Single-cell ATAC and RNA sequencing reveal pre-existing and persistent cells associated with prostate cancer relapse.

Prostate cancer is heterogeneous and patients would benefit from methods that stratify those who are likely to respond to systemic therapy. Here, we employ single-cell assays for transposase-accessible chromatin (ATAC) and RNA sequencing in models of early treatment response and resistance to enzalutamide. In doing so, we identify pre-existing and treatment-persistent cell subpopulations that possess regenerative potential when subjected to treatment. We find distinct chromatin landscapes associated with enzalutamide treatment and resistance that are linked to alternative transcriptional programs. Transcriptional profiles characteristic of persistent cells are able to stratify the treatment response of patients. Ultimately, we show that defining changes in chromatin and gene expression in single-cell populations from pre-clinical models can reveal as yet unrecognized molecular predictors of treatment response. This suggests that the application of single-cell methods with high analytical resolution in pre-clinical models may powerfully inform clinical decision-making.

Androgen receptor expression in circulating tumour cells from castration-resistant prostate cancer patients treated with novel endocrine agents.

BACKGROUND: Abiraterone and enzalutamide are novel endocrine treatments that abrogate androgen receptor (AR) signalling in castration-resistant prostate cancer (CRPC). Here, we developed a circulating tumour cells (CTCs)-based assay to evaluate AR expression in real-time in CRPC and investigated nuclear AR expression in CTCs in patients treated with enzalutamide and abiraterone. METHODS: CTCs were captured and characterised using the CellSearch system. An automated algorithm to identify CTCs and quantify AR expression was employed. The primary aim was to evaluate the association between CTC AR expression and prior treatment with abiraterone or enzalutamide. RESULTS: AR expression in CTCs was evaluated in 94 samples from 48 metastatic CRPC patients. We observed large intra-patient heterogeneity of AR expression in CTCs. Prior exposure to abiraterone or enzalutamide was not associated with a change in CTCs AR expression (median intensity and distribution of AR-positive classes). In support of this, we also confirmed maintained nuclear AR expression in tissue samples collected after progression on abiraterone. AR staining also identified additional AR-positive CD45-negative circulating cells that were CK-negative/weak and therefore missed using standard protocols. The number of these events correlated with traditional CTCs and was associated with worse outcome on univariate analysis. CONCLUSIONS: We developed a non-invasive method to monitor AR nuclear expression in CTCs. Our studies confirm nuclear AR expression in CRPC patients progressing on novel endocrine treatments. Owing to the significant heterogeneity of AR expression in CTCs, studies in larger cohorts of patients are required to identify associations with outcome.

Distribution of Galanin and Galanin Receptor 2 in the Pre-optic Area of the Female Guinea Pig.

This study describes the distribution of galanin (Gal) and galanin receptor 2 (GalR2) in the pre-optic area (POA) of the female guinea pig. Frozen sections were undergone for a routine immunofluorescence labelling. Gal and GalR2 display immunoreactivity in all parts of the pre-optic area. Gal shows reactivity both in perikarya and fibres, whereas GalR2 was observed only in perikarya. Gal- and GalR2-immunoreactive (-ir) perikarya were the most numerous in the medial pre-optic area (MPA) with the highest reactivity in its dorsal part. In the median pre-optic nucleus (MPN) and periventricular pre-optic nucleus (PPN), only single Gal- and GalR2-ir neurons were observed. The highest density of Gal-ir fibres was revealed in the PPN and the lowest in the lateral pre-optic area (LPA). The results of this study indicate that the distribution pattern of Gal containing neurons overlaps well with the distribution pattern of GalR2-positive neurons, especially in the MPA. This may suggest GalR2-dependent activity in this brain region.

A morphometric comparative study of the medial geniculate body of the rabbit and the fox.

