Quantitative Comparison of Conventional and t-SNE-guided Gating Analyses.

Dimensionality reduction using the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm has emerged as a popular tool for visualizing high-parameter single-cell data. While this approach has obvious potential for data visualization it remains unclear how t-SNE analysis compares to conventional manual hand-gating in stratifying and quantitating the frequency of diverse immune cell populations. We applied a comprehensive 38-parameter mass cytometry panel to human blood and compared the frequencies of 28 immune cell subsets using both conventional bivariate and t-SNE-guided manual gating. t-SNE analysis was capable of stratifying every general cellular lineage and most sub-lineages with high correlation between conventional and t-SNE-guided cell frequency calculations. However, specific immune cell subsets delineated by the manual gating of continuous variables were not fully separated in t-SNE space thus causing discrepancies in subset identification and quantification between these analytical approaches. Overall, these studies highlight the consistency between t-SNE and conventional hand-gating in stratifying general immune cell lineages while demonstrating that particular cell subsets defined by conventional manual gating may be intermingled in t-SNE space.

Cat satellite DNA. Isolation using netropsin with CsCl gradients.

The absence of centromeric bands in the karyotype of Felis catus is confirmed. It is also confirmed that no satellite band is visible in CsCl density gradients. However, a satellite is observed both by recentrifuging the fraction of the DNA that bands at high density in CsCl and by using netropsin to enhance the resolution of a CsCl gradient containing total F. catus DNA. The satellite, about 0.5% of total DNA, was isolated by repeated centrifugation in CsCl alone and in CsCl with netropsin. Netropsin was removed and a pure satellite DNA obtained. The reassociation kinetics (C0t1/2 less than 10(-3) M . s) show that the satellite is of the simple sequence type and hence a candidate for centromeric heterochromatin. Its cytological localisation awaits in situ hybridisation experiments.

Gene structure and promoter function of a teleost ribosomal protein: a tilapia (Oreochromis mossambicus) L18 gene.

We have cloned and characterized a tilapia (Oreochromis mossambicus) L18 ribosomal protein gene, including the complete transcribed region and 488 bp of upstream regulatory sequences. We have also isolated two L18 cDNAs from another tilapia (Oreochromis niloticus) with a few conservative nucleotide differences. Our results suggest the presence of two genes in both species. Reporter constructs were tested for transient expression in CV1 cells and in microinjected zebrafish and tilapia embryos. The tilapia L18 promoter was able to drive expression of the reporter gene in all three experiments, with no apparent preference for a particular tissue. The tilapia L18 promoter is therefore likely to be a powerful tool to drive tissue-independent gene expression in fish.

Transgenic tilapia and the tilapia genome.

The tilapia fish (Oreochromis niloticus) has an important place in the aquaculture of the developing world. It is also a very useful laboratory animal, and readily lends itself to the transgenic technology. Through the use of reporter genes, a range of potential gene promoters have been tested in tilapia, both through transient and stable expression of the reporter construct. Using the transgenic technology, growth enhanced lines of tilapia have been produced. These fish have no abnormalities and offer a considerable growth advantage for future exploitation. It is however crucial that transgenic fish, to be exploited in aquaculture, be sterile, and various methods of achieving sterility are considered. These include triploidy, gene knock out of crucial hormone encoding genes via homologous recombination, and knock down of the function of the same genes via ribozyme or antisense technologies. Transgenic tilapia also offer the potential for exploitation as biofactories in the production of valuable pharmaceutical products, and this is also discussed.

A calcium-activated nuclease endogenous to Xenopus erythrocytes.

An endogenous nuclease is found in purified Xenopus erythrocyte nuclei. This enzyme is activated specifically by Ca2+ in the presence of which chromatin is digested into discrete fragments. Both 10 bp and 200 bp ladders are detected upon electrophoresis of DNA. This endogenous enzyme therefore resembles mammalian and chicken liver endonucleases.

Cloning and expression analysis of an inducible HSP70 gene from tilapia fish.

We isolated and characterized the tilapia (Oreochromis mossambicus) HSP70 gene, highly homologous to other HSP70 genes. A dramatic increase of tilapia HSP70 mRNA levels was observed after heat shock of whole animals in all organs tested. Reporter constructs were tested for transient expression in carp cells and in microinjected zebrafish embryos. The entire isolated regulatory region (-851/+157) was able to mediate heat shock inducible expression of the reporter gene, with no preference for a particular tissue. Our studies represent the first transcriptional analysis of a HSP70 promoter from fish, revealing a powerful tool to direct controlled, tissue-independent gene expression in fish.

