Chromothripsis orchestrates leukemic transformation in blast phase MPN through targetable amplification of DYRK1A.

Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a common event in cancer. Whether chromothripsis might constitute an actionable molecular event amenable to therapeutic targeting remains an open question. We describe recurrent chromothripsis of chromosome 21 in a subset of patients in blast phase of a myeloproliferative neoplasm (BP-MPN), which alongside other structural variants leads to amplification of a region of chromosome 21 in ∼25% of patients ('chr21amp'). We report that chr21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. The chr21amp event is highly clonal and present throughout the hematopoietic hierarchy. DYRK1A , a serine threonine kinase and transcription factor, is the only gene in the 2.7Mb minimally amplified region which showed both increased expression and chromatin accessibility compared to non-chr21amp BP-MPN controls. We demonstrate that DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development, including DNA repair, STAT signalling and BCL2 overexpression. DYRK1A is essential for BP-MPN cell proliferation in vitro and in vivo , and DYRK1A inhibition synergises with BCL2 targeting to induce BP-MPN cell apoptosis. Collectively, these findings define the chr21amp event as a prognostic biomarker in BP-MPN and link chromothripsis to a druggable target.

Exposure of neonatal rats to anti-androgens induces penile mal-developments and infertility comparable to those induced by oestrogens.

We previously reported that oestrogen exposure in neonatal rats induced permanent infertility and malformed penis characterized by fat accumulation, which replaced most of the smooth muscle cells and cavernous spaces in the body of the penis, structures essential for erection. The objective of this study was to determine if reduced androgen production/action in the neonatal period, in the absence of exogenous oestrogen exposure, induces penile deformities similar to those caused by oestrogen. Male rats were treated from postnatal days 1-6 with GnRH antagonist antide (A, 10 mg/kg) or androgen receptor (AR) antagonist flutamide (F, 50 mg/kg) or F + A, with or without AR agonist dihydrotestosterone (DHT, 20 mg/kg). For comparison, pups received diethylstilbestrol (DES, 0.1 mg/kg), with or without DHT. Tissues were collected at ages 7 and 12 days and at adulthood. Flutamide alone decreased penile length and weight significantly (p < 0.05), but it caused neither fat accumulation, nor affected fertility (80% vs. 87% in controls). Antide alone reduced penile length and weight significantly, and induced fat accumulation in 4/11 rats and infertility in 13/14 rats. Conversely, all 11 F + A-treated rats, similar to all nine DES-treated rats, had fat accumulation and loss of smooth muscle cells and cavernous spaces in the body of the penis and were infertile. In addition, reductions in penile length and weight were higher than in rats treated with F or A alone. DHT co-administration mitigated penile deformities in the DES group, but did not in the F + A group. Testicular testosterone was reduced by 70-95% at 7 or 12 days of age in all treated groups, except in the F group, which had threefold higher testosterone than controls. Collectively, data unequivocally show that reduced androgen production/action in the neonatal period, in the absence of oestrogen exposure, induces permanent infertility and malformed penis similar to that caused by oestrogen.

Estrogen-induced developmental disorders of the rat penis involve both estrogen receptor (ESR)- and androgen receptor (AR)-mediated pathways.

This study tested the hypothesis that the estrogen receptor (ESR) pathway, androgen receptor (AR) pathway, or both mediate estrogen-induced developmental penile disorders. Rat pups received diethylstilbestrol (DES), with or without the ESR antagonist ICI 182,780 (ICI) or the AR agonist dihydrotestosterone (DHT) or testosterone (T), from Postnatal Days 1 to 6. Testicular T concentration, penile morphology and morphometry, and/or fertility was determined at age 7, 28, or 150 days. DES treatment alone caused 90% reduction in the neonatal intratesticular T surge; this reduction was prevented by ICI coadministration, but not by DHT or T coadministration. Unlike the T surge, coadministration of ICI and coadministration of DHT or T mitigated penile deformities and loss of fertility. Generally, ICI, DHT, or T treatment alone did not alter penile morphology; however, fertility was 20% that of controls in ICI-treated rats vs. 70%-90% in DHT- or T-treated rats. The lower fertility in the rats treated with ICI alone could be due to altered sexual behavior, as these males did not deposit vaginal plugs. In conclusion, observations that both an ESR antagonist and AR agonists prevent penile deformities and infertility suggest that both pathways are involved in estrogen-induced penile disorders. Observations that coadministration of ICI, but not DHT or T, prevents the DES-induced reduction in the neonatal T surge suggest that, although ICI exerts its mitigating effect both at the level of Leydig cells and penile stromal cells, DHT and T do so only at the level of stromal cells.

