Integrating EM and Patch-seq data: Synaptic connectivity and target specificity of predicted Sst transcriptomic types.

Neural circuit function is shaped both by the cell types that comprise the circuit and the connections between those cell types 1 . Neural cell types have previously been defined by morphology 2, 3 , electrophysiology 4, 5 , transcriptomic expression 6-8 , connectivity 9-13 , or even a combination of such modalities 14-16 . More recently, the Patch-seq technique has enabled the characterization of morphology (M), electrophysiology (E), and transcriptomic (T) properties from individual cells 17-20 . Using this technique, these properties were integrated to define 28, inhibitory multimodal, MET-types in mouse primary visual cortex 21 . It is unknown how these MET-types connect within the broader cortical circuitry however. Here we show that we can predict the MET-type identity of inhibitory cells within a large-scale electron microscopy (EM) dataset and these MET-types have distinct ultrastructural features and synapse connectivity patterns. We found that EM Martinotti cells, a well defined morphological cell type 22, 23 known to be Somatostatin positive (Sst+) 24, 25 , were successfully predicted to belong to Sst+ MET-types. Each identified MET-type had distinct axon myelination patterns and synapsed onto specific excitatory targets. Our results demonstrate that morphological features can be used to link cell type identities across imaging modalities, which enables further comparison of connectivity in relation to transcriptomic or electrophysiological properties. Furthermore, our results show that MET-types have distinct connectivity patterns, supporting the use of MET-types and connectivity to meaningfully define cell types.

Haematopoietic stem cell transplantation in South Africa: Current limitations and future perspectives.

The growing need for haematopoietic stem cell transplantation (HSCT) is reflected in the increasing number of transplants performed globally each year. HSCT provides life-changing and potentially curative therapy for a range of pathologies including haematological malignancies; other indications include certain congenital and acquired disorders of the haematopoietic system, autoimmune conditions and hereditary diseases. The primary goals of HSCT are either to replace haematopoietic stem and progenitor cells (HSPC) following myeloablative chemotherapy or to cure the original pathology with allogeneic HSPCs. Success depends on optimal outcomes at various stages of the procedure including mobilisation of marrow stem/progenitor cells for harvesting from the patient or donor, long-term and sustainable engraftment of these cells in the recipient, and prevention of graft-versus-host disease in the case of allogeneic HSCT. Challenges in South Africa include high cost, limited infrastructure and lack of appropriately trained staff, as well as limitations in securing suitable haematopoietic stem cell donors. This review aims to provide an overview of HSCT and some of the challenges that are faced in the South African context.

The pharmacokinetic and bactericidal characteristics of oral cefixime.

The pharmacokinetics of cefixime (FK 027), a broad-spectrum cephalosporin, were assessed in 12 normal subjects after single oral doses of 50, 100, 200, and 400 mg. Mean peak serum concentrations were 1.02, 1.46, 2.63, and 3.85 micrograms/ml after the four respective doses. Respective mean serum levels at 12 hours were 0.16, 0.33, 0.72, and 1.13 micrograms/ml. Volumes of distribution averaged 0.1 L/kg body weight, and the elimination t1/2 was 3 hours for all doses. The AUC was 7.01, 11.4, 22.5, and 36.4 micrograms X hr/ml for the four doses, respectively. Serum clearance averaged 0.4 mg/min/kg and mean 24-hour urinary recovery was 21%, 19%, 20%, and 16% for the four respective doses. Serum bactericidal titers at 4 hours exceeded 1:16 for Streptococcus pneumoniae, S. pyogenes, Hemophilus influenzae, and Branhamella catarrhalis. Urine bactericidal titers exceeded 1:8 for Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae resistant to the available oral cephalosporins.

Ticarcillin plus clavulanic acid in the treatment of pneumonia and other serious infections.