Spatial and temporal genetic variation in British field populations of the grain aphid Sitobion avenae (F.) (Hemiptera: Aphididae) studied using RAPD-PCR.

The grain aphid Sitobion avenae (F.) was collected from winter wheat and adjacent cocksfoot grass at two locations in southern England and at four times in the year (April-July). Genetic variation between individual aphids was then investigated using random amplified polymorphic DNA polymerase chain reaction. Individuals caught in wheat and cocksfoot during April provided very different and highly diagnostic banding patterns that were independent of location. This host-based genetic differentiability was less evident as the season progressed, largely as a result of genetic drift and local movement between adjacent host species, which appeared to be predominantely in the direction from cocksfoot to wheat. The diversity of putative clones fell significantly, the mean number of individuals per clone rose and clones became more exclusively associated with certain sites which suggests that long-distance migration may have less of a homogenizing effect than hitherto thought for this species.

Compartmentalization of regulatory proteins in the cell nucleus.

The cell nucleus is increasingly recognized as a spatially organized structure. In this review, the nature and controversies associated with nuclear compartmentalization are discussed. The relationship between nuclear structure and organization of proteins involved in the regulation of RNA polymerase II-transcribed genes is then discussed. Finally, very recent data on the mobility of these proteins within the cell nucleus is considered and their implications for regulation through compartmentalization of proteins and genomic DNA are discussed.

Detection of neuron-specific protein gene product (PGP) 9.5 in the rat and zebrafish using anti-human PGP9.5 antibodies.

Protein gene product (PGP) 9.5 is a developmentally regulated neuron- and neuroendocrine cell-specific ubiquitin carboxy-terminal hydrolase (UCHL1) expressed throughout the mammalian central and peripheral nervous systems. We have compared the use of monoclonal and polyclonal antibodies raised against human PGP9.5 for immunodetection of the protein in tissues of the zebrafish and rat. We show that a monoclonal antibody 13C4, which recognises an N-terminal epitope, detects PGP9.5 on Western blots as a single 27 kDa band present at high levels in zebrafish and rat brain. By contrast, the polyclonal antisera recognises multiple tissue-specific proteins in the rat and fails to detect PGP9.5 in the zebrafish. Finally, we have developed a specific ELISA assay for detection of cellular PGP9.5 using MAb13C4 and have employed the assay to show that PGP9.5 is not upregulated during nerve growth factor (NGF)-induced differentiation of rat PC12 cells.

A critical review of the concept of patient motivation in the literature on physical rehabilitation.

Rehabilitation professionals have long suspected that a patient's motivation plays an important role in determining the outcome of therapy, despite the lack of a clear definition of the phenomenon. The fact that such a subjective concept is commonly used by clinicians prompted this investigation into the range and nature of professional understandings of patient motivation. The literature dealing with physical rehabilitation and motivation was reviewed and was found to fall into three broad groups. One group of mainly clinical articles conceives of motivation as an internal 'personality trait' of the individual patient, and explains the nature and causes of motivation purely in terms of internal dispositions. Another body of literature considers motivation to be a quality which is affected by social factors, and stresses the importance of awareness of such factors in explaining motivation. The third approach considers social factors in combination with personality or clinical characteristics. It is argued that the personality-based approach facilitates moralising in the therapeutic encounter, a problem which is both highlighted and critiqued by the methodology which emphasises the importance of social factors. The practical implications of the relative merits of these different theories of motivation are considered.

An investigation of the differential performance of clones of the aphid Sitobion avenae on two host species.

Individuals of the grain aphid Sitobion avenae were collected from wheat and cocksfoot stands around Hampshire, UK, during March and April 1994. Eight wheat and eight cocksfoot aphid clones were chosen on the basis of readily distinguishable RAPD-PCR fingerprint profiles. The performances (weight, fecundity and survival) of successive generations of each of these clones were then carefully monitored in the laboratory as new generations of aphids were transferred either to winter wheat or to cocksfoot in planned sequences. Even those clones that were originally caught on the same host showed significant variability in performance. Clones generally performed better on their host of origin than they did on the alternate host, and they performed less well on the alternate host compared to the clones that had originated there. A comparison of the performance of third generation aphids with first generation aphids showed that the experience of the mother in the second generation often influences the subsequent performance of their offspring. As the sequence of host transfers had more effect on the performance of wheat clones than cocksfoot clones, it is likely that wheat clones are more specialised, such that wheat is a satisfactory host for cocksfoot clones but not vice versa. The study provides evidence of genetic variation in performance on host and evidence for clonal adaptation to particular host species. This adaptation may well be a major cause of the observed consistent genetic differentiability of populations of S. avenae found on wheat and roadside grasses in early spring in southern England.

