ENDO-Pore: high-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing.

Mapping the precise position of DNA cleavage events plays a key role in determining the mechanism and function of endonucleases. ENDO-Pore is a high-throughput nanopore-based method that allows the time resolved mapping single molecule DNA cleavage events in vitro. Following linearisation of a circular DNA substrate by the endonuclease, a resistance cassette is ligated recording the position of the cleavage event. A library of single cleavage events is constructed and subjected to rolling circle amplification to generate concatemers. These are sequenced and used to produce accurate consensus sequences. To identify the cleavage site(s), we developed CSI (Cleavage Site Investigator). CSI recognizes the ends of the cassette ligated into the cleaved substrate and triangulates the position of the dsDNA break. We firstly benchmarked ENDO-Pore using Type II restriction endonucleases. Secondly, we analysed the effect of crRNA length on the cleavage pattern of CRISPR Cas12a. Finally, we mapped the time-resolved DNA cleavage by the Type ISP restriction endonuclease LlaGI that introduces random double-strand breaks into its DNA substrates.

Triple therapy in early active rheumatoid arthritis: a randomized, single-blind, controlled trial comparing step-up and parallel treatment strategies.

OBJECTIVE: The Tight Control of Rheumatoid Arthritis study previously demonstrated that an intensive step-up disease-modifying antirheumatic drug (DMARD) treatment strategy targeting persistent disease activity was superior to routine care in the management of early rheumatoid arthritis (RA). We undertook this study to test the hypothesis that early parallel triple therapy achieves better outcomes than step-up therapy within an intensive disease management regimen. METHODS: Ninety-six patients with early RA (mean disease duration 11.5 months) were randomized to receive step-up therapy (sulfasalazine [SSZ] monotherapy, then after 3 months, methotrexate [MTX] was added, and when the maximum tolerated dosage of MTX was reached, hydroxychloroquine [HCQ] was added) or parallel triple therapy (SSZ/MTX/HCQ). All patients were assessed monthly for 12 months. If their disease activity score in 28 joints (DAS28) was > or =3.2, the dosage of DMARDs was increased according to protocol, and swollen joints were injected with triamcinolone acetonide (maximum dosage 80 mg per month). A metrologist who was blinded to the treatment allocation performed assessments every 3 months. The primary outcome measure was the mean decrease in the DAS28 score at 12 months. RESULTS: Both groups showed substantial improvements in disease activity and functional outcome. At 12 months, the mean decrease in the DAS28 score was -4.0 (step-up therapy group) versus -3.3 (parallel therapy group) (P = 0.163). No significant differences in the percentages of patients with DAS28 remission (step-up therapy group 45% versus parallel triple therapy group 33%), DAS28 good response (60% versus 41%, respectively), or American College of Rheumatology criteria for 20% improvement (ACR20) (77% versus 76%, respectively), ACR50 (60% versus 51%, respectively), or ACR70 (30% versus 20%, respectively) responses were seen. Radiologic progression was similar in both groups. CONCLUSION: This study confirms that highly effective control of disease activity can be achieved using conventional DMARDs as part of an intensive disease management strategy. Within this setting, step-up therapy is at least as effective as parallel triple therapy.

Audit of chest X-rays in general practice--a case for local guidelines?

OBJECTIVE: To investigate the use of the chest X-ray (CXR) in a typical urban practice with reference to the Royal College of Radiology (RCR) guidelines. DESIGN: A retrospective review of all requests over a two-year period. SETTING: Urban general practice with 9,463 registered patients. RESULTS: A total of 569 CXRs were performed in 482 patients. Thirty per cent of the CXRs were assessed as being "not indicated" and a significant percentage of requests did not come within the RCR guidelines. The guidelines advise a six weeks' delay following onset of chest wall pain prior to chest X-ray. We assessed the application of the rule in this patient group. A large percentage of those X-rayed were referred at less than the recommended six weeks' interval from time of presentation and the vast majority of CXRs were entirely normal. The "six weeks rule" was developed further in patients with cough, differentiating between simple and productive cough. Those with productive cough who were otherwise well had no significant findings in contrast to the unwell group where several abnormalities were noticed. Radiology-recommended follow-up films were further studied. Where the CXR was carried out after a time interval of less than three weeks, several showed incomplete resolution and a further film was required. CONCLUSION: The RCR guidelines might be adapted to apply to those with simple cough or to those with productive cough who are otherwise well. This simple recommendation, in addition to the RCR guidelines, we feel would reduce the number of unnecessary CXRs performed in general practice.

A regional integrated information system to assure maternal-child health services: a progress report.

Priorities for improving the health of our nation include rebuilding the public health infrastructure through increasing assessment, policy development, and assurance capacities in our communities. Capacity building necessitates formalizing and strengthening public health practice linkages to achieve Year 2000 objectives. Determining progress in achieving objectives requires development of public health infrastructure surveillance and data system capabilities. The Tracking Center of Tracking and Outreach Program for St. Louis (TOPS), through unique collaboration among academic and practice partners, laid the foundation for a Regional Integrated Information System (RIIS) by developing a centralized maternal-child health data base for prenatal and pediatric care providers. The RIIS model provides an example of a capacity building system designed to provide public health surveillance, assessment, planning, and evaluation capabilities. Medical Subject Headings (MeSH): health information systems, prenatal care, immunization, maternal-child health care, public health surveillance.

Doppler ultrasound in pelviureteric junction obstruction in infants and children.

