M2WISH: An easy and efficient protocol for whole-mount mRNA in situ hybridization that allows 3D cell resolution of gene expression in Arabidopsis thaliana.

Gene expression analysis is essential for understanding the mechanisms involved in plant development. Here, we developed M2WISH, a protocol based on MicroWave treatment for Wholemount mRNA In Situ Hybridization in Arabidopsis. By permeabilizing tissues without damaging cellular organization this protocol results in high and homogeneous hybridization yields that enable systematic analysis of gene expression dynamics. Moreover, when combined with cellular histochemical staining, M2WISH successfully provides a cellular resolution of gene expression. Thus, we demonstrate the robustness of M2WISH with 10 genes on roots, aerial meristems, leaves, and embryos in the seed. We applied M2WISH to study the spatial dynamics of WUSCHEL (WUS) and CLAVATA3 (CLV3) expression during in vitro meristematic conversion of roots into shoot apical meristems. Thus, we showed that shoot apical meristems could arise from two different types of root structures that differed by their CLV3 gene expression patterns. We constructed 3D cellular representations of WUS and CLV3 gene co-expression pattern and stressed the variability inherent to meristem conversion. Thus, this protocol generates a large amount of data on the localization of gene expression, which can be used to model complex systems.

Chemokine signaling via the CXCR2 receptor reinforces senescence.

Cells enter senescence, a state of stable proliferative arrest, in response to a variety of cellular stresses, including telomere erosion, DNA damage, and oncogenic signaling, which acts as a barrier against malignant transformation in vivo. To identify genes controlling senescence, we conducted an unbiased screen for small hairpin RNAs that extend the life span of primary human fibroblasts. Here, we report that knocking down the chemokine receptor CXCR2 (IL8RB) alleviates both replicative and oncogene-induced senescence (OIS) and diminishes the DNA-damage response. Conversely, ectopic expression of CXCR2 results in premature senescence via a p53-dependent mechanism. Cells undergoing OIS secrete multiple CXCR2-binding chemokines in a program that is regulated by the NF-kappaB and C/EBPbeta transcription factors and coordinately induce CXCR2 expression. CXCR2 upregulation is also observed in preneoplastic lesions in vivo. These results suggest that senescent cells activate a self-amplifying secretory network in which CXCR2-binding chemokines reinforce growth arrest.

Direct conversion of root primordium into shoot meristem relies on timing of stem cell niche development.

To understand how the identity of an organ can be switched, we studied the transformation of lateral root primordia (LRP) into shoot meristems in Arabidopsis root segments. In this system, the cytokinin-induced conversion does not involve the formation of callus-like structures. Detailed analysis showed that the conversion sequence starts with a mitotic pause and is concomitant with the differential expression of regulators of root and shoot development. The conversion requires the presence of apical stem cells, and only LRP at stages VI or VII can be switched. It is engaged as soon as cell divisions resume because their position and orientation differ in the converting organ compared with the undisturbed emerging LRP. By alternating auxin and cytokinin treatments, we showed that the root and shoot organogenetic programs are remarkably plastic, as the status of the same plant stem cell niche can be reversed repeatedly within a set developmental window. Thus, the networks at play in the meristem of a root can morph in the span of a couple of cell division cycles into those of a shoot, and back, through transdifferentiation.

Regulation of ploidy and senescence by the AMPK-related kinase NUAK1.

Senescence is an irreversible cell-cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss-of-function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK-related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant-negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.

Encapsulating peritoneal sclerosis post liver transplant and peritoneal dialysis: case report and literature review.

Encapsulating peritoneal sclerosis (EPS) is a rare and debilitating condition. A fibrocollagenous membrane, which promotes encasement of the small intestine leaving a cocoon-like appearance, takes place. It is mainly associated with peritoneal infections, medications, peritoneal dialysis and systemic inflammatory diseases. Diagnosis is based on clinical history, intestinal obstruction and imaging exam. We report a case of EPS in a 68-year-old man with a medical history of liver transplantation and peritoneal dialysis, complaining of obstructive bowel symptoms.

Pneumatosis cystoides intestinalis with pneumoperitoneum in an 87-years-old male patient: a case report.