Taper: an important feature of Y-bifurcations in porcine renal arteries and human cerebral arteries.

The geometry of arterial bifurcations has been shown to alter fluid flow and the propagation of both pressure and flow waves. Here we provide a more complete description of the renal artery bifurcation geometry and show that the geometry of the bifurcation is more complex than was believed previously. The objective of this study was to quantify changes in cross-sectional luminal area in systemic arterial bifurcations using the method developed by Macfarlane [Ph.D. thesis, University of Western Ontario, Ontario (1985)] to study the geometry of human cerebral bifurcations. Porcine renal arterial bifurcations from seven young (6-14 weeks) and six old (greater than 52 weeks) animals were pressure-fixed (P = 140 mmHg) for 3 h with 10% formalin. Bifurcations were embedded in a block of frozen latex paint. Serial sections were cut at 20 microns (+/- 0.01 microns) using a sledge microtome while the block face was scanned with a video camera and the images were stored on a videotape. The luminal area was measured digitally upon playback. Bifurcations from the two age groups changed in cross-sectional area not only at the flow divider but also along the so-called straight regions. Proximal linear increases in cross-sectional area were observed proximal to the apex of the bifurcation in both young and old vessels, while linear luminal area decreases were measured in the daughter branches of both young and old porcine renals. Taper was defined as the change in luminal area per unit length of parent and daughter branches, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrocarbon transport in chylomicrons and high-density lipoproteins in rat.

A lipoprotein system is described that transports gut hydrocarbons of low polarity in chylomicrons of intestinal lymph and plasma to plasma high density lipoproteins (HDL) in rat. Four highly lipophilic aryl and alkyl hydrocarbons [benzo(alpha)pyrene; 1,1,1-trichloro-2,2-bis(p-chlorophenol)ethane (DDT), hexadecane and octadecane] were selected to give a graded range of polarity. Chylomicrons were labeled doubly with radioisotopes in triacylglycerol and a single hydrocarbon by feeding [3H]-glycerol and [14C]hydrocarbon. All hydrocarbons were transported in the triacylglycerol oil phase of chylomicrons. Injected chylomicron triacylglycerol and 3 of 4 hydrocarbons were cleared simultaneously from plasma consistent with lipoprotein-lipase dependent hydrocarbon clearance but DDT was cleared more rapidly. HDL was the major plasma acceptor of all labelled hydrocarbons. Plasma chemical fluxes were measured for octadecane and DDT and both showed net fluxes from chylomicrons to HDL. HDL selectively concentrated chylomicron hydrocarbons from chylomicron triacylglycerol. Lipoprotein lipase stimulation by intravenous heparin significantly increased transfer of alkanes from chylomicrons to HDL. These results indicate that (a) chylomicrons transport gut-derived hydrocarbons with a wide range of structure and polarity as triacylglycerol solutes; (b) HDL are a major plasma acceptor of all these hydrocarbons, demonstrating both selective solute uptake from triacylglycerol and net chemical uptake for the 2 hydrocarbons studied and (c) efflux of these chylomicron hydrocarbons from plasma and into HDL is regulated partly by hydrolysis of chylomicron triacylglycerol.

The effect of semirigid dressings on below-knee amputations.

BACKGROUND AND PURPOSE: The effect of using semirigid dressings (SRDs) on the residual limb of individuals who have had below-knee amputations as a consequence of peripheral vascular disease was investigated, with the primary question being: Does the time to readiness for prosthetic fitting for patients treated with the SRDs differ from that of patients treated with soft dressings? SUBJECTS: Forty patients entered the study and were alternately assigned to one of two groups. Nineteen patients were assigned to the SRD group, and 21 patients were assigned to the soft dressing group. METHODS: The time from surgery to readiness for prosthetic fitting was recorded for each patient. Kaplan-Meier survival curves were generated for each group, and the results were analyzed with the log-rank test. RESULTS: There was a difference between the two curves, and an examination of the curves suggests that the expected time to readiness for prosthetic fitting for patients treated with the SRDs would be less than half that of patients treated with soft dressings. CONCLUSION AND DISCUSSION: The results suggest that a patient may be ready for prosthetic fitting sooner if treated with SRDs instead of soft dressings.

