosis (bTB) caused by d humans ,2. Cattlorldwide , while morldwide . Aerosolorldwide ,4. Otherorldwide . Informaorldwide .Bovine tuberculosis is a disease with potential public health and economic importance  since itM. bovis has been isolated from human lymph biopsies [Previously published information indicates that bTB is endemic in Tanzania’s cattle, with regional prevalences ranging from 0.2% to 13.2% ,9-15 sugbiopsies ,19, cow’biopsies ,19 and fbiopsies . In theibiopsies  revealedbiopsies  and proxbiopsies -26.transhumance, which refers to a pattern of seasonal movement between dry season and wet season pastures. Given the husbandry practices and proximity to wildlife, studies are needed to explore the disease status, dynamics in the ecosystem and risk factors for bTB infection in animals and ultimately also humans. Here we present findings of a study conducted to determine the prevalence of bTB and risk factors associated with bTB infection in pastoral and agro-pastoral communities at the livestock-wildlife interface in the Serengeti ecosystem in Tanzania.The Serengeti ecosystem comprises an ecosystem defined by annual movement of herds of ungulates  interact2) and 176,609 (16.1 persons per km2), respectively [The study was carried out in the Serengeti and Bunda districts of the Mara region, and the Ngorongoro district of Arusha region in northern Tanzania, from October to December 2011  in the Ngorongoro district (E35° and 36°E and S 2° and 4°), of the Arusha region is part of the Serengeti ecosystem extending from the plains of Serengeti National Park (SNP) in the north-west, to the eastern arm of the Great Rift Valley. Both SNP and NCA are part of the UNESCO  Biosphere Reserve. The Ngorongoro district has a population of 129,776 (9.6 persons per km2) [Acacia xanthonhloea and Rauvolfia caffra. The conservation area is dominated by pastoralists of the Maasai ethnic group who constantly move livestock in search of pasture and water where interaction with wildlife is common. Infection of cattle with bTB poses a great risk to infection in wildlife in Ngorongoro due to great interaction between cattle and wildlife as the Maasai pastoralists are found within the conservation area [The Ngorongoro Conservation Area (NCA) 8,288 km8 km2 in ion area . RainfalThe sample size for the study was calculated by using StatsDirect statistical software version 2.7.8 at 95% confidence interval based on 13.2% as the highest prevalence of bTB in indigenous cattle in Tanzania  with 2% nd animal was tested for bTB, every 4th animal was tested in the case of herds of 51–200 animals and every 12th animal was tested for bTB in cattle enclosures with herd size ≥201. This was done by gathering the cattle into an enclosure  and allowing the cattle to exit one at a time. Every 2nd, 4th or 12th animal was then selected for study. The procedure resulted into 292, 289 and 522 cattle in each group , which were finally tested for bTB leading to a total number of 1111 cattle. Readings from eight animals (8) animals could not be obtained for post PPD injection reading as the animals were not available. Regardless of gender all cattle older than 6 months were restrained by using ropes with different restraint techniques depending on the prevailing situation. Body condition in individual animals was assessed using a modified guideline described by Msangi et.al. [The study was a cross sectional study conducted in villages bordering the Serengeti ecosystem in Tanzania. A complete list by names of all villages bordering the Serengeti ecosystem in Bunda and Serengeti districts was obtained from the District Agriculture and Livestock authorities. Likewise, a list of all villages inside the Ngorongoro Conservation Area was obtained from Ngorongoro Conservation Area Authority (NCAA) veterinary unit. A convenience sample of 11 villages was selected based on their proximity to wildlife, the willingness of the pastoral community to participate in the study and availability of Veterinary Services staff experienced to perform TB testing of cattle. In some areas, experience of the local veterinary service staff with the community and nomadic lifestyle of the pastoral communities dictated the selection of herds for inclusion. A total number of 32 herds were thus available for tuberculin skin testing. For herds with cattle ranging between 1–50 herds, every 272 – BOV0) – (AV72 – AV0) and (AV72 – AV0) - (BOV72 – BOV0) respectively, where BOV0 and AV0 and BOV72 and AV72 indicate skin thicknesses prior and 72 hrs post injection of bovine and avian tuberculin respectively [Tuberculin skin testing was performed using aliquots of 0.1 mL of 2500 IU/mL bovine purified protein derivative (PPD) and 0.1 mL of 2500 IU/mL avian PPD . Bovine and avian PPDs were injected intradermally at two sites approximately 12 centimetres apart at the border of the anterior and middle thirds of one side of the neck. This was done after shaving the two sites by using a razor blade. The skin thickness was measured with callipers prior to and 72 hours after PPD injection and recorded. For young animals where there is insufficient space to inject both tuberculin PPDs into the same side of the neck, the tuberculin PPDs were injected on different sides of the neck. Clinically sick animals and cows one month pre-and post-partum were excluded from the skin test considering the expected clearly weaker cellular immune response that might