Pneumatosis cystoides intestinalis (PCI) is a rare condition, characterized by gas-filled cysts in the intestinal wall. The mesentery and intra-abdominal ligaments can be affected. PCI is classified as primary or secondary and associated with multiple predisposing factors. An asymptomatic 87-year-old man underwent an abdominal tomography for follow-up of bladder carcinoma. The examination revealed intestinal and mesenteric pneumatosis associated with pneumoperitoneum. At laparoscopy, intestinal and mesenteric pneumatosis without intestinal infarction was identified. He was discharged on the fifth postoperative day. PCI is a benign condition that can be confused with mesenteric ischemia. Treatment is conservative, with periodic clinical evaluations. Surgical procedure is unnecessary for its diagnosis or management.

Gallbladder agenesis a rare and underdiagnosed congenital anomaly: a case report and literature review.

Gallbladder agenesis (GA) is a rare congenital anomaly with conflicting epidemiology described in the literature. When present, it is misinterpreted as cholelitiasis, a highly prevalent condition. Nevertheless, surgeons and radiologists must be aware of it since it can lead to unnecessary invasive procedures. Diagnosis of GA is challenging due to the anatomical structures that sometimes resemble a shrunken gallbladder. We report the case of a 55-year-old man with preoperative diagnosis of cholelitiasis and further intraoperative find of GA. Since cholecystectomy is one of the most common surgeries worldwide, it demonstrates how relevant this case is to emphasize the need to recognize this diagnosis and be aware of its management to avoid unnecessary surgery.

Septic Thrombophlebitis of the Internal Jugular Vein in an Immunocompromised Patient with Lemierre Syndrome: A Case Report.

Lemierre syndrome is a rare complication of oropharyngeal infection, especially acute pharyngotonsillitis, associated with septicemia and thrombophlebitis of the internal jugular vein (IJV). We present the case of a 52-year-old patient who underwent liver transplantation and returned with symptoms of pain, redness and left cervical bulging 1 month after surgery. After investigation, the diagnosis of septic thrombophlebitis of the IJV was made. The patient responded well to treatment with antibiotic therapy and full anticoagulation. To the best of our knowledge, the present report is the first report of Lemierre syndrome in a post-liver transplant patient.

The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development.

Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation. Conversely, the expression of PHS1 was able to complement the developmental defects and the accumulation of long chain bases of the pas2-1 mutant. The pas2-1 mutant was characterized by a general reduction of VLCFA pools in seed storage triacylglycerols, cuticular waxes, and complex sphingolipids. Most strikingly, the defective elongation cycle resulted in the accumulation of 3-hydroxy-acyl-CoA intermediates, indicating premature termination of fatty acid elongation and confirming the role of PAS2 in this process. We demonstrated by in vivo bimolecular fluorescence complementation that PAS2 was specifically associated in the endoplasmic reticulum with the enoyl-CoA reductase CER10, the fourth enzyme of the elongase complex. Finally, complete loss of PAS2 function is embryo lethal, and the ectopic expression of PHS1 led to enhanced levels of VLCFAs associated with severe developmental defects. Altogether these results demonstrate that the plant 3-hydroxy-acyl-CoA dehydratase PASTICCINO2 is an essential and limiting enzyme in VLCFA synthesis but also that PAS2-derived VLCFA homeostasis is required for specific developmental processes.

Biliary tract melanoma metastasis mimicking hilar cholangiocarcinoma: a case report.

Malignant melanoma is the 19th leading cause of cancer worldwide. It is an aggressive neoplastic disease in which pathophysiological understanding and management has been in constant evolution in recent decades. The primary site is the skin, uvea and mucous membranes and has the capacity to metastasize to any organ. There are few reports of primary or secondary involvement of the biliary tract. We present the case of a 73-year-old woman with a bile duct lesion suggestive of cholangiocarcinoma and a final diagnosis of a single melanoma metastasis. Surgical treatment was performed due to oligometastatic stage IV melanoma with possibility of R0 resection followed by immune checkpoint therapy.

Feasibility of Routine Ambulatory Laparoscopic Cholecystectomy in Brazil.

BACKGROUND AND OBJECTIVES: In several developed countries, most laparoscopic cholecystectomies (LCs) are performed as an ambulatory operation (ALC) with a high rate of success. In Latin America, the experience with this procedure is still limited. Our objective is to evaluate the feasibility to implement ALC in a Brazilian teaching hospital. METHODS: Data obtained from electronic medical records and study protocols of all patients who underwent an LC between January 2011 and March 2018 were evaluated. All patients with chronic or acute cholecystitis were initially considered for an ALC. RESULTS: Of a total of 1645 patients who underwent LC, 1577 (95.9%) were discharged on the same day of the operation. The main reasons for hospital admission after ALC were patient refusal to be discharged (n = 23; 1.4%), nausea and vomiting (n = 15; 0.9%), and complicated acute cholecystitis. No patient was excluded from consideration for ALC based only on age, history of previous upper abdominal operation, and presence of comorbidity. Patient age ranged from 12 to 100 years, with a mean of 50.23 ± 15.35 years. Intraoperative and postoperative complication rates were 0.4% and 5.5%, respectively. Most perioperative complications were because of technical surgical difficulties and complications common to most abdominal operations (surgical site, pulmonary, urinary, and venous complications). Thirteen (0.8%) patients were readmitted to the hospital because of abdominal pain and fever (n = 4), pneumonia (n = 3), deep venous thrombosis (n = 3), or urinary retention (n = 3). CONCLUSIONS: ALC may be performed in Brazil with low rates of morbidity, mortality, and hospital readmission. Its implementation should be stimulated in Latin America.