Abortion for fetal abnormality.

PIP: I wish to thank Dr. Pauline Bennett for her reply (NZ Med J, 13 June). She has demonstrated well that in dealing with sensitive difficult issues such as abortion for fetal abnormality, the one thing the doctor is not recommended to do is to speak the truth] I am prompted to write this letter for 2 reasons. Firstly, the excellent letter written by Dr. A. M. Rutherford (NZ Med J, 13 June) on the subject of abortion stated, "The most disturbing feature about the whole controversy is the 'blunting of our conscience'." When the doctors are not encouraged to be honest with patients then indeed our conscience has been blunted. Secondly, I watched Holocaust last night, and cannot refrain from stating that I see frightening parallels between our liberal abortion policy and the activities of the Nazis. As I watched the "mental patients" being herded into the shed for gassing by the polite, tidy, white coated medical staff, and then heard the compassionate, sensitive, letter of the hospital authorities to the relatives of the deceased, the parallel became obvious. The mental patients were weak, defenseless, burdensome, and uneconomic; the unborn are weak, defenseless, burdensome, and uneconomic. The hospital authority's letter was acceptable in many ways, acceptable except that its words bore no relation to the truth. It is said that the "first casualty of war is the truth". Whether that war involves the Jews, or the insane, or the unborn, the statement would seem correct.^ieng

Perinatal mortality in Southland: an ongoing review.

A review of Southland's perinatal mortality was undertaken from 1978 to 1983 and included 124 deaths with a postmortem rate of 90%. The perinatal mortality rate has continued to fall during this period, and has more than halved since 1971. Prematurity remains the single most important factor, and there are still a large number of avoidable deaths. Eighty percent of confinements are attended solely by general practitioner obstetricians.

Perinatal mortality in Southland.

A review of Southland's perinatal mortality was undertaken from 1971 to 1977 and included 237 deaths with a post mortem rate of 90 percent. During this period, the perinatal mortality rate fell to below the national average. Prematurity remains the most important single factor. Seventy-four percent of confinements are attended solely by general practitioner obstetricians. Fetal monitoring of high risk pregnancies in labour markedly reduces the intrapartum death rate of mature babies.

Diagnosis of fetal hydrocephalus by ultrasound.

An ultrasound scan of a fetus at 26 weeks gestation suggested abnormal dilatation of a lateral cerebral ventricle. Serial scans confirmed progressive dilatation of both lateral ventricles. Clinical hydrocephalus causing obstructed labour occurred and required craniotomy. Post mortem upon the fresh still born infant confirmed internal hydrocephalus.

Laparoscopy in Southland.

A review was made of 625 consecutive gynaecological laparoscopies carried out in the Southland Hospital. Forty-one point seven percent were for diagnostic reasons, mainly in the diagnosis of pelvic pain and infertility, and 57.3 percent were for sterilisation. Complications occurred with 2.2 percent and 5.6 percent respectively, but the serious complications and those requiring laparotomy only came to 1.1 percent. Four women (1.1 percent) became pregnant after sterilisation, all four occurring during the first year of the procedure. Overall, laparoscopy is confirmed to be a valuable diagnostic technique and a safe method of sterilisation.

PIP: A retrospective review of 625 consecutive laparoscopies performed in Southland Hospital from 2/74 to 10/78 revealed that 41.7% (262) were carried out for diagnostic purposes (pelvic pain and infertility), 57.3% (357) for sterilization, and 1% (6) for a combination of both. Complications rate for each were observed in 2.2% and 5.6% respectively; 1.1% had serious complications and required laparotomy. Complication rate for each year decreased since the beginning of the study (from 6.7% in 1974 to 2.2% in 1978). The introduction of laparoscopic sterilization in 1975 resulted in an increase in number of sterilizations (from 142 in 1974 to 310 in 1975). 1/3 of the total sterilizations each year consisted of laparoscopic sterilization; abdominal tubal ligation, and postpartum tubal ligation. Of the sterilizations 97.2% (353) were interval procedures, 2.2% (8) were done at the time of termination of pregnancy, and .6% (2) were after spontaneous abortion. Because of lower complication rates associated with laparoscopy; shorter hospital stay; and rapid recovery, it was concluded that laparoscopic sterilization is a valuable diagnostic technique and a safe method of sterilization.