The role of the dermatologist in Raynaud's phenomenon: a clinical challenge.

Raynaud's phenomenon (RP) is a functional vascular disorder involving extremities. In his practice, the dermatologist may frequently encounter RP which affects mainly women and is categorized into a primary benign form and a secondary form associated with different diseases (infections, drugs, autoimmune and vascular conditions, haematologic, rheumatologic and endocrinologic disorders). Still today, the differential diagnosis is a clinical challenge. Therefore, a careful history and a physical examination, together with laboratory tests and nailfold capillaroscopy, is mandatory. RP is generally benign, but a scheduled follow-up for primary RP patients should be established, due to risk of evolution to secondary RP. A combination of conservative measures and medications can help in the management of RP. The importance of avoiding all potential physical, chemical and emotional triggers, as well as quitting smoking, should be strongly suggested to the patient. As first-line treatment, dihydropyridine calcium channel blockers should be used. If this approach is not sufficient, prostacyclin derivatives, phosphodiesterases inhibitors and endothelin receptor antagonists can be considered as second-line treatment. In cases of acute ischaemia, nifedipine and intravenous prostanoids are helpful. In refractory cases, botulinum injections have shown a significant benefit. The approach to the RP patients requires therefore a coordinated care of specialists together with the primary care physician.

HER2 expression in oesophageal carcinoma and Barrett's oesophagus associated adenocarcinoma: An Australian study.

BACKGROUND: Several studies have evaluated the prognostic value of HER2 in oesophageal cancer, but the prognostic influence of HER2 overexpression in oesophageal cancer remains uncertain. The aim of this study was to assess the incidence of HER2 positivity and relationship with clinicopathological features in patients with oesophageal cancer. DESIGN: The study cohort consisted of 269 patients diagnosed with oesophageal carcinoma in a single institution. HER2 expression was analysed by immunohistochemistry (IHC) and silver in situ hybridization (SISH) in 152 archival oesophageal cancer specimens. Survival analysis was assessed using Hazard models. RESULTS: HER2 expression was IHC3+ in 14 (9.2%), IHC2+ in 14 (9.2%), IHC1+ in 57 (37.5%), and IHC0 in 67 (44.1%) cases. SISH results confirmed that 15 specimens (9.9%) were HER2 gene amplified. Among 27 squamous cell carcinomas (SCCs) only 3.7% were HER2 positive whereas 11.2% of 125 adenocarcinomas were HER2 positive. The HER2 positive tumours were more likely to occur in men (OR: 5.00, 95% CI: 1.69-14.29), smokers (OR: 10.00, 95% CI: 4.17-25) and in patients with Barrett's oesophagus (OR: 8.33, 95% CI: 3.71-20.00). There was no significant difference in survival between the (HER2 +ve, 14.3 months vs HER2 -ve, 24.6 months, p = 0.42) CONCLUSION: A HER2 prevalence rate of 9.9% was found among patients with oesophageal cancer and no correlation with survival was detected overall.

Role of decompressive hemicraniectomy in extensive middle cerebral artery strokes: a meta-analysis of randomised trials.

BACKGROUND: Prognosis for patients with 'malignant' or space-occupying oedema post middle cerebral artery infarct remains poor despite maximal medical therapy delivered in the intensive care setting. AIM: We performed a meta-analysis to evaluate the value of surgical decompression versus medical management alone in patients suffering from malignant middle cerebral artery infarct. METHODS: A systematic search was conducted using MEDLINE, PubMed, EMBASE, Current Contents Connect, Cochrane library, Google Scholar, Science Direct and Web of Science. Original data was abstracted from each study and used to calculate a pooled odds ratio (OR) and 95% confidence interval (95% CI). RESULTS: The overall OR for mRS 6 (death) at 6 months for decompressive surgery as compared with standard medical management revealed a statistically significant reduction with OR of 0.19 (95% CI: 0.10-0.37). The frequency of patients with mRS 2, 3 and 5 outcomes was higher in the decompressive surgery cohort; however, these outcomes did not reach statistical significance. On the other hand, the number of patients with a mRS score of 4 was significantly higher in the decompressive surgery cohort with an OR of 3.29 (95% CI: 1.76-6.13). The overall OR for mRS 6 (death) at 12 months for decompressive surgery as compared with standard medical management revealed a statistically significant reduction with OR of 0.17 (95% CI: 0.10-0.29). The frequency of patients with mRS 3 and 5 outcomes was higher in the decompressive surgery cohort; however, these outcomes did not reach statistical significance. On the other hand, the number of patients with a mRS score of 4 was significantly higher in the decompressive surgery cohort with an OR of 4.43 (95% CI: 2.27-8.66). In the long run it was also observed that the number of patients with a mRS score of 2 was significantly higher in the decompressive surgery cohort an OR of 4.51 (95% CI: 1.06-19.24). CONCLUSIONS: Our results imply that surgical intervention decreased mortality of patients with fatal middle cerebral artery infarct at the expense of increasing the proportion suffering from substantial disability at the conclusion of follow up.