Clavulanic acid is a potent inhibitor of bacterial beta-lactamases, and ticarcillin is a potent antipseudomonal penicillin. The combination of ticarcillin disodium and clavulanate potassium provides an excellent spectrum of activity against the majority of bacterial pathogens responsible for serious infections in both normal and abnormal hosts. Eighteen courses of therapy were administered to 16 patients; 35 percent of the patients were in poor or critical condition, and all but one had severe underlying disease. Thirteen separate episodes of pneumonia were treated, of which nine were in patients with cystic fibrosis, and 11 involved Pseudomonas aeruginosa. Of the 13 cases of pneumonia, 11 showed clinical cure or improvement, whereas only three showed bacteriologic cure. Of the four nonpulmonary cases, three showed clinical improvement or cure, and one showed a bacteriologic cure. In two patients, phlebitis developed at the site of intravenous infusion. The combination of ticarcillin and clavulanic acid is safe and effective therapy for pneumonia in anatomically compromised hosts.

(2,6-Dimethylphenylsulphonyl)nitromethane: a new structural type of aldose reductase inhibitor which follows biphasic kinetics and uses an allosteric binding site.

Many of the complications of diabetes seem to be due to aldose reductase (aldehyde reductase 2, ALR2) catalysing the increased conversion of glucose to sorbitol. Therapy with aldose reductase inhibitors (ARIs) could, therefore, decrease the development of diabetic complications. (2,6-Dimethylphenylsulphonyl)nitromethane (ICI 215918) is an example from a newly discovered class of ARIs, and we here describe its kinetic properties. Preparations of bovine lens ALR2 exhibit biphasic kinetics with respect to glucose and various inhibitors including ICI 215918. The inhibitor sensitive form (ALR2S) has a higher affinity for glucose than does the inhibitor insensitive form (ALR2I). Only ALR2S was characterized in detail because ALR2I activity is very low at physiological levels of glucose and is difficult to measure with accuracy. Aldehyde reductase (ALR1) is the most closely related enzyme to ALR2. Inhibition of ALR1 was, therefore, investigated in order to assess the specificity of ICI 215918. The values of Ki and Kies (dissociation constants for inhibitor from enzyme-inhibitor and enzyme-inhibitor-substrate complexes, respectively) for ICI 215918 with bovine kidney ALR1 and bovine lens ALR2S have been determined. When glucose is varied, the compound is an uncompetitive inhibitor of ALR2S (Kies = 0.10 microM and Ki is much greater than Kies), indicating that ICI 215918 associates with an allosteric site on the enzyme. These kinetic characteristics would cause a decrease in the concentration required to give 50% inhibition when glucose levels rise during hyperglycaemia. ICI 215918 is a mixed noncompetitive inhibitor of ALR1 (Ki = 10 microM and Kies = 1.8 microM) when glucuronate is varied. Thus, the compound has up to 100-fold specificity in favour of ALR2S relative to ALR1. Therapeutic interest has now centred upon at least three distinct structural types of ARIs: spirohydantoins, acetic acids and sulphonylnitromethanes. Using one representative of each type, we have demonstrated kinetic competition for inhibition of ALR2S. This observation strongly suggests that the different inhibitors use overlapping binding sites.

Kinetic characteristics of ZENECA ZD5522, a potent inhibitor of human and bovine lens aldose reductase.

