In this prospective study carried out in 44 PD patients and 25 neurological controls we wanted to verify whether the combination of CSF α-synuclein species—t-α-syn and o-α-syn—and classical AD biomarkers may help in differentiating PD from neurological controls, and if these biomarkers may predict cognitive decline. The median of follow-up duration was 3 years (range: 2–6 years). Mini Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) were used for monitoring cognitive changes along time, being administered once a year. Oligo/total α-syn ratio (o/t-α-syn ratio) confirmed its diagnostic value, significantly contributing to the discrimination of PD from neurological controls. A greater diagnostic accuracy was reached when combining o/t-α-syn and Aβ42/tau ratios . Low CSF Aβ42 level was associated with a higher rate of MMSE and MoCA decline, confirming its role as independent predictive factor for cognitive decline in PD. None of the other biomarkers assessed  showed to have prognostic value. We conclude that combination of CSF o/t-α-syn and Aβ42/tau ratios improve the diagnostic accuracy of PD. PD patients showing low CSF Aβ42 levels at baseline are more prone to develop cognitive decline.There is a great interest in developing cerebrospinal fluid (CSF) biomarkers for diagnosis and prognosis of Parkinson's disease (PD). CSF alpha synuclein (α-syn) species, namely total and oligomeric α-syn (t-α-syn and o-α-syn), have shown to be of help for PD diagnosis. Preliminary evidences show that the combination of CSF t-α-syn and classical Alzheimer's disease (AD) biomarkers—β-amyloid 1–42 (Aβ Parkinson's disease (PD) is a common neurodegenerative disorder evolving, in a substantial proportion of patients, to dementia. Although it is defined as a typical movement disorder and its diagnosis is mainly based on motor-related clinical criteria, other functional domains are also involved. Accordingly, post-mortem findings of alpha-synuclein (α-syn) pathology—the histopathologic hallmark of PD—show that the involvement of the dorsal motor nucleus of the vagal nerve and the olfactory bulb takes place much earlier before midbrain involvement  and phosphorylated tau (p-tau) are state markers of Alzheimer's disease (AD), as they reliably reflect AD pathology also in pre-dementia phases. This knowledge has been translated into operational diagnostic criteria  composition, the CSF represents an ideal source for biomarkers of different pathophysiological processes characterizing the early phases of the disease, when the clinical diagnosis is more challenging. For example, Aβ42 levels as a prognostic factor for cognitive impairment in PD. Data on the possible relationship between α-syn species and the risk of dementia in PD are still scanty.Currently, detection of reliable CSF biomarkers for PD is under intensive investigation. Several recent studies have explored the potential use of CSF total α-syn (t-α-syn) as a putative PD biomarker. A clear trend of lower CSF t-α-syn levels in PD and other synucleinopathies has been consistently reported  and α-syn species  in PD patients and neurological controls with a median follow up duration of 3 years.In this study we evaluated both the diagnostic accuracy and the capability in predicting cognitive decline of CSF AD biomarkers (Aβn = 15), postural instability (n = 3), seizures (n = 2) and polyneuropathy (n = 5). Follow-up visits included clinical examination and neuropsychological testing carried out yearly by means of MMSE  were consecutively recruited between 2007 and 2011 and followed-up. They underwent a baseline clinical examination by experienced neurologists, detailed neuropsychological testing, blood chemistry, neuroimaging (computed tomography and/or magnetic resonance imaging), and lumbar puncture. CSF was collected according to the hospital standard protocol and with the local ethical committee approval, after informed written consent was given by the patient. All PD patients fulfilled the United Kingdom Parkinson's Disease Society Brain Bank clinical diagnostic criteria. All of them were treated with L-DOPA and the great majority (39 out of 44) were also taking DA-agonists. PD patients had good control of motor symptoms (mean UPRDS-III 25.39 ± 12.97), and were functionally independent or minimally dependent . Disease duration was calculated from the onset of the first motor symptoms to the time of lumbar puncture. As a control group, 25 cognitively normal age-matched subjects who underwent lumbar puncture as a part of diagnostic work up for other neurological conditions (OND) were recruited to our study. The OND group included: primary headache (3 were not included in the study). CSF Aβ42, t-tau and p-tau were measured with ELISA assays . For CSF α-syn, 0.5 mL samples were thawed on ice and then divided into aliquots of 110 μL in siliconized tubes containing a cocktail of protease inhibitors, including AEBSF, aprotinin, E-64, EDTA, and leupeptin , and 0.05% Tween 20, and the samples were then stored at −80°C until used in the immunoassay for α-syn. While CSF t-α-syn is expressed as ng/mL, CSF o/t-α-syn ratio is expressed in CPS (counts per second). All the samples were obtained at the Section of Neurology, Perugia General Hospital, according to the protocol approved by the Regional Ethical Committee (Prot. N. 19369/AV), after informed written consent was obtained.Lumbar puncture was performed between 8:00 and 10:00 A.M., after an overnight fast. CSF (10 mL) was collected in sterile polypropylene tubes, centrifuged for 10 min at 2000× g, and 0.5-mL aliquots were immediately frozen at −80°C. None of the samples was contaminated by blood during the procedure . The accuracy of the diagnostic value of the biomarkers was assessed by area under the curve (AUC) of the receiver operating characteristic (ROC) curve  and after follow-up . Follow-up observations refer to the last visit carried out.Demographic data, clinical features, and biomarkers values are listed in Table 42 levels were higher as opposite to lower median t-tau levels. As a consequence, Aβ42/t-tau ratio was significantly increased in the PD group with respect to OND subjects  and an increase of o-α-syn levels (p = 0.041) were found in PD group  and α-syn species  in a cohort of PD patients and neurological controls followed up for 2–6 years (median follow-up duration: 3 years).In this investigation we assessed both the diagnostic accuracy and the performance in predicting cognitive decline of the combination of CSF AD biomarkers (Aβ42/t-tau and o/t-α-syn ratios showed good sensitivity (0.82) but low specificity . While the usefulness of o/t-α-syn ratio in discriminating PD and controls has already been reported in recent investigations  confirmed that low CSF Aβ42 levels are independent predictor of cognitive decline in PD, either measured by MMSE or MOCA.About the predictive value of CSF biomarkers for cognitive decline in PD, our study confirmed the specific role of low CSF levels of Aβ42 levels as independent predictor of cognitive decline in PD. Longitudinal studies measuring biomarkers and clinical parameters over several years represent a major contribution in this field. Analogously to the longitudinal AD Neuroimaging Initiative  in AD, the Parkinson's Progression Markers Initiative  will give important knowledge in the field of PD, thanks to the measurement of several CSF, blood, and imaging biomarkers in early de novo PD followed up for several years.In conclusion, this study further contributes to the evidence of the usefulness of CSF biomarkers for PD diagnosis and prognosis. Major points are the need to combine several CSF biomarkers for improving the diagnostic accuracy, and the confirmed role of low CSF AβThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Of the various options for patients with end stage renal disease, kidney transplantation is the treatment of choice for a suitable patient. The kidney for transplantation is retrieved from either a cadaver or a live donor. Living donor nephrectomy has been developed as a method to address the shortfall in cadaveric kidneys available for transplantation. Laparoscopic living donor nephrectomy (LLDN), by reducing postoperative pain, shortening convalescence, and improving the cosmetic outcome of the donor nephrectomy, has shown the potential to increase the number of living kidney donations further by removing some of the disincentives inherent to donation itself. The technique of LLDN has undergone evolution at different transplant centers and many modifications have been done to improve donor safety and recipient outcome. Virtually all donors eligible for an open surgical procedure may also undergo the laparoscopic operation. Various earlier contraindications to LDN, such as right donor kidney, multiple vessels, anomalous vasculature and obesity have been overcome with increasing experience. Laparoscopic live donor nephrectomy can be done transperitoneally or retroperitoneally on either side. The approach is most commonly transperitoneal, which allows adequate working space and easy dissection. A review of literature and our experience with regards to standard approach and the modifications is presented including a cost saving model for the developing countries. An assessment has been made, of the impact of LDN on the outcome of donor and the recipient. End stage renal disease (ESRD) patients have the option of life long maintenance haemodialysis, continuous ambulatory peritoneal dialysis (CAPD) or kidney transplantation. Kidney transplantation is the treatment of choice for a suitable patient with ESRD. The first cadaveric renal transplant was performed in 1945 and first live related renal transplant was performed in 1953. performeLive donor nephrectomy is a unique surgical challenge because it is performed on healthy donors. It is of great importance to keep the morbidity and mortality of live donors as low as possible and to harvest the kidney in optimal condition for transplantation. The traditional method of open live donor nephrectomy is associated with significant trauma to the thoracoabdominal wall with a long flank incision, more pain, possible pleural injury, pseudohernia, longer hospital stay, long term wound complications  and relatively long convalescence for otherwise healthy volunteer donors.7 For a lLaparoscopic live donor nephrectomy minimizes the drawbacks of open live donor nephrectomy and makes the prospects of live donor nephrectomy more appealing to prospective donors by reducing post operative pain, shortening convalescence and improving the cosmetic outcome of donor nephrectomy. LLDN has shown the potential to increase the number of living kidney donations by removing some of the disincentives inherent to kidney donation itself.Virtually all donors eligible for an open surgical procedure may also undergo the laparoscopic operation. Various earlier contraindications to LDN, such as right donor kidney, multiple vessels, anomalous vasculature and obesity have been overcome with increasing experience. The contLaparoscopic live donor nephrectomy can be done transperitoneally or retroperitoneally on either side. The operative technique of LLDN has undergone many refinements since it was first described. The approach is most commonly transperitoneal, which allows adequate working space and easy dissection.All the donors are hydrated overnight with 1.5 L of intravenous fluids. After general anaesthesia, a Foley's catheter is inserted. The donor is placed in a modified lateral decubitus position. Pneumoperitoneum is created by insufflation of carbon dioxide using a veress needle or after introduction of a laparoscopic 12–mm port at umbilicus by open Hassan technique. The umbilical port is primarily used as a camera port. Another 12–mm laparoscopic port is placed between umbilical port and anterior superior iliac spine . A 5–mm port is placed in line with the camera port about 3 cm below the costal margin and 3 cm lateral to the midline. Fourth 5 mm laparoscopic port may be used for retraction, if needed and is placed 4 cm below the costal margin in anterior axillary line  is placed through a 6 cm pfannenstiel incision. By placing forceps between previously mobilized renal artery and vein, excellent exposure of renal hilum is possible. The renal artery and vein are sequentially ligated with a GI vascular stapler or 13mm Weck Clips . The kidney is then placed in the retrieval bag and carefully extracted from the pfannenstiel incision.We do not use the Endocatch organ retrieval bag or the stapler because of the high cost. A small amount of perirenal fat is left intact on the lateral and inferior border of the kidney and this is helpful in holding the graft during subsequent manual retrieval. We use 13mm Weck Clips for the ligating renal artery and vein & the kidney is extracted by a two finger technique.