Association between vitamin B12 deficiency and long-term use of acid-lowering agents: a systematic review and meta-analysis.

BACKGROUND: Vitamin B12 (cobalamin) deficiency can result in irreversible structural brain changes if not treated appropriately. Long-term use of acid-lowering agents (ALA) has been linked to vitamin B12 deficiency, but results are inconsistent. AIM: To evaluate the association between prolonged ALA use and vitamin B12 deficiency by performing a meta-analysis. METHODS: A systematic search was conducted using MEDLINE, PubMed, EMBASE, Current Contents, Cochrane Library, Google Scholar, Science Direct and Web of Science. Original data were abstracted from each study and used to calculate a pooled odds ratio and 95% confidence interval (95% CI). RESULTS: Of the articles reviewed, four case-control studies (4254 cases and 19,228 controls) and one observational study met full criteria for analysis. The long-term ALA use was significantly associated with development of vitamin B12 deficiency (hazard ratio 1.83, 95% CI: 1.36-2.46, P-value 0.00). CONCLUSION: Chronic use of ALA is a risk factor for developing vitamin B12 deficiency. Judicious prescribing of ALA and regular monitoring of vitamin B12 in patients who are inevitably on long-term ALA therapy are recommended.

Systematic review with meta-analysis: the relationship between chronic Salmonella typhi carrier status and gall-bladder cancer.

BACKGROUND: Carcinoma of the gall-bladder is the fifth commonest gastrointestinal tract cancer and is endemic in several countries. An association of chronic typhoid carriage and carcinoma of the gall-bladder has been reported. AIM: To clarify whether chronic Salmonella typhi carrier state is associated with carcinoma of the gall-bladder. METHODS: A systematic search was conducted using MEDLINE, PubMed, EMBASE, Current Contents, Cochrane library, Google Scholar, Science Direct and Web of Science. Original data were abstracted from each study and used to calculate a pooled odds ratio (OR) and 95% confidence interval (95% CI). RESULTS: Of the articles selected, only 17 studies met full criteria for analysis. The overall OR for chronic S. typhi carrier state was 4.28(95% CI: 1.84-9.96). Most of the studies were from South Asia especially India and China. When a subgroup analysis was performed according to region, a significant association was observed in South-East Asia (OR: 4.13, 95% CI: 2.87-5.94, P value <0.01). Chronic S. typhi carrier state was associated with carcinoma of the gall-bladder based on detection methods of S. typhi antibody levels (OR: 3.52, 95% CI: 2.48-5.00, P value <0.01) and even more so on culture (OR: 4.14, 95% CI: 2.41-7.12, P value <0.01). The association was prominent in controls without gallstones (OR: 5.86, 95% CI: 3.84-8.95, P value <0.01) when compared with controls with gallstones (OR: 2.71, 95% CI: 1.92-3.83, P value <0.01). CONCLUSIONS: Chronic S. typhi carrier state is an important risk factor among patients with carcinoma of the gall-bladder. Given the high risk associated with this carrier state, management options should include either elective cholecystectomy or careful monitoring using ultrasound.

Is complete lymph node dissection after a positive sentinel lymph node biopsy for cutaneous melanoma always necessary? A meta-analysis.

