Moxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosis through inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosis H37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatis GyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of <5 nM, an MIC of 0.03 μg/ml against M. tuberculosis H37Rv, and an MIC90 of <0.25 μg/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was ∼10−6 to 10−8, and the point mutation mapped to the M. tuberculosis GyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivo efficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents.
In this study we adapted a Mud-based delivery system to construct a random yfp reporter gene (encoding the yellow fluorescent protein) insertion library in the genome of Salmonella Typhimurium LT2, and used fluorescence activated cell sorting and fluorescence microscopy to screen for translational fusions that were able to clearly and specifically label the bacterial nucleoid. Two such fusions were obtained, corresponding to a translational yfp insertion in iscR and iolR, respectively. Both fusions were further validated, and the IscR::YFP fluorescent nucleoid reporter together with time-lapse fluorescence microscopy was subsequently used to monitor nucleoid dynamics in response to the filamentation imposed by growth of LT2 at high hydrostatic pressure (40–45 MPa). As such, we were able to reveal that upon decompression the apparently entangled LT2 chromosomes in filamentous cells rapidly and efficiently segregate, after which septation of the filament occurs. In the course of the latter process, however, cells with a “trilobed” nucleoid were regularly observed, indicative for an imbalance between septum formation and chromosome segregation.
In this study, we examined the intracellular whereabouts of Mrr, a cryptic type IV restriction endonuclease of Escherichia coli K12, in response to different conditions. In absence of stimuli triggering its activity, Mrr was found to be strongly associated with the nucleoid as a number of discrete foci, suggesting the presence of Mrr hotspots on the chromosome. Previously established elicitors of Mrr activity, such as exposure to high (hydrostatic) pressure (HP) or expression of the HhaII methyltransferase, both caused nucleoid condensation and an unexpected coalescence of Mrr foci. However, although the resulting Mrr/nucleoid complex was stable when triggered with HhaII, it tended to be only short-lived when elicited with HP. Moreover, HP-mediated activation of Mrr typically led to cellular blebbing, suggesting a link between chromosome and cellular integrity. Interestingly, Mrr variants could be isolated that were specifically compromised in either HhaII- or HP-dependent activation, underscoring a mechanistic difference in the way both triggers activate Mrr. In general, our results reveal that Mrr can take part in complex spatial distributions on the nucleoid and can be engaged in distinct modes of activity.
To study the clinicopathological profile, recurrence and malignant potential of the inverted papilloma of nose and paranasal sinuses in relation to the definitive diagnosis and the management undertaken. A total number of 52 cases of histologically proven inverted papilloma managed in our department of otolaryngology over a 10-year period (May 1998–April 2008) were followed up (until October 2009) and the results were analyzed retrospectively to find out any incidence of recurrence of these tumours. A total of 52 inverted papilloma cases were managed with different surgical interventions. Male:female ratio was found to be 10:1. The mean follow up period was 74 months (range 16 months–11 years). Recurrence was observed in 20 (38%) cases. 23% had recurrence without any specific histological pattern, 12% had recurrence with focal dysplasia and 4% had recurrence with malignant transformation into squamous cell carcinoma (SCC). Post-operative radiotherapy was given to 8% (four cases) with malignant transformation. The management of inverted papilloma depends on its size and extension. Recurrence can be minimized by an appropriate surgical planning. Careful endoscopic assessment is essential to detect early recurrence. Recurrent inverted papilloma should be treated more aggressively. Malignant transformation in inverted papilloma should be managed like any aggressive sino-nasal malignancy.
Inverted papilloma; Recurrence; Malignant transformation; Management
The Mrr protein of Escherichia coli is a laterally acquired Type IV restriction endonuclease with specificity for methylated DNA. While Mrr nuclease activity can be elicited by high-pressure stress in E. coli MG1655, its (over)expression per se does not confer any obvious toxicity. In this study, however, we discovered that Mrr of E. coli MG1655 causes distinct genotoxicity when expressed in Salmonella typhimurium LT2. Genetic screening enabled us to contribute this toxicity entirely to the presence of the endogenous Type III restriction modification system (StyLTI) of S. typhimurium LT2. The StyLTI system consists of the Mod DNA methyltransferase and the Res restriction endonuclease, and we revealed that expression of the LT2 mod gene was sufficient to trigger Mrr activity in E. coli MG1655. Moreover, we could demonstrate that horizontal acquisition of the MG1655 mrr locus can drive the loss of endogenous Mod functionality present in S. typhimurium LT2 and E. coli ED1a, and observed a strong anti-correlation between close homologues of MG1655 mrr and LT2 mod in the genome database. This apparent evolutionary antagonism is further discussed in the light of a possible role for Mrr as defense mechanism against the establishment of epigenetic regulation by foreign DNA methyltransferases.
To present four rare cases of peripheral primitive neuroectodermal tumors of different sites of head and neck region. Four cases of different age (range 8–40 years) and sex (three female, one male) with rare primitive neuroectodermal tumor of sinonasal region and neck are presented. Treatment options, biological behavior and prognostic outcome are discussed herewith. Two patients succumbed to the disease within four to six months of treatment; other two patients are still under follow-up depicting the aggressive nature of the tumor. Primitive neuroectodermal tumor belongs to the class of malignant round cell tumor. Immunohistochemistry plays a pivotal role in differentiating this tumor entity. Chemoradiation was tried, but local and systemic spread occurs early and holds poor prognosis. This case series is an attempt to describe the aggressive behavior of this rare tumor with high mortality.