–12 A fanThe transperitoneal laparoscopic approach may be used similar to that on the left side. Modifications have been made in right LLDN to compensate for shorter vessels and to prevent thrombotic complications. In the recipient, the iliac vein is completely mobilized by dividing all of its posterior branches to facilitate making a tension free anastomosis. The dissInsertion of an Endo-GIA stapler through the port positioned in the right lower abdominal quadrant allows control of the renal vein at its junction with vena cava, preserving the maximum possible length of renal vein. To maximth rib. The underlying muscles are split till the thoracolumbar fascia is seen, which is incised sharply and retroperitoneal space is entered. Blunt finger dissection of pararenal fatty tissue is done to separate the fatty tissue from the abdominal wall to create an initial small retroperitoneal space for the placement of dilating balloon. Retroperitoneal space is now created by a PBD-1000 balloon device . A 12 mm trocar replaces the balloon device and it is used as a camera port. Two more trocars (one 12mm & other 10 mm) are placed under vision. During operation, a pressure of about 10–12 mm of Hg is enough to create a sufficient retroperitoneal working space.[The donor is given general anaesthesia, catheterization done with a Foley catheter and is placed in an overextended flank position. The firsng space. Hook monAfter identifying the psoas muscle, one of the most important landmarks, Gerota's fascia is incised laterally, and retroperitoneal space is freed in cranial and caudal direction. It is important to keep the psoas muscle horizontally on the video screen to remain oriented. The ureter is identified in the area of medial line of psoas muscle, is carefully freed, care being taken not to dissect the periureteric sheath and hence damage the nutritive vessels of the ureter, which will result in necrosis after transplantation. Perirenal fatty tissue is identified and renal vessels are freed of surrounding lymphatic tissue. Gentle and meticulous dissection is necessary to avoid injuries to the renal vessels. The gonadal vein is dissected adjoining the renal vein & may be clipped and divided if required. The renal artery is dissected and freed at its origin from the aorta. The adrenal vein is identified, dissected near the renal vein, clipped and divided. The dissection proceeds to separate the adrenal gland from the upper pole of left kidney. The surrounding fatty tissue is freed from the renal capsule. The gonadal vein is clipped and divided at the pelvic brim if not already divided. The ureter is clipped distally and transected. The kidney can be retrieved by any of the incisions: muscle splitting lumbotomy incision, illiac fossa incision inferior to the anterior port or by a pfannenstiel incision after creating space extraperitoneally.nd port is placed 2–3 cm behind the first post below the 12th rib and a third port 10 mm anterior to the first port  and leave the better functioning kidney with the donor. Right LDN is done only retroperitoneoscopally at our centre as this gives better access to the hilum with good length of vein and artery.st and anterior ports. Only superficial layer of the muscles is incised and a sponge is kept to prevent air leak. The posterior port is used to clip and cut the renal artery. Subsequently, an incision is given, the anterior port is removed and an open surgical Satinsky clamp is inserted and applied on the IVC under vision. The vein is divided with a cuff of the IVC, the graft is retrieved and the IVC is sutured using a long needle holder. The final knot is placed by throwing the knot and using the right angle to guide and tighten it as there is no space for the hand to go in.Our modification of right retroperitoneal approach Initial part of the procedure is similar to that described above and just before the removal of the graft, an incision is made between the 1The limited working space makes dissection difficult due to repeated fogging of lens and retroperitoneal fat keeps coming in the way. We could overcome this difficulty by inserting a fourth port and using a 5 mm fan retractor to retract the fat and at the same time giving gentle traction at the kidney to facilitate hilar dissection. However, retraction of the kidney with 10mm/5mm retractors results in small subcapsular hematomas with could be seen in 60% of our patients. They are common on the posterior aspect near the hilar area and near the upper pole where maximum retraction is used while dissecting the hilar structures and mobilizing the kidney. These hematomas did not affect the functional graft outcome.Hand assisted LDN has been described both transperitoneally and retroperitoneally. Ruiz-Deya et alSundqvist et al comparedAt many centers, the number of individuals willing to undergo LLDN today would not have donated a kidney if open surgery was the only option available for organ donation. This is so because LLDN offers decreased postoperative pain, marked cosmetic benefit, and better convalescence compared to open donor nephrectomy.P < 0.05) in LDN series compared with open donor nephrectomy series.[In most reported experiences, the operating time at the beginning of the LDN learning curve was generally longer than for open live donor nephrectomy. In a mety series. However,y series. Our operLDN and open donor nephrectomy are comparable in the estimated blood loss and postoperative transfusion requirement.28–30 In 24The reported frequency of open conversion during LDN ranges from 0% to 13%. The mostP < 0.05). Similar results have been found in other series .[vs e” 3 minutes and WIT d” 5, 5–10, and e” 10 min. Prolonged WIT did not appear to have an effect on early graft function or the rate of decline in serum creatinine during the first 3 months post-transplantation.This represents a major concern as it has always been slightly longer in the laparoscopic group when compared with open donor nephrectomy due to the longer extraction time. It has been thought that any increase in this would translate into a poor graft function. This notion has been disproved by various studies suggesting no bearing of this small difference on the recipient outcome. The warm ischemia time during LDN may range between 95 and 300 seconds.28 At the< 0.001). Similar < 0.001). The effe< 0.001). AnalysisWe evaluated the impact of warm ischemia time in LDN. It did not correlate with the incidence of delayed graft function, acute rejection or allograft or recipient survival Tables  and 3. IThere have been concerns for vessel and ureteral length which could be harvested in LDN as it would have a direct bearing on the recipient operation. In a randomized controlled trial studyingvs open donor nephrectomy group .[Several clinical trials, have shown, no significant difference in the serum creatinine level between open and LDN at 3 days, 30 days, and 3 months after transplantation. The longp =0.26).vs 1.3 mg/dL, respectively, p = 0.9), and the graft survival rate  has also been similar.[vs laparoscopic), live donor kidney transplantation was associated with a delayed graft function rate of 5% to 10%.[In a comparison between open donor nephrectomy and LDN in patients with multiple renal arteries, the postoperative serum creatinine level after 1 year of follow-up has been found to be similar  and open donor nephrectomy (0% to 32%). Similar vs 0% to 6%, respectively).[In initial LDN series, ureteral injuries occurred more frequently than during open donor nephrectomy (0% to 11% ctively). Subsequectively). Universictively).Wolf et al reportedSince the start of live donor program in 1953, there has been a constant effort by the health care professionals to reduce the donor morbidity and improve their outcome. LDN has come as an answer to this, especially with refinements in equipment and surgical techniques. It provides all the benefits of minimal invasive surgery to the most unique patient that is the donor while maintaining results equivalent to the ODN in terms of graft function and recipient outcome. LDN has now stood the test of time. The results are equal and morbidity is less, hence its increasing popularity and becoming the standard of care.


The protective effects of granulocyte colony-stimulating factor (G-CSF) have been demonstrated in a variety of renal disease models. However, the influence of G-CSF on diabetic nephropathy (DN) remains to be examined. In this study, we investigated the effect of G-CSF on DN and its possible mechanisms in a rat model.Otsuka Long-Evans Tokushima Fatty (OLETF) rats with early DN were administered G-CSF or saline intraperitoneally. Urine albumin creatinine ratio (UACR), creatinine clearance, mesangial matrix expansion, glomerular basement membrane (GBM) thickness, and podocyte foot process width (FPW) were measured. The levels of interleukin (IL)-1β, transforming growth factor (TGF)-β1, and type IV collagen genes expression in kidney tissue were also evaluated. To elucidate the mechanisms underlying G-CSF effects, we also assessed the expression of G-CSF receptor (G-CSFR) in glomeruli as well as mobilization of bone marrow (BM) cells to glomeruli using sex-mismatched BM transplantation.p<0.05), as were mesangial matrix expansion, GBM thickness, and FPW (p<0.05). In addition, the expression of TGF-β1 and type IV collagen and IL-1β levels was lower in the G-CSF treatment group (p<0.05). G-CSFR was not present in glomerular cells, and G-CSF treatment increased the number of BM-derived cells in glomeruli (p<0.05).After four weeks of treatment, UACR was lower in the G-CSF treatment group than in the saline group (G-CSF can prevent the progression of DN in OLETF rats and its effects may be due to mobilization of BM cells rather than being a direct effect. Diabetes mellitus (DM) is a multisystem disorder that affects various organs. Almost 30% of DM patients develop diabetic nephropathy (DN), despite control of blood glucose and/or blood pressure. DN is widely recognized as a common cause of end-stage renal disease Granulocyte colony-stimulating factor (G-CSF) is frequently used to mobilize hematopoietic stem cells from the bone marrow (BM) into the peripheral blood The experiments were performed in compliance with the ARRIVE guidelines on animal research n = 12) received water containing 30% sucrose ad libitum to facilitate the development of DN. After DN had been induced, they received no more sucrose water. LETO rats (n = 7), as normal controls, received tap water without 30% sucrose. The DN model was confirmed by serum fasting glucose level exceeding 200 mg/dl, urine albumin creatinine ratio (UACR) exceeding 300 mg/g, and histologically-confirmed GBM thickening and mesangial matrix expansion Starting at 16 weeks of age, all male OLETF rats , DN was confirmed in the female rats (data not shown). The experimental design is outlined in Starting at 6 weeks of age, female OLETF rats received lethal γ-irradiation  from a Gammacell 1000 Elite  n = 4 each), treated daily for 5 days with G-CSF  or an equal volume of saline , and observed for 4 weeks OLETF rats in which DN had been induced were divided randomly into two groups , triglyceride (TG), UACR, and creatinine clearance (CrCl). Animals were transferred to individual metabolic cages for urine sample collection, and 24-hours urine samples were collected to measure UACR levels Kidney tissue from each animal was fixed in 10% formalin solution (pH 7.4) and embedded in paraffin. The embedded tissue was cut into 3- µm-thick sections and subjected to periodic acid-Schiff (PAS) staining for light microscopic evaluation. Mesangial matrix expansion in the glomeruli was evaluated in PAS-stained sections using Image-Pro Plus 4.5  For electron microscope evaluation, the kidneys were fixed in 2.5% glutaraldehyde  and embedded in epoxy resin. Ultrathin sections were double-stained with 1.25% uranium acetate and 0.4% lead citrate and then observed with an electron microscope . GBM thickness was determined in the glomeruli where the epithelial and endothelial cells were clearly visible. A perpendicular line of GBM was drawn from the endothelial to the epithelial edge, and it was measured by image analysis using Image-Pro Plus 4.5  Kidneys were frozen in liquid nitrogen and stored at −80°C for the RT-PCR assays. Total RNA was extracted using TRIzol , and then reverse transcribed using SuperScript II reverse transcriptase  according to the manufacturer’s protocol. We performed RT-PCR to analyze G-CSFR and β-actin. The sequences of the rat primer pairs for G-CSFR and β-actin and the original clones are listed in TGF-β1), type IV collagen, and interleukin (IL)-1β (IL-1β)  was performed using SYBR Green qPCR Mix  and analyzed on a LightCycler 1.5 . The genes selected were transforming growth factor (TGF)-β1 ( (IL-1β) . qPCR amTo detect donor BM cells in recipient kidneys, the Y chromosome was detected by FISH in female rats. Cy3-labeled rat Y chromosomes  were provided in the supplier’s hybridization mix. FISH analysis was performed using IDetect™ according to the manufacturer’s recommendations. The Y-chromosome positive cells were calculated for ten randomly selected glomeruli of each kidney section. Images were obtained on an ECLIPSE 80i microscope equipped with an iAi progressive scan camera  and Cytovision software .