result into false negative for the tuberculin test in this group of animals. Bovine positive reactors and avian reactors were identified using the following formulae , Tanzania (Reference number NIMR/HQ/R.8a/Vol.IX/1299).http://vl.academicdirect.org/applied_statistics/binomial_distribution/ref/CIcalculator.xls). Herd level prevalence was calculated as the number of herds with at least one-reactor positive animal divided by the total number of herds tested. General linear models was used to assess the association between the different risk factors  and results of single intradermal comparative tuberculin test (SICTT) of individual animals using STATA for Windows  with a p-value of <0.05 considered statistically significant. In both univariate and multivariate analysis, a random effect logistic regression analysis was performed with herd treated as a random effect to account for the difference in herd sizes and also for the fact that animals within herd could be considered as forming a cluster. In univariate analysis variables with p-value ≤ 0.25 [The data from the questionnaire were entered and analysed using SPSS for Windows . The apparent prevalence of bovine tuberculosis was defined as the number of positive reactors divided by the number of cattle in which the test was read. True prevalence was calculated based on formula described by Rogan and Gladen , TP =  cattle were not found during follow up for the second reading after 72 hours. The apparent individual animal prevalence of tuberculin reactors was 2.4% , 1.7 – 3.5%, whereas the true prevalence was 0.6% (95% CI), 0.6 – 0.7%. The apparent prevalence was comprised of 3.17% (12 out of 379) males and 2.07% (15 out of 724) females  had a high prevalence of M. bovis compared to other groups. The questionnaire results  therefore, we could not predict how many herds we might expect to be positive of the 32 considered.The variation of tuberculin skin reaction in relation to age and sex is shown in Figure ts Table  indicatets Table , particuThis study has shown an overall bTB apparent prevalence of 2.4% in cattle around the Serengeti ecosystem. Generally the prevalence in this study is consistent with studies conducted in other regions of Tanzania ,11,13-15The study has also shown that most of the cattle owners interviewed in villages had poor knowledge on bTB. Moreover, the majority of the respondents had never heard of bovine tuberculosis and were not aware of bTB presence in wildlife. The infection of pastoral and agro-pastoral cattle with bTB, poor community knowledge on the mode of transmission for bTB among people poses serious risks of infection with zoonotic diseases including bTB to people in the Serengeti ecosystem. Therefore, there is a great need for awareness creation and community involvement in planning and implementation of disease control programmes especially zoonotic diseases in the Serengeti ecosystem.The prevalence of bTB among the three districts varied considerably with significant differences between the three districts Table . AlthougM. bovis in buffalo, wildebeest and lion [M. bovis and its social behaviour of living in large herds provides favourable conditions for aerosol transmission of M. bovis to other members of the same herd [M. bovis, it is said that age alone is not enough to account for susceptibility to infection but continued opportunities to exposure to the bacterium. Previously published studies conducted in the Southern Highlands and northern regions of Tanzania [Previous studies conducted in wildlife in Serengeti have confirmed the presence of and lion . The bufame herd . The migame herd . While yTanzania ,17 indicTanzania ,17,24. Set al. [et al. [et al. [Findings from this study have indicated no significant association between sex and intrademal skin positivity Table . The finet al.  and Cadm [et al.  reported [et al. , where met al. [et al. [Our results have shown that about 64.8% of respondents reported presence of very thin and emaciated animals in their herds. However, through physical observation, most of thin and emaciated animals in those herds tested negative for bTB as compared to positive reactors whose body condition was good. This supports previous findings by Amen et al.  and Muny [et al.  in which [et al. ,48. In oet al. [et al. [et al. [et al. [M. avium and M. fortuitum are shed in meat or milk from cattle as it happens in case of M. bovis. Atypical mycobacteria which are commonly environmental have been reported in cattle farms [The study has shown a high prevalence of atypical mycobacteria in cattle, which signifies environmental contamination. Cattle could have acquired the infection through contaminated environment during grazing or at water sources. The high prevalence of atypical mycobacteria in this study concurs with previous findings in Tanzania whereby Shirima et al.  and Durn [et al. , reporte [et al.  and Durn [et al.  found a  [et al. . The prele farms  with widM. gordonae, M. smegmatis, M. fortuitum, M. phlei, M. flavescenes and M. avium intracellular to be shed in cattle milk samples in Morogoro region and Kibaha, Tanzania [M. avium is said to be clearly a human pathogen in infants and also in HIV-infected adults; but the others are arguably less virulent, with M. fortuitum very rarely causing disease in individuals with genetic immune system problems. Others are regarded to be commensals. However, the non-specific reactions are