Complex population history of two Anopheles dirus mosquito species in Southeast Asia suggests the influence of Pleistocene climate change rather than human-mediated effects.

Anopheles dirus and Anopheles baimaii are closely related species which feed on primates, particularly humans, and transmit malaria in the tropical forests of mainland Southeast Asia. Here, we report an in-depth phylogeographic picture based on 269 individuals from 21 populations from mainland Southeast Asia. Analysis of 1537 bp of mtDNA sequence revealed that the population history of A. baimaii is far more complex than previously thought. An old expansion (pre-300 kyr BP) was inferred in northern India/Bangladesh with a wave of south-eastwards expansion arriving at the Thai border (ca 135-173 kyr BP) followed by leptokurtic dispersal very recently (ca 16 kyr BP) into peninsular Thailand. The long and complex population history of these anthropophilic species suggests their expansions are not in response to the relatively recent (ca 40 kyr BP) human expansions in mainland Southeast Asia but, rather, fit well with our understanding of Pleistocene climatic change there.

High levels of population structure caused by habitat islands in the malarial vector Anopheles scanloni.

The genetic structure of four populations of the malarial vector Anopheles scanloni in Thailand was studied using mitochondrial DNA sequences. Four highly divergent lineages were observed, all with signals of population expansion. Since An. scanloni is restricted to 'islands' of limestone karst habitat, this suggests there is a metapopulation-type dynamic in this species, with restricted gene flow, extinctions and drift all contributing to lineage divergence. Historical environmental change and marine transgressions may also have contributed to population extinction, expansion and divergence. Although there is some current gene flow inferred between nearby populations, it is extremely restricted between the northern and southern populations, which also differed by one fixed polymorphism at the ITS2 rDNA locus. Crossing experiments showed no post-mating barriers existing between the north and the south, but the lack of gene flow between these populations could ultimately result in speciation and has implications for malaria control strategies.

Molecular identification of mosquito species in the Anopheles annularis group in southern Asia.

The Anopheles annularis group of subgenus Cellia Theobald (Diptera: Culicidae) includes five currently recognized species in southern Asia: An. annularis Van der Wulp, Anopheles nivipes (Theobald) and Anopheles philippinensis Ludlow, which are widespread in the region, Anopheles pallidus Theobald, which is known in Sri Lanka, India and Myanmar, and Anopheles schueffneri Stanton, which occurs in Java and Sumatra. Identification of the four mainland species based on morphology is problematic. In view of the fact that the three widespread species are variously involved in malaria transmission in different parts of the region, we developed a species-specific polymerase chain reaction assay based on rDNA internal transcribed spacer 2 (ITS2) sequences to facilitate entomological and epidemiological studies of the four species. The method proved to be reliable when tested over a wide geographical area.

Mycosis fungoides--a review of the management of 28 patients and of the recent literature.

BACKGROUND: Mycosis fungoides is an uncommon cutaneous T-cell lymphoma characterized by malignant monoclonal proliferation of T-helper lymphocytes. Its course is variable with a potential for lymphatic and hematogenous involvement. We report the investigations, staging, treatment, follow-up, and outcome of 28 patients. This is the first such study reported from Ireland. METHODS: Twenty-eight patients with mycosis fungoides (14 women, 14 men; average age, 52.5 years) were reviewed over 12 years in the dermatology clinic which assesses an average of 4500 patients per year. All mycosis fungoides patients were referred from their family physicians. The diagnosis was made in all cases from a combination of clinical findings, histology, and immunohistochemistry. TNM staging revealed 11 patients at diagnosis stage IA (T1), 12 at stage IB (T2), four at stage IIB (T3), and one at stage III (T4). RESULTS: The usual male preponderance was not found. Eight patients needed multiple biopsies to establish the diagnosis. Detailed investigations were not useful in the early stages. Patients were followed up over a 12-year period. Thirteen patients died as a result of cutaneous lymphoma. Two patients with stage IA disease progressed rapidly and died, a feature reported in only 10% of patients at this stage. Five patients showed unusual features, including a long history prior to presentation, the development of the rarely reported bullous mycosis fungoides, and aggressive disease beginning at a young age. CONCLUSIONS: Mycosis fungoides is rare; we reviewed 28 patients over 12 years. The prognosis is poor at the later stages; 13 patients died. Two patients who died were unusual in that they rapidly progressed from stage IA disease; however, in the majority of patients with this stage, the prognosis is excellent. Detailed investigations were unhelpful in early stage disease. Close clinical follow-up is essential to identify disease progression.