SUMMARY: Unbiased stereological methods were used to morphometrically examine and compare the medial geniculate body (MGB) of two species from different mammalian orders. The MGB had a similar nuclear pattern, and it was parcelled into three major cytoarchitectural areas: the dorsal nucleus (MGd), the ventral nucleus (MGv) and the medial nucleus (MGm). The MGd was predominant in the fox, where it contributed nearly 50% to the total MGB volume, while in the rabbit, the MGv was insignificantly larger than the MGd. In both species, the percentage contribution of the MGm was the lowest. The MGd in the fox was also characterized by twice as many neurons per mm(3) as in the rabbit, whereas a reverse proportion was observed in the MGm, although the numerical density in the MGv was very similar in both species. The total number of MGB neurons in the fox was over twice higher than that in the rabbit. The variability in the percentage contribution of the MGd, MGv and MGm cells to the total neuronal population of the MGB was different in both mammals. In the rabbit, there was a larger contribution from the MGv and MGm, while in the fox, the MGd was predominant. These data demonstrate that the main areas of the MGB complex differ in terms of the morphometric characteristics in both species. Our results also show that the negative correlation between the volume and numerical density in the sensory centres of the brain might not be as distinct as in the non-sensory brain structures.

The cocaine- and amphetamine-regulated transcript (CART) immunoreactivity in the amygdala of the pig.

The distribution and morphology of neurons containing cocaine- and amphetamine-regulated transcript (CART) was investigated in the pig amygdala. CART- immunoreactive (CART-IR) cell bodies were rarely observed in the pig amygdala and most often they were present in the posterior (small-celled) parts of the basolateral and basomedial nuclei. In all other subdivisions only a small number of randomly scattered pericarya were present. In every region studied the CART-IR neurons formed a heterogeneous population consisting mostly of small, rounded or slightly elongated cell bodies, with a few poorly branched, smooth dendrites. In general, the morphological features of these cells clearly resembled non-pyramidal Golgi type II interneurons. Some randomly scattered CART-IR cell bodies were significantly larger and they demonstrated features of pyramidal-like Golgi type I projecting neurons. The highest densities of CART-IR fibres were evident within the central and medial nuclei. Moderate to high expression was found within the large-celled part of the basolateral nucleus and moderate to low levels in the lateral, basomedial and cortical nuclei. The routine double-labelling studies with antisera directed against CART and somatostatin (SOM), or neuropeptide Y (NPY), or cholecystokinin (CCK), or vasoactive intestinal peptide (VIP), or substance P (SP) demonstrated that, in general, these peptides do not co-exist in the CART-IR neurons. However, small subpopulations of the CART-IR fibres contained SOM, CCK, VIP or SP together.

A morphometric study of the preoptic area of the guinea pig.

The aim of the study was to provide the topography and morphometric characteristics of the preoptic area (POA) of the guinea pig. The study was carried out on the brains of sexually mature guinea pigs of both sexes. A uniform procedure was followed in the study of the paraffin-embedded brain tissue blocks of males and females. The blocks were cut in the coronal plane into 50 mm sections and stained according to the Nissl method. The guinea pig POA consists of four parts: the medial preoptic area (MPA), lateral preoptic area (LPA), periventricular preoptic nucleus (PPN), and median preoptic nucleus (MPN). The topography and general structure of POA parts are similar in males and females. However, the PPNa cells of females are more intensely stained and are more densely packed than the PPNa cells of males. For morphometric analysis, the MPA and LPA as well as PPN and MPN were considered respectively as uniform structures, namely MPA-LPA and PPN-MPN. The statistical analysis showed that the volume of the PPN-MPN was larger in males than in females, whereas the MPA-LPA volume did not differ between the sexes. Moreover, the numerical density and the total number of neurons were statistically larger in males than in females in both the MPA-LPA and PPN-MPN. The parameters describing POA neurons were larger for MPA-LPA neurons in comparison with the PPN-MPN neurons. However, in this respect no sex differences were observed in both studied complexes.

A morphometric comparative study of the lateral geniculate body in selected placental mammals: the common shrew, the bank vole, the rabbit, and the fox.