not always caused by atypical mycobacteria. Sometimes, other closely related bacteria species , as well as some other factors can cause the unspecific reactions .Available reports revealed atypical mycobacteria to include Tanzania . M. aviuThe study has shown that older cattle >4 years) had a high response to PPD and doubtful reaction as compared to young cattle, with more doubtful reaction in females as compared to males (Figure  years haIn conclusion, findings from this study add useful epidemiological data/information regarding bTB infection at the livestock-wildlife interface in Tanzania. It was revealed that agro-pastoral and pastoral communities in the study area have low knowledge on bTB and its presence in other animals including wildlife. Moreover, majority of the community engage in husbandry practises such as free movement of cattle, sharing of water sources and pasture which increase chances of diseases transmission to livestock. This information is useful and could be used by respective authorities in designing appropriate control measures against bTB. Therefore, we propose that education programs should be implemented in pastoral communities to raise awareness on preventive measures against bTB and other infectious diseases. The education programs at livestock-wildlife interface areas should primarily focus on minimizing contact between cattle and wildlife. However, in order to be more certain whether strains are circulating between cattle and wild animals, we need to cultivate mycobacteria and use genetic strain typing methods to characterise them accurately.The authors declare that they have no competing interest related to this article.BZK designed study, conducted field work, analyzed the data, drafted the manuscript. EVM participated in designing the study, supervised the field work and contributed in drafting and initial review of the manuscript. EDK assisted in initial design of the study and reviewed the manuscript. JDK contributed to conception and commented the manuscript. SK assisted in conceptualization and critically reviewed the manuscript. GSK assisted in critical review of the manuscript and commented the manuscript. PGF had an important contribution and commented the manuscript. ALM contributed to conception and critically commented the manuscript. RRK contributed in outset and review of the manuscript. PVH made conceptual contribution commented the manuscript and critically revised it. MIM coordinated the field work and helped in drafting the manuscript. All authors have read and approved the final manuscript.


The dispersed MWCNTs have been found to be stable in hexafluoroisopropanol for several months without precipitation. A MWCNTs/Nylon-6 nanofiber composite based chemical sensor has demonstrated its responsiveness towards a wide range of solvent vapours at room temperature and only mg quantities of MWCNTs were expended. The large surface area and porous nature of the electrospun Nylon-6/MWCNT nanofibers facilitates greater analyte permeability. The experimental analysis has indicated that the dipole moment, functional group and vapour pressure of the analytes determine the magnitude of the responsiveness.We have prepared electrospun Nylon-6 nanofibers via electrospinning, and adsorbed multi-walled carbon nanotubes (MWCNTs) onto the surface of Nylon-6 fibers using Triton HigheFillers have been successful in imparting electrical conductivity to non-conducting polymers, but the conductivity is dependent on the type of filler used . MoreoveFirstly, aggregation and poor interfacial interaction are the major problems encountered when conductive fillers are used in a host polymer matrix -21. AggrSecondly, unless the MWCNTs are uniformly dispersed in the nylon-6 matrix, the full advantage of the electronic properties of individual MWCNTs will not be realized. Researchers have developed methods to disperse the CNTs, such as functionalizing CNTs on the polymer , solutio® X-100 as surfactant and also impregnating (im) them within the Nylon-6 nanofibers. Their performance for chemical gas sensing has then been investigated.In this study, the MWCNTs/Nylon-6 nanofiber composites have been prepared by adsorbing the MWCNTs on the surface of Nylon-6 nanofibers with the aid of Triton2.2.1.N,N,-dimethylformamide (DMF), methanol, acetone, and trichloromethane have been purchased from Sigma-Aldrich (Germany). N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), ethyl acetate (EA), dichloromethane (DCM), trichloromethane (TCM), hexane and toluene have been procured from Merck (Darmstadt). Ethanol has been bought from Fisher Scientific (UK). MWCNTs (95% purity), each of 10-30 nm in diameter, 1-2 μm in length, 40-300 m2/g of surface-area, have been obtained from Shenzhen Nanotech Port Co Ltd, (P.R. China).Nylon-6 polymer  was obtained from UBE Industries Ltd (Japan). Hexafluoroisopropanol , 2.2.The dispersibility of MWCNTs has been examined in various solvents: dimethylformamide (DMF), methanol, acetone, HFIP, trichloromethane, DMA, THF, ethanol and water. HFIP has been found to be the best solvent for dispersing the MWCNTs effectively. The solution has also been found to be stable at room temperature for over several months without precipitation .Nylon-6 polymer was dissolved in HFIP solvent to form the Nylon-6 solution for electrospinning. Microscopic glass-slides were cleaned before use and then cut to a size of 25 × 20 mm. Gold as