Six infants and seven children with known pelviureteric junction (PUJ) obstruction (11 unilateral, 2 bilateral) were assessed with Doppler ultrasound before and after diuresis. After intravenous frusemide, there was a significant increase in the resistance index in the interlobar renal arteries of the obstructed kidneys. No such change was detected in the normal kidneys. Five patients were reassessed after pyeloplasty. The Doppler indexes facilitated prediction of those cases with a satisfactory outcome after surgery. Doppler ultrasound with diuresis is useful in the assessment of PUJ obstruction in infants and children and in the postoperative follow-up.

Optic foraminal radiography--a redundant investigation?

A retrospective study of all plain radiographs taken of optic foramina during the six years 1984-9 inclusive, comprising a total of 318 examinations, was carried out in order to determine the role of this investigation in patients with ophthalmic disorders. All but one of the radiographs were reported as showing no abnormality. However, for this exception, review of the radiographs showed that the optic foramina were asymmetrical but fell within the normal range. This study indicates that plain radiographs of the optic foramina have little or no useful function. Abandoning the use of these views as a routine will result both in a reduction in radiation hazard and in considerable financial saving.

Comparison of colour Doppler ultrasound with venography in the diagnosis of axillary and subclavian vein thrombosis.

The primary imaging technique in suspected venous occlusive disease has for many years been contrast venography. Recent studies have shown ultrasound with the addition of colour Doppler imaging to be a suitable alternative method in the diagnosis of lower limb venous thrombosis. We have applied these techniques to the upper limb venous system, and have performed a prospective study of 19 patients (30 limbs) comparing colour Doppler ultrasound with venography in the diagnosis of axillary and subclavian vein thrombosis, for which colour Doppler ultrasound has a sensitivity and specificity of 100%. If vein stenosis is included, the sensitivity falls to 89%. We propose that colour Doppler ultrasound is a suitable first-line alternative to venography in the diagnosis of axillary and subclavian vein thrombosis. In addition to showing the major venous drainage of the upper limb, ultrasound routinely assesses patency of the internal jugular vein, which is, on occasion, of clinical relevance when determining possible future sites of venous access. If, however, colour Doppler ultrasound is normal then bilateral upper limb venography is indicated to exclude a more central venous problem or localized stenotic lesion.

Two patients illustrating lymphoma transition and response to therapy in Sjögren's syndrome.

Two patients with Sjögren's syndrome (SS) who subsequently developed malignant B-cell lymphomas are reported in detail. The first patient had both benign- and malignant-appearing lymphoid infiltrates on the same submandibular gland specimen and was successfully treated with combined chemotherapy and irradiation. The second patient developed cutaneous lymphoid infiltrates difficult to diagnose by light microscopy but containing a monoclonal IgM-Kappa population revealed by immunoperoxidase staining and immunoglobulin gene rearrangement studies. Her lesions resolved rapidly and completely on cyclophosphamide, recurred rapidly when this drug was discontinued, and resolved again on a second course of cyclophosphamide which is currently maintained at 50 mg daily. Both patients are doing well without recurrence two and three years after initial treatment. This clinical experience is presented to emphasize: (1) the clinical use of molecular biologic techniques to define the earliest appearance of malignant transformation in Sjögren's syndrome, and (2) the successful outcome that can be achieved with prompt institution of appropriate treatment. The phenomenon of lymphoma development in SS is discussed with regard to immunoregulatory abnormalities predisposing to malignancy in the setting of autoimmune disease.

Regulation of natural killer cell activity by macrophages in the rheumatoid joint and peripheral blood.

Recently, in another study, we observed that indomethacin, a prostaglandin synthetase inhibitor, significantly increased NK activity in both normal and rheumatoid arthritis (RA) peripheral blood (PB) but not in RA synovial fluid (SF). Because macrophages are a major source of prostaglandins, we examined the effect of macrophage-enriched adherent cells (AC) on NK activity as measured by a 3-hr Cr-release assay with K 562 cells. The removal of AC resulted in increased (p less than 0.01) NK activity in both normal and RA PB. In contrast, the removal of AC from RA SF resulted in a significant decrease (p less than 0.001) of NK activity. By using only nonadherent cells (NAC), NK activity in RA SF and synovial tissue (ST) was significantly reduced when compared to autologous RA PB (p less than 0.001). Enhancement of NK activity of SF NAC by both poly I:C and IL 2 was not dependent on AC. Mixing experiments demonstrated that the addition of synovial AC for 16 hr increased NK activity of synovial NAC to a level similar to that of unseparated mononuclear cells, whereas autologous PB AC suppressed NK activity of PB NAC. PB AC, when added to SF NAC, also increased NK activity. Supernatants from synovial mononuclear cells were stimulatory of synovial NAC NK activity, whereas normal PB mononuclear supernatants were suppressive. These observations document 1) a significant reduction of NAC-mediated NK activity in the rheumatoid joint as compared to PB from the same patient, and 2) that AC modulate NK activity differently in the rheumatoid joint as compared to RA or normal PB.

Serum ferritin assay and bone-marrow iron stores in patients on maintenance hemodialysis.

Fifty-five patients on maintenance hemodialysis underwent bone-marrow aspirations for evaluation of iron stores that were to be compared to concomitant measurements of hematocrit, red blood cell volume, serum iron concentration, total iron binding capacity, transferrin saturation, and serum ferritin concentration. In 42 patients (76.4%), the bone marrow iron stores were found to be absent or deficient. Mean hematocrit for the total group was 26.4%, and red blood cell volume measurement showed a mean value of 41.1% of predicted normal. Results also indicated that serum ferritin was the best predictor of iron storage levels, with diagnostic thresholds of 80 to 350 ng/ml derived from statistical analysis of the data. Other hematologic parameters studied had significantly less correlation with bone-marrow iron stores.