Genomic Complexity as a Biomarker to De-Escalate Adjuvant Imatinib Treatment in High-Risk Gastrointestinal Stromal Tumor.

PURPOSE: Adjuvant imatinib treatment is recommended for patients with localized gastrointestinal stromal tumor (GIST) at high risk of recurrence. Almost half of high-risk patients are cured by surgery alone, indicating a need for improved selection of patients for adjuvant therapy. The aim of this study was to investigate if genomic tumor complexity could be used as a prognostic biomarker. METHODS: The discovery cohort consisted of patients who underwent resection of primary GIST at Oslo University Hospital between 1998 and 2020. Karyotypes were categorized as simple if they had ≤ 5 chromosomal changes and complex if there were > 5 chromosomal aberrations. Validation was performed in an independent patient cohort where chromosomal imbalances were mapped using comparative genomic hybridization. RESULTS: Chromosomal aberrations were detected in 206 tumors, of which 76 had a complex karyotype. The most frequently observed changes were losses at 14q, 22q, 1p, and 15q. The 5-year recurrence-free survival (RFS) in patients classified as very low, low, or intermediate risk was 99%. High-risk patients with a simple tumor karyotype had an estimated 5-year RFS of 94%, and patients with a complex karyotype had an estimated 5-year RFS of 51%. A complex karyotype was associated with poor RFS in patients with and without adjuvant imatinib treatment and in multivariable analysis adjusted for tumor site, size, mitotic count, and rupture. The prognostic impact of genomic complexity was confirmed in the validation cohort. In both cohorts, the 5-year disease-specific survival was > 90% for high-risk patients with genomically simple tumors. CONCLUSION: Genomic tumor complexity is an independent prognostic biomarker in localized, high-risk GIST. Recurrences were infrequent for tumors with simple karyotypes. De-escalation of adjuvant imatinib treatment should be explored in patients with cytogenetically simple GISTs.

Mitigating greenhouse gas emissions from irrigated rice cultivation through improved fertilizer and water management.

Greenhouse gas (GHG) emissions from agriculture sector play an important role for global warming and climate change. Thus, it is necessary to find out GHG emissions mitigation strategies from rice cultivation. The efficient management of nitrogen fertilizer using urea deep placement (UDP) and the use of the water-saving alternate wetting and drying (AWD) irrigation could mitigate greenhouse gas (GHG) emissions and reduce environmental pollution. However, there is a dearth of studies on the impacts of UDP and the integrated plant nutrient system (IPNS) which combines poultry manure and prilled urea (PU) with different irrigation regimes on GHG emissions, nitrogen use efficiency (NUE) and rice yields. We conducted field experiments during the dry seasons of 2018, 2019, and 2020 to compare the effects of four fertilizer treatments including control (no N), PU, UDP, and IPNS in combination with two irrigation systems- (AWD and continuous flooding, CF) on GHG emissions, NUE and rice yield. Fertilizer treatments had significant (p < 0.05) interaction effects with irrigation regimes on methane (CH4) and nitrous oxide (N2O) emissions. PU reduced CH4 and N2O emissions by 6% and 20% compared to IPNS treatment, respectively under AWD irrigation, but produced similar emissions under CF irrigation. Similarly, UDP reduced cumulative CH4 emissions by 9% and 15% under AWD irrigation, and 9% and 11% under CF condition compared to PU and IPNS treatments, respectively. Across the year and fertilizer treatments, AWD irrigation significantly (p < 0.05) reduced cumulative CH4 emissions and GHG intensity by 28%, and 26%, respectively without significant yield loss compared to CF condition. Although AWD irrigation increased cumulative N2O emissions by 73%, it reduced the total global warming potential by 27% compared to CF irrigation. The CH4 emission factor for AWD was lower (1.67 kg ha-1 day-1) compared to CF (2.33 kg ha-1 day-1). Across the irrigation regimes, UDP increased rice yield by 21% and N recovery efficiency by 58% compared to PU. These results suggest that both UDP and AWD irrigation might be considered as a carbon-friendly technology.