Aldose reductase (aldehyde reductase 2) catalyses the conversion of glucose to sorbitol, and methylglyoxal to acetol. Treatment with aldose reductase inhibitors (ARIs) is a potential approach to decrease the development of diabetic complications. The sulphonylnitromethanes are a recently discovered class of aldose reductase inhibitors, first exemplified by ICI215918. We now describe enzyme kinetic characterization of a second sulphonylnitromethane, 3',5'-dimethyl-4'-nitromethylsulphonyl-2-(2-tolyl)acetanilide (ZD5522), which is at least 10-fold more potent against bovine lens aldose reductase in vitro and which also has a greater efficacy for reduction of rat nerve sorbitol levels in vivo (ED95 = 2.8 mg kg-1 for ZD5522 and 20 mg kg-1 for ICI 215918). ZD5522 follows pure noncompetitive kinetics against bovine lens aldose reductase when either glucose or methylglyoxal is varied (K(is) = K(ii) = 7.2 and 4.3 nM, respectively). This contrasts with ICI 215918 which is an uncompetitive inhibitor (K(ii) = 100 nM) of bovine lens aldose reductase when glucose is varied. Against human recombinant aldose reductase, ZD5522 displays mixed noncompetitive kinetics with respect to both substrates (K(is) = 41 nM, K(ii) = 8 nM with glucose and K(is) = 52 nM, K(ii) = 3.8 nM with methylglyoxal). This is the first report of the effects of a sulphonylnitromethane on either human aldose reductase or utilization of methylglyoxal. These results are discussed with reference to a Di Iso Ordered Bi Bi mechanism for aldose reductase, where the inhibitors compete with binding of both the aldehyde substrate and alcohol product. This model may explain why aldose reductase inhibitors follow noncompetitive or uncompetitive kinetics with respect to aldehyde substrates, and X-ray crystallography paradoxically locates an ARI within the substrate binding site. Aldehyde reductase (aldehyde reductase 1) is closely related to aldose reductase. Inhibition of bovine kidney aldehyde reductase by ZD5522 follows uncompetitive kinetics with respect to glucuronate (K(ii) = 39 nM), indicating a selectivity greater than 5-fold for bovine aldose reductase relative to aldehyde reductase.

The pharmacokinetics and serum and urine bactericidal activity of ciprofloxacin.

Ciprofloxacin is an investigational quinolone agent possessing an impressive antibacterial spectrum. Its pharmacokinetics were studied in six volunteers after 250-mg and 500-mg single oral doses, and its bactericidal activity compared to that of trimethoprim-sulfamethoxazole given to the same volunteers. Mean peak serum levels were 1.45 micrograms/mL and 2.46 micrograms/mL for 250-mg and 500-mg doses, and time to peak was 1 and 1.3 hours. The 12-hour levels were 0.12 micrograms and 0.22 microgram. Half-life (T1/2)alpha were 0.32 and 0.43 with T1/2 beta were 3.97 and 4.15 and volume of distribution (area) were 80L and 90L, respectively. Area under the concentration curve (AUC) was 5.65 h X micrograms/mL and 10.37h X micrograms/mL. Serum clearance was 23L for both doses. Approximately 49% of the 250-mg dose and 43% of the 500-mg dose was recovered in the urine. Bactericidal levels were determined against clinical isolates. Sera at 1.5 hours after the 500-mg dose averaged bactericidal levels of 1:20 or better for an Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and beta-lactamase producing Haemophilus influenzae and Branhamella catarrhalis. Urinary bactericidal levels at eight to 12 hours were greater than or equal to 1:157 for E coli, K pneumoniae, gentamicin-piperacillin resistant P aeruginosa, Staphylococcus aureus, and 1:20 for Streptococcus faecalis. Serum bactericidal levels were superior, and urine bactericidal levels were superior or equal to the bactericidal levels obtained with trimethoprim-sulfamethoxazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Spacer oligonucleotide typing of bacteria of the Mycobacterium tuberculosis complex: recommendations for standardised nomenclature.

Spacer oligonucleotide typing (spoligotyping) is widely used for differentiation of bacteria of the Mycobacterium tuberculosis complex. However, the absence of any standardised method for concise description of spoligotypes makes it difficult to compare the results from different laboratories. This paper describes unambiguous, interconvertible systems for the designation of spoligotype patterns, the adoption of which will be beneficial to mycobacterial research.

Erythromycin.

Erythromycin is the only macrolide antibiotic to have gained widespread use in the United States. Introduced in 1952, it rapidly gained a popularity that it enjoys to this day. Numerous other antimicrobial agents have been marketed since that time: Whole new classes of antibiotics, both natural and synthetic, have been discovered, studied, and released for general use. Many of these newer agents boast a broader spectrum of antimicrobial activity, yet erythromycin's place in the clinician's arsenal is unthreatened because erythromycin remains the drug of first choice for a number of pathogens against which the new drugs are inactive. It is one of the safest antibiotics available for use today and when used against susceptible organisms and in indicated clinical situations, its effectiveness is unquestioned.

Spacer oligotyping of Mycobacterium bovis isolates compared to typing by restriction fragment length polymorphism using PGRS, DR and IS6110 probes.