the electrode was sputtered on an area of 25 × 3 mm size on the two opposite ends of glass-slide for the electrical connection. Then the glass-slide has been carefully masked with paraffin film so that a blank space of 10 × 20 mm area is available for the deposition of electrospun polymer nanofibers.2.2.1.The electrospinning setup has possessed a high-voltage power supply, a syringe pump, a syringe, and a grounded collector . Nylon-6®-100 surfactant solution. The entire setup has been kept on a shaker  at ∼75 rpm for a period of 1 min in order to allow for uniform adsorption of MWCNTs on the surface of the Nylon-6 nanofiber composite adhered-slide. A uniform dispersion of MWCNTs on the surface of the Nylon-6 nanofibers was achieved for the 1 wt % concentration of Triton® X-100 surfactant. The adhered-nanofiber composite slide was then rinsed with de-ionized water and sonicated repeatedly until the excess of surfactant, as well as the loosely bound MWCNTs are removed. As a result, the sa MWCNT/ Nylon-6 nanofibers mat (The prepared Nylon-6 nanofiber composite mat was immersed in a bath containing dispersed MWCNTs (5 mg) and investigated in different concentrations  of Triton Xbers mat  was obtaim) of the MWCNTs inside the Nylon-6 polymer nanofibers in order to compare the sensing performance with the sa MWCNT/Nylon-6 nanofibers composite mat. A 1 wt % MWCNTs and Nylon-6 polymer/HFIP solution were blended using an ultrasonicator. The resulting mixture was electrospun, keeping the same above mentioned electrospinning conditions to form im MWCNTs/Nylon-6 nanofibers. It should be mentioned that for im MWCNTs/Nylon-6 nanofibers, the Triton® X-100 surfactant was not used whereas for the preparation of sa MWCNTs/Nylon-6 nanofibers it was used because the role of surfactant is critical for the adsorption of MWCNTs onto the surface of Nylon-6 nanofibers. The presence of surfactant not only prevents the MWCNTs from aggregating, which results from a substantial van der Waals interaction between MWCNTs, but also induces the interaction of MWCNTs and Nylon-6 polymer that causes them to adhere on the surface and self assemble. The Triton®-X surfactant was not employed in the preparation of im MWCNTs/Nylon-6 nanofibers because the use of surfactant would lead to nanofiber pore blockage and act as a barrier for the effective interaction of analytes and the MWCNTs/Nylon-6 nanofibers. The adhered-im MWCNT/Nylon-6 nanofiber slide  was placed in a conical glass flask, and sealed with a two holed rubber stopper. The sensing device was attached . The dis3.sa MWCNTs/Nylon-6 nanofibers have been characterized using a Quanta 200F scanning electron microscope (SEM) which operates at 10 kV, whereas the im MWCNTs/Nylon-6 nanofibers have been examined using a JEM 2010F transmission electron microscope (TEM) operated at an accelerating voltage of 200 kV.The et al. [® X-100 as surfactant, however they encountered blockage of pores by the MWCNTs that resulted in a lower responsiveness. Pore blockage has been prevented in our study by optimizing the electrospinning conditions used to obtain nanofibers of Nylon-6 polymer of well-defined 1-D nanostructures and high porosity. Cross-sectional examination of our MWCNTs/Nylon-6 nanofiber composite mat using SEM  towards various analytes has been calculated using the equations 1 and 2 [Nylon-6, a polymer that possesses a repeating unit of amide and carbonyl groups in its structure could interact with MWCNTs upon surface adsorption through hydrogen bonding. An increase in the interconnectedness of the MWCNTs on the surface of Nylon-6 nanofibers, in turn provides a continuous conducting path, known as ‘hreshold  and thus 1 and 2 , and the0R and 0I are the initial resistance and current at time t = 0 and R and I are the resistance and current obtained at maximum steady state value. For 1 volt, the above equation transformed as follows:sa MWCNTs/Nylon-6 nanofiber composite towards various polar organic analytes, clearly indicates that the nanofiber composite is responding to the various type of analytes used.All the experiments have been run in triplicate and it was observed that the overall variations of the responsiveness are within 10%. sa MWCNTs/Nylon-6 nanofiber based device and neat Nylon-6 nanofiber based device (control) towards acetone vapor. In the current work, all the analyte molecules have been involved in a small level of charge transfer, suggesting a weaker interaction between analyte and MWCNTs. Desorption of analyte molecules from the sensor device has been carried out by keeping the device open under ambient air conditions overnight and at room temperature. The reversibility has been checked and confirmed for three repeated measurement cycles, indicating that the detachment of gas molecules is possible at room temperature because of the weaker interaction between MWCNTs and analyte molecules.sa MWCNTs/Nylon-6 nanofiber based gas sensor has shown promise during the repeated tests, which are shown in The reusability and stability of our 3.1.sa MWCNTs/Nylon-6 nanofiber based sensor exhibited the smaller response towards the hexane and toluene, which are apolar in nature. These results suggest that the nature of the analyte plays a vital role in controlling the performance of the sa MWCNTs/Nylon-6 nanofiber composite sensor. Our further analysis of the physical properties of analytes has hinted that analyte volatility also contributes