In vivo biological activity of the histone deacetylase inhibitor LAQ824 is detectable with 3'-deoxy-3'-[18F]fluorothymidine positron emission tomography.

Histone deacetylase inhibitors (HDACI) are emerging as growth inhibitory compounds that modulate gene expression and inhibit tumor cell proliferation. We assessed whether 3'-deoxy-3'-[(18)F]fluorothymidine-positron emission tomography ([18F]FLT-PET) could be used to noninvasively measure the biological activity of a novel HDACI LAQ824 in vivo. We initially showed that thymidine kinase 1 (TK1; EC2.7.1.21), the enzyme responsible for [18F]FLT retention in cells, was regulated by LAQ824 in a drug concentration-dependent manner in vitro. In HCT116 colon carcinoma xenograft-bearing mice, LAQ824 significantly decreased tumor [18F]FLT uptake in a dose-dependent manner. At day 4 of treatment, [18F]FLT tumor-to-heart ratios at 60 minutes (NUV60) were 2.16 +/- 0.15, 1.86 +/- 0.13, and 1.45 +/- 0.20 in vehicle, and 5 and 25 mg/kg LAQ824 treatment groups, respectively (P < or = 0.05). LAQ825 at 5 mg/kg also significantly reduced both TK1 levels and [18F]FLT uptake at day 10 but not at day 2 (P < or = 0.05). [18F]FLT NUV60 correlated significantly with cellular proliferation (r = 0.68; P = 0.0019) and was associated with drug-induced histone H4 hyperacetylation. Of interest to [18F]FLT-PET imaging, both TK1 mRNA copy numbers and protein levels decreased in the order vehicle >5 mg/kg LAQ824 > 25 mg/kg LAQ824, providing a rationale for the use of [18F]FLT-PET in this setting. We also observed increases in Rb hypophosphorylation and p21 levels, factors that could have contributed to the alteration in TK1 transcription in vivo. In conclusion, we have shown the utility of [18F]FLT-PET for monitoring the biological activity of the HDACI, LAQ824. Drug-induced changes in tumor [18F]FLT uptake were due, at least in part, to reductions in TK1 transcription and translation.

Surgical Management of Solid Pseudopapillary Tumor of the Pancreas.

BACKGROUND AND OBJECTIVES: Although solid pseudopapillary tumor (SPT) of the pancreas is rare, its diagnosis has increased severalfold in the past decades. We present our experience in the management of SPT, including a patient who experienced tumor rupture during laparoscopy pancreatic resection. METHODS: Data on all patients with SPT who were subjected to surgical treatment were retrospectively obtained. RESULTS: Of 20 patients evaluated, 17 (85%) were females. The mean age was 31 years. Tumor size varied from 2.7 × 1.5 to 13.5 × 10.0 cm, with a mean of 6.4 × 7.6 cm. The most common location was the tail and/or body of the pancreas (14 patients [70%]). Pancreatic tumor resection was performed in 19 patients (50%). The type of resection depended on tumor location and size: distal pancreatectomy (n = 13), pancreatoduodenectomy (n = 5), and central pancreatectomy (n = 1) Pancreatic resection was performed via laparoscopy in 7 patients who underwent distal pancreatectomy. Tumor resection was not performed in only 1 patient (5%), due to invasion of mesenteric vessels and presence of liver metastases. One patient had tumor rupture during laparoscopic resection, with no apparent macroscopic dissemination of the tumor. All 19 patients who underwent SPT resection had no tumor recurrence, including a patient with capsule invasion and another patient with tumor rupture during surgical dissection. The mean follow-up time was 38 months (range, 6-72 months). CONCLUSION: Complete SPT resection is possible in most patients, with a low recurrence rate. Because of its large size, laparoscopic resection of SPT's should be performed only by experienced surgeons to avoid tumor rupture.