The lateral geniculate body (LGN) was morphometrically examined and compared in representatives of four mammalian orders (Insectivora, Rodentia, Lagomorpha, and Carnivora). In each studied species, the lateral geniculate body was divided into two distinct parts: the dorsal nucleus (LGNd) and the ventral nucleus (LGNv). The lateral geniculate body of the common shrew and the bank vole are very similar in appearance and nuclear pattern. The dorsal and ventral nuclei of these two species also have the most similar statistical characteristics. The lateral geniculate body of the fox has the most complicated morphology and multilayered structure. A significant disproportion was observed between the sizes of both geniculate nuclei in the fox, where the dorsal nucleus definitely surpassed the ventral nucleus in terms of volume. With the exception of the fox, the neuronal density of the LGN nuclei was negatively correlated with the volumes of the LGN. The mean neuronal size of the LGNd and LGNv, which was the resultant of the length, width, area, and circumference of the soma, grew correlatively to the volumes of these nuclei. In all examined species, somas of the LGNd neurons are distinctly larger and have more similar shapes than the LGNv perikarya. In addition, the numerical density of neurons in the ventral nucleus is significantly higher than in the dorsal nucleus. All these morphometric parameters clearly differentiate the LGNd from the LGNv.

Distribution of cocaine- and amphetamine-regulated transcript in the hippocampal formation of the guinea pig and domestic pig.

This study provides a detailed description concerning the distribution of cocaineand amphetamine-regulated transcript (CART) subunits - CART(61-102) and rhCART(28-116) - in the hippocampal formation (HF) of the guinea pig and domestic pig, focussing on the dentate gyrus (DG) and hippocampus proper (HP). Although in both studied species CART-immunoreactive (CART-IR) neuronal somata and processes were present generally in the same layers, some species-specific differences were still found. In the granular layer (GL) of both species, the ovalshaped neurons and some thick varicose fibres were encountered. In the guinea pig there was an immunoreactive "band of dots", probably representing crosssectioned terminals within the DG molecular layer (MOL), whereas in the domestic pig, some varicose fibres were detected, thus suggesting a different orientation of, at least, some nerve terminals. Furthermore, some CART-positive cells and fibres were observed in the hilus (HL) of the guinea pig, whereas in the analogical part of the domestic pig only nerve terminals were labelled. In both species, in the pyramidal layer (PL) of the hippocampus proper, CART-IR triangular somata were observed in the CA3 sector, as well as some positive processes in MOL; however, a few immunoreactive perikarya were found only in the CA1 sector of the guinea pig. As regards the localization patterns of two isoforms of CART in the guinea pig, both peptide fragments were present simultaneously in each of the labelled neurons or fibres, whereas in the domestic pig three types of fibres may be distinguished within the area of the DG. In the hilus and MOL of the dentate gyrus, there were fibres expressing both isoforms of CART in their whole length (fibres of the first type). Fibres of the second type (in GL) coexpressed both peptides only on their short segments, and the last ones (in MOL) expressed solely rhCART(28-116). These results indicate that the distribution of the two CART isoforms are specifically related, thus the relationship between the two CART isoforms may imply different metabolic profiles of CART-expressing neurons.

The morphometric study of the amygdala in the rabbit.

Volumetric measurements of the individual nuclei in the amygdala (CA) of the rabbit reveal poor development of the basolateral (BL) and lateral olfactory tract (NLOT) and medial (ME) nuclei. On the other hand, the volumes of the lateral (LA), basomedial (BM), central (CE) and cortical (CO) nuclei are remarkable in this species. A comparison of the densities of neurons in the individual nuclei with the mean numerical density of cells in the rabbit CA indicates that the densities of neurons in LA, BL and BM are significantly lower than the mean (p < 0.05), whereas in CE, CO, ME and NLOT these values are significantly higher than the mean (p < 0.05). It should be noted, however, that of all the nuclei studied those in CE show the greatest similarity in density to CA as a whole. To some extent a similar division of the rabbit CA may be made using the size parameters of the amygdaloid neurons as a marker. The large neurons populate less densely organised CA areas such as LA, BL and BM, whereas the small cells create ME and NLOT, where the neurons are densely arranged. The CE and CO occupy intermediate positions, with the neurons similar in size to the mean for the total rabbit CA. These morphometric data from CA in the rabbit, when compared with the similar data for the common shrew and guinea pig (see our previous papers), lead to the conclusion that the amygdalae in all three species are very similar with respect to the distribution of neurons in relation to density and size and that, when volumetric measurements are taken into account, CA in the rabbit is much more similar to that of the guinea pig than that of the common shrew.