to the differences in the responsiveness. The vapor pressure, a good indicator of solvent volatility, can be defined here as the condition required to become vapor from the container to the sensing zone in sensor. According to the National Technical Information Service (NTIS), any chemical with a vapor pressure of greater than 13.33 kPa (>100 mm Hg) is said to have high volatility. The vapor pressure of EA is ca. three times lower than acetone  of the composite matrix is expected to increase due to swelling, which results in lower resistance (i.e. higher conductance) according to the following correlation:L is the thickness of the conducting matrix and ρ is the resistivity in Ohm-m.Interaction between Nylon-6 and analyte molecules are also equally possible and this occurs especially in a region where poor dispersion of MWCNTs exists. Swelling is a common phenomenon that is encountered when a polymer is exposed to strong polar analytes , 48. Sanet al.  have remsa MWCNTs/Nylon-6 composite sensor only arises from the MWCNTs and hence, the conductivity of the Nylon-6 composite depends on the interconnectivity of MWCNTs. Swelling of the polymer matrix increases the distance between the MWCNTs, thus loosing their interconnectivity that eventually leads to a decrease in the conductivity. This could also account for the larger increase in responsiveness of the sa MWCNT composite towards DCM, TCM and acetone.Since the Nylon-6 polymer is an insulator, the conductivity in the im MWCNT/Nylon-6 nanofiber composite sensor has been investigated by exposing it to ethanol and comparing the results seen with the sa MWCNTs/Nylon-6 nanofiber based sensor. Given the identical analysis time, temperature and the same concentration of MWCNTs and ethanol analyte, the responsiveness of the im MWCNTs/Nylon-6 nanofibers has been found to be much smaller than that of the sa MWCNTs/Nylon-6 nanofiber based device (sa MWCNTs/Nylon-6 based sensor as the MWCNTs are infused in the Nylon-6 nanofibers, which could accounted for its poor gas responsiveness (® X-100 surfactant the MWCNTs in im MWCNT/Nylon-6 nanofibers have been found more aggregated (im MWCNT/Nylon-6 nanofiber based device.As another part of our study, the sensing performance of the d device . The anasiveness . This cogregated  due to t4.sa MWCNTs/Nylon-6 nanofiber materials for gas sensing at room temperature has been demonstrated. The nanofiber based device has shown the significant reproducibility and responsiveness to organic analytes, especially to polar and non-polar molecules. It is expected that polar molecules with a carbonyl group might withdraw electrons from MWCNTs and hence the sa MWCNTs/Nylon-6 nanofiber composite has shown higher responsiveness towards acetone, compared to non polar molecules without those groups, like hexane and toluene. Overall, the dipole moment, nature of functional groups and vapor pressure of analytes are the key factors that influence the responsiveness of sensor device at room temperature. The im MWCNTs/Nylon-6 nanofiber based sensor has produced lower responsiveness compared to a sa MWCNTs/Nylon-6 nanofiber composite, suggesting that the analyte interaction with the MWCNT is important for increased responsiveness. Currently, our research group has been investigating the effects of temperature and analyte concentration to gain a better perspective of the wider applications of the sa MWCNTs/electrospun Nylon-6 composite. Recently, there has been growing interest in adopting well-aligned nanofiber materials for fabricating electrode systems in sensor devices [aligned Nylon-6 nanofibers in the sensing device could allow for maximum dispersion of MWCNTs, thereby resulting in enhancement in the sensitivity, which is also under investigation in our laboratory.The feasibility of using  devices , 51 to i


Plasmodium falciparum. This protocol utilizes a modified commercial buffer to improve speed of assay and consistency in the bioluminescence signal measured by reducing the manipulation steps required to release the cytoplasmic fraction. The utility of this improved assay protocol is demonstrated in typical assays that explore absolute and temporal gene expression activity.This report describes the optimization and evaluation of a simple single-step lysis protocol to measure luciferase bioluminescence from genetically modified  Plasmodium falciparum have proven an invaluable tool in the functional characterization of cis-acting sequences that govern the promoter and terminator activities of gene flanking sequences [cat) gene or a Photinus pyralis luciferase (luc) gene [luc, however, offers additional attributes such as speed of assay (no need for lengthy incubations for the end-point of the cat assay), easier handling and waste disposal characteristics, and ability to exploit recent advances in instrumentation for the detection of bioluminescence. One final advantage of the luc assay, its flexibility, has recently been evidenced in the malarial field by the development of a high throughput anti-malarial drug screening assay using transgenic parasites expressing luc [The use of reporter assay systems in the human malarial parasite equences -3. The tuc) gene ,5. Both sing luc -9.luc reporter assay system. Whilst valuable in demonstrating the effect of modified gene flanking sequences in determining the absolute level of gene expression, the concatamerization of episomal plasmids, unequal