Ninety-two Mycobacterium bovis isolates from cattle, deer and badgers in Northern Ireland and the Republic of Ireland were genotyped by spacer-oligotyping (spoligotyping) and 67 of these were analysed by restriction fragment length polymorphism (RFLP). RFLP analysis was performed using three DNA probes, PGRS, DR and IS6110. Forty-seven of the M. bovis isolates were from 45 different sources; these were typed using both RFLP and spoligotyping. These 47 isolates could be differentiated into 24 different RFLP types and 15 distinct spoligotypes. Although RFLP was found to be more discriminatory compared to the present spoligotyping technique, spoligotyping was able to differentiate 21 RFLP type 'ACA' isolates into three different patterns. The remaining 45 M. bovis isolates were from a small case study, involving infected cattle, deer and badgers from the same geographic region. All these isolates were analysed by spoligotyping and a selection of 20 isolates were RFLP typed. All the isolates in the case study had the same spoligotype pattern with the exception of one cervine isolate. Similarly all the isolates typed by RFLP had the same pattern. Consequently, the predominant strain in the case study was not host restricted. The consistency between the results obtained using the two techniques indicates the potential value of both techniques for epidemiological studies. Spoligotyping was found to be a much more rapid technique and easier to perform, requiring less sophisticated computer software for strain typing. Spoligotyping results were more readily documented and analysed and the technique was also more suitable than RFLP analysis for large-scale screening studies.

Inhibition of aldose reductase by (2,6-dimethylphenylsulphonyl)nitromethane: possible implications for the nature of an inhibitor binding site and a cause of biphasic kinetics.

Aldose reductase (aldehyde reductase 2, ALR2) is often isolated as a mixture of two forms which are sensitive (ALR2S), or insensitive (ALR2I), to inhibitors. We show that ICI 215918 ((2-6-dimethylphenylsulphonyl)-nitromethane) follows either noncompetitive, or uncompetitive kinetics with respect to aldehyde for ALR2S, or the closely related enzyme, aldehyde reductase (aldehyde reductase 1, ALR1). Similar behaviour is exhibited by two other structural types of aldose reductase inhibitor (ARI), spirohydantoins and acetic acids, when either aldehyde, or NADPH is varied. For ALR2S, we have demonstrated kinetic competition between a sulphonylnitromethane, an acetic acid and a spirohydantoin. Thus, different ARIs probably have overlapping binding sites. Published studies imply that ALR2 follows an ordered mechanism where coenzyme binds first and induces a reversible conformation change (E.NADPH-->E*.NADPH). Reduction of aldehyde appears rate-limited by the step E*.NADP+-->E.NADP+. Spontaneous activation converts ALR2S into ALR2I and increases kcat. This must be associated with acceleration of the rate-determining step. We now propose the following hypothesis to explain characteristics of ARIs. (1) Inhibitors preferentially bind to the E* conformation. (2) The ARI binding site contains residues in common with that for aldehyde substrates. When aldehyde is varied, uncompetitive inhibition arises from association at the site for alcohol product in the E*.NADP+ complex which has little affinity for the substrate. Any competitive inhibition arises from use of the aldehyde site in the E*.NADPH complex. (3) Acceleration of the E*.NADP+-->E.NADP+ step upon activation of ALR2 reduces steady state levels of E* and so decreases sensitivity to ARIs.

The pharmacology of orally administered ciprofloxacin.

Ciprofloxacin pharmacokinetics were studied in 6 volunteers after 250 and 500 mg single oral doses. Mean peak serum levels were 1.45 micrograms/ml and 2.5 micrograms/ml for 250 and 500 mg doses. The 12-h levels were 0.12 micrograms and 0.22 micrograms. T1/2 alpha values were 0.32 and 0.43 h; T1/2 beta was 4 h and Vd (area) values were 80L and 90L for the two doses respectively. AUC was 5.65 h. micrograms/ml and 10.37 h. micrograms/ml. Serum clearance was 23L for both doses. Approximately 49% of the 250 mg dose and 43% of the 500 mg dose was recovered in the urine. Ciprofloxacin's in vitro activity and human pharmacology should permit a twice or once-daily dosing schedule for systemic infections due to most Enterobacteriaceae, Haemophilus, Branhamella and Pseudomonas and S. aureus, and once-daily doses for urinary and gastrointestinal infections.