The nerve cells of the neostriatum in the common shrew (Sorex araneus) and bank vole (Clethrionomys glareolus): a Golgi comparative study.

The studies were carried out on 12 brains derived from adult representatives of two mammalian orders, Insectivora and Rodentia. The neostriatum was compared in the common shrew (Sorex araneus) and bank vole (Clethrionomys glareolus). Three main types of striatal neuron were distinguished in the common shrew and five types of neurons in the bank vole. The fifth type of bank vole neurons was additionally divided into two subtypes with respect to dendritic pattern.

The neuronal structure of the dorsal nucleus of the lateral geniculate body in the common shrew (Sorex araneus) and the bank vole (Clethrionomys glareolus): Golgi and Nissl studies.

The topography and neuronal structure of the dorsal nucleus of the lateral geniculate body (GLd) of the common shrew and the bank vole are similar. The lateral geniculate body of both the species examined has a homogeneous structure and no observable cytoarchitectonic lamination. On the basis of the shape of the dendritic arbours as well as the pattern of dendritic arborisations the following two types of neurons were distinguished. Type I "bushy" neurons that have multipolar or round perikarya (common shrew perikarya 9-12 microm, bank vole perikarya 10-13 microm), with 4-6 short thick dendritic trunks that subdivide into many bush-like branches. The dendritic trunks are smooth, in contrast to the distal branches, which are covered with numerous spine-like protrusions of different lengths and forms. An axon emerges from the soma, sometimes very close to one of the primary dendrites. The type I neurons are typically projection cells that send their axons to the primary visual cortex. These neurons predominate in the GLd of both species. Type II neurons, which have an elongated soma with primary dendrites arising from opposite poles of the perikaryon (common shrew perikarya 8-10 microm, bank vole perikarya 9-11 microm). The dendritic arbours of these cells are less extensive and their dendrites have fewer spines than those of the type I neurons. Axons were seldom observed. The type II neurons are presumably interneurons and are definitely less numerous than the type I neurons.

The neuronal structure of the preoptic area in the mole and the rabbit: Golgi and Nissl studies.

The present studies were carried out on the brains of the adult mole and rabbit. The preparations were made by means of the Golgi technique and the Nissl method. Two types of neurons were distinguished in the preoptic area (POA) of both species: bipolar and multipolar. The bipolar neurons have oval, fusiform or round perikarya and two dendritic trunks arising from the opposite poles of the cell body. The dendrites bifurcate once or twice. The dendritic branches have swellings, single spine-like and filiform processes. The multipolar neurons usually have triangular and quadrangular perikarya and from 3 to 5 dendritic trunks. The dendrites of the mole neurons branch sparsely, whereas the dendrites of the rabbit neurons display 2 or 3 divisions. On the dendritic branches varicosities and different protuberances were observed. The general morphology of the bipolar and multipolar neurons is similar in the mammals studied, although the neurons of the rabbit POA display a more complicated structure. Their dendritic branches show more divisions and possess more swellings and different processes than the dendrites of the neurons of the mole POA. Furthermore, of the multipolar neurons only the dendrites in POA of the rabbit were observed to have a rosary-like beaded appearance.

The neuronal structure of the dorsal lateral geniculate nucleus in the guinea pig: Golgi and Klüver-Barrera studies.