Evolutionary patterns of chromosomal instability and mismatch repair deficiency in proximal and distal colorectal cancer.

AIM: Colorectal carcinomas (CRCs) progress through heterogeneous pathways. The aim of this study was to analyse whether or not the cytogenetic evolution of CRC is linked to tumour site, level of chromosomal imbalance and metastasis. METHOD: A set of therapy-naïve pT3 CRCs comprising 26 proximal and 49 distal pT3 CRCs was studied by combining immunohistochemistry of mismatch repair (MMR) proteins, microsatellite analyses and molecular karyotyping as well as clinical parameters. RESULTS: A MMR deficient/microsatellite-unstable (dMMR/MSI-H) status was associated with location of the primary tumour proximal to the splenic flexure, and dMMR/MSI-H tumours presented with significantly lower levels of chromosomal imbalances compared with MMR proficient/microsatellite-stable (pMMR/MSS) tumours. Oncogenetic tree modelling suggested two evolutionary clusters characterized by dMMR/MSI-H and chromosomal instability (CIN), respectively, for both proximal and distal CRCs. In CIN cases, +13q, -18q and +20q were predicted as preferentially early events, and -1p, -4 -and -5q as late events. Separate oncogenetic tree models of proximal and distal cases indicated similar early events independent of tumour site. However, in cases with high CIN defined by more than 10 copy number aberrations, loss of 17p occurred earlier in cytogenetic evolution than in cases showing low to moderate CIN. Differences in the oncogenetic trees were observed for CRCs with lymph node and distant metastasis. Loss of 8p was modelled as an early event in node-positive CRC, while +7p and +8q comprised early events in CRC with distant metastasis. CONCLUSION: CRCs characterized by CIN follow multiple, interconnected genetic pathways in line with the basic 'Vogelgram' concept proposed for the progression of CRC that places the accumulation of genetic changes at centre of tumour evolution. However, the timing of specific genetic events may favour metastatic potential.

Expression of the Neural REST/NRSF-SIN3 Transcriptional Corepressor Complex as a Target for Small-Molecule Inhibitors.

The repressor element 1 (RE1) silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) modulates the expression of genes with RE1/neuron-restrictive silencing element (RE1/NRSE) sites by recruiting the switch independent 3 (SIN3) factor and the REST corepressor (COREST) to its N and C-terminal repressor domain, respectively. Both, SIN3 and COREST assemble into protein complexes that are composed of multiple subunits including a druggable histone deacetylase (HDAC) enzyme. The SIN3 core complex comprises the eponymous proteins SIN3A or SIN3B, the catalytically active proteins HDAC1 or HDAC2, the histone chaperone retinoblastoma-associated protein 46/retinoblastoma-binding protein 7 (RBAP46/RBBP7) or RBAP48/RBBP4, the SIN3-associated protein 30 (SAP30), and the suppressor of defective silencing 3 (SDS3). Here, we overcome a bottleneck limiting the molecular characterization of the REST/NRSF-SIN3 transcriptional corepressor complex. To this end, SIN3 genes were amplified from the complementary DNA of neural stem/progenitor cells, and expressed in a baculovirus/insect cell expression system. We show that the isolates bind to DNA harboring RE1/NRSE sites and demonstrate that the histone deacetylase activity is blocked by small-molecule inhibitors. Thus, our isolates open up for future biomedical research on this critical transcriptional repressor complex and are envisioned as tool for drug testing.

Mitigation of greenhouse gas emissions and reduced irrigation water use in rice production through water-saving irrigation scheduling, reduced tillage and fertiliser application strategies.