Genetic typing of ruminant pestivirus strains from Northern Ireland and the Republic of Ireland.

A study was performed to investigate the genotypes and sub-groups of pestiviruses present in ruminants in Ireland. These comprised one ovine and eighteen bovine pestiviruses from Northern Ireland and six bovine pestiviruses from the Republic of Ireland. A 288 base pair (bp) portion of the 5'-non coding region (5'-NCR) from each of 25 pestiviruses collected over a period of 31 years was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) and the product directly sequenced. From each pestivirus, nucleotide sequences corresponding to bases 130 to 374 of the 5'-NCR of NADL were aligned and compared with each other and with the corresponding sequences of a number of reference, field or vaccinal strains of BVDV types I and II, border disease virus and classical swine fever virus. All of the 25 sequenced pestiviruses were found to be BVDV type Ia. These were closely related to the constituent viruses of the 2 inactivated vaccines currently licensed for use in Northern Ireland and to recent bovine isolates from England.

Identifying reinforcers for persons with profound handicaps: staff opinion versus systematic assessment of preferences.

We evaluated a systematic means of determining stimulus preferences among seven profoundly handicapped persons. Preferences were determined by observing student approach responses to individual stimuli. Results indicated that there were differential stimulus preferences across the multiply handicapped participants. However, results of the systematic assessment did not coincide with the results of a more traditional, caregiver-opinion method of assessing student preferences. A second experiment was then conducted with five participants to evaluate whether stimuli that were assessed to consistently represent preferences would function as reinforcers in skill training programs. Results indicated that stimuli that were systematically assessed to represent student preferences typically functioned as reinforcers when applied contingently. However, preferred stimuli as reflected by caregiver opinion did not function as reinforcers unless those stimuli were also preferred on the systematic assessment. Results are discussed in terms of assisting profoundly handicapped persons by (a) improving the effectiveness of training programs by increasing the likelihood of using stimuli that have reinforcing value and (b) increasing the overall quality of life by providing preferred stimuli in the routine living environment.

Haematopoietic stem cell transplantation in South Africa: Current limitations and future perspectives

The growing need for haematopoietic stem cell transplantation (HSCT) is reflected in the increasing number of transplants performed globally each year. HSCT provides life-changing and potentially curative therapy for a range of pathologies including haematological malignancies; other indications include certain congenital and acquired disorders of the haematopoietic system, autoimmune conditions and hereditary diseases. The primary goals of HSCT are either to replace haematopoietic stem and progenitor cells (HSPC) following myeloablative chemotherapy or to cure the original pathology with allogeneic HSPCs. Success depends on optimal outcomes at various stages of the procedure including mobilisation of marrow stem/progenitor cells for harvesting from the patient or donor, long-term and sustainable engraftment of these cells in the recipient, and prevention of graft-versus-host disease in the case of allogeneic HSCT. Challenges in South Africa include high cost, limited infrastructure and lack of appropriately trained staff, as well as limitations in securing suitable haematopoietic stem cell donors. This review aims to provide an overview of HSCT and some of the challenges that are faced in the South African context

Establishing an educational programme for nurses to supply emergency hormonal contraception (combined method) to protocol.

This paper gives an account of an innovative educational programme developed by the Department of Midwifery Studies at the University of Central Lancashire (UCLAN) in 1995. The North West Regional Health Authority (NWRHA) approached the Department of Midwifery Studies to develop an educational programme for family planning nurses to supply the combined method of emergency hormonal contraception (EHC) under protocol when a doctor was not present. The purpose was to increase the availability and accessibility of EHC for young people in the North West region. The 3-day programme was designed to complement previous ENB 901/900 training, and also to provide the nurses with the specific skills and knowledge required to undertake this new role. One hundred and thirty-nine nurses from the North West area attended the programme between 1995-1998. Students were assessed both theoretically and clinically. Extending the role of family planning nurses to supply EHC gives purchasers and providers of sexual health care the potential to offer a wider range of accessible services. The recently published interim Crown Report1 on the supply and administration of medicines under group protocols states that protocols should specify clear arrangements for professional responsibility and accountability. Appropriate training is essential to ensure that the extended role of the nurse in family planning is fully understood.