BACKGROUND: The current recommendation for patients with cutaneous melanoma and a positive sentinel lymph node (SLN) biopsy is a complete lymph node dissection (CLND). However, metastatic melanoma is not present in approximately 80% of CLND specimens. A meta-analysis was performed to identify the clinicopathological variables most predictive of non-sentinel node (NSN) metastases when the sentinel node is positive in patients with melanoma. METHODS: A systematic search was conducted using MEDLINE, PubMed, EMBASE, Current Contents Connect, Cochrane library, Google scholar, Science Direct, and Web of Science. The search identified 54 relevant articles reporting the frequency of NSN metastases in melanoma. Original data was abstracted from each study and used to calculate a pooled odds ratio (OR) and 95% confidence interval (95% CI). FINDINGS: The pooled estimates that were found to be significantly associated with the high likelihood of NSN metastases were: ulceration (OR: 1.88, 95% CI: 1.53-2.31), satellitosis (OR: 3.25, 95% CI: 1.86-5.66), neurotropism (OR: 2.51, 95% CI: 1.39-4.53), >1 positive SLN (OR: 1.77, 95% CI: 1.2-2.62), Starz 3 (old) (OR: 1.83, 95% CI: 0.89-3.76), Angiolymphatic invasion (OR: 2.46, 95% CI: 1.34-4.54), extensive location (OR: 2.22, 95% CI: 1.74-2.81), macrometastases >2 mm (OR: 1.95, 95% CI: 1.61-2.35), extranodal extension (OR: 3.38, 95% CI: 1.79-6.40) and capsular involvement (OR: 3.16, 95% CI: 1.37-7.27). There were 3 characteristics not associated with NSN metastases: subcapsular location (OR: 0.51, 95% CI: 0.38-0.67), Rotterdam Criteria <0.1 mm (OR: 0.29, 95% CI: 0.17-0.50) and Starz I (new) (OR: 0.44, 95% CI: 0.22-0.91). Other variables including gender, Breslow thickness 2-4 mm and extremity as primary site were found to be equivocal. INTERPRETATION: This meta-analysis provides evidence that patients with low SLN tumor burden could probably be spared the morbidity associated with CLND. We identified 9 factors predictive of non-SLN metastases that should be recorded and evaluated routinely in SLN databases. However, further studies are needed to confirm the standard criteria for not performing CLND.

Nasoenteric tube complications.

The use of nasoenteric tubes (NETs) is ubiquitous, and clinicians often take their placement, function, and maintenance for granted. NETs are used for gastrointestinal decompression, enteral feeding, medication administration, naso-biliary drainage, and specialized indications such as upper gastrointestinal bleeding. Morbidity associated with NETs is common, but frequently subtle, mandating high index of suspicion, clinical vigilance, and patient safety protocols. Common complications include sinusitis, sore throat and epistaxis. More serious complications include luminal perforation, pulmonary injury, aspiration, and intracranial placement. Frequent monitoring and continual re-review of the indications for continued use of any NET is prudent, including consideration of changing goals of care. This manuscript reviews NET-related complications and associated topics.

Reversible polyelectrolyte capsules as carriers for protein delivery.

A reversible drug delivery system based on spontaneous deposition of a model protein into preformed microcapsules has been demonstrated for protein delivery applications. Layer-by-Layer assembly of poly(allylamine hydrochloride) (PAH) and poly(methacrylic acid) (PMA) onto polystyrene sulfonate (PSS) doped CaCO3 particles, followed by core removal yielded intact hollow microcapsules having a unique property to induce spontaneous deposition of bovine serum albumin (BSA) at pH below its isoelectric point of 4.8, where it was positively charged. These capsules showed reversible pH dependent open and closed states to fluorescence labeled dextran (FITC-Dextran) and BSA (FITC-BSA). The loading capacity of BSA increased from 9.1 x 10(7) to 2.03 x 10(8) molecules per capsule with decrease in pH from 4.5 to 3. The loading of BSA-FITC was observed by confocal laser scanning microscopy (CLSM), which showed homogeneous distribution of protein inside the capsule. Efficient loading of BSA was further confirmed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The interior capsule concentration was as high as 209 times the feeding concentration when the feeding concentration was increased from 1 to 10 mg/ml. The deposition was initially controlled by spontaneous loading mechanism at lower BSA concentration followed by diffusion controlled loading at higher concentration; which decreased the loading efficiency from 35% to 7%. Circular dichroism (CD) measurements and Fourier transform infrared spectroscopy (FTIR) confirmed that there was no significant change in conformation of released BSA in comparison with native BSA. The release was initially burst in the first 0.5 h and sustained up to 5 h. The hollow capsules were found to be biocompatible with mouse embryonic fibroblast (MEF) cells during in vitro cell culture studies. Thus these pH sensitive polyelectrolyte microcapsules may offer a promising delivery system for water soluble proteins and peptides.

Genome-wide analysis of the intrinsic terminators of transcription across the genus Mycobacterium.