Rice production systems are the largest anthropogenic wetlands on earth and feed more than half of the world's population. However, they are also a major source of global anthropogenic greenhouse gas (GHG) emissions. Several agronomic strategies have been proposed to improve water-use efficiency and reduce GHG emissions. The aim of this study was to evaluate the impact of water-saving irrigation (alternate wetting and drying (AWD) vs. soil water potential (SWP)), contrasting land establishment (puddling vs. reduced tillage) and fertiliser application methods (broadcast vs. liquid fertilisation) on water-use efficiency, GHG emissions and rice yield. The experiment was laid out in a randomised complete block design with eight treatments (all combinations of the three factors) and four replicates. AWD combined with broadcasting fertilisation was superior to SWP in terms of maintaining yield. However, seasonal nitrous oxide (N2O) emissions were significantly reduced by 64% and 66% in the Broadcast-SWP and Liquid fertiliser-SWP treatments, respectively, compared to corresponding treatments in AWD. The SWP also significantly reduced seasonal methane (CH4) emissions by 34 and 30% in the broadcast and liquid fertilisation treatments, respectively. Area-scaled GWPs were reduced by 48% and 54% in Broadcast-SWP and Liquid fertiliser-SWP treatments respectively compared to the corresponding treatments in AWD. Compared to AWD, the broadcast and liquid fertilisation in SWP irrigation treatments reduced yield-scaled GWPs by 46% and 37%, respectively. In terms of suitability, based on yield-scaled GWPs, the treatments can be ordered as follows: Broadcast-SWP < Broadcast-AWD = Liquid fertiliser-SWP < Liquid fertiliser-AWD. Growing-season water use was 15% lower in the SWP treatments compared with the water-saving AWD. Reduced tillage reduced additional water use during land preparation. The conclusions of this study are that improved water management and timely coordination of N fertiliser with crop demand can reduce water use, N loss via N2O emissions, and CH4 emissions.

E-cadherin to P-cadherin switching in lobular breast cancer with tubular elements.

Loss of E-cadherin expression due to mutation of the CDH1 gene is a characteristic feature of invasive lobular breast cancer (ILBC). Beta-catenin, which binds to the cytoplasmic domain of E-cadherin, is simultaneously downregulated, reflecting disassembly of adherens junctions (AJs) and loss of cell adhesion. E-cadherin to P-cadherin expression switching can rescue AJs and cell adhesion. However, P-cadherin has not been implicated in ILBC, so far. We aimed to characterize 13 ILBCs with exceptional histomorphology, which we termed ILBCs with tubular elements. The CDH1 mutational status was determined by next generation sequencing and whole-genome copy number (CN) profiling. Expression of cadherins was assessed by immunohistochemistry. ILBCs with tubular elements were ER-positive (13/13) and HER2-negative (13/13) and harbored deleterious CDH1 mutations (11/13) accompanied by loss of heterozygosity due to deletion of chromosome 16q22.1 (9/11). E-cadherin expression was lost or reduced in noncohesive tumor cells and in admixed tubular elements (13/13). Beta-catenin expression was lost in noncohesive tumor cells, but was retained in tubular elements (11/13), indicating focal rescue of AJ formation. N-cadherin and R-cadherin were always negative (0/13). Strikingly, P-cadherin was commonly positive (12/13) and immunoreactivity was accentuated in tubular elements. Adjacent lobular carcinoma in situ (LCIS) was always P-cadherin-negative (0/7). In a reference cohort of LCIS specimens, P-cadherin was constantly not expressed (0/25). In a reference cohort of invasive mammary carcinomas, P-cadherin-positive cases (36/268, 13%) were associated with triple-negative nonlobular breast cancer (P < 0.001). Compared with ILBCs from the reference cohort, P-cadherin expression was more common in ILBCs with tubular elements (12/13 versus 7/84, P < 0.001). In summary, E-cadherin to P-cadherin switching occurs in a subset of ILBCs. P-cadherin is the molecular determinant of a mixed-appearing histomorphology in ILBCs with tubular elements.

Effects of water management on greenhouse gas emissions from farmers' rice fields in Bangladesh.