In vitro activity of Ro 23-6240, a new fluorinated 4-quinolone.

The in vitro activity of Ro 23-6240 (AM833), 6,8-difluoro-1-(2-fluoroethyl)-1,4-dihydro-4-oxo-7(4-methyl-1-piper azinyl) quinolone-3-carboxylic acid, was compared with those of norfloxacin, enoxacin, ofloxacin, and ciprofloxacin. Ro 23-6240 inhibited the majority of Enterobacteriaceae isolates at a concentration of less than or equal to 0.5 microgram/ml. It was especially active against Shigella sp., Salmonella sp., Escherichia coli, and Yersinia enterocolitica, with an MIC for 90% of the strains of less than or equal to 0.12 microgram/ml. The MIC for 90% of the strains was 1 microgram/ml for Serratia marcescens and 8 micrograms/ml for Pseudomonas aeruginosa. Staphylococcus aureus isolates, including methicillin-resistant strains, were inhibited by less than or equal to 1 microgram/ml. Streptococcal and anaerobic species were inhibited by 8 to 16 micrograms/ml. Ro 23-6240 inhibited beta-lactamase-producing bacteria resistant to broad-spectrum cephalosporins. The overall activity of Ro 23-6240 was similar to those of enoxacin, norfloxacin, and ofloxacin, but less than that of ciprofloxacin. The frequency of spontaneous resistance was low, although resistant bacteria could be isolated by repeated subculture. The activity of Ro 23-6240 was decreased in the presence of magnesium at concentrations similar to those present in urine.

Genomic fingerprinting of Mycobacterium bovis from cattle by restriction fragment length polymorphism analysis.

Two insertion sequences, IS6110 and IS1081, specific to the tuberculosis complex mycobacteria and a highly reiterated DNA element (pTBN12) cloned from Mycobacterium tuberculosis were systematically used to identify restriction fragment length polymorphism (RFLP) types among bovine isolates of Mycobacterium bovis in Northern Ireland. In a sample of 109 isolates, probes IS6110, IS1081, and pTBN12 identified 10, 2, and 12 distinct patterns, respectively. By combining the patterns generated by the three probes it was possible to identify 28 distinct RFLP types. The standard protocol advocated for RFLP analysis of M. tuberculosis was used and would facilitate computer-based gel documentation and image analysis to establish a database of M. bovis types for large-scale epidemiological studies. These procedures will facilitate interlaboratory comparisons of M. bovis isolates and will help to elucidate the precise epidemiology of bovine tuberculosis in different countries.

Differentiation of Mycobacterium bovis isolates from animals by DNA typing.

The insertion sequence IS6110 and the direct repeat (DR) specific to tuberculosis complex mycobacteria and the highly repeated DNA sequence, the polymorphic GC-rich repeat sequence (PGRS), were systematically used to identify restriction fragment length polymorphisms (RFLPs) within 210 isolates of Mycobacterium bovis. The isolates were primarily of bovine origin, but isolates from badgers, feral deer, sheep, humans, and a pig were included. The RFLP probes IS6110, DR, and PGRS individually identified 17, 18, and 18 different RFLP types, respectively, but in combination these probes identified a total of 39 different M. bovis RFLP types. The recommendations (J. D. A. van Embden, M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach, B. Gicquel, P. W. M. Hermans, C. Martin, R. McAdam, T. M. Shinnick, and P. M. Small, J. Clin. Microbiol. 31:406-409, 1993) for a standardized RFLP analysis for M. tuberculosis were adapted to facilitate gel documentation, image analysis, and construction of a database of RFLP types. In the present study the same M. bovis RFLP types were evident in the various animal species included, indicating that the strains were not host restricted. Application of these techniques to defined field studies should help elucidate more accurately aspects of the epidemiology of bovine tuberculosis in different countries.