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.

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

Mycobacterium tuberculosis complex DNA from an extinct bison dated 17,000 years before the present.

In order to assess the presence of tuberculosis in Pleistocene bison and the origin of tuberculosis in North America, 2 separate DNA extractions were performed by 2 separate laboratories on samples from the metacarpal of an extinct long-horned bison that was radiocarbon dated at 17,870+/-230 years before present and that had pathological changes suggestive of tuberculosis. Polymerase chain reaction amplification isolated fragments of tuberculosis DNA, which were sequenced, and on which spoligotyping was also performed to help determine its relationship to the various members of the Mycobacterium tuberculosis complex. Extensive precautions against contamination with modern M. tuberculosis complex DNA were employed, including analysis of paleontologic and modern specimens in 2 geographically separate laboratories.

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.

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.

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.

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.

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.

Relationship of ventricular tachycardia to sleep/wakefulness in a model of sudden cardiac death.

Death from some childhood disorders such as sudden infant death syndrome and the congenital long QT syndrome are associated with specific behaviors such as sleep or emotional stress. We studied young German shepherd dogs that die suddenly during presumed sleep. These dogs have inherited ventricular tachycardia (VT) which is most frequent during sinus bradycardia and sinus arrhythmia. We hypothesized that the number of VT complexes (three or more consecutive ectopic complexes) would be greatest during sleep. Moreover, we hypothesized that pauses in the sinus rhythm of greater than 1000 ms would be greatest in the behavior with the most frequent VT. Behavioral states [excited, ambulatory, sitting, lying, rapid eye movement (REM) sleep] were quantified from 24-h video recordings of seven dogs. VT and pauses were quantified for each behavior using simultaneously recorded ECGs. A multivariate model was used to analyze the results. After controlling for time of day, lying, and REM sleep were significantly (p < 0.02) associated with VT, whereas more active behaviors were not. Time of day also independently affected the number of VT complexes. However, behavior and time of day did not account for all of the variability in the number of VT complexes. Pauses were significantly associated with behavior, with the highest number of pauses occurring during lying and REM sleep. However, pauses were not always associated with VT, indicating that a pause was a necessary, but not sufficient, condition for the development of VT. These results suggest that modulation of VT incidence in these animals is multifactorial and that the highest number of VT complexes is associated with the bradycardia that accompanies REM sleep.

Phenylephrine-induced ventricular arrhythmias in dogs with inherited sudden death.

INTRODUCTION: Dogs with an inherited predisposition to sudden death display ventricular arrhythmias having certain characteristics, such as pause dependence, that are suggestive of early afterdepolarization-induced triggered activity. We hypothesized that alpha-adrenergic stimulation may facilitate the development of these arrhythmias by inducing a reflex bradycardia and by exerting a direct myocardial effect. METHODS AND RESULTS: Twenty affected dogs and 7 unaffected dogs were studied. The incidence and severity of ventricular arrhythmias were determined after administration of phenylephrine (0.01 mg/kg IV), with or without pretreatment with propranolol (0.1 to 0.3 mg/kg IV), atropine (0.04 mg/kg IV), or prazosin (0.5 mg/kg IV). Third-degree heart block was induced by AV nodal ablation in 4 affected dogs. Phenylephrine increased ventricular arrhythmias in affected dogs, with or without pretreatment with propranolol, but did not induce ventricular arrhythmias in unaffected dogs. In dogs with intact AV nodal conduction, atropine increased sinus rate, which suppressed baseline and phenylephrine-induced arrhythmias. In dogs with heart block, arrhythmias were increased during baseline and after phenylephrine, with or without pretreatment with atropine. Prazosin and overdrive ventricular pacing suppressed phenylephrine-induced arrhythmias. CONCLUSION: Phenylephrine increases ventricular arrhythmias in dogs with inherited sudden death via both an induction of reflex bradycardia and a direct myocardial effect. Superimposition of heightened alpha-adrenergic and vagal tone may facilitate the development of sudden death in these animals.

Fast food arthritis--a clinico-pathologic study of post-Salmonella reactive arthritis.

OBJECTIVE: To study the clinical presentation, immunogenetics, and serum immune response to lipopolysaccharide (LPS) in a cohort of patients with post-Salmonella reactive arthritis (ReA). METHODS: A validate ReA screening questionnaire (Quest 2) was mailed to 919 individuals reporting symptoms of gastroenteritis to the health department after eating at a single restaurant. Three hundred twenty-one persons returned questionnaires; 170 reported symptoms outside the gastrointestinal tract; 23 of those 170 reporting persistent joint symptoms were seen 4 to 16 weeks after the outbreak and 5 of the 23 were seen in followup 12 to 20 weeks later. Clinical features, HLA Class I typing, serum soluble CD8 levels, and serum antibodies to gram negative LPS by ELISA were determined. RESULTS: Joint complaints were reported more frequently by individuals with a longer duration of diarrhea. Upper extremity joints were frequently involved, and 66% reported one or more extraarticular symptoms of Reiter's syndrome. Three of 5 typed individuals were HLA-B27 positive, including 3 of the 4 most severely involved. Serum soluble CD8 levels correlated poorly with disease activity measured either clinically or by C-reactive protein. Antibodies to Klebsiella and Shigella LPS rose over time, while antibodies to Salmonella LPS fell. CONCLUSION: The clinical picture of post-Salmonella ReA is less stereotyped than often assumed, although severity correlated with HLA-B27 status. The association of joint symptoms with duration of diarrhea and the kinetics of the anti-LPS antibody response support the hypothesis that abnormal gut permeability plays a role in the pathogenesis of post-Salmonella ReA.

Use of surgical intrauterine insemination to manage infertility in a colony of research German shepherd dogs.

A colony of German shepherd dogs with inherited ventricular arrhythmias and sudden death had infertility that was resolved by intrauterine insemination. Mating of German shepherd males to related German shepherd females (40 cycles) by vaginal artificial insemination resulted in a low pregnancy rate of 35% and a small median litter size of 3.5. When these same German shepherd males were bred to female beagles by vaginal artificial insemination, the pregnancy rate of 100% (P = 0.02) and median litter size of 7.0 were significantly (P = 0.04) greater. Therefore, inadequate fertility existed when the German shepherds were mated. Because matings between these dogs were necessary, surgical intrauterine insemination of fresh semen was instituted in eight German shepherd females over nine cycles. In bypassing the cervix with this method, German shepherd fecundity increased significantly, with a pregnancy rate of 100% (P = 0.002) and median litter size of 8.0 (P = 0.001). Surgical intrauterine insemination may be an important method for management of canine infertility in the research environment.

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.

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.

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.

(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.

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.

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.