Craniospinal Irradiation in Medulloblastoma using High Energy Medical Linear Accelerator: an Innovative Approach to Planning Technique.

BACKGROUND: Craniospinal irradiation (CSI) of medulloblastoma poses technological challenges due to the involvement of large treatment volume. Commonly, the whole treatment length is covered with two different isocentric plans in which the junction is shifted after every five fractions to overcome the possibility of hot and cold spot. OBJECTIVE: This study aims to evaluate dosimetrically and clinically the innovative planning technique for the CSI which doesn't need re-planning and re-setup of patients after every five fractions. MATERIAL AND METHODS: Computed tomography was done for fifteen (ten children and five adults) patients diagnosed with medulloblastoma. Treatment planning for 36 Gray (Gy) in 20 fractions (#) at the rate of 1.8Gy/# was done on the treatment planning system. A single plan for children was created with two bilateral fields of 6 Mega Voltage (MV) energy for cranium and one posterior field of 6 MV for spinal cord (C1-S2). Two plans for adult patients were created, first plan was with two bilateral fields of 6 MV for cranium and two posterior oblique fields of 6 MV for cervical and the part of thoracic spinal cord (up to T8-T9). The second plan was with a single posterior field of 15 MV covering remaining thoracic (T8-T9 to T12), lumbar and sacrum (up to lower border of S2) spine. After careful evaluation of all the plans, treatment was delivered; acute toxicities were recorded. RESULTS: 95% of prescribed dose was received by more than 95% of planning target volume in all the plans with the acceptable hot spot and good homogeneity index. All the patients reported common radiation induced acute toxicities (headache, vomiting, weakness) during radiotherapy. CONCLUSION: The new planning technique for CSI has acceptable dosimetric and acute clinical possibilities; therefore it can be used for CSI for improved homogeneous dose delivery.

Intraluminal brachytherapy boost following external beam radiotherapy with concurrent chemotherapy of oesophagus carcinoma: Results of a prospective observational study.

PURPOSE: The main objective of our study is to evaluate response and toxicity profile in patients receiving external beam radiotherapy with concurrent chemotherapy followed by intraluminal brachytherapy boost for a carcinoma of the oesophagus. MATERIAL AND METHODS: Twenty patients with biopsy-proven carcinoma of the oesophagus received external beam radiotherapy (50Gy in 25 fractions) with concurrent chemotherapy (cisplatin: 40mg/m2). After a gap of two to three weeks, intraluminal brachytherapy (10Gy in two fractions each 1 week apart by a high dose rate 60Co source) was given. Response was evaluated at 1 month and at 1 year of completion of treatment. In addition, acute and chronic toxicity was evaluated at 1 month and 6 months of treatment. RESULTS: Complete response were seen in 80% of patients and partial response in 20% at 1 month. Moreover, there were 65% complete response, 10% local recurrences, 15% patients showed local control with distant metastasis and 10% patients died at 1 year. Grade 1, grade 2 and grade 3 oesophagitis were seen in 10%, 70% and 20% of patients respectively. Stricture was seen in 40% of patients and fistula in 10% of patients. There was no spinal cord, cardiac and nephrotoxicity found. CONCLUSIONS: With the concept that high tumoricidal dose for adequate tumor control achieved by intraluminal brachytherapy as a mean of dose escalation, while sparing surrounding normal tissue and potentially improving therapeutic ratio, external beam radiotherapy followed by intraluminal brachytherapy could be a better choice for oesophagus carcinoma.

Study of anemia in nondialysis dependent chronic kidney disease with special reference to serum hepcidin.