On the basis of Golgi and Klüver-Barrera preparations we have distinguished four types of neurons in the dorsal lateral geniculate nucleus of the guinea pig: 1. Fusiform neurons with 1-3 thick dendritic trunks arising from each pole of the soma. The dendritic trunks branch twice dichotomically. The branches sometimes show varicosities. 2. Pear-shaped cells. From one pole of the perikaryon one or two thick dendritic trunks arise, from the opposite pole an axon emerges. The ends of the dendritic branches divide in a tuft-like manner (a characteristic feature of the interneurons). 3. Rounded neurons with 4-7 dendritic trunks without cones. The dendritic trunks branch once or twice dichotomically and give finally 2-3 thin ramifications which show a varicose course and knob-like protuberances. 4. Triangular cells with 3 thick, chronically arising dendritic trunks. They bifurcate dichotomically. The surface of the dendritic trunks and of their branches is smooth.

The neuronal structure of the ventromedial and infundibular nuclei in the guinea pig: Nissl and Golgi study.

The studies were carried out on the mesencephalons of adult guinea pigs. On the basis of the Golgi technique, as well as the Nissl and Klüver-Barrera methods, four types of neurons were distinguished in the ventromedial nucleus (VMH) and infundibular nucleus (Ni): 1. Rounded neurons (perikarya 12-18 microns) with 3-4 dendritic trunks, which divide once, twice or not at all. The dendritic branches possess varicosities and knob-like spines. These neurons predominate in VMH. 2. Fusiform neurons (perikarya 15-28 microns) with 2 dendritic trunks, which arise from the opposite poles of the cell body. Bead-like protuberances and knob-like processes are observed on the dendrites. These neurons are the most numerous in Ni. 3. Triangular neurons (perikarya 15-22 microns) possess three thick, conical dendrites, which bifurcate dichotomically. Bead-like appendages and knob-like processes were seen on the dendritic surface. 4. Multipolar neurons (perikarya 18-22 microns) with 4-5 dendritic trunks, which are poorly ramified. The dendritic branches are smooth, but varicosities can be observed on their surface. In all types of neurons an axon was observed to arise either from the dendritic trunk or from the soma.

The neuronal structure of the substantia nigra in the guinea pig: Nissl and Golgi study.

The studies were carried out on the mesencephalos of adult guinea pigs. The preparations were made by means of the Golgi technique, as well as the Nissl and Klüver-Barrera methods. Four types of neurons were distinguished in the substantia nigra (SN) of the guinea pig: 1. Bipolar neurons of two kinds: the neurons of the first kind have elongated, fusiform perikarya (25-40 microns), whereas the cells of the second kind have rounded and oval perikarya (15-22 microns). These neurons possess two dendritic trunks which arise from the opposite poles of the cell body and run for a relatively long distance. The bipolar neurons are the most numerous in the pars compacta of SN. 2. Triangular neurons with three primary dendrites arising conically from a perikaryon (20-35 microns). They are the most often observed type of neurons in the pars reticulata of SN. 3. Multipolar neurons with quadrangular or oval perikarya (22-35 microns) and 4-5 dendritic trunks which spread out in all directions. 4. Pear-shaped neurons (perikarya 15-25 microns), which have one or two primary dendritic trunks arising from one pole of the cell body. In all the types of neurons an axon originates either from the dendritic trunk or from the soma and is observed only in its initial segment.

The cytoarchitectonic and neuronal structure of the red nucleus in guinea pig: Nissl and Golgi studies.

The present studies were carried out on the brains of adult guinea pigs, Dunkin-Hartley strain. On the basis of preparations, they were stained according to the Nissl and the Klüver-Barrera method's; a short description of the cytoarchitectonics and the characteristics of the rubral cells were written. The red nucleus (RN) of the guinea pig is 1.2 mm in length. Three cellular parts in RN, and three classes (A, B, C) of the rubral cells were distinguished. Taking into consideration the predominant cell size, RN was divided into magnocellular part (RNm), parvocellular part (RNp) and intermediate part (RNi). On the basis of Golgi impregnated preparations four neuronal types (I, II, III, IV) were distinguished. To sum up, in the guinea pig were observed: the large, mainly multipolar (type I) and bipolar (type II) spiny being coarse (class A) in Nissl material; the medium-sized, triangular, aspiny (type III) corresponding to the fine cells (class B); and the small, both spiny and aspiny neurons (type IV), which are the fine or achromatic cells (classes B or C) in Nissl stained slices. The highest degree of dendritic branching was observed in type I, whereas the lowest in cells of types III and IV.