Genetic diversity and molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of rDNA.

The species diversity and genetic structure of mosquitoes belonging to the Anopheles maculatus group in Southeast Asia were investigated using the internal transcribed spacer 2 (ITS2) of ribosomal DNA (rDNA). A molecular phylogeny indicates the presence of at least one hitherto unrecognised species. Mosquitoes of chromosomal form K from eastern Thailand have a unique ITS2 sequence that is 3.7% divergent from the next most closely related taxon (An. sawadwongporni) in the group. In the context of negligible intraspecific variation at ITS2, this suggests that chromosomal form K is most probably a distinct species. Although An. maculatus sensu stricto from northern Thailand and southern Thailand/peninsular Malaysia differ from each other in chromosomal banding pattern and vectorial capacity, no intraspecific variation was observed in the ITS2 sequences of this species over this entire geographic area despite an extensive survey. A PCR-based identification method was developed to distinguish five species of the group (An. maculatus, An. dravidicus, An. pseudowillmori, An. sawadwongporni and chromosomal form K) to assist field-based studies in northwestern Thailand. Sequences from 187 mosquitoes (mostly An. maculatus and An. sawadwongporni) revealed no intraspecific variation in specimens from Thailand, Cambodia, mainland China, Malaysia, Taiwan and Vietnam, suggesting that this identification method will be widely applicable in Southeast Asia. The lack of detectable genetic structure also suggests that populations of these species are either connected by gene flow and/or share a recent common history.

Multidimensional analysis of the insoluble sub-proteome of Oceanobacillus iheyensis HTE831, an alkaliphilic and halotolerant deep-sea bacterium isolated from the Iheya ridge.

We report the first proteomic analysis of the insoluble sub-proteome of the alkaliphilic and halotolerant deep-sea bacterium Oceanobacillus iheyensis HTE831. A multidimensional gel-based and gel-free analysis was utilised and a total of 4352 peptides were initially identified by automated MS/MS identification software. Automated curation of this list using PROVALT reduced our peptide list to 467 uniquely identified peptides that resulted in the positive identification of 153 proteins. These identified proteins were functionally classified and physiochemically characterised. Of 26 proteins identified as hypothetical conserved, we have assigned function to all but four. A total of 41 proteins were predicted to possess signal peptides. In silico investigation of these proteins allowed us to identify three of the five bacterial classes of signal peptide, namely: (i) twin-arginine translocation; (ii) Sec-type and (iii) lipoprotein transport. Our proteomic strategy has also allowed us to identify, at neutral pH, a number of proteins described previously as belonging to two putative transport systems believed to be of importance in the alkaliphilic adaptation of O. iheyensis HTE831.

Multidimensional proteomic analysis of the soluble subproteome of the emerging nosocomial pathogen Ochrobactrum anthropi.

We report the first large-scale gel-free proteomic analysis of the soluble subproteome of the emerging pathogen Ochrobactrum anthropi. Utilizing our robust offline multidimensional protein identification protocol, a total of 57 280 peptides were initially identified utilizing automated MS/MS analysis software. We describe our investigation of the heuristic protein validation tool PROVALT and demonstrate its ability to increase the speed and accuracy of the curation process of large-scale proteomic datasets. PROVALT reduced our peptide list to 8517 identified peptides and further manual curation of these peptides led to a final list of 984 uniquely identified peptides that resulted in the positive identification of 249 proteins. These identified proteins were functionally classified and physiochemically characterized. A variety of typical "housekeeping" functions identified within the proteome included nucleic acid, amino and fatty acid anabolism and catabolism, glycolysis, TCA cycle, and pyruvate and selenoamino acid metabolism. In addition, a number of potential virulence factors of relevance to both plant and human disease were identified.

A combined shotgun and multidimensional proteomic analysis of the insoluble subproteome of the obligate thermophile, Geobacillus thermoleovorans T80.

To further our understanding of the biology of the thermophilic bacterium Geobacillus thermoleovorans T80, we now report the first proteomic analysis of the insoluble subproteome of this isolate. A combination of both shotgun and multidimensional methodologies were utilized, and a total of 8628 peptides was initially identified by automated MS/MS identification software. Curation of these peptides led to a final list of 184 positive protein identifications. The proteins from this insoluble subproteome were functionally classified, and physiochemical characterization was carried out. Of 15 hypothetical conserved proteins identified, we have assigned function to all but four. A total of 31 proteins were predicted to possess signal peptides. In silico investigation of these proteins allowed us to identify four of the five bacterial classes of signal peptide, namely, (i) twin-arginine translocation; (ii) Sec-type; (iii) lipoprotein, and (iv) ABC transport. In addition, a number of proteins were identified that are known to be involved in the transport of compatible solutes, known to be important in microbial stress responses.