We studied the role of serum hepcidin in anemia of chronic kidney disease (CKD) in a hospital-based cross-sectional study. Serum hepcidin, ferritin, and high-sensitivity C-reactive protein (hsCRP) levels were evaluated in patients of CKD. Hepcidin levels were increased in patients as compared to healthy adults. Hepcidin levels increased as CKD progressed through stage 3-5 (P trend = 0.015) but did not correlate with estimated glomerular filtration rate. Hepcidin correlated positively with ferritin (P < 0.0001) and transferrin saturation (TSAT) (P = 0.0217) and negatively with erythropoietin (EPO) levels (P = 0.0258) but did not correlate with either hsCRP or estimated glomerular filtration rate. Iron status influenced hepcidin levels of patients. Patients were divided according to iron status on the basis of TSAT and serum ferritin levels. We observed that while absolute iron deficiency (transferrin saturation <20%, ferritin <40 ng/ml) is associated with downregulation of hepcidin, hepcidin is elevated in other two categories of CKD patients (P = 0.0039). Iron status of patients also influenced interaction between hepcidin and hemoglobin (Hb). Hepcidin correlated negatively with Hb in patients with sufficient iron status (r = -0.7452, P < 0.0001) but nearly correlated positively with Hb in patients with absolute iron deficiency (r = 0.9428, P = 0.0572). Almost similar association persisted when cutoff value for serum ferritin was raised to 100 ng/ml as per NKF/KDOQI 2006 clinical practice guidelines except that no association was observed in absolute iron deficiency category. Cutoff value for hepcidin for differentiating absolute iron deficiency from other categories in our study population is ≤ 34 ng/ml (area under curve = 0.836, P < 0.0001). In conclusion, serum hepcidin level is increased in nondialysis CKD patients as compared to healthy adults possibly due to associated inflammation and decreased renal clearance. Furthermore, iron status modifies hepcidin level and its association with Hb. Raised hepcidin can predict the need for parenteral iron therapy and need for higher dose of recombinant human EPO to overcome iron-restricted erythropoiesis.

Estrogen-induced abnormal accumulation of fat cells in the rat penis and associated loss of fertility depends upon estrogen exposure during critical period of penile development.

We previously reported that diethylstilbestrol (DES) or estradiol valerate (EV) exposure at a dose of 0.10-0.12 mg/kg, or higher, per day, on alternate days, from postnatal days 2-12, resulted in abnormal penis development and infertility (H. O. Goyal et al., 2005, J. Androl. 26, 32-43). The objective of this study was to identify a critical developmental period(s) during which EV exposure results in the observed penile abnormalities. Male pups received EV at a dose of 0.10-0.12 mg/kg on postnatal day(s) 1, 1-3, 4-6, 1-6, 7-12, 13-18, 19-24, or 25-30. Fertility was tested at 102-115 days of age and tissues were examined at 117-137 days. Both penile morphology and fertility were unaltered in rats treated with EV after 12 days of age. Conversely, except in rats treated on postnatal day 1 only, none of the males treated prior to 12 days of age sired pups, and all had abnormal penises, including varying degrees of abnormal accumulation of fat cells and loss of cavernous spaces and smooth muscle cells in the corpora cavernosa penis, which were maximal in the 1-6-day group. Also, the preputial sheath was partially released or its release was delayed, and the weight of the bulbospongiosus muscle was significantly reduced. Plasma testosterone (T) in the 1-6- and 4-6-day groups and intratesticular T in the 4-6-day group were significantly lower. The testosterone surge, characteristic of controls in the first week of life, was suppressed in the 1-3-day group. Estrogen receptor alpha mRNA expression was enhanced in the body of the penis in the 1-3-day group, but not in the 13-18-day group. Hence, EV exposure prior to 12 days of age (as short as 1-3 days postnatal), but not after 12 days of age, results in long-term abnormal penile morphology, characterized by abnormal accumulation of fat cells in the corpora cavernosa penis and, consequently, loss of fertility.

Exposure of neonatal male rats to estrogen induces abnormal morphology of the penis and loss of fertility.