Types of neurons of the subthalamic nucleus and zona incerta in the guinea pig--Nissl and Golgi study.

The studies were carried out on the subthalamus of adult guinea pigs. Golgi impregnation, Nissl and Klüver-Barrera methods were used for the study. In Nissl stained sections the subthalamic neuronal population consists of multipolar, fusiform, oval and pear-shaped perikarya. In two studied areas: nucleus subthalamicus (STN) and zona incerta (ZI) three types of neurons were distinguished. Type I, multipolar neurons with quadrangular, triangular or oval perikarya. They have 3-6 primary dendrites which run slightly wavy and spread out in all directions. Type II, bipolar neurons with fusiform or semilunar perikarya, they have two primary dendrites. Type III, pear-shaped neurons with 1-2 dendritic trunks arising from one pole of the neuron. In all types of neurons axon emerges from the perikaryon or initial segment of a dendritic trunk and can be followed at a maximum distance of about 50 microns.

[Evaluation of treatment and preventive care for recurrent urolithiasis in children]. / Ocena skutecznosci leczenia i profilaktyki kamicy nawrotowej ukladu moczowego u dzieci.

Among 425 children with urolithiasis treated in the Paediatric Clinical Department of the National Research Institute of Mother and Child in Warsaw between 1976-1997, 50 of them i.e. 11.7% (26 boys and 24 girls) had recurrent urolithiasis. Patients' age was from 10 months to 16 years and 5 months. The number of recurrences of uroliothiasis before treatment in the Institute was from 1 to 8. Most of the children had numerous surgical operations, some of them excreted stones spontaneously. The etiology was determined in all cases. A metabolic cause of urolithiasis was found in 34 cases, i.e. 68% of the analysed group. They were as follows: idiopathic hypercalcuria--24 cases, uric acid urolithiasis--5 cases, cystynuria--4 cases, and incomplete distal renal tubular acidosis--1 case. Other reasons for urolithiasis were: infection--7 cases, idiopathic urolithiasis--8 cases, ren spongiosum--1 case. Prevention of recurrences depending on the etiology was successful. In 45 cases no recurrences were found. Recurrent urolithiasis was observed in 4 cases of cystynuria and in one case of incomplete tubular acidosis. The observation period was from 3-19 years.

The morphology and cell structure of some nuclei in tuberal region of bison bonasus.

The topography, shape and cell structure of the ventromedial, dorsomedial and infundibular nuclei in aurochs hypothalamus were studied on the basis of standard histological preparations. These nuclei are uniform cell bands. Nucleus infundibularis is the longest centre in tuberal region and enter the mamillary area. Its posterior part has maximum size. The ventromedial nucleus is outlined and developed best. It is typical ring-shaped in anterior half of its length. The dorsomedial nucleus is developed best in its middle sector. There is no clear border between Dm and Ni. Comparing the topography and structure of Ni, Vm and Dm of bison with analogic centres of other mammals, it was found that have more common features with these nuclei in breeding domestic animals.

[Clinical evaluation of Uro-Vaxom in treatment of recurrent urinary tract infections in girls]. / Kliniczna ocena preparatu Uro-Vaxom w leczeniu nawracajacych zakazen ukladu moczowego u dziewczynek.

Uro-Vaxom was used in the treatment of recurrent urinary tract infections in 28 girls. Most of them (27/28) tolerated the drug very well, no side effects were observed. We stopped administration of the Uro-Vaxom in one girl during the first month of treatment because of vomiting. Uro-Vaxom efficiency was, therefore, evaluated in 27 girls. Uro-Vaxom was found to be a valuable drug, supplementing antibiotic therapy in recurrent urinary tract infections caused by E. coli.