segregation of plasmids during mitotic division, and frequent use of mis-matched 5' and 3' gene flanking sequences, limit their use in the exploration of molecular mechanisms that govern temporal patterns of gene expression [bxb1 integrase system to ensure production of homogenous populations of parasites that each contain a single reporter gene cassette [There are, however, inherent limitations in the utilization of the pression ,10. Thescassette ,12. HoweP. falciparum is described. This revised protocol uses a simple single-step lysis protocol with an improved lysis buffer formulation to generate a more consistent bioluminescent signal output. The utility of this modified assay is demonstrated by showing its ease of use in generating a comprehensive portrait of temporal gene expression and how consistency in data can provide significant information from a multi-variant promoter deletion study where previously only trends had been described.Here the optimization and validation of a modified luciferase reporter gene assay protocol for the asexual intraerythrocytic stages of P. falciparum clone expressing luciferase (Pfluc) used here has previously been described [attB) using the mycobacteriophage bxb1 integration system [luc reporter gene is flanked by 1417 bp and 647 bp of 5' and 3' flanking sequences, respectively of Pfpcna (PF13_0328). Clones Δ2 and Δ3 contain 1160 bp and 990 bp of 5' Pfpcna flanking sequence, respectively [2, 3% CO2, and 96% N2) and maintained using 5 nM WR99210 and 2.5 μg/ml BSD (blasticidin S) drug selection [The genetically modified escribed . This cln system ,12. In PPfpcna PF_0328. ClPlasmodium falciparum-infected erythrocytes (IE) were collected by centrifugation  and 20 μl samples of packed IE lysed using 10 volumes of 1× phosphate buffered saline (PBS)/0.1% saponin at room temperature for 5 min. Following centrifugation, the parasite pellet was washed three times in 1× PBS and re-suspended in 100 μl of 1× Reporter Lysis Buffer (Promega), and subjected to three cycles of freeze (liquid nitrogen) and thaw (37°C) with a final centrifugation step of 13000 rpm for 2 min to clear the parasite supernatant. Bioluminescence readings were measured using the single-tube luminometer  from 20 μl of cleared parasite supernatant and 100 μl of Luciferase Substrate Reagent (Promega).Analysis for significance in the bioluminescent signals determined for the reporter deletion series was carried out using n = 6 independent measurements for each construct at each developmental stage and analysed using one-way analysis of variants (ANOVA), with Tukey's post tests for significance, on MiniTab version 14. All other experiments were carried out using n = 5 independent measurements. Where indicated, t-tests were carried out in GraphPad Prism version 5.0 .P. falciparum requires release of the parasite from the host erythrocyte, extensive washing of the parasite pellet to remove haemoglobin, and then a repeated process of freeze-thaw lysis of the parasite to release the cytoplasmic fraction [The standard luciferase reporter assay protocol for fraction ,12. Thist-test, p < 0.05).Throughout this study, a standard of five repeats of 20 μl of packed trophozoite-IE were prepared to investigate each set of parameters under investigation. Following lysis, 20 μl of IE or isolated-parasite supernatant fractions were mixed with 100 μl of luciferase assay substrate  and the bioluminescent signal measured for 10 sec using a Glomax 20/20 luminometer (Promega). The signals obtained from the standard lysis protocol were initially compared with those from a single-step lysis of IE using a commercially available reporter lysis buffer . Here, packed IE were re-suspended in a range of pellet volumes of 1 × RLB , mixed using a bench-top vortex and incubated at room temperature for 5 min. A 20 μl volume of IE supernatant was then removed for detection of the bioluminescent signal. This single-step lysis protocol is thus dramatically less complex and time-consuming than the previous standard protocol, which typically takes 30-45 min. Whilst the standard lysis protocol, irrespective of the volumes of RLB used, always generated a higher bioluminescent signal, the variation in signal obtained for the four matched samples was high; such that no significant difference could be determined between each of the conditions used Figure . This ist-test, p < 0.005). Comparison of the bioluminescent signal using the different buffers shows a clear preference for the modified PLB/0.1% saponin buffer. A revised assay was thus adopted using this modified buffer as the data would indicate that some 65% of the bioluminescent signal lost on adoption of the single-step assay can be recovered without adversely compromising the consistency of the data produced.To explore the possibility of improving the bioluminescent signal obtained using the single-step lysis protocol, two variables were explored. First was the use of different lysis buffers, and second the pellet-volume of lysis buffer used. A range of commercial lysis buffers optimized for bioluminescence assays are available; here the performance of the previously used RLB was compared against cell culture lysis reagent  and passive lysis buffer , in each case assessing its use in the presence or absence of a 0.1% saponin supplement. Using five-pellet volumes (i.e. 100 μl) of each lysis buffer, the bioluminescent activity of the same IE