Alternate wetting and drying (AWD) irrigation in lowland rice cultivation increases water use efficiency and could reduce greenhouse gas (GHG) emissions compared to the farmers' practice of continuous flooding (CF). However, there is a dearth of studies on the impacts of water management on methane (CH4) and nitrous oxide (N2O) emissions in Bangladesh. Multi-location field experiments were conducted during the dry seasons of 2018 and 2019 to determine the baseline emissions of CH4 and N2O from rice fields and compare the emissions from AWD irrigation and CF. CH4 and N2O emissions were measured using the closed chamber technique and their concentrations were determined using a gas chromatograph. CH4 and N2O emissions varied across water management schemes and sites. AWD irrigation significantly (p < 0.05) reduced cumulative CH4 emissions (37%, average across sites) without affecting grain yields compared to CF. The CH4 emission factor for AWD was lower (1.39 kg ha-1 day-1) compared to CF (2.21 kg ha-1 day-1). Although AWD irrigation increased seasonal cumulative N2O emissions by 46%, it did not offset reduced CH4 emissions. AWD reduced the total global warming potential (GWP) by 36% compared to CF. Similarly, GHG intensity (GHGI) in AWD was 34% smaller compared to that in CF. Emissions varied across sites and the magnitudes of seasonal cumulative CH4 and N2O emissions were higher at the Gazipur site compared to the Mymensingh site. AWD, which saves irrigation water without any yield penalty, could be considered a promising strategy to mitigate GHG emissions from rice fields in Bangladesh.

Optimal thresholds of risk parameters for gastrointestinal stromal tumors.

BACKGROUND: Gastrointestinal stromal tumors (GIST) are the most frequent mesenchymal neoplasms of the gastrointestinal tract with highly variable potential for relapse. Tumor size and mitotic index (MI) are major risk factors that predict the outcome of GIST patients. Recent risk stratification schemes include some or all of the empirical size thresholds of 2 cm, 5 cm, and 10 cm and MI thresholds of 5 per 50 high-power fields (hpf) and 10 per 50 hpf. However, data that verify these numbers are sparse. METHODS: By exhaustive regression tree analysis, maximally selected rank statistics and survival difference analysis with bootstrap sampling on a naive GIST population of 161 patients with a mean follow-up of 44 months, current stratification schemes using tumor size and MI were analyzed herein. RESULTS: /Conclusions: Thresholds that optimally stratify the risk of recurrence are observed at tumor sizes of 4-5 cm and 10-11 cm and at mitotic indices of about 5 per 50 hpf and 10 per 50 hpf, respectively. While these data validate the canonical thresholds for size and MI used in risk stratification of GIST, transition regions as well as differences in the implementation of these thresholds between the different classification schemes proposed in the recent years should be considered when classifying GIST.

The FlpTRAP system for purification of specific, endogenous chromatin regions.

The repressor element 1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) binds to repressor element 1/neuron-restrictive silencer element (RE1/NRSE) sites in the genome and recruits effector proteins to repress its target genes. Here, we developed the FlpTRAP system to isolate endogenously assembled DNA-protein complexes such as the REST/NRSF complex. In the FlpTRAP system, we take advantage of the step-arrest variant of the Flp recombinase, FlpH305L, which, in the presence of Flp recognition target (FRT) DNA, accumulates as FRT DNA-protein adduct. The FlpTRAP system consists of three elements: (i) FlpH305L-containing cell extracts or isolates, (ii) a cell line engineered to harbor the DNA motif of interest flanked by FRT sites, and (iii) affinity selection steps to isolate the target chromatin. Specifically, 3×FLAG-tagged FlpH305L was expressed in insect cell cultures infected with baculovirus, and cell lysates were prepared. The lysate was used to capture the FRT-SNAP25 RE1/NRSE-FRT chromatin from a human medulloblastoma cell line, and the target RE1/NRSE chromatin was isolated by anti-FLAG immunoaffinity chromatography. Using electrophoretic mobility shift assays (EMSAs) and chromatin immunopurification (ChIP), we show that FlpH305L recognized and bound to the FRT sites. Overall, we suggest the FlpTRAP system as a tool to purify endogenous, specific chromatin loci from eukaryotic cells.