β1 levels were measured under high magnification x200 in ten images of each kidney section by image analysis using Image-Pro Plus 4.5  To confirm the presence of macrophages in glomeruli, immunohistochemical staining was performed on the kidneys of female rats that had received BM from male rats. We used a mouse monoclonal anti-monocyte/macrophage (ED-1)  or rabbit polyclonal anti-TGF-β1  antibodies as the primary antibody. ED-1 and TGF-β1 levels were detected with streptavidin-peroxidase and peroxidase substrate solution . For ED-1, the numbers of positively stained macrophages were counted under high magnification (x400) in 120 to 130 glomeruli of each kidney section and an average score was calculated and expressed as positive cells/glomerulus. TGF-P<0.05 were considered statistically significant.All data are presented as mean±SD, except for histological data, which are presented as mean±SE. Statistical differences were determined with the Statistical Package for the Social Sciences (SPSS) 18.0 software . Data were analyzed using Mann Whitney U-tests (for single comparisons) or Kruskal-Wallis nonparametric ANOVA (for multiple comparisons). Values of P<0.05) in the OLETF rats than the control LETO rats during the first 12 weeks of sucrose feeding (to 28 weeks of age) but was significantly lower (P<0.05) after 22 weeks of sucrose feeding (at 38 weeks of age) (P<0.05). Serum glucose levels exceeded 200 mg/dl during the first 12 weeks of sucrose feeding and UACR levels exceeded 300 mg/g during the first 16 weeks of sucrose feeding (at 32 weeks of age) (P<0.05). CrCl levels did not differ between the two groups. Body weight was lower in OLETF rats than the control LETO rats (P<0.05). TC and TG levels were higher in OLETF rats than the control LETO rats (P<0.05) (P<0.05) (Body weight was significantly higher ( of age) . Serum g of age) . Kidney/(P<0.05) . Histolo(P<0.05) . These cP<0.05) but still higher than the control LETO group. TC levels were lower in the G-CSF-treated group than in the saline-treated group (P<0.05) and not significantly different from that of the control LETO group. Serum glucose, TG level, and body weight were not significantly different from in the saline-treated group. CrCl levels did not differ significantly within the three groups  but still higher than the control LETO group (P<0.05) (P<0.05) and not significantly different from that of the control LETO group (P<0.05) but still higher than the control LETO group (P<0.05)  . In addi(P<0.05) . GBM thiTO group . FPW was(P<0.05) .P<0.05) but still higher than the control LETO group (P<0.05) Levels of type IV collagen and IL-1β mRNA were also lower in the G-CSF-treated group than in the saline-treated group (P<0.05) and not significantly different from those of the control LETO group (P<0.05)  .P<0.05)  . In addiP<0.05) .We confirmed the presence of G-CSFR in kidney tissue by RT-PCR . HoweverOur data show that G-CSF treatment can prevent progression of early DN in OLETF rats, and that mobilization of BM cells, rather than a direct effect of G-CSF, is the likely explanation for the observed effect.Previous studies have demonstrated that the characteristic glomerular changes in DN include mesangial matrix expansion, GBM thickening, and podocyte foot process effacement According to a previous study, early DN is characterized by increased kidney/body weight and UACR levels and unchanged CrCl level The pathophysiology of DN involves the accumulation of microvascular matrix protein and inflammation. Previous studies indicated that TGF-β1 played a key role in the development of renal hypertrophy and the accumulation of extracellular matrix components in diabetes, as well as stimulation of the synthesis of extracellular matrix molecules such as type IV collagen Previous studies suggested several possible mechanisms for the general effect of G-CSF on renal disease. First, G-CSF may affect the kidney directly. A direct effect of G-CSF on cardiomyocytes and endothelial cells has been reported As another possible mechanism, increased bone marrow cells homing to injured renal cells could induce trans-differentiation or trophic effects, thereby contributing to renal cell repair. Previous reports suggested that BM cells might trans-differentiate into renal tubular cells that repair kidneys Sugimoto et al. have demonstrated that administration of pioglitazone for 6 months ameliorates renal injury, and Ko et al. showed that treatment with enalapril for 32 weeks had beneficial effects on renal damage due to diabetes This study has three limitations. First, investigating the main mechanism underlying the effect of G-CSF on early DN will need further investigations. To clarify the mechanism, illustration of a BM-dependent effect of G-CSF and analysis about downstream G-CSFR targets such as hematopoietic cell-specific Lyn substrate (HCLS)1, HCLS1-associated protein X (HAX)1, and lymphoid-enhancer binding factor (Lef)-1 in order to confirm no direct effect of G-CSF on early DN are needed. Second, the effects of G-CSF on the different stages of DN were not demonstrated. Finally, we did not establish the optimum dosage and regimen of G-CSF. In order to overcome this limitation, future studies should involve a larger number of animals.In summary, we demonstrate that G-CSF could prevent the progression of early DN in OLETF rats. In addition, we speculate that mobilization of BM cells, rather than a direct effect of G-CSF, could be the mechanism of the observed G-CSF effect. This is the first report to show beneficial effects of G-CSF on early DN in an animal model. Our findings suggest that G-CSF has potential as a novel therapeutic drug in early DN patients.


Medium spiny neurons (MSNs) are the principal neurons in the striatum. The recurrent connections among them mediate weak feedback inhibition, whereas fast-spiking interneurons (FSIs) project extensively to the MSNs and provide strong feedforward inhibition. The striatum is innervated by massive excitatory afferents from various regions of the neocortex via the corticostriatal projection neurons. Interestingly, despite their strong convergence, corticostriatal projections are structured in such a way that neighboring MSNs in the striatum are not likely to share their presynaptic inputs .To understand the functional consequences of such a corticostriatal connectivity structure we studied the representation of cortical inputs in a spiking neural network model of the striatum. Activation of a fraction of MSNs resulted in a corresponding decrease in the spiking of unstimulated neurons, due to the overall inhibitory nature of the striatum network. This was similar to the neuronal activity recorded in behaving rats . FurtherMSNs can display short epochs of rapid firing, which may change their overall response to cortical inputs. Our simulation results suggest that the bursting activity of the MSNs did not affect the correlation dependence of the signal representation. Rather, it modulated the representation in a qualitative manner. In addition to the correlation of the excitatory inputs, synchrony within the inhibitory striatal network also influenced the signal representation. Specifically, we show that correlated feedforward inhibition mediated by FSIs, which might arise due to gap junction couplings, impaired the signal representation in the striatum. To date, experimental  and compIn summary, we showed that uncorrelated excitation and uncorrelated inhibition in the striatum support optimal signal representation, which, based on the anatomical and physiological findings, may be utilized by the striatum to enhance the encoding of cortical information for the execution of different cognitive functions.


Biogenesis of eukaryotic ribosomes occurs mainly in a specific subnuclear compartment, the nucleolus, and involves the coordinated assembly of ribosomal RNA and ribosomal proteins. Identification of amino acid sequences mediating nucleolar localization of ribosomal proteins may provide important clues to understand the early steps in ribosome biogenesis. Human ribosomal protein S9 (RPS9), known in prokaryotes as RPS4, plays a critical role in ribosome biogenesis and directly binds to ribosomal RNA. RPS9 is targeted to the nucleolus but the regions in the protein that determine its localization remains unknown. Cellular expression of RPS9 deletion mutants revealed that it has three regions capable of driving nuclear localization of a fused enhanced green fluorescent protein (EGFP). The first region was mapped to the RPS9 N-terminus while the second one was located in the proteins C-terminus. The central and third region in RPS9 also behaved as a strong nucleolar localization signal and was hence sufficient to cause accumulation of EGFP in the nucleolus. RPS9 was previously shown to interact with the abundant nucleolar chaperone NPM1 (nucleophosmin). Evaluating different RPS9 fragments for their ability to bind NPM1 indicated that there are two binding sites for NPM1 on RPS9. Enforced expression of NPM1 resulted in nucleolar accumulation of a predominantly nucleoplasmic RPS9 mutant. Moreover, it was found that expression of a subset of RPS9 deletion mutants resulted in altered nucleolar morphology as evidenced by changes in the localization patterns of NPM1, fibrillarin and the silver stained nucleolar organizer regions. In conclusion, RPS9 has three regions that each are competent for nuclear localization, but only the central region acted as a potent nucleolar localization signal. Interestingly, the RPS9 nucleolar localization signal is residing in a highly conserved domain corresponding to a ribosomal RNA binding site. The nucleolus is a distinct subnuclear compartment first described a few hundred years ago While the nuclear import of proteins usually relies on one or several NLS, a consensus motif for nucleolar targeting has not been found The amino acid sequence motifs required for nucleolar localization are known in the literature as nucleolar targeting signals, nucleolar binding domains, nucleolar retention motifs, or nucleolar localization signals (NoLS). The choice of term depends upon how one sees the nucleolar assembly mechanism. As mentioned, nucleolar proteins including RPs can associate with, and dissociate from, nucleolar components in a dynamic exchange with the surrounding nucleoplasm A second model for how NoLSs may function exists. A class of acidic nucleolar proteins can shuttle between the nucleus and cytoplasm Ribosomal protein S9 , RPS4 in prokaryotes (GI:323376825), was previously shown to be a novel NPM1 interacting protein RPS9 can be detected in the nucleoli and cytoplasm of mammalian cells U2OS osteosarcoma cells were obtained from American Type Culture Collection (ATCC) and cultured in Iscovés medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and penicillin-streptomycin in a +37°C humidified incubator. RPS9-FLAG3C, EGFP (C1)-RPS9, RPS9-EGFP (N1), and RPS9-FLAG3C deletion and point mutants were created by PCR and site directed mutagenesis PCR. NPM1 (B23) expression plasmids  were described previously For immunofluorescence studies, cells were seeded on coverslips in 6-well plates and transfected. At 24 hours after transfection, the cells were washed in PBS and fixed in formalin solution containing 4% w/v formaldehyde (Sigma-Aldrich), permeabilized with 0.2% Triton X-100 for three minutes, and kept for 30 minutes in blocking buffer . Next, the cells were incubated 60 minutes with primary antibodies in blocking buffer followed by three washes in PBS and a 30 minutes exposure to FITC- or Texas Red-conjugated anti-mouse or anti-rabbit secondary IgG (Vector). After three PBS washes and one final wash in distilled water, the coverslips were mounted onto glass slides with Vectashield anti-fading agent (Vector) containing DAPI.Immunostained cells were analyzed by using a Zeiss Axioplan II microscope equipped with Plan-Apochromat 63×/1.4 and Plan-Neofluar 100x/1.30 objectives, under the control by Axiovision 3.1 software. An Olympus microscope equipped with a Leica DFC320 camera and a PlanApo 40 objective (Olympus) was dedicated to