culture was assayed and compared to a control using the standard lysis protocol and a five-pellet volume of RLB Figure . Saponin2 = 0.90) relationship between the bioluminescent signal per standardised parasite number and the volume of lysis buffer, suggesting that larger buffer volumes are more efficient in releasing luciferase from the IE pellet  were subject to single-step lysis using PLB/0.1% saponin and the bioluminescent signal plotted against parasite number Figure . To explluc reporter gene in Pfluc is flanked by 1417 bp and 647 bp of 5' and 3' flanking sequences, respectively, of the Pfpcna (PF13_0328) gene [Pfpcna encodes the proliferating cell nuclear antigen, a protein essential for efficient DNA polymerase δ activity during S-phase. These flanking sequences have previously been demonstrated to be sufficient to completely reconstitute the trophozoite-specific temporal pattern of intraerythrocytic expression of Pfpcna over the luc reporter gene [Pfpcna-mediated bioluminescent signal was linked to the development of early trophozoite stage parasites and peaked in mature trophozoites; entirely coincident with the expected demand for Pfpcna during schizogony [Pfpcna steady-state mRNA levels  gene . Pfpcna ter gene ,15,16. Phizogony ,15. The Pfpcna promoter has identified two distal regions that affect promoter activity, both of which were shown to competitively bind trans-acting nuclear factors [Pfpcna promoter, only its absolute activity . Whilst comparisons in the other samples were not significant, this presumably results from what appears to be the very low, probably background, bioluminescent signals at these time points.Functional characterization of the  factors ,16. Thes factors ) used iny Figure . And, asP. falciparum promoters is demonstrated. It is anticipated, however, that this protocol could be more widely applied to bioluminescence reporter gene assays in other intraerythroctytic pathogens.Here a revised luciferase reporter gene assay protocol is described, based on the use of a single lysis-step, which offers a simpler and quicker procedure whilst delivering a more consistent bioluminescent signal. Reduction in the bioluminescent signal due to the presence of haemoglobin does not affect the specificity of the assay, and this loss in signal can be ameliorated using a saponin-supplementation of a commercial buffer. The utility of this revised assay in typical studies that examine the absolute and temporal properties of The authors declare that they have no competing interests.SH and PH designed the study which was predominantly carried out by SH. EHW provided initial material and performed the statistical analyses. All authors read and approved the final manuscript.


Patients with human immunodeficiency virus infection are known to have increased risk of various neoplasms, including Kaposi sarcoma, which classically involves the skin and mucosal locations. The anaplastic variant of Kaposi sarcoma is rare and poorly documented in the literature. It is characterised clinically by a more aggressive behaviour and increased metastatic potential, and histologically by increased cellularity, mitotic rate, and rarely by epithelioid angiosarcoma-like morphology. We report herein a 64-year-old man with a long-standing history of human immunodeficiency virus infection who developed a right adrenal tumor with a high-grade anaplastic angiosarcoma-like morphology. Immunohistochemistry for human herpes virus-8 was strongly positive in the tumor cells. To the best of our knowledge, this is the first report of an anaplastic Kaposi sarcoma in the adrenal gland. Kaposi sarcoma (KS) is a rare low-grade malignant vascular neoplasm caused by human herpes virus-8 (HHV-8) and commonly affects the skin of lower extremities, face, trunk, and genitalia. Lymph nodes and other visceral organs are also frequently involved . KS has Anaplastic transformation of KS, which is defined histologically as areas of increased cellular pleomorphism and mitosis  and clinWe report herein the case of 64-year-old man with a long-standing history of human immunodeficiency virus (HIV), treated with antiviral therapy, who developed a right adrenal mass. Excision revealed a high-grade spindle and epithelioid malignant vascular neoplasm that was positive for both vascular markers and HHV-8 immunostaining, consistent with anaplastic KS. To the best of our knowledge, this is the first case report in the literature of an anaplastic KS resembling angiosarcoma in the adrenal gland.A 64-year-old man with a long-standing history of HIV diagnosed over 20 years ago, presented in the early 1990s with Kaposi sarcoma of the right and left legs and left hand. He was treated with vinblastine alternating with vincristine and had a good response and control of the disease at that point. Subsequently, the patient had multiple relapses of his cutaneous KS, for which he received different modalities of treatments including radiation therapy, chemotherapy, and updated antiretroviral therapy. He appeared to have a reasonably good response. Apart from KS, his past medical history was also significant for multiple skin cancers including basal cell carcinomas on his head, and leg, and melanoma on his back , Ménière's disease, multiple actinic keratoses, and recurrent cellulitis. In the three years prior to development of the current tumor, the patient received kaletra (lopinavir and ritonavir) and