The research objectives are to determine whether estrogen-induced infertility is associated with abnormal morphology of the penis and if morphological alterations can be reversed by testosterone (T). Male pups received diethylstilbestrol (DES) on alternate days from postnatal days 2 to 12. They received T or empty implants at 180 days, were tested for fertility at 188 days, and terminated at 200 days. While 5/7 control males sired pups, only 1/6 did in the DES group, and 0/8 in the DES plus T group. In addition to reductions in penile length and weight, the novel structural change induced by DES, and not reversed by T, was a replacement of cavernous spaces by fat cells in the penis body. Hence, T substitution for 8 days at adulthood did not reverse infertility in rats treated neonatally with DES and provided evidence that infertility probably resulted from absence of cavernous spaces and/or accumulation of fat cells in the penis body.

Histoquantitative effects of orchiectomy with and without testosterone enanthate treatment on the bovine epididymis.

The histoquantitative effects of orchiectomy with and without testosterone enanthate treatment on the bovine epididymis as relates to epithelial height, luminal diameter, and thickness of smooth muscle wall were determined. With androgen deprivation (induced by orchiectomy), epithelial heights in epididymal regions I, II, III, IV, V, and VI were reduced to 25%, 47%, 46%, 39%, 39%, and 79% of the controls respectively. In the orchiectomized bulls treated with testosterone, the epithelial height in region I increased to only 29% of the controls and the heights reached 58% to 67% of the controls in regions II to V and as much as 111% in region VI. These data indicate that circulating testosterone does not have an important role in maintaining the epithelial height in region I, whereas it seems to be solely responsible for its maintenance in region VI. The epithelial heights in regions, II, III, IV, and V are probably dependent on both circulating and luminal androgens. Similar to the epithelial height, the luminal diameter in all regions was markedly reduced by orchiectomy; however, testosterone treatment failed to restore the diameter to the control levels. In contrast to the epithelial height and luminal diameter, the thickness of the smooth muscle wall in all regions was significantly increased following orchiectomy. Although it decreased in the orchiectomized bulls treated with testosterone, it was still thicker than that of controls. The relative significance of luminal and circulating androgens in maintaining the structure and function of the epididymal epithelium is discussed.

Light microscopic and ultrastructural evidence of epithelial phagocytosis of sperm in the rete testis and ductuli efferentes in the bull.

Light microscopic and ultrastructural observations were made in the bull rete testis and the ductuli efferentes with emphasis on the presence of sperm in the epithelium. Phagocytosed sperm in various stages of degeneration were found in the epithelial cells lining the rete testis and in the nonciliated cells of the ductuli efferentes. Phagocytosis was more prevalent in the rete testis than in the ductuli efferentes. Besides the epithelial cells, degenerating sperm components and residual bodies were in the luminal macrophages of the rete testis. The degeneration of sperm heads presumably progressed in the following order: (i) disruption of the cell membrane, (ii) aggregation of small vesicles, probably of Golgi origin, between the disrupted cell membrane and an outer acrosomal membrane, (iii) loss of the acrosomal matrix, and finally (iv) disintegration of the nuclear chromatin. These degenerative changes probably resulted from increased lysosomal activity of phagocytosing cells. The possible importance and causes of spermiophagy are discussed.

Histochemical activity of alkaline phosphatase and acid phosphatase in the epididymis of mature intact and androgen-deprived bulls.

The histochemical localization of alkaline phosphatase (ALP) and acid phosphatase (ACP) was determined in regions I to VI of the epididymis in mature intact, orchidectomized, and orchidectomized testosterone-treated bulls. The intensity of ALP activity was essentially the same in all regions; however, its localization varied depending upon the region. Although the stereocilia and luminal border of epithelial cells were strongly-positive in regions I to III, these were negative in regions IV to VI. The basement membrane and circumtubular smooth muscle cells were strongly ALP reactive in all regions. Epithelial ALP activity was abolished completely by orchidectomy; however, it was restored to normal concentration by testosterone treatment implying its dependence on circulating testosterone. The ACP activity was present in the epithelial cells of all regions with the strongest activity in the supranuclear region. Similar to ALP, ACP activity was markedly reduced in the epithelial cells by orchidectomy. However, in contrast to ALP activity, lost ACP activity was only minimally increased by testosterone treatment in all regions, except in region VI where it was restored to a normal concentration. These observations imply that although the epithelial ACP activity of region VI was mainly under the influence of circulatory testosterone, there may be other factors such as luminal androgens, testicular fluid, and sperm that may be important in regulating the ACP activity of regions I to V of the epididymis. The importance of ALP and ACP in the epididymal epithelium was discussed.

Effects of age and season on the type and occurrence of sperm abnormalities in Nubian bucks.

Effects of age and season on type and occurrence of sperm abnormalities were examined in semen samples collected from 3 groups of Nubian bucks at ages of 4 to 9 months, 10 to 21 months, and 39 to 50 months. The average total percentage of sperm abnormalities at the onset of puberty (141 +/- 4 days) was 64.6 +/- 14.8% (head, 19.5 +/- 13.6%; middle piece, 17.2 +/- 9.3%; and proximal protoplasmic droplets, 14.6 +/- 10.5%), but this improved rapidly and was reduced to 12.5 +/- 7.5% by 8 months of age (head, 1.9 +/- 4.5%; middle piece, 4.6 +/- 2.8%). Further increase in age, at least up to 4 years, did not reveal a significant effect (P less than 0.05) on the type or percentage of total abnormalities. Similar to age, a comparison of data among seasons did not reveal a significant effect on the type or occurrence of sperm abnormalities in 10- to 21-month-old or 39- to 50-month-old bucks. Seemingly, Nubian bucks started producing good quality semen at 8 months of age, and season did not influence sperm abnormalities.

Right precordial mapping. Diagnostic sensitivity and specificity of leads V3R to V6R in different intercostal spaces in cases with right ventricular infarction.

Twelve lead conventional electrocardiography and right precordial mapping including precordial leads V2R to V6R in the 2nd to 5th intercostal spaces were obtained in 150 cases with acute myocardial infarction. There were 27 (18%) cases with right ventricular infarction: 24 associated with inferior wall infarction and 3 with anterior wall infarction. In the right precordial mapping, leads V3R to V6R in all intercostal spaces had equal sensitivity of 100%; 3rd intercostal space recording, however, had the maximum specificity of 92.6%.

Growth rates at the extremities of limb bones in young horses.

Measurements were made of growth at the extremities of all the long bones of the left limbs of 23 horses between 52 and 104 days of age. Growth rates were more rapid in the younger than in the older animals. Growth was more rapid in the hind limb than in the forelimb. Growth rates were greater for males than in females. Although this age group represents only two of the 60 or so months necessary for maturity to be reached, oxytetracycline bone-labelling produced results which are more precise than measurements obtained by other methods. This study provides a substantial amount of data for an age group of horses whose growth rate has not been well documented.

Role of estrogen in induction of penile dysmorphogenesis: a review.

In this review, we report permanent dysmorphogenesis of the penis and loss of fertility in adult rats treated neonatally with estrogen. Specifically, we report replacement of smooth muscle cells and cavernous spaces by fat cells in the corpus cavernosum penis, but not in the adjoining corpus spongiosum. Induction of these novel, region-specific phenotypes is dose-dependent, requires a critical window of exposure and associated with decreased testosterone and up-regulation of estrogen receptor alpha (ER alpha). The resistance of ER alpha knockout mice to develop these abnormalities implies an unequivocal role for ER alpha in mediating maldevelopment of the penis. Additionally, the prevention of estrogen-inducible penile abnormalities by ER antagonist ICI 182 780 implies that a functional ER-mediated pathway is essential for inducing penile abnormalities. Likewise, the ability of testosterone or dihydrotestosterone to negate these abnormalities suggests a role for an androgen receptor (AR)-mediated pathway. Taken together, these observations led us to hypothesize that neonatal estrogen exposure, via an ER-mediated pathway (direct action) or an AR-mediated pathway (indirect action through decreased testosterone) or both pathways, up-regulates ER alpha expression in stromal cells of the penis, which are then reprogrammed such that their differentiation into smooth muscle cells is inhibited and their differentiation into adipocytes is stimulated.

Estrogen receptor alpha mediates estrogen-inducible abnormalities in the developing penis.