Termination of transcription in eubacteria is achieved by a region of the nascent transcript. In Escherichia coli, this intrinsic terminator consists of a hairpin followed by a U-stretch. Absence of the typical terminators in several genes of Mycobacterium tuberculosis led us to develop an accurate and efficient algorithm to identify putative terminators in all sequenced microbial genomes. In addition to the typical Escherichia coli type of terminators, several variant terminator structures were predicted by the algorithm and their existence was experimentally verified. We have now analysed 17 Mycobacterium genomes to obtain a comprehensive picture of the transcription terminators in mycobacteria. Our results show that the terminators that lack a U-trail, variant from the typical E. coli intrinsic terminators, are overwhelmingly predominant in all members of the genus. Most terminator structures are concentrated within 50 base pairs downstream of the stop codon. A large number of these terminators occur at the end of experimentally verified or predicted transcription units. We have observed inter-species variations in DeltaG and positioning of the terminators downstream of specific genes amongst closely related mycobacterial species suggesting differences in gene expression. The analysis would be useful in furthering our understanding of genome organization and gene expression in mycobacteria, in addition to the improvement in the annotation of the new genomes.

Analysis of DNA curvature distribution in mycobacterial promoters using theoretical models.

In this paper, 125 different mycobacterial promoters are analyzed for their DNA curvature distribution using several di- and tri-nucleotide dependent models of DNA curvature. Different models give similar behavior and therefore qualitative validation of the results. Mycobacterial promoters resembling the E. coli sigma(70) type have almost 81% (85%) sequences having medium and high curvature profiles using dinucleotide-dependent models. Non-E. coli sigma(70) type mycobacterial promoters have comparatively higher percent of low curvature profiles. Very few extended -10 promoters have low curvature profiles. Mycobacterial promoters having A(n)T(m) (n+m > or =3) tract in the upstream region of -35 box and repeated in phase with each other have high curvature profiles. M. smegmatis promoters have high curvature profiles compared to M. tuberculosis promoters.

DNA topoisomerase I from mycobacteria--a potential drug target.

DNA topoisomerases are ubiquitous group of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. The enzymes are classified based on the pattern of DNA cleavage. Type IA enzymes found in all bacteria nick the DNA and attach themselves covalently to the 5' side of the nick during the first transesterification reaction. Most of the information on this group of enzymes comes from studies with E. coli topoisomerase I and III. Members of type IA group are single subunit Zn(++) metalloenzymes recognizing single stranded DNA without high degree of sequence specificity during relaxation reaction of negatively super coiled DNA. So far no inhibitors are known for this group of enzymes inspite of their important role in maintaining homeostasis of DNA topology. Molecular characterization of DNA topoisomerase I from mycobacteria has revealed some of the important features of type IA enzymes hitherto unknown and provide scope for identifying novel inhibitors. The present review describes the recent developments in the area summarizing the distinctive features of mycobacterial topoisomerase I. The enzyme has several properties not shared by either type IA or IB enzymes with respect to DNA binding, recognition, sequence specificity and interaction pattern. The physiological basis of the unusual features is discussed. The unique properties described would aid in developing the enzyme as a target molecule in pharmaceutical design. In addition, the findings lead to address some fundamental questions on the intracellular role of topoisomerase I in the biology of mycobacteria which are one of the most formidable group of pathogenic organisms.

Functional characterisation of mycobacterial DNA gyrase: an efficient decatenase.

A rapid single step immunoaffinity purification procedure is described for Mycobacterium smegmatis DNA gyrase. The mycobacterial enzyme is a 340 kDa heterotetrameric protein comprising two subunits each of GyrA and GyrB, exhibiting subtle differences and similarities to the well-characterised Escherichia coli gyrase. In contrast to E.coli gyrase, the M.smegmatis enzyme exhibits strong decatenase activity at physiological Mg2+ concentrations. Further, the enzymes exhibited marked differences in ATPase activity, DNA binding characteristics and susceptibility to fluoroquinolones. The holoenzyme showed very low intrinsic ATPase activity and was stimulated 20-fold in the presence of DNA. The DNA-stimulated ATPase kinetics revealed apparent K0.5 and kcat of 0.68 mM and 0.39 s(-1), respectively. The dissociation constant for DNA was found to be 9.2 nM, which is 20 times weaker than that of E.coli DNA gyrase. The differences between the enzymes were further substantiated as they exhibited varied sensitivity to moxifloxacin and ciprofloxacin. In spite of these differences, mycobacterial DNA gyrase is a functionally and mechanistically conserved enzyme and the variations in activity seem to reflect functional optimisation for its physiological role during mycobacterial genome replication.

An orphan gyrB in the Mycobacterium smegmatis genome uncovered by comparative genomics.