Protein tyrosine phosphatase genes downregulated in melanoma.

Phospho-tyrosine levels are increased in melanoma, apparently consistent with reports of elevated protein tyrosine kinase activity. Some protein tyrosine kinases are encoded by oncogenes and have been implicated in melanoma genesis. Decreased protein tyrosine phosphatase activity may also increase phospho-tyrosine. Protein tyrosine phosphatase genes are candidate tumor suppressors and loss of expression may contribute to melanoma genesis. Here we survey protein tyrosine phosphatase expression in pigment cells. Protein tyrosine phosphatase genes were cloned by reverse transcriptase polymerase chain reaction using degenerate primers based upon conserved sequences within the phosphatase catalytic domain. Reaction products were cloned and sequenced: 118 and 113 partial protein tyrosine phosphatase products were isolated from normal melanocytes and melanoma cells, respectively. Northern blotting analysis was used to study expression of 15 protein tyrosine phosphatase genes. Expression of PTP-kappa and PTP-pi was absent or downregulated in more than 20% of melanoma cell lines and in some unmanipulated melanoma biopsies. These closely related enzymes are members of the 2B receptor protein tyrosine phosphatase family previously implicated in contact inhibition. Loss of protein tyrosine phosphatase expression may contribute to the abnormal tyrosine phosphorylation seen in melanoma; these genes are candidate tumor suppressors.

Suspected skin malignancy: a comparison of diagnoses of family practitioners and dermatologists in 493 patients.

BACKGROUND: In the Irish health system, dermatology patients present to their family practitioner for diagnosis and treatment, and are referred to a dermatologist for a second opinion where diagnosis is in doubt or when there has been therapeutic failure. The level of expertise in dermatology amongst family practitioners varies considerably. AIM: To compare the diagnoses of general practitioners and dermatologists over a selected period in patients with a possible diagnosis of skin cancer. METHODS: Four hundred and ninety-three patients were seen by one of two dermatologists over a 1-year period at a rapid referral clinic for patients suspected by their family practitioners of having unstable or possibly malignant skin lesions; 213 of these patients had a diagnosis made on clinical examination by the dermatologist, while 264 had diagnostic or therapeutic biopsies performed; 16 patients defaulted on surgery. RESULTS: The diagnoses of the family practitioners agreed with the diagnoses of the dermatologists on patients diagnosed clinically in 54% of cases. Thirty-eight patients had histologically proven skin malignancy. These were diagnosed accurately by the referring family practitioner in 22% of patients, while the dermatologists made the correct diagnosis prior to biopsy in 87%. CONCLUSIONS: In over 50% of cases diagnosed clinically, the dermatologist and family practitioner agreed. Histologically proven skin cancers were diagnosed accurately in only 22% of cases by family practitioners, compared to 87% of cases by dermatologists. Specific areas of diagnostic difficulty for family practitioners include benign pigmented actinic and seborrheic keratoses, squamous cell carcinoma, and melanoma. Postgraduate education for family practitioners should be directed towards these areas of deficiency. Dermatologists had difficulty distinguishing pigmented actinic keratoses from melanoma.

Choice of management strategy for colorectal cancer based on a diagnostic immunohistochemical test for defective mismatch repair.

BACKGROUND: Despite intensive research into the molecular abnormalities associated with colorectal cancer (CRC), no diagnostic tests have emerged which usefully complement standard histopathological assessments. AIMS: To assess the feasibility of using immunohistochemistry to detect replication error (RER) positive CRCs and determine the incidence of RER positivity within distinct patient subgroups. METHODS: 502 CRCs were analysed for RER positivity (at least two markers affected) and/or expression of hMSH2 and hMLH1. RESULTS: There were 15/30 (50%) patients with metachronous CRCs, 16/51 (31%) with synchronous CRCs, 14/45 (31%) with a proximal colon carcinoma, and 4/23 (17%) who developed a CRC under the age of 50 showed RER positivity. However, 0/54 patients who developed a solitary carcinoma of the rectum/left colon over the age of 50 showed RER positivity. Immunohistochemical analysis revealed that 66/66 (100%) RER positive carcinomas were associated with complete lack of expression of either hMSH2 or hMLH1. This correlation was confirmed using a further 101 proximal colon carcinomas. Patients with a mismatch repair defective carcinoma showed improved survival but a 5.54 times relative risk of developing a metachronous CRC. A prospective immunohistochemical study revealed 13/117 (11%) patients had a mismatch repair defective carcinoma. A fivefold excess of hMLH1 defective cases was noted. CONCLUSIONS: All RER positive carcinomas were identified by the immunohistochemical test. This is the first simple laboratory test which can be performed routinely on all CRCs. It will provide a method for selecting patients who should be investigated for HNPCC, offered long term follow up, and who may not respond to standard chemotherapy regimens.