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5.

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individual proteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.

Hemoglobin polymerization via disulfide bond formation in the hypoxia-tolerant turtle Trachemys scripta: implications for antioxidant defense and O2 transport.

The ability of many reptilian hemoglobins (Hbs) to form high-molecular weight polymers, albeit known for decades, has not been investigated in detail. Given that turtle Hbs often contain a high number of cysteine (Cys), potentially contributing to the red blood cell defense against reactive oxygen species, we have examined whether polymerization of Hb could occur via intermolecular disulfide bonds in red blood cells of freshwater turtle Trachemys scripta, a species that is highly tolerant of hypoxia and oxidative stress. We find that one of the two Hb isoforms of the hemolysate HbA is prone to polymerization in vitro into linear flexible chains of different size that are visible by electron microscopy but not the HbD isoform. Polymerization of purified HbA is favored by hydrogen peroxide, a main cellular reactive oxygen species and a thiol oxidant, and inhibited by thiol reduction and alkylation, indicating that HbA polymerization is due to disulfide bonds. By using mass spectrometry, we identify Cys5 of the αA-subunit of HbA as specifically responsible for forming disulfide bonds between adjacent HbA tetramers. Polymerization of HbA does not affect oxygen affinity, cooperativity, and sensitivity to the allosteric cofactor ATP, indicating that HbA is still fully functional. Polymers also form in T. scripta blood after exposure to anoxia but not normoxia, indicating that they are of physiological relevance. Taken together, these results show that HbA polymers may form during oxidative stress and that Cys5αA of HbA is a key element of the antioxidant capacity of turtle red blood cells.

The effective mitigation of greenhouse gas emissions from rice paddies without compromising yield by early-season drainage.

Global rice production systems face two opposing challenges: the need to increase production to accommodate the world's growing population while simultaneously reducing greenhouse gas (GHG) emissions. Adaptations to drainage regimes are one of the most promising options for methane mitigation in rice production. Whereas several studies have focused on mid-season drainage (MD) to mitigate GHG emissions, early-season drainage (ED) varying in timing and duration has not been extensively studied. However, such ED periods could potentially be very effective since initial available C levels (and thereby the potential for methanogenesis) can be very high in paddy systems with rice straw incorporation. This study tested the effectiveness of seven drainage regimes varying in their timing and duration (combinations of ED and MD) to mitigate CH4 and N2O emissions in a 101-day growth chamber experiment. Emissions were considerably reduced by early-season drainage compared to both conventional continuous flooding (CF) and the MD drainage regime. The results suggest that ED+MD drainage may have the potential to reduce CH4 emissions and yield-scaled GWP by 85-90% compared to CF and by 75-77% compared to MD only. A combination of (short or long) ED drainage and one MD drainage episode was found to be the most effective in mitigating CH4 emissions without negatively affecting yield. In particular, compared with CF, the long early-season drainage treatments LE+SM and LE+LM significantly (p<0.01) decreased yield-scaled GWP by 85% and 87% respectively. This was associated with carbon being stabilised early in the season, thereby reducing available C for methanogenesis. Overall N2O emissions were small and not significantly affected by ED. It is concluded that ED+MD drainage might be an effective low-tech option for small-scale farmers to reduce GHG emissions and save water while maintaining yield.

Subclonal analysis in a lobular breast cancer with classical and solid growth pattern mimicking a solid-papillary carcinoma.

Recently, a new variant of invasive lobular breast cancer (ILBC) with solid-papillary-like growth pattern has been described. We present a case of ILBC with solid-papillary-like growth pattern in the main tumour mass and classical invasive lobular growth pattern in adjacent satellite foci. The two tumour components were subjected to comprehensive molecular analyses. Both components were ER/PR-positive, HER2-negative, and showed a complete loss of E-cadherin and beta-catenin protein expression, as determined by immunohistochemistry. Gene expression profiling classified the main tumour and