DNA gyrase is an essential topoisomerase found in all bacteria. It is encoded by gyrB and gyrA genes. These genes are organized differently in different bacteria. Direct comparison of Mycobacterium tuberculosis and Mycobacterium smegmatis genomes reveals presence of an additional gyrB in M. smegmatis flanked by novel genes. Analysis of the amino acid sequence of GyrB from different organisms suggests that the orphan GyrB in M. smegmatis may have an important cellular role.

A versatile in vivo footprinting technique using 1,10-phenanthroline-copper complex to study important cellular processes.

A number of reagents have been used to define the sequence-specific protein-DNA contacts by footprinting analysis. We report a new in vivo technique using the complex of 1,10-phenanthroline and copper [(OP(2))Cu] as a probe to study various intracellular DNA-protein interactions in whole cells. The versatility of the protocol is demonstrated by applying the technique to address various processes. The protocol is applied to (i) detect structural alterations in DNA as a result of single base substitution, (ii) footprint site-specific DNA-binding proteins, (iii) analyze promoter occupancy by RNA polymerase and (iv) analyze molecular interactions during transcription initiation. The results demonstrate that in vivo (OP)(2)Cu probing is a useful tool in studying important cellular processes involving DNA-protein interactions and has potential applications in post-genomic research.

Effect of different classes of inhibitors on DNA gyrase from Mycobacterium smegmatis.

Quinolones, coumarins, cyclothialidines, CcdB and microcin B17 inhibit DNA gyrase. Information regarding these various inhibitors comes from studies performed with the enzyme from Escherichia coli, and subsequent analyses have also primarily been confined to this system. We have carried out a detailed analysis of the effect of various groups of inhibitors on Mycobacterium smegmatis gyrase and demonstrate differential susceptibility of the E. coli and M. smegmatis gyrases. Interestingly, M. smegmatis gyrase was refractory to the plasmid-borne proteinaceous inhibitors CcdB and microcin B17. Ciprofloxacin, a fluoroquinolone, showed a 10-fold reduction in efficacy against M. smegmatis compared with E. coli gyrase. We have also shown that etoposide, an antineoplastic drug, inhibits DNA gyrase activity by trapping the gyrase-DNA complex. DNA gyrases from both E. coli and M. smegmatis were susceptible to etoposide at comparable levels.

DNA unwinding mechanism for the transcriptional activation of momP1 promoter by the transactivator protein C of bacteriophage Mu.

Transcription factor-induced conformational changes in DNA are one of the mechanisms of transcription activation. C protein of bacteriophage Mu appears to transactivate the mom gene of the phage by this mode. DNA binding by C to its site leads to torsional changes that seem to compensate for a weak momP1 promoter having a suboptimal spacing of 19 bp between the poor -35 and -10 elements. The C-mediated unwinding could realign the promoter elements for RNA polymerase recruitment to the reoriented promoter. In this study, the model has been tested by mutational analysis of the spacer region of momP1 and by assessing the strength of the mutant promoters. The response to C-mediated transactivation was dependent on the spacer length of the promoters. Mutants with 17-bp spacing between the two promoter elements showed reduced activity in the presence of the transactivator as compared with their basal level. A synthetic promoter with near consensus promoter elements and optimal 17-bp spacing was also tested to evaluate the model. The results imply a role for C-mediated unwinding in mom transcription activation.

A novel bipartite mode of binding of M. smegmatis topoisomerase I to its recognition sequence.

We have investigated interaction of Mycobacterium smegmatis topoisomerase I at its specific recognition sequence. DNase I footprinting demonstrates a large region of protection on both the scissile and non-scissile strands of DNA. Methylation protection and interference analyses reveal base-specific contacts within the recognition sequence. Missing contact analyses reveal additional interactions with the residues in both single and double-stranded DNA, and hence underline the role for the functional groups associated with those bases. These interactions are supplemented by phosphate contacts in the scissile strand. Conformation specific probes reveal protein-induced structural distortion of the DNA helix at the T-A-T-A sequence 11 bp upstream to the recognition sequence. Based on these footprinting analyses that define parameters of topoisomerase I-DNA interactions, a model of topoisomerase I binding to its substrate is presented. Within the large protected region of 30 bp, the enzyme makes direct contact at two locations in the scissile strand, one around the cleavage site and the other 8-12 bases upstream. Thus the enzyme makes asymmetric recognition of DNA and could carry out DNA relaxation by either of the two proposed mechanisms: enzyme bridged and restricted rotation.