tenofovir; viral load was undetectable (<40 copies/ml) and CD4 count was 220/uL around the time of diagnosis. The patient subsequently developed cough and had a CT scan of the chest, which disclosed bilateral pleural thickening, and an incidental right adrenal mass lesion, with no other abnormalities detected. The patient was then referred for a core needle biopsy of the right adrenal mass, performed under CT guidance. Following pathological diagnosis (see below), the patient underwent clinical staging via CT scan of the abdomen and pelvis, and a PET scan. As shown in The core needle biopsy revealed a mixture of spindle and epithelioid cells admixed with lymphoplasmacytic infiltrate, occasional slit-like vascular spaces, and focal necrosis. Immunohistochemistry was positive for CD31 and HHV-8, and negative for melanoma markers .The excision specimen was a 10 × 7 × 3 cm soft tissue mass with unremarkable outer surface. Cut section revealed a hemorrhagic well-delineated mass measuring 7 × 5 × 2.5 cm, variegated in appearance, and showing an admixture of solid tan-coloured areas and hemorrhagic areas with cystic degeneration. No residual adrenal gland tissue was identifiable grossly.Multiple formalin-fixed sections of the neoplasm were made and stained with hematoxylin and eosin. Histopathological examination showed total replacement of the adrenal gland by a cellular, pleomorphic and nodular spindle and epithelioid neoplasm. Spindled areas disclosed solid sheets of cells growing in interlacing fascicles and bundles, with a high mitotic count (12/10 high power fields), extravasated red blood cells, occasional slit-like vascular spaces, lymphoplasmacytic infiltrates, and few intracytoplasmic eosinophilic hyaline globules . The epiImmunohistochemistry was performed using standard immunoperoxidase methods: tissue sections were stained with an automated immunostainer (Ventana) following antigen retrieval using an EDTA-based solution (CC1) or a citrate-based solution at pH 6. The primary antibodies used were CD31 , CD34 , FLI-1 , HHV-8 , D2-40  S-100 (U of Toronto), MelanA,, HMB45 , keratin (DakoCytomation), desmin , smooth muscle actin , and p53 . The primary and secondary antibodies and avidin-enzyme conjugate were visualised using the precipitating enzyme DAB (diaminobenzidine). Immunohistochemistry was positive for CD31, CD34, and D2-40 in a strong and diffuse membranous pattern . FLI-1, On the basis of the given clinical history, histological findings and immunohistochemical results, a diagnosis was made of high-grade Kaposi sarcoma, anaplastic variant.We report herein the case of a 64-year-old male with long standing history of AIDS and previous Kaposi sarcoma, who developed a high-grade anaplastic Kaposi sarcoma of the adrenal gland, demonstrated by both histological features and immunohistochemistry. To the best of our knowledge, this is the first case report in the literature of anaplastic KS of the adrenal gland.KS is a vascular neoplasm of low malignant potential often occurring in the setting of HIV infection, first described by Kaposi in 1872. Typically, it affects mucocutaneous sites, such as the skin of the lower extremities, face, trunk, genitalia, and oropharyngeal mucosa . There aThe histological appearance of these various clinical types of KS does not differ greatly from one group to another. The earliest patch stage of the disease is characterized by proliferation of small slit-like or ectatic vascular spaces, which dissect between dermal collagen, mostly in the upper dermis , 5. ThesVery few cases of KS have been reported in the adrenal gland, or similar unusual visceral locations ; most of Anaplastic KS is a rare and poorly documented variant , and hasThe Kaposi sarcoma herpes virus (KSHV), or HHV-8, described by Cheuk et al. in 2004 , is consOther variants of KS reported in the literature include: lymphedematous KS, lymphangioma-like KS, lymphangiectatic KS, telangiectatic KS, bullous KS, hyperkeratotic KS, keloidal KS, micronodular KS, pyogenic granuloma-like KS, ecchymotic KS, and intravascular KS .The principal differential diagnoses in the present case included both melanoma  and angiosarcoma (on the basis of the above-mentioned microscopic findings). For melanoma, the histological features of having spindled and epithelioid cells with occasional slit-like and large irregular vascular spaces, lymphoplasmacytic infiltrate, and extravasated red blood cells, bolstered by the immunohistochemical findings of positive vascular markers evident by CD31, CD34, FLI-1, and D2-40, and negative melanoma markers , overwhelmingly favour KS over melanoma. Distinguishing anaplastic KS from angiosarcoma is more difficult to ascertain on the basis of the histological findings alone, and expression of vascular markers is of no help. However, the aforementioned clinical history, as well as positive immunostaining for HHV-8 strongly, supports the diagnosis of anaplastic KS over that of primary adrenal angiosarcoma. Some studies have demonstrated that P53 protein mutation is implicated in the pathogenesis and progression, but not initiation of KS , 14. IndIn summary, we report the 64-year-old man with a long-standing history of HIV who developed a right adrenal anaplastic KS resembling high-grade epithelioid angiosarcoma. To the best of our knowledge, this is the first case report in the literature of an adrenal anaplastic KS resembling epithelioid angiosarcoma.