Primitive neuroectodermal tumor (PNET); Ewing’s sarcoma; Paranasal sinuses; Head-neck; Immunostaining; Chemotherapy; Radiation therapy
To report a case of disseminated cutaneous metastasis from malignant melanoma of sino-nasal region. A 53-year-old man from rural parts of West Bengal presented with progressive nasal obstruction. CT scanning was done to know the extent of the mass and punch biopsy from the mass was performed. Malignant melanoma of sino-nasal region was diagnosed and chemotherapy was started. The patient developed cutaneous deposits after two cycles of chemotherapy. The patient developed cutaneous deposits during the course of chemotherapy. Excision biopsy from cutaneous deposits revealed malignant melanoma. A rare case of diffuse cutaneous metastasis of malignant melanoma is presented here along with review of literature.
Malignant melanoma; Cutaneous metastasis; Chemotherapy
Rhinosporidiosis is an infection caused by Rhinosporidium seeberi that frequently presents as polypoidal nasal lesions. Here, we report two cases of rhinosporidiosis with unusual presentations. The first case presented in our department with chronic dacryocystitis of left side for endoscopic dacryocystorhinostomy (endoscopic DCR) operation. The second case presented as a long hanging mass arising from the right side of nasal septum. The diagnosis was established on the morphological basis by the identification of endospores and sporangia. The clinicopathological and immunologic features were discussed and the literature was reviewed.
Rhinosporidium seeberi; Rhinosporidiosis; Lacrimal sac; Endoscopic DCR
38 cases of sarcoma of head and neck region were analysed in a retrospective way in relation to age, anatomic location, histological, clinical profile, and surgical approaches. Compared to other types of head and neck neoplasms, such as squamous cell carcinoma, soft tissue sarcomas have low rates of regional metastases. However the biological behaviour of soft tissue sarcoma is more aggressive specially in paediatric age group. In the present series, CT scan was considered as the primary modality of investigation. Surgery generally has been recommended as the primary method of treatment for achieving local control, except in those high-grade tumours arising in sites not amenable to resection. 3-year and 5-year survival rates in this present series 50% and 31.6% respectively.
Soft tissue sarcoma; CT Scan; Excisional Biopsy; Chemoradiation; Survival rate; Prognostic factors
The E6 protein from high-risk human papillomaviruses (HPVs) targets the p53 tumor suppressor for degradation by the proteasome pathway. This ability contributes to the oncogenic potential of these viruses. However, several aspects concerning the mechanism of E6-mediated p53 degradation at the cellular level remain to be clarified. This study therefore examined the role of cell localization and ubiquitination in the E6-mediated degradation of p53. As demonstrated within, following coexpression both p53 and high-risk HPV type 18 (HPV-18) E6 (18E6) shuttle from the nucleus to the cytoplasm. Mutation of the C-terminal nuclear export signal (NES) of p53 or treatment with leptomycin B inhibited the 18E6-mediated nuclear export of p53. Impairment of nuclear export resulted in only a partial reduction in 18E6-mediated degradation, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation. This was also consistent with the observation that 18E6 mediated the accumulation of polyubiquitinated p53 in the nucleus. In comparison, a p53 isoform that localizes predominantly to the cytoplasm was not targeted for degradation by 18E6 in vivo but could be degraded in vitro, arguing that nuclear p53 is the target for E6-mediated degradation. This study supports a model in which (i) E6 mediates the accumulation of polyubiquitinated p53 in the nucleus, (ii) E6 is coexported with p53 from the nucleus to the cytoplasm via a CRM1 nuclear export mechanism involving the C-terminal NES of p53, and (iii) E6-mediated p53 degradation can be mediated by both nuclear and cytoplasmic proteasomes.
The development of cancer is a multistep process involving mutations in proto-oncogenes, tumor suppressor genes, and other genes which control cell proliferation, telomere stability, angiogenesis, and other complex traits. Despite this complexity, the cellular pathways controlled by the p53 tumor suppressor protein are compromised in most, if not all, cancers. In normal cells, p53 controls cell proliferation, senescence, and/or mediates apoptosis in response to stress, cell damage, or ectopic oncogene expression, properties which make p53 the prototype tumor suppressor gene. Defining the mechanisms of regulation of p53 activity in normal and tumor cells has therefore been a major priority in cell biology and cancer research. The present study reveals a novel and potent mechanism of p53 regulation originating through alternative splicing of the human p53 gene resulting in the expression of a novel p53 mRNA. This novel p53 mRNA encodes an N-terminally deleted isoform of p53 termed p47. As demonstrated within, p47 was able to effectively suppress p53-mediated transcriptional activity and impair p53-mediated growth suppression. It was possible to select for p53-null cells expressing p47 alone or coexpressing p53 in the presence of p47 but not cells expressing p53 alone. This showed that p47 itself does not suppress cell viability but could control p53-mediated growth suppression. Interestingly, p47 was monoubiquitinated in an Mdm2-independent manner, and this was associated with its export out of the nucleus. In the presence of p47, there was a reduction in Mdm2-mediated polyubiquitination and degradation of p53, and this was also associated with increased monoubiquitination and nuclear export of p53. The expression of p47 through alternative splicing of the p53 gene thus has a major influence over p53 activity at least in part through controlling p53 ubiquitination and cell localization.