A clinicopathological correlation of 134 stage 1 and 79 non-invasive cutaneous melanomas presenting over a decade (1984-1993) at the Mater Misericordiae Hospital, Dublin.

In a study of malignant melanoma during the period 1984-1993, 134 (63 per cent) had invasive melanoma and in 79 (37 per cent) melanoma was confined to the epidermis (in situ). There was female predominance, F: M = 2.4 : 1, a family history of melanoma in 1.5 per cent, a mean age at diagnosis of 50 yr. Females presented a decade earlier than males on average. Over half of invasive melanomas in females occurred on lower limbs; 40 per cent of lesions in males occurred on the trunk. Almost one third of lesions in males and over two thirds in females occurred in sun exposed area. Sixty per cent of invasive lesions were of the superficial spreading type and half of all lesions were histologically thin [less than 1.5 mm vertical depth]. Surprisingly, median lesion thickness was lower in males, probably reflecting the greater frequency of nodular lesions in females compared to males (36 per cent -v- 24 per cent). The marked increase in the number of invasive melanoma patients presenting in the second half of the decade studied (treble that of the first half) probably reflects an increase in melanoma incidence. Over the decade no change in invasive melanoma type, anatomical site or histological thickness was noted, the latter suggesting a failure to diagnose melanoma at an increasingly earlier stage. An official melanoma public education programme is required, particularly as half of the patients delayed 1 yr or more before seeking medical advice. However it is encouraging that, of the invasive melanomas, 30 per cent were small (< 10 mm), 50 per cent were histologically thin and that 37 per cent of all melanomas were in situ. The melanoma-in-situ group had a similar gender ratio and mean age at diagnosis to the invasive melanoma patients but lesions were smaller, were predominantly on the head, neck and limbs with lentigo melanoma as the commonest type.

Persistent infection of the chin with an unusual skin pathogen (Streptococcus milleri): a sign of intraoral carcinoma.

Streptococcus milleri is a commensal of the oropharynx and gastrointestinal tract which is not generally associated with skin disease. We now report a patient who presented with a pustular mass of the chin with lower lip anaesthesia. He was initially thought to have sycosis barbae, but response to treatment was poor and lesional swabs repeatedly cultured S. milleri. After some delay, squamous cell carcinoma of the mouth, involving the mandible and overlying skin, was detected. We consider that the S. milleri either invaded through the tumour from the mouth or root canal or colonized the skin from saliva dribbled over the numb lower lip. Isolation of an unusual organism and numbness of the chin are features that should suggest the need for early radiography.

Linear IgA disease--a review of four patients.

Four cases of Linear IgA Disease (LAD) seen over a five year period are reviewed and our most recent LAD patient is described in detail. We summarise data on our patients and outline clinicopathologic features, aetiology and management of this unusual but important condition.

Dermatitis Herpetiformis: a review of fifty-four patients.

A review of 54 patients with dermatitis herpetiformis presenting between 1984-1993 to The Regional Centre of Dermatology, Mater Misericordiae Hospital was undertaken. All patients had skin lesions clinically and histologically typical of dermatitis herpetiformis. Deposition of granular IgA at the dermoepidermal junction on direct immunofluorescence was present in each case. The average age of onset was 41.8 yr, patients having symptoms for an average of 1.6 yr before diagnosis. Six patients had a prior history of coeliac disease. Two patients had a family history of dermatitis herpetiformis, a father and son who were both propositi in this study. Small bowel biopsy was performed on 35 patients, 71.4 per cent of them showing evidence of villous atrophy. All patients were controlled on a gluten free diet or by dapsone or a combination of these. None of the patients experienced serious adverse effects of therapy, nor did any develop lymphoma of the small bowel with a mean follow up period of 4.2 yr (range 1-10 yr).

Idiopathic anaphylaxis.

Three cases of idiopathic anaphylaxis are presented. This condition represents a rare but important cause of collapse. The development of atrial fibrillation, reported here for the first time, may lead to diagnostic difficulties. Early recognition and treatment is essential and will result in a good prognosis.