Oral glutamine and the healing of colonic anastomoses in rats.

BACKGROUND: Recent evidence has suggested that glutamine is one of the primary energy sources of the colon. The aim of this study was to evaluate the effects of oral glutamine supplementation on the healing of colonic anastomoses in rats. METHODS: Forty-eight adult male Wistar rats, weighing 174.41 +/- 37.39 g, were housed in individual cages. All rats had free access to water and standard rat chow. The rats were randomized to receive daily, for 7 days before the operation and during the postoperative period, 10% L-glutamine (GLN group) or 10% glycine (GLY group) in isonitrogenous and isovolumetric solutions (1.5 g/kg per day), through an orogastric tube. On the eighth day, rats were anesthetized and subjected to 2 colonic transections, one 6 cm distal from the ileocecal valve and another 5 cm distal from the first transection. Bowel continuity was restored by 2 end-to-end, single layer, everted, anastomoses with 8 interrupted sutures (6-0 nylon). After the operation, the rats were kept in individual cages and had free access to water and rat chow. One-half of the rats in each group were killed either on postoperative day 3 or 8, and the 2 colonic anastomoses of each animal were resected and stored in 0.9% saline and 10% formalin for tensile strength and histologic (hematoxylineosin and collagen densitometry) studies, respectively. Student's t-test and Kruskal Wallis tests were used for statistical analysis. RESULTS: Total rupture strength was significantly higher in the GLN group (GLN: 0.068 +/- 0.045 kgf versus GLY: 0.042 +/- 0.027 kgf, p = .04). The mean monocytes infiltrate was significantly smaller in the GLN group (p = .04). The collagen densitometry analysis demonstrated greater percent area of type I (mature) in the GLN group compared with GLY (58.65 +/- 11.70% versus 41.79 +/- 10.54%, p = .0000), respectively. Subgroup analyses according to the day of rat death were still significant: GLN 3: 54.22 +/- 10.02% versus GLY 3: 41.92 +/- 13.31% (p = .04) and GLN 8: 62.63 +/- 12.13% versus GLY 8: 41.67 +/- 7.69% (p = .0004). Type III collagen (immature) percent area was significantly smaller in the GLN group's colonic anastomoses (GLN: p = .0000; GLN 3: p = .04 and GLN 8: p = .0003, respectively). CONCLUSIONS: Perioperative oral glutamine supplementation increases total rupture strength and improves the percent area of mature collagen at the anastomoses sites on postoperative days 3 and 8.

Liver transplantation in a patient with complex anomaly of the inferior vena cava.

BACKGROUND: After the introduction of noninvasive imaging exams, congenital anomalies of the inferior vena cava (IVC) have become more commonly recognized. We report the first successful orthotopic liver transplantation (OLT) performed in an asymptomatic adult with complex IVC anomaly: duplication of the infrarenal IVC, azygos continuation of the IVC, agenesia of the hepatic portion of the IVC and presence of several anomalous veins communicating the common iliac vein and the IVC of one side with the contralateral side. METHODS: This complex anomaly was diagnosed with a venous abdominal angio CT. RESULTS: At liver transplantation, the short suprahepatic portion of the IVC was identified and clamped. The right, middle, and left hepatic veins were sectioned and joined in a single, wide cuff, using venoplasty. This single orifice was anastomosed to the suprahepatic IVC of the new liver. No venovenous bypass was employed. The patient had an uneventful postoperative course. A post transplantation venous abdominal angio CT showed normal blood flow at the anastomosis of the hepatic veins of the receptor and the IVC of the new liver. CONCLUSIONS: This report is important to alert liver transplant teams of the possibility of complex IVC in asymptomatic adult individuals. Identification of these anatomical anomalies is vital to reduce the risk of serious hemorrhage and other operative complications during OLT.

[The importance of the contextual approach in the teaching of biosafety]. / A importância da abordagem contextual no ensino de biossegurança.

Biosafety is a field of knowledge that raises questions geared to genetically modified organisms that are linked to social and job-related employee protection. The educational process involves seeking to create a participative and transforming agent and must therefore transcend the simple concept of teaching. Thus, it is important to contextualize biosafety within a constructive teaching strategy by identification of its core concepts - risk, hazard and accident - which allows each individual to understand how risk is perceived within society and dealt with in academia in order to add multiple skills to tackle the situation. Understanding how the relationship between work and health and its consequences and effects are constructed over the course of time, makes it possible to train more critical and well prepared citizens to participate in decisions of a political and social nature that can influence their future.