Previously, we reported an association between estrogen receptor-alpha (ERalpha) upregulation and detrimental effects of neonatal diethylstilbestrol (DES) exposure in the rat penis. The objective of this study was to employ the ERalpha knockout (ERalphaKO) mouse model to test the hypothesis that ERalpha mediates DES effects in the developing penis. ERalphaKO and wild-type C57BL/6 mice received oil or DES at a dose of 0.2 microg/pup per day (0.1 mg/kg) on alternate days from postnatal days 2 to 12. Fertility was tested at 80-240 days of age and tissues were examined at 96-255 days of age. DES caused malformation of the os penis, significant reductions in penile length, diameter, and weight, accumulation of fat cells in the corpora cavernosa penis, and significant reductions in weight of the bulbospongiosus and levator ani muscles in wild-type mice. Conversely, ERalphaKO mice treated with DES developed none of the above abnormalities. While nine out of ten male mice sired pups in the wild-type/control group, none did in the wild-type/DES group. ERalphaKO mice, despite normal penile development, are inherently infertile. Both plasma and intratesticular testosterone levels were unaltered in the DES-treated wild-type or DES-treated ERalphaKO mice when compared with controls, although testosterone concentration was much higher in the ERalphaKO mice. Hence, the resistance of ERalphaKO mice to developing penile abnormalities provides unequivocal evidence of an obligatory role for ERalpha in mediating the harmful effects of neonatal DES exposure in the developing penis.

Morphology of the bovine epididymis.

The epididymis of the bull was divided into six regions, and morphological differences between regions were studied. The epithelium of all regions contained four cell types: principal and basal epithelial cells, and intraepithelial lymphocytes and macrophages. The epithelium of regions II-V also contained a few apical cells. Principal cells of all regions possessed an endocytotic apparatus including stereocilia underlain by canaliculi, coated vesicles, and subapical vacuoles (up to 1 micron in diameter); however, large vacuoles with a flocculent content and multivesicular bodies (up to 5 microns in diameter) were most numerous in regions II, III, and IV. The unique features of principal cells of region I were the presence of well-developed Golgi bodies, few lipid droplets, and whorls of smooth endoplasmic reticulum in the supranuclear cytoplasm. Numerous mitochondria, distended cisternae of rough endoplasmic reticulum, and dense granules characterized the infranuclear cytoplasm of the principal cells of regions II-VI; however, these features were more developed in region V. Apical cells were characterized by the apical location of the nucleus, many mitochondria in the apical cytoplasm, and few microvilli at the luminal border. Basal cells with few cytoplasmic lipid droplets were present throughout the length of the epididymis but appeared more numerous in region V. Intraepithelial lymphocytes were present at all levels of the epithelium but were never seen in the lumen. Intraepithelial macrophages containing heterogeneous granules, eccentric nuclei, and pseudopods were invariably seen near the basal area of the epithelium in all regions. These observations are discussed in an effort to define the role of each cell type in the epididymal epithelium.

Appendix epididymidis and aberrant ductules of the bull: light-microscopic and ultrastructural study.

The appendix epididymidis and aberrant ductules possessed similar morphological characteristics. The epithelium was 31 +/- 3 microns in height and consisted primarily of ciliated and nonciliated cells, although a few lymphocytes were also present. The ultrastructure of major cell types showed most cell organelles in their cytoplasm. However, these organelles were poorly developed, suggesting that neither cell type performed either a secretory or an absorptive function. Although the vestigial organs and ductuli efferentes were similar in epithelial height and epithelial cell types, there were important morphological differences that were reliably used to differentiate between the two. First, the luminal diameter was significantly smaller in the vestigial organs (60 +/- 12 vs. 146 +/- 44 microns in the ductuli efferentes). Second, the nonciliated cells of the vestigial organs, unlike those of the ductuli efferentes, lacked both dense granules and vacuoles in the cytoplasm. Finally, the tubular cross-sections of the vestigial organs were closely packed and were located at the tip of the caput epididymidis in the case of the appendix epididymidis, and between the lobules of the ductuli efferentes in the case of the aberrant ductules.