the analysis and documentation of AgNOR and Toluidine Blue stained cells. For live cell imaging, an inverted Nikon Eclipse TS100 (S Plan Fluor 30x/0.60 and LWD 20x/0.40 objectives) with an Infinity camera was used. Images were assembled in Adobe Photoshop CS5.The following antibodies were used: mouse anti-Myc , rabbit anti-Myc 9E10 epitope (Abcam), mouse anti-FLAG , rabbit anti-FLAG (Sigma-Aldrich), mouse anti-β-actin , mouse anti-BrdU , mouse anti-NPM1 (Invitrogen), rabbit anti-fibrillarin (Abcam), mouse anti-GFP (Clontech), and rabbit anti-RPS9 (S9-162) Transcription inhibitor Actinomycin D was prepared as a 1 mM stock solution in DMSO and used at a final concentration of 5 nM. Proteasome inhibitor MG132 was dissolved in ethanol and used at 10 µM final concentration. All compounds were purchased from Sigma-Aldrich.For silver staining of nucleolar organizer regions (Ag-NOR staining), cells grown on coverslips were fixed in 2% glutaraldehyde in PBS for 10 minutes at room temperature, rinsed with PBS and once in distilled water, followed by fixation for 5 minutes with methanol:acetic acid 3∶1, again rinsed in distilled water and subsequently stained with a mix of 1 part A and 2 parts B for 45 minutes in the dark at room temperature (solution A: 2% gelatin in 1% formic acid and solution B: 50% silver nitrate in ultrapure distilled water) as described For estimation of DNA replication activity, U2OS cells were seeded at an equal sub-confluent cell density in 6-well plates. Cells were transfected with wt or mutant RPS9-FLAG plasmids and after 18 hours bromodeoxyuridine (BrdU) (Sigma–Aldrich) was added (10 µg/ml) to the culture medium and cells were incubated in it for an additional 24 hours. Cells were fixed in methanol–acetone (1∶1 vol/vol), stained for FLAG using a rabbit polyclonal antibody, and then a secondary FITC anti-rabbit antibody. Stained cells were then treated for 15 min with 1.5 M HCl and extensively washed. Next, cells were stained for one hour with a mouse monoclonal anti-BrdU antibody, followed by a Texas Red-conjugated anti-mouse antibody.3, 1 mM dithiothreitol, 1x protease inhibitor cocktail (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride-PMSF). Samples were prepared by mixing with SDS-sample buffer and loaded on 10 or 12% Bis-Tris gels (Life Technologies). Proteins were blotted onto PVDF membranes, incubated in blocking solution with primary antibodies overnight followed by three washes in PBS. Membranes were subsequently incubated with secondary horse radish peroxidase conjugated antibody at room temperature for two hours and after three additional washes in PBS, proteins were visualized by using ECL reagent (Amersham Biosciences) or West Pico (Pierce).Cells were harvested in Nonidet P-40 lysis buffer . For in vitro binding assays individually translated proteins were mixed with GST-NPM1 expressed by the plasmid pGEX3X-NPM1 in BL21 bacteria. Expression and purification of GST fusion proteins were done according to the manufacturer's instructions (Amersham Biosciences). GST fusion proteins bound to glutathione-Sepharose 4B beads (Amersham Biosciences) were incubated and rotated overnight at +4°C with in vitro translated protein in binding buffer . Following three extensive washes with rotation and shaking in binding buffer the beads were boiled and re-suspended in SDS-sample buffer, and the proteins resolved by SDS-PAGE, followed by staining of the gels with Coomassie Brilliant Blue, drying, and autoradiography.The coupled http://www.compbio.dundee.ac.uk/nod). Nuclear localization signals were predicted using PSORT II (http://www.psort.org/). NetNES 1.1 predicts leucine-rich nuclear export signals (NES) in eukaryotic proteins (http://www.cbs.dtu.dk/services/NetNES/). Multiple sequence alignments were made with ClustalW .NoD, nucleolar localization sequence detector, provides predictions of nucleolar localization sequences in eukaryotic proteins  and the localization of the fusion protein was determined. RPS9-EGFP was predominantly nucleolar with a weaker but distinct cytoplasmic staining, excluding the nucleoplasm . This wa1–70 (81% of transfected cells) and also in cells expressing EGFP-RPS91–106 (95%) -RPS9, a panel of RPS9-EGFP (N1) deletion constructs was made. The localization was determined in living non-fixed U2OS cells  . EGFP-RPOS cells . Express1–70-FLAG and RPS91–106-FLAG were localized predominantly throughout the nucleoplasm, but it should be emphasizes that the RPS91–70-FLAG protein was not excluded from the nucleolus and in fact could be detected at the nucleolar periphery in quite a few of the transfected cells. RPS9140–194-FLAG mutant was predominantly nucleoplasmic while RPS91–40-FLAG was evenly distributed throughout the cells in majority of the transfected cells (87%), being excluded from the nucleoli. An intense nucleolar staining was seen in cells expressing RPS9100–194-FLAG, RPS91–140-FLAG and RPS9100–140-FLAG, suggesting that a potent NoLS is to be found within the central domain of RPS9, which would be in agreement with the data obtained with RPS9-EGFP fusion proteins. Intriguingly, RPS970–140-FLAG displayed a peculiar fine dotty cytoplasmic staining  antibody was less than when using the Myc antibody. In addition, RPS9 protein ends with an acidic amino acid patch (DDEEED). It was thought that the FLAG tag may work in harmony with this patch because the FLAG tag is acidic itself (DYKDDDDK). Because of this and the low FLAG antibody background, expression plasmids were constructed containing various RPS9 sequences fused in frame with a C-terminal 3x FLAG-tag. The RPS9-FLAG constructs were transfected into U2OS cells and monitored for expression 24 hours post-transfection by immunofluorescence staining . Localizstaining  that corstaining . A simil194-FLAG . Both ofresidues . In addiresidues .100–194-FLAG (but not RPS9140–194-FLAG), and EGFP-RPS9106–140 (but not EGFP-RPS91–106 or EGFP-RPS9140–194) were targeted to nucleoli suggested that a central motif within aa 106–140 is important for the nucleolar localization of RPS9. This motif “LERRLQTQVFKLGLAKSIHHARVLIRQRHIRVRKQ” contains several conserved basic residues. Predicting NoLS in proteins is even more difficult but based on experimental data sets and computer programming advances have been made. One program that can be used to predict NoLS is the NoD program That RPS91–194-FLAG led to an altered cellular localization. The RPS91–194(R109A R127A)-FLAG mutant protein displayed an even nuclear staining without any cytoplasmic staining in the majority (97%) of transfected cells   . In contnucleoli . The tranucleoli .100–194-FLAG revealed that fibrillarin, a nucleolar protein expressed in the nucleolar dense fibrillar centers 100–194-FLAG , presumably EGFP has a shielding effect. The impact on cell proliferation in cultures transfected with the mutant construct was severe. Cells expressing RPS9100–194-FLAG had a dramatically reduced incorporation of BrdU   are composed of hundreds of repeated copies of rDNA genes. NORs include regions that are highly condensed and regions that are de-condensed, the latter corresponding to regions at which associated proteins stain intensively with silver (Ag-NORs) and where active rRNA gene transcription is thought to occur rillarin . Next, urillarin . Importarequency , but was p<0.01) . Moreove content , and didRibosome biogenesis is thought to require highly efficient nuclear import of ribosomal proteins Following nuclear import, the majority of RPs must travel to the nucleolus in order to be able to participate in ribosome subunit assembly. Nucleolar localization could occur by diffusion through the nucleoplasm and retention by interaction with other nucleolar components or it could be a more active process that relies on specific nucleolar shuttling proteins. RPS9 localizes to nucleoli similar to other RPs NoLSs in RPS6, RPS25 and RPS7 reside in evolutionary highly conserved sequences The N-terminus of RPS9 (aa 1–70) interacted with NPM1 in vitro, conferred nuclear localization and had some affinity for the nucleolus. Interestingly, according to NoD this N-terminal domain in RPS9 has a rather high probability of nucleolar localization. However, this domain appeared rather weak in its nucleolar targeting activity and I therefore do not consider it to be a genuine NoLS. Nonetheless, the predominantly nucleoplasmic RPS9 mutant 1–70 was more efficiently targeted to the nucleolus by co-expression of NPM1. Conceivably, NPM1 could play a role in promoting the nucleolar localization of RPS9, reminiscent of NPM1 mediated nucleolar targeting of viral proteins and the p14ARF tumor suppressor There are a number of pitfalls that one has to keep in mind when evaluating RPS9 mutants. RPS9 is a structurally complex protein and with such an extended RNA binding surface, manipulation of its structure by sequential deletions is likely to have major impact on its folding, rRNA binding and association with NPM1. Therefore, the behaviour of individual RPS9 mutants might therefore not always accurately describe the situation in the context of the wt protein. Moreover, several of the RPS9 mutants displayed localization patterns  or had a fairly variable localization pattern not observed with the endogenous protein.100–194-FLAG construct, overexpression of the mutant resulted in nucleolar enlargement and altered nucleolar organization. A key component of the nucleolar dense fibrillar center, fibrillarin, was dispersed in a few small spots or ring like structures surrounding the enlarged nucleoli. These spots were also intensely stained with silver. Intriguingly, NPM1 appeared trapped in these enlarged nucleoli. It is possible that the expanding nucleolar area contains an aggregate of the mutant protein mixed with other nucleolar proteins and RNA resulting in structural changes The increasing interest concerning RPs and their role in cell growth and proliferation through the RP-MDM2-p53 pathway 100–194-FLAG mutant, as well as fragments 1–140 and 70–194 tagged with FLAG, associated with NPM1 and an alternative explanation is that RPS9 mutants directly inhibit NPM1, similar to what has been described for a subset of NPM1 binding peptides Expression of the RPS9-FLAG mutant severely impaired cell proliferation as shown by a reduced BrdU incorporation, lowered amounts of total protein, and by the failure to establish stable cell lines. Truncated versions of RPS9 may disrupt ribosome biogenesis by acting as dominant negative mutants. By interfering with rRNA synthesis or pre-ribosome export the mutant may induce nucleolar stress Figure S1RPS9 multiple sequence alignment. RPS9 sequences from different organisms were aligned using Clustal W and the NLS-1 and NLS-2 motifs as predicted by PSORT II are indicated in green. The NoLS containing domain is indicated in purple. Abbreviations: Hs- Homo Sapiens, Rn – Rattus Norvegicus, Mm – Mus Musculus, Xt- Xenopus Tropicalis, Dr – Danio Rerio, Bt – Bos Taurus, Dm – Drosophila Melanogaster, Ce- Caenorhabditis Elegans, Sp –. Schizosaccharomyces Pombe, Nc – Neurospora Crassa.(TIF)Click here for additional data file.Figure S2Prediction of NoLS and NES in RPS9. A.) Diagram showing the NoLS prediction score per residue in human RPS9 using NoD. B.) Schematic representation showing nuclear export sequence probability per residue using NetNES.(TIF)Click here for additional data file.Figure S3Partial mitochondrial localization of RPS9-FLAG deletion mutant. U2OS cells were transfected with wt RPS9-FLAG or RPS9-FLAG70–140 deletion mutants. Cells were incubated with Mitotracker Red, fixed, and stained for FLAG. Merged images show co-localization of the FLAG and Mitotracker Red signals in the case of the mutant protein but not in wt expressing cells. Bar 10µM. Cell nuclei were stained by DAPI.(TIF)Click here for additional data file.Figure S41–70-FLAG induced by co-expression of NPM1.Nucleolar translocation of RPS9 A.) Nucleolar localization of RPS91–70-FLAG was induced by co-expressed Myc-NPM1 (wt), whereas a cytoplasmic NPM1 mutant did not. Cells were stained for expression of wt and mutant Myc-NPM1 using a rabbit anti-Myc polyclonal antibody and a monoclonal FLAG antibody (M2). Corresponding phase contrast images are shown.(TIF)Click here for additional data file.Table S1Prediction of nuclear (NLS) and nucleolar localization signals (NoLS) in small subunit ribosomal proteins. An overall NLS score according to PSORT II is given for each protein, as is the number of predicted NLS, percentage of basic amino acid residues, and the number of predicted NoLS. Note that for some ribosomal proteins the repetitive nature of basic amino acid residues results in a high number of NLS in the case of e g RPS6, RPS8, RPS27A and RPS27.(PDF)Click here for additional data file.


Adenocarcinoma of the rete testis is very rare. There is still little knowledge about its etiology and pathogenesis. Herein, we present a case of rete testis adenocarcinoma in a 36-year-old Chinese male. The tumor was predominantly composed of irregular small tubules and papillary structures with cuboidal or polygonal cells. In peripheral area of the tumor, the remaining normal rete testis and adenomatous hyperplasia of the rete testis could also be seen, indicating the possible relationship between adenomatous hyperplasia and adenocarcinoma. In addition, the patient underwent a left hydrocelectomy because of the existence of hydrocele 3 years ago. But, it is unclear whether hydrocele and hydrocelectomy is its cause or just the early clinical presentation of the adenocarcinoma.http://www.diagnosticpathology.diagnomx.eu/vs/6757609119625499The virtual slide(s) for this article can be found here:  Adenocarcinoma of the rete testis is a very uncommon malignancy with approximately 60 cases reported in the literatures . BecauseA 36-year-old male referred to our hospital for complaining of a painful swelling in the left testis 1 year ago. Physical examination demonstrated that the left testis apparently enlarged, and felt firm. Laboratory examination revealed values of serum alpha-fetoprotein (AFP), alkaline phosphatase (AP), CA19-9, CA125 and prostate specific antigen (PSA) were in normal level. Scrotal ultrasound revealed that there was an irregular, solitary mass about 7.5 × 4.3 × 4.0 cm in the lower region of the left testis. No lesions in other organs including lung, prostate and rectum were detected. The patient reported had undergone a hydrocelectomy for hydrocele and slight enlargement of the testis 3 years ago. However, after the first surgery, the testis still gradually enlarged, and increased in size rapidly for the past six months. A second surgery was then performed in our hospital. At surgery, there was a gray-yellow mass in the testis, and the testis with the mass was removed, and underwent diagnostic examination. According to the morphological and immunohistochemical findings, the tumor was diagnosed as an adenocarcinoma of the rete testis. Then the patient underwent BEP  chemical therapy two times. He was alive with no tumor recurrence or metastasis within 15 months of follow-up.The resected specimens were fixed with 10% neutral-buffered formalin and embedded in paraffin blocks. Tissue blocks were cut into 4-μm slides, deparaffinized in xylene, rehydrated with graded alcohols, and immunostained with the following antibodies: cytokeratin , cytokeratin 5/6 , cytokeratin7 , Vimentin , CD30 , carcino embryonic antigen , α-Fetoprofein , human chorionic gonadotropin beta , thyroid transcription factor 1 , epithelial membrane antigen , Prostate Specific Antigen , CA19-9 , CA125 , Calretinin ,α-inhibin , PLAP , CD117  and Ki67 . Sections were stained with a streptavidin-peroxidase system . The chromogen used was diaminobenzidine tetrahydrochloride substrate , slightly counterstained with hematoxylin, dehydrated and mounted. For the negative controls, the primary antibody was replaced with PBS. This study was prospectively performed and approved by the institutional Ethics Committees of China Medical University and conducted in accordance with the ethical guidelines of the Declaration of Helsinki.Grossly, the testis was approximately 8.3 × 5.1 × 4.9 cm, was involved by a firm, irregular 7.1 × 4.2 × 4.1 cm tumor. The tumor was relatively well circumscribed, mainly located in the region of testicular hilum. The cut face of the tumor was grey-yellow or grey-white in color. The tunica of the testis was lost.Histologically, the tumor was mainly confined to testicular hilum. The tumor was predominantly composed of irregular small tubules and complicated papillary structures with cuboidal or polygonal cells. Focally, the cells were arranged into solid sheets or masses with apparent necrosis. Amidst the tumor cells, little fibrovascular stroma could be seen. The cells had marked cellular atypia, with dark staining chromatin and conspicuous nucleoli. The mitosis was very common. In the peripheral area of the tumor, the remaining normal rete testis could be seen. In addition, an area of adenomatous hyperplasia which consisted of irregular tubule also presented, the adenomatous hyperplasia was located within the normal rete testis. In several distended tubules, the papillary hyperplasia of epithelial cells could be seen Figure .Immunohistochemical staining showed that the tumor cells including area of adenomatous hyperplasia were diffusely positive for EMA, CK, and CK7, focally positive for CEA and Vimentin, negative for CK 5/6, CD30 , AFP, HCG-β, TTF-1, PSA, CA19-9, CA125, Calretinin, PLAP, CD117 and α-inhibin. Ki67 index was approximately 40%, In contrast, in the area of adenomatous hyperplasia, the Ki67 proliferative index was less than 5% (Figure The rete testis is a relatively inconspicuous structure. Histologically, it is an anastomosing network consisted of delicate tubules. Adenocarcinoma of the rete testis is very rare, thus its etiology and histogenesis is still unclear . It was The first case of adenomatous adenomatous hyperplasia of the rete testis was reported by Nistal and Paniagua in 1988 . In the The reported immunohistochemical staining findings are not completely consistent -14. In gAlthough the diagnostic criteria seem to be well established, the correct diagnosis of this rare tumor is still a hard work. The differential diagnosis includes metastatic adenocarcinoma , malignaCarcinoma of the rete testis is a highly aggressive tumor with a reported survival of 13% at 5 years . Our casBecause of the rarity, etiology and histogenesis of adenocarcinoma of the rete testis is still not completely clear. Our reported case demonstrates the apparent transition from normal rete testis to adenomatous hyperplasia and adenocarcinoma. It suggests that the adenomatous hyperplasia may be the precancerous lesion of adenocarcinoma of the rete testis.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in Chief of this Journal.The authors declare that they have no competing interests.LXY participated in the histopathological evaluation, performed the literature review, acquired photomicrographs and drafted the manuscript. YJH and XHT carried out the immunohistochemical stains evaluation. WL FCH and LY conceived and designed the study. WEH gave the final histopathological diagnosis and revised the manuscript. All the authors read and approved the final manuscript.


Batrachochytrium dendrobatidis, in North American bullfrogs (Rana catesbeiana) farmed for the international restaurant trade. Our findings suggest that international trade may play a key role in the global dissemination of this and other emerging infectious diseases in wildlife.Chytridiomycosis is an emerging disease responsible for global decline and extinction of amphibians. We report the causative agent,  Batrachochytrium dendrobatidis, which develops solely within keratinized cells , 46 km from Montevideo, Uruguay. The farm is a commercially managed operation, with trained employees under permanent, technical supervision. The farm produces approximately 150,000 tadpoles each summer and 30,000 metamorphs. Mortality rates during the days before the outbreak were approximately 0.5% per week, which is considered normal for this type of farm. Frog growth rates were also within normal parameters for these conditions (Mass deaths occurred at a large rearing facility for North American bullfrogs (Eisenia foetida) cultured at the farm. Tanks are cleaned every day to eliminate excess feed and feces, and animals are sorted to maintain homogeneous sizes in each tank. Dead animals are removed twice a day.At this farm, spawning occurs in 50-L tanks from October to February (spring and summer), tadpoles grow until March, and metamorphosis occurs from March to May . Newly metamorphosed frogs (5–8 g) are harvested from tadpole ponds and stocked in tanks with circulating water until resorption of the tail is complete. Frogs are then transferred to large growing tanks, in which the frogs are reared at a density of 500 per square meter while standing in shallow water with only their bodies immersed  in these three tanks. To reduce the risk of bacterial infections, poly-hexamethylene-biguanide-hydrochloride  was added to the tanks to give a final concentration of 500 ppm. The deaths continued and spread to other tanks; 15,000 frogs died during the first week. An antifungal therapy   were performed on liver, kidney, and heart blood collected from a sample of euthanized frogs, but these tests did not implicate causative pathogens. Necropsies were performed systematically on 30 frogs, but no gross abnormalities of the viscera were found. Internal organs  from 10 of these frogs were examined by histologic testing, but no lesions were detected. Examination of skin smears , and histologic and ultrastructural analyses of fixed skin indicated subspherical developing sporangia and flask-shaped mature sporangia within the superficial layer of the epidermis . Septa, porangia . Slight In a basic transmission experiment, two groups of five apparently healthy frogs from affected tanks were placed in separate aquaria, one contained five frogs from an unaffected farm in Uruguay and the other empty. Deaths began on day 4, with death occurring 24–48 h after first signs of disease. Within 8 days, all 15 frogs were dead. A group of five control frogs from the unaffected farm remained healthy.B. dendrobatidis has been repeatedly observed in skin smears of healthy frogs from the farm at which the outbreak occurred. Similar results have since been obtained from six other bullfrog farms in Uruguay . Histologic examination of infected frogs on which necropsies were performed since the 1999 epidemic shows focal mild hyperplasia of the superficial epidermis associated with sporangia of B. dendrobatidis. No unusual death rates have occurred at the index site since the 1999 incident.After the incident, death rates returned to levels before the event. As a result of this event, monthly surveillance for cutaneous chytrid infection was instigated; ventral hind limbs, feet, and digits are examined from five healthy frogs and from any sick or dead frogs. B. dendrobatidis had been performed before the deaths, this organism has been repeatedly observed in skin examinations of healthy frogs in follow-up studies. Second, although not proved efficacious against cutaneous chytridiomycosis, the use of benzalkonium chloride, a recognized therapy for infections with nonhyphal fungi, failed to reduce mortality rates.Cutaneous chytrid infection was detected during an investigation into mass deaths in farmed North American bullfrogs. Although chytridiomycosis has been reported as an important cause of mass deaths in wild amphibians, whether chytrid infection was the cause of deaths in the disease outbreak described in this paper is unclear. First, while no examinations for B. dendrobatidis in culture  suggest that disease is a major economic threat to this industry in South America. This situation is exacerbated by a lack of veterinary diagnostic capacity, management protocols, and treatment or prophylaxis.B. dendrobatidis without developing signs of disease  infections in farmed bullfrogs suggest that this species may act as a carrier of chytridiomycosis, which has serious implications for conservation of biodiversity. Recently, guidelines for amphibian translocations have been published that include specific quarantine and testing recommendations for chytridiomycosis  have been observed in the greenhouses where indoor rearing tanks are located. Whatever the origins of B. dendrobatidis in farmed bullfrogs in Uruguay, such open-plan farms are likely sources of infection and disease for wild amphibian species. In view of this risk, surveillance for chytrid infection and for unusual levels of deaths in endemic amphibian species should be established in the vicinity of frog farms in Uruguay and elsewhere in the world.The farm we studied was opened in October 1998 with stock that originated from Brazil. The Brazilian farmers originally imported 300 pairs of frogs from Canada to set up the first South American farms approximately 30 years ago. The pathogen has been present in bullfrogs in North America since at least the 1970s (B. dendrobatidis in an international food animal trade has implications for amphibian conservation and for disease emergence in general. Our study adds to reports of chytridiomycosis in the international pet trade (Global trade and commerce are regularly cited as drivers of disease emergence in humans, domestic animals, wildlife, and plants (With a continued rise in the international air transport volume (


In 8 other subjects without any evidence of CSF leak, the dural substitute was placed intradurally and fibrin glue was injected intradurally to hold the material in place.During the 12-months period evaluated, we performed an endoscopic endonasal transsphenoidal operation for a variety of pituitary lesions on 102 consecutive patients. Among these, in 12 patients (9.4%) harboring a pituitary adenoma, the implant of the pericardium sheet was used. Four patients (33.3%) presented a small intraoperative cerebrospinal fluid (CSF) leak; in these cases the Lyomesh®) as dura mater substitute in transsphenoidal surgery is safe and biocompatible, as compared with other dural substitutes.Even if based on a relatively small patient series, our experience demonstrated that the use of equine pericardium sheet (Lyomesh The widespread use of the endoscopic techniques has widened the possibilities of the transsphenoidal surgery, providing access to several areas of the skull base. Notwithstanding a consistent number of advantages, still there are several drawbacks, among those the postoperative CSF leakage. It has to be minded that this risk is different between so called “standard” procedures, targeted to the sellar area and the “extended” procedures, targeted mostly to the whole midline skull base. Indeed, in recent series reporting outcomes of the transsphenoidal approach, either endoscopic or microscopic, for the removal of sellar or intra-suprasellar infradiaphragmatic lesions, the rate of postoperative CSF leak is low, ranging from 0% to 5.1%; on the other side, in the so called “extended” procedures this rate is higher, ranging from 0% to 21% –12Therefore, in recent years, different sellar and skull base reconstruction techniques have been described, accounting on an extreme variety of strategies and materials, i.e. autologous, heterologous and synthetic. There is not a univocal consensus concerning the best reconstruction option and strategies and materials should be tailored according to every single case.® is an equine pericardium membrane, used as dural substitute, recently introduced in the market; it is made of polypeptides chains, forming the elementary fibers, interwoven and shaped as cords, then anastomosed in complanate networks.The use of autologous materials  might be preferred, because they are vital and do not engender any immune and/or inflammatory response. However, many of these substances require a separate surgical incision, either on the abdomen or on the thigh. With the modern minimally invasive conceptual way of thinking and the reduction of morbidity and mortality, the patients’ requests are leant toward the avoidance of a second skin incision. For these reasons, and for the possibilities offered by these products of reliable sterility, the use of non-autologous dural substitutes, both heterologous and synthetic, has received a tremendous boost. In this environment, pericardium-based heterologous products are becoming increasingly popular. LyomeshThe aim of this study is to evaluate the safety and the effectiveness of this material for sellar reconstruction after a standard endoscopic endonasal approach for the removal of a pituitary adenoma.We retrieved data from the analysis of 102 patients that underwent surgery for pituitary lesions, from May 2012 to May 2013.®dural foil was used in 12 cases harboring a pituitary adenoma: in four of them it was adopted to repair an intraoperative CSF leakage - grade I according to Kelly’s paradigm -, whereas in eight cases to protect a thinned suprasellar cistern - grade 0 according to Kelly’s paradigm [The Lyomeshradigm -.We reported the use of such dural substitute in three PRL-secreting macroadenomas, two ACTH–secreting macroadenomas and seven non-functioning macroadenomas.There were five males and seven females . The postoperative follow-up ranged from 3 to 12 months.The technique of reconstruction was different according to the entity of leakage to the anatomical and/or lesion features.®.All patients underwent a three months postoperative endoscopic endonasal exploration of the sino-nasal cavities and a sellar MRI scan to evaluate the integration of LyomeshAll patients underwent surgery by means of an endoscopic endonasal approach to sellar region for the removal of a pituitary adenoma, according to technique already described in the main literature –27For the reconstruction phase of the standard procedure, we usually don’t harvest any autologous material such as abdominal fat graft or fascia lata, nor remove the middle turbinate to be used as free mucoperichondrium flap.®foil was placed intradurally in the eight cases presenting a CSF leak grade 0, when the suprasellar cistern was intact, prolapsed into the sellar cavity but thinner; fibrin glue was injected intradurally to hold the material in place.In case of small intraoperative CSF weeping leak (grade I) or also to protect a thinner and/or prolapsed suprasellar cistern (grade 0) , we pe®was placed intradurally with fibrin glue and, thereafter, several layers were positioned in the extradural space to ensure the watertighteness.In four cases with grade 1 intraoperative CSF leak, a single layer of LyomeshIn two cases of this latter group the sphenoid sinus was filled with fibrin glue.®is a processed equine pericardium deproteinized with enzymatic method that preserves only purified collageneous network. The pericardium foil is made up of complanate multi-layer networks, rendered thinner by machinery process. Before its use the dural substitute must be washed at least 10 minutes in 50cc physiological saline solution. A full load of sheet measures is available.LyomeshThere were two main reasons that required the use of the dural substitute: the first one was an intraoperative CSF leak  might be preferred because of their biocompatibility and the lesser risk of immune or inflammatory response, they are harvested through a separate surgical incision, either on the abdomen or on the thigh that prolongs the surgical times and could be aesthetically disfiguring. On the other hand, it has to be said that, during endoscopic endonasal surgery, autologous materials could be harvested from the nasal structures i.e. middle turbinate and/or septal mucosa.Heterologous materials, conversely, should be preferred to the synthetic ones because they are not absorbable and their inertness is still not absolute, with the possibility of causing a cell-mediated immunoallergic response .®has never been studied before for the use in this type of surgery. Thanks to the data collected along our series we have drawn some considerations in regards:Watertightness: it provides a fluid-tight barrier against CSF leakage;safety and biocompatibility: differently from bovine-derived dural substitute, this one is taken from horse, the only animal BSE safe, so there is no risk of infection transmission to human. It is biologically tested, non cytotoxic, non sensitizing, non mutageneous, with zero intracutaneous reactivity. The device is biocompatible and releases molecules that are metabolized without flogistic response. Nevertheless it should be used in patients allergic to horse meat;easiness to handle: can be molded and shaped according to different needs of the repairs;®enables the surgeon to optimally inspect the underlying tissues;transparency: the transparency of Lyomesh®shows the mechanical resistance/thickness best ratio if compared with other biologic tissues of animal origin and to the artificial collageneous sheets;mechanical resistance: the sheet traction and pressure resistance indexes are far higher than the one of the collagen tissues obtained through purification and artificial reticulation. Lyomeshslow resorption: it remains unchanged while fibroblasts and endotelial cells enter it, acting as a guide for connective regeneration while it is slowly digested and replaced by autologous connective tissue.LyomeshSince no previous studies are currently available to assess its security and effectiveness in transsphenoidal pituitary surgery, we used this new dural substitute only in cases of small CSF weeping, to protect the suprasellar cistern after the tumor removal or to close the sellar floor.In fact, especially in cases of large or invasive adenomas, the suprasellar arachnoidal membrane could be thinned and the diaphragma sellae could be incompetent: even if there is not intraoperative evidence of CSF leak, this latter may occur later. Therefore, in these cases, the graft was kept in place by the sole fibrin glue that has proven in preliminary studies to be sufficient for this purpose –30Due to the limited experience we would not yet recommend this material as sole substitute in case of large intraoperative CSF leak. In these cases, we followed the paradigm of graded sellar repair with multiple materials, combined to seal the different compartments of the defect .®, presents advantages at least comparable with previous studies on similar materials [Our preliminary results, even on a small patient series, demonstrate that the pericardium sheet, i.e. the Lyomesherials : it is


Since rupture-prone plaques commonly contain high amounts of activated macrophages, imaging of the macrophage content may provide a useful tool for the evaluation of plaque vulnerability. The purpose of this study was to explore the uptake of 68Ga was investigated in atherosclerotic LDLR-/-ApoB100/100 and C57BL/6N control mice at 3 h after injection. The ex vivo biodistribution of the 68Ga was assessed and autoradiography of aortic cryosections was defined. In vivo imaging of 68Ga was performed using a small animal positron emission tomography PET/CT scanner.Uptake of ionic 68Ga-radioactivity was higher in atherosclerotic plaques than in healthy vessel wall  and adventitia . The autoradiography signal co-localized with macrophages prominently as demonstrated by Mac-3 staining. In both mice strains, the highest level of radioactivity was found in the blood.Our results revealed that the uptake of 68Ga-radioactivity uptake in the aortic plaques of atherosclerotic mice, especially at the sites rich in macrophages. While the uptake of 68Ga was promising in this animal model, the slow blood clearance may limit the usability of 68Ga as a PET tracer for clinical imaging of atherosclerotic plaques.We observed a moderate but significantly elevated  Atherosclerosis is a chronic inflammatory disease of artery wall with infiltration of monocytes into subendothelial space and their differentiation into macrophages. Macrophages express pro-inflammatory and chemotactic molecules thus playing an essential role in the formation, progression, and pathogenicity of atherosclerotic plaques. The population of macrophages in atherosclerotic lesions is heterogeneous. The majority of macrophages transform into foam cells by accumulating lipids in their cytoplasm, while other macrophages may have vascular protective and repair properties. Furthermore, some macrophages may secrete enzymes that degrade the extracellular matrix and fibrous cap, possibly leading to a rupture of the atherosclerotic plaque. Significantly higher quantities of macrophage-rich plaque areas have been reported in atherosclerotic patients with acute coronary syndromes than in patients with stable angina. Since the rupture-prone plaques commonly contain high amounts of activated macrophages, imaging of the macrophages may provide a useful tool for determining plaque vulnerability -5.68Gallium  is a generator-produced, positron-emitting radionuclide with a high positron yield (89%) possessing suitable physical properties for in vivo PET imaging. 67Gallium (67Ga) has traditionally been used to image inflammation with gamma cameras. Since 68Ga owns the same chemical characteristics as 67Ga, it is assumed that ionic 68Ga could accumulate at the sites of macrophage-rich areas in inflammatory lesions. Furthermore, 68Ga-based PET may have some added advantages compared to 67Ga-based SPECT, such as a better resolution for the detection of inflammatory plaques fluoro-D-glucose (18F-FDG), we have measured the plaque/wall ratio of 2.7 ± 1.1 approximately in the same aged LDLR-/-ApoB100/100 mice as used in the present study [68Ga-tracers  and 18F-tracers  are not necessarily very much different from each other [68Ga has a higher kinetic energy compared to 18F, which could decrease the imaging spatial resolution, particularly when a small plaque lesion is imaged with a high resolution small PET/CT. Based on these earlier observations, 18F-FDG seems to be more promising PET tracer for imaging of atherosclerotic plaques than 68Ga.In the present study, the plaque/wall ratio of -deoxy-2-Ffluoro-Dch other . However68Ga between the atherosclerotic mice and controls was found in bone and liver. The uptake of 68Ga-radioactivity in the whole aorta of atherosclerotic mice was higher than that in the control mice, but the difference was not significant. The uptake in the inflammatory plaques could partly explain the lower uptake of 68Ga in the liver of atherosclerotic mice. More likely this may be due to liver failure caused by high fat content, which can occur in older LDLR-/-ApoB100/100 mice fed with a Western-type diet. Concerning the uptake of 68Ga in the bone, we assume that in the young control mice (aged 2.5 months), active bone remodeling may have contributed to the higher bone uptake as compared to the older LDLR-/-ApoB100/100 mice (aged 10 to 11 months).Clinical observations indicate that in normal circumstances, the major proportion of the gallium retained in the body is localized in the bone and liver, and that inflammatory lesions alter this biodistribution, frequently causing decreased hepatic uptake . In the 68Ga-radioactivity in inflammatory lesions is complex, and the majority of 68Ga is probably delivered into the inflammatory lesions through the increased permeability at the site of atherosclerotic arteries. In the present study, accumulated 68Ga-radioactivity was detected in the macrophages-rich plaques suggesting that 68Ga accumulates at the site of inflammation. In addition to that, calcified areas of atherosclerotic plaques were also detected by 68Ga, which may be explained by the competitive binding of gallium ions to the Ca2+ and Mg2+ binding sites [68Ga to distinguish macrophages-rich plaques and calcified plaques.The accumulation of ng sites . Since t68Ga in inflamed atherosclerotic plaques was promising as measured by autoradiography, the slow blood clearance limits the use of ionic 68Ga for in vivo imaging of atherosclerotic plaques.To some extent, gallium may also be taken up by atherosclerotic plaques due to its binding to the circulating transferrin and avidly to the transferrin receptors at the site of atherosclerotic arteries. Transferrin receptor expression has been shown to correlate positively with macrophage infiltration in human carotid atherosclerotic lesions . Moreove68Ga-radioactivity uptake in the aortic plaques of atherosclerotic mice, especially at the sites rich in macrophages, as compared to areas of healthy vessel wall. While the uptake of 68Ga-radioactivity was prominent in the atherosclerotic plaques of LDLR-/-ApoB100/100 mice, the slow blood clearance limits the feasibility of ionic 68Ga for clinical imaging of atherosclerotic plaques especially in coronary arteries.Autoradiography analysis showed a moderate, but significantly higher The authors declare that they have no competing interests.68gallium and participated in writing of the manuscript. VJOL involved in designing the analysis and interpretation of data, and in drafting and revising the manuscript. PL involved in designing the experiments and revising the manuscript. SY-H, JK, and AR contributed the conception and design, critically contributed and revised the manuscript and enhanced its intellectual content. All authors have approved the final content of the manuscript.JMUS contributed the conception and design, performed the experiments, analyzed and interpreted data and drafted the manuscript. IL performed the experiments, acquired, analyzed and interpreted data and participated in writing of the manuscript. HJS prepared the


CRWN genes, which were originally named LINC (LITTLE NUCLEI).Plant nuclei superficially resemble animal and fungal nuclei, but the machinery and processes that underlie nuclear organization in these eukaryotic lineages appear to be evolutionarily distinct. Among the candidates for nuclear architectural elements in plants are coiled-coil proteins in the NMCP (Nuclear Matrix Constituent Protein) family. Using genetic and cytological approaches, we dissect the function of the four NMCP family proteins in Arabidopsis encoded by the CRWN genes. Mutants deficient in different combinations of the four CRWN paralogs exhibit altered nuclear organization, including reduced nuclear size, aberrant nuclear shape and abnormal spatial organization of constitutive heterochromatin. Our results demonstrate functional diversification among CRWN paralogs; CRWN1 plays the predominant role in control of nuclear size and shape followed by CRWN4. Proper chromocenter organization is most sensitive to the deficiency of CRWN4. The reduction in nuclear volume in crwn mutants in the absence of a commensurate reduction in endoreduplication levels leads to an increase in average nuclear DNA density.CRWN proteins are essential for viability as evidenced by the inability to recover mutants that have disruptions in all four Our findings indicate that CRWN proteins are important architectural components of plant nuclei that play diverse roles in both heterochromatin organization and the control of nuclear morphology. The cellular components and processes that specify nuclear size, shape and internal organization are poorly understood, particularly in flowering plants. Despite similarities at the gross morphological level among all eukaryotic nuclei, such as a double-membrane boundary perforated with nuclear pores, most of the proteins known to affect nuclear structure in animals are not evolutionarily conserved and are consequently difficult to recognize or absent entirely in plant proteomes -3. TheseLINC4, another of the four paralogous genes in this family, leads to reduced nuclear size and loss of elongated nuclear shape in differentiated cells, mirroring the phenotype of linc1 mutants [We demonstrated previously ,6 that t mutants . These p mutants . Althoug mutants ,10. More mutants , howevercrwn mutants is not matched by a commensurate reduction in endopolyploid levels, resulting in increased nuclear DNA densities (mass per unit volume) up to four-fold higher than wild type levels.Here, we extend our reverse genetic analysis to encompass all four members of the Arabidopsis NMCP-related protein family, which we have renamed CRWN (CROWDED NUCLEI) to avoid confusion with the acronym LINC (LINKER of NUCLEOSKELETON and CYTOSKELETON) that refers to SUN-KASH protein linkages that bridge the inner and outer nuclear membrane -16. Our We performed a phylogenetic analysis of Arabidopsis CRWN proteins and their homologues in other species to begin our investigation of the potential diversification within this family. The predicted Arabidopsis proteome contains four closely related CRWN proteins (CRWN1 through 4) that share 30-40% amino acid identity; no other Arabidopsis proteins with extended regions of significant amino acid identity to CRWN proteins were found. Similar proteins were found in other plant species, but interestingly, no fungi or animal CRWN homologues were identified from searches of protein databases. In addition, CRWN homologues were absent in the predicted proteome of the green algae Chlamydomonas and Volvox.Selaginella moellendorffii and Physcomitrella patens. One clade includes three of the Arabidopsis paralogs, CRWN1, CRWN2 and CRWN3, while CRWN4 belongs to the other clade. Within each clade, the monocot proteins, represented by maize, sorghum and rice, group independently from the dicot proteins. Only two CRWN paralogs exist in these monocots – one CRWN1-like and one CRWN4-like. However, certain dicot species, such as Arabidopsis, poplar, grape, and castor bean, contain multiple copies of CRWN1-like proteins. The dicot CRWN4-like proteins are also distinct from their monocot counterparts in lacking a conserved amino acid motif at the extreme C-terminus  [The inference that CRWN4 and related proteins are divergent from members of the CRWN1-containing clade was supported by genetic analyses to dissect the functions of the WN genes . Previounsertion . Here, wWeb.cgi) .CRWN gene had phenotypes similar to wild-type Columbia plants, as did the double crwn2 crwn3 and crwn3 crwn4 mutants. The crwn2 crwn4 and crwn1 crwn4 double mutants exhibited slightly smaller rosettes, while the remaining double mutants, crwn1 crwn2 and crwn1 crwn3, displayed markedly smaller rosette sizes. We were able to recover only two of the four triple mutants - crwn1 crwn2 crwn4 and crwn1 crwn3 crwn4, both of which were extremely stunted and set few seed.Mutant plants carrying single insertions were intercrossed and progeny carrying homozygous insertions in different combinations were recovered. Figure CRWN genes indicates that at least one functional CRWN protein is required for viability. Triple mutant plants carrying only CRWN2 or CRWN3 were extremely stunted, but still viable. This result suggests that CRWN2 or CRWN3 alone can cover the minimum requirements for the entire CRWN protein family. However, plants carrying only CRWN1 or only CRWN4 were not recovered, suggesting that CRWN1 and CRWN4 are specialized and that neither protein alone can express the full range of functions of the CRWN protein family.Our inability to isolate a mutant combining alleles in all four crwn mutant nuclei from adult leaf tissue to determine the role of different CRWN proteins in specifying nuclear size and shape. Among the single mutants, a deficiency of CRWN1 or CRWN4 reduced nuclear size . A similar pattern was observed in double mutants containing the crwn4 mutation  (see Methods), to characterize the distribution of visible DAPI-bright spots within interphase nuclei. The value of this index ranges from 0 to 1, reflecting both the number of distinct chromocenter spots and the uniformity of their size distribution. The expected AI for wild-type nuclei containing 10 equally sized chromocenters is 0.1, while clustering of chromocenters into fewer but larger aggregates will lead to a higher AI value. A dispersal of chromocenters into smaller heterochromatic satellites will push the AI lower. For a given chromocenter number, a skewed CC size distribution is associated with a larger AI compared to when each CC is equally sized. As shown in Figure crwn1 crwn2 and crwn4 mutants. Arabidopsis chromocenters are comprised of large segments of repetitive DNA such as the tandemly-arrayed centromeric and 5S RNA repeats located within pericentromeric regions [in situ hybridization (FISH), we examined the spatial organization of the major 180-bp centromeric tandem repeat and the 5S RNA gene arrays in both large and small nuclei from wild-type, crwn1 crwn2 and crwn4 plants  [crwn1 loss-of-function mutation with a deficiency in any of the remaining three paralogs causes a further reduction in nuclear size in leaf cells. The additive effect on nuclear size indicates that CRWN2 and CRWN3 share overlapping functions with CRWN1, although loss of CRWN2 and/or CRWN3 has no effect on nuclear size or endopolyploidy in leaf cells when a functional CRWN1 allele is present. We previously reported that the crwn2-1 allele caused a reduction in nuclear size in leaf cells [crwn2 mutant lines. Regardless, it is clear that CRWN1 plays the major role in adult leaf tissue among the three paralogs in the CRWN1-like clade. This situation might reflect the higher level of expression of CRWN1 in leaf tissue compared to CRWN2 and CRWN3 – ca. 30 FPKM (fragment per kilobase/million mapped RNA-seq reads) for CRWN1, with 4× and 2× less expression from CRWN2 and CRWN3, respectively (data not shown). Alternatively, the different contributions of CRWN1-like genes could also result from structural differences among the encoded proteins.The phenotypic effects of combining mutations in different y Figure . Loss ofy Figure  as was re Figure , despiteWeb.cgi) , suggestaf cells ; the reacrwn mutants is a reduction in nuclear size and that the reduction in endopolyploidy observed in double and triple crwn mutants is a secondary effect in response to reduced nuclear size. First, mutation of either CRWN1 or CRWN4 had an effect on nuclear size in leaf cells without affecting endopolyploidy , as seen in animal cells when lamin is disrupted [crwn4 nuclei are consistent with this structural integrity model. These predictions, however, are not consistent with the smaller, round nuclei in crwn1 mutants that do not adopt the elongated spindle shapes typical of wild-type nuclei in many cell types. These considerations suggest alternative models wherein the CRWN1 protein might establish flexible hinge regions or expansion zones in the nucleoskeleton, facilitating nuclear size changes that accompany endopolyploidy and other developmental transitions. We note that both nucleoporin 136 [The large coiled-coil domains that comprise the central region of all four CRWN proteins point toward a structural role in the nucleus. Recent analysis based on secondary structure for analogues of Arabidopsis coiled-coil proteins indicates that CRWN proteins are candidates for paramyosin homologues . Paramyo muscles . The strisrupted  or down-isrupted . The irrorin 136 ,36 and Lcrwn1 crwn2 nuclei [CRWN gene family. One unexpected finding was the wide variation in chromocenter number in crwn4 nuclei and the direct correlation between chromocenter number and nuclear size. The reduction in chromocenter number in crwn1 crwn2 mutants, as well as crwn4 mutants with smaller nuclei, is consistent with aggregation of individual chromocenters. The in situ hybridization data shown in Figure crwn4 nuclei as evidenced by the dispersed signals seen for the 5S RNA genes, and to a less extent, the centromere repeat arrays. Considering that chromocenters are localized primarily to the nuclear periphery [Deficiency of CRWN proteins also affects chromocenter organization Figures  and 7. W2 nuclei , and we eriphery ,26 . Our findings also demonstrated that CRWN4 plays a role in maintenance of heterochromatin organization in interphase nuclei. The specificity of the nuclear morphological and higher-order chromatin organization defects seen in crwn mutants reveals the interplay between nuclear morphology and the three-dimensional packaging of the genome.This study addresses fundamental questions about how plant cells specify and control the morphology of their nuclei and its relationship with internal chromatin organization. We conducted a comprehensive reverse genetics study of the crwn1-1), 5′-TGC CTT CTC CTC GCT TTT CAA-3′ and 5′-TGC GTG AAT GGG AAA GAA AGT TG-3′; SALK_076653 (crwn2-1), 5′-GAA GCT CAT TGC TAG AGA AGG GG-3′ and 5′-AAC GCT GAT CGT TCA TGT TCC A-3′; SALK_099283 (crwn3-1), 5′-TTC TGC ATC TTG ACA CCA TCC AA-3′ and 5′-TCG TCG ACT AGT TAA CAA AAT CA-3′; SALK_079296 (crwn4-1), 5′-CGC AAA GCC TTC GAA GAC AAA-3′ and 5′-GCT TCA GCC AGC ATT TCA AGC-3′.All T-DNA insertion alleles used in this study are from the SALK collection  in straiAmino acids similar to CRWN1 were downloaded from public databases , and guard cell nuclei were imaged using laser scanning confocal microscopy (Leica SP5). Images were taken at the focal plane with the maximum nuclear area, and the resulting images were processed using ImageJ software.Nuclei were isolated from the fifth true leaf of adult plants harvested after initiation of flowering stem elongation (stem height ≥ vegetative rosette diameter). Therefore, the tissues were developmentally matched across the different genotypes. Mesophyll cells predominated but other cell types were present. Each harvested leaf was bisected and one half used for the nuclear area measurement, while the remaining half leaf was processed for flow cytometry measurements (see below). Leaf tissue was fixed using a 3:1 acetic acid:ethanol solution and tissues were rehydrated in 100 mM sodium citrate buffer pH 4.8 for 15 min followed by incubation in digestion buffer  for 2 hours at 37°C. Digested tissue was carefully homogenized by pipetting, centrifuged briefly at low speed, and resuspended with 100 mM sodium citrate buffer pH 4.8; this cycle was repeated three times and the final pellet was resuspended in 3:1 acetic acid:ethanol. The resulting suspension of nuclei were pipetted onto microscope slides, dried for ca. 1 min, and stained with 10 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). A Leica DM 5500 epifluorescence microscope was used to image the nuclei, and the nuclear area was measured from digital images using ImageJ software after manual tracing of nuclear boundaries. Note that the chromocenter number versus leaf cell nuclear area scatter plots shown in Figure in situ hybridization step, nuclei were stained with DAPI and imaged using optical sectioning microcopy. Projections of the processed images were analyzed using ImageJ software to identify chromocenters. Briefly, the images were manually manipulated when necessary to adjust the local threshold and chromocenter area and number were assigned using the Analyze Particle function of the software. The aggregation index (AI) was calculated using the following equation: AI = Σ 2; where i = 1, …, n; Si = the area of chromocenter i; Stotal = the total area of all chromocenters in the nucleus.Tissue from adult leaves was harvested and prepared as described for FISH (see below). Rather than performing the Bisected tissue from the fifth true leaf of adult plants were harvested (see above) and immersed in magnesium sulfate buffer  and chopin situ hybridization as described by Golubovskaya et al. (2002) [Fully expanded adult leaf tissue was harvested less than a week after flowering stem elongation, and fixed in Buffer A  with 4% . (2002) . The cenThe authors declare that they have no competing interests.HW planned and performed the experiments, prepared the figures, and contributed to the preparation of the manuscript. TAD provided technical advice, generated genetic reagents and contributed to the preparation of the manuscript. EJR supervised the project and wrote the manuscript. All authors read and approved the final manuscript.CRWN-like proteins used in this study. The first column shows the abbreviated name of the protein used in alignment to construct the tree shown in Figure Click here for fileAmino acid sequences comprising the extreme C-termini of 28 CRWN-like proteins, including ten CRWN4-like proteins. The similarity in this region, which falls outside of the coiled-coil domains, reinforces the topology of the tree shown in Figure Click here for fileTranscript analysis of the crwn3-1 and crwn4-1 alleles used in this study. Reverse transcription-PCR results investigating the effect of T-DNA insertions on the transcription of CRWN3 and CRWN4. Panel A shows that a CRWN3 transcript is produced from the wild-type allele but not from the crwn3-1 allele using primers spanning the T-DNA insertion site. Panel B demonstrates that some transcription can be detected downstream of the insertion site from the crwn3-1 allele using RT-PCR and a primer set recognizing sequences 3’ of the insertion site. Panel C indicates that the T-DNA insertion in the crwn4-1 allele blocks transcription. Amplification of cDNA from cyclophilin and Actin2 were used as positive controls. M, marker lanes; + RT (plus reverse transcriptase); - RT (no reverse transcriptase). Information on the oligonucleotide primers used in these experiments is shown at the bottom of the figure. Our previous data [crwn1-1 and crwn2-1 alleles block transcription downstream of the T-DNA insertion site in the sixth exon of both genes.ous data  indicateClick here for filecrwn mutants used to construct Figure The nuclear phenotype data for Click here for fileNuclear shape changes in crwn1 and crwn4 mutants. Images of representative DAPI-stained adult leaf cell nuclei from wild type, crwn1 and crwn4 mutants were processed by ImageJ software to determine the circularity index (4π · Area/(perimeter)2), as well as a shape index (perimeter/π · major axis)2. Nuclei that deviate from a perfect circle (1.0) show a lower circularity index. The shape index highlights different types of deviations from the round shape. Nuclei in the crwn1 sample show a shape index close to 1.0, indicating consistently round nuclei. The reduced (relative to 1.0) shape indices in the wild-type sample across all nuclear sizes indicate uniformly elongated nuclear shapes. The elevated shape indices characteristic of larger crwn4 nuclei result from the presence of thin projections from the surface of otherwise round nuclei.Click here for fileLeaf nuclear preparation and confocal imaging reveals a consistent nuclear thickness across a range of nuclear sizes. Mature leaves were harvested from five individuals in a F2 population segregating both crwn1 and crwn2 mutations (F2 plants of a crwn1 crwn2 x wild type cross). Consequently, the sample captured a range of nuclear shapes and sizes. The nuclei were fixed, isolated, and prepared for imaging as described for Figure Click here for fileChromocenter changes in crwn double mutants. Nuclei were harvested from developmentally matched rosette leaves from approximately one month-old plants, stained with DAPI, and imaged using epifluorescence microscopy. The area and chromocenter number of randomly-selected individual nuclei (n = 41–52) were determined for each genotype, and chromocenter number was plotted against nuclear area. A linear regression line showing the relationship between chromocenter number and nuclear size (as a proxy for endopolyploidy level) was plotted for each genotype.Click here for filein situ hybridization experiment. Representative nuclei from wild type (Movie S1), crwn1 crwn2(Movie S2) and crwn4(Movie S3) leaf cells were processed by FISH as described in Figure Three-dimensional reconstruction of nuclei imaged in the fluorescence Click here for file