Prevalence of cholelithiasis in patients subjected to liver transplantation for cirrhosis.

BACKGROUND AND AIMS: The prevalence of cholelithiasis in patients subjected to liver transplantation has not been evaluated yet. This study examines the prevalence of cholelithiasis in patients subjected to liver transplantation due to cirrhosis compared to an age- and sex-matched control group. METHODS: The electronic study protocols of 400 consecutive cirrhotic patients aged between 20 and 69 years who had undergone liver transplantation for cirrhosis were evaluated to determine the presence of gallstones. RESULTS: The overall prevalence of cholelithiasis was higher in transplant recipients (96 patients; 24%) than in controls (38 patients; 9.5%) (p<0.001). There was no increase in the prevalence of cholelithiasis with age in the transplant recipients (p=0.332). Conversely, the prevalence of cholelithiasis increased with age in the control group (p<0.001). There was no difference in the gallstone prevalence between sexes in the transplant recipient group (p=0.102). However, the gallstone prevalence was 2.2 times higher in females (14.8%) than in males (6.8%) in the control group (p=0.009). CONCLUSION: Prevalence of cholelithiasis is higher in patients subjected to liver transplantation for cirrhosis. In contrast with the general population, the prevalence of cholelithiasis in cirrhotic patients is similar in both sexes and does not increase with age.

A genetic screen identifies topoisomerase 1 as a regulator of senescence.

Normal cell growth can be permanently blocked when cells enter a state known as senescence. This phenomenon can be triggered by various stresses, such as replicative exhaustion, oncogenic stimulation, or oxidative stress. Senescence prevents transmission of aberrant signals to daughter cells and thus prevents irreversible damage that could favor cancer development. To identify new genetic events controlling senescence, we have performed a loss-of-function genetic screen on normal human cells. We report that knockdown of topoisomerase I (Top1) results in an increased replicative potential associated with a decrease in senescence markers and a diminished DNA damage response. In addition, Top1 depletion also favors a bypass of oncogene-induced senescence. Conversely, Top1 constitutive expression induces growth arrest, the appearance of a senescence marker, and an activation of the DNA damage response. Altogether, these results reveal an unanticipated function of Top1 in regulating senescence.

Latent protein LANA2 from Kaposi's sarcoma-associated herpesvirus interacts with 14-3-3 proteins and inhibits FOXO3a transcription factor.

The Kaposi's sarcoma-associated herpesvirus latent protein LANA2 has been suggested to have an important role in the transforming activity of the virus based on its capacity to inhibit p53 and PKR-dependent apoptosis as well as the interferon-dependent response. Here, we describe a novel interaction between LANA2 and both the phosphoserine/phosphothreonine-binding 14-3-3 proteins and the transcription factor FOXO3a. In addition, our results indicate that LANA2 inhibits the transcriptional activity of FOXO3a and blocks the G2/M arrest induced by 14-3-3 protein overexpression. These results suggest a novel mechanism by which LANA2 may promote tumorigenesis.

FOXO3a induces differentiation of Bcr-Abl-transformed cells through transcriptional down-regulation of Id1.

Leukemic transformation often requires activation of protein kinase B (PKB/c-Akt) and is characterized by increased proliferation, decreased apoptosis, and a differentiation block. PKB phosphorylates and inactivates members of the FOXO subfamily of Forkhead transcription factors. It has been suggested that hyperactivation of PKB maintains the leukemic phenotype through actively repressing FOXO-mediated regulation of specific genes. We have found expression of the transcriptional repressor Id1 (inhibitor of DNA binding 1) to be abrogated by FOXO3a activation. Inhibition of PKB activation or growth factor deprivation also resulted in strong down-regulation of Id1 promoter activity, Id1 mRNA, and protein expression. Id1 is highly expressed in Bcr-Abl-transformed K562 cells, correlating with high PKB activation and FOXO3a phosphorylation. Inhibition of Bcr-Abl by the chemical inhibitor STI571 resulted in activation of FOXO3a and down-regulation of Id1 expression. By performing chromatin immunoprecipitation assays and promoter-mutation analysis, we demonstrate that FOXO3a acts as a transcriptional repressor by directly binding to the Id1 promoter. STI571 treatment, or expression of constitutively active FOXO3a, resulted in erythroid differentiation of K562 cells, which was inhibited by ectopic expression of Id1. Taken together our data strongly suggest that high expression of Id1, through PKB-mediated inhibition of FOXO3a, is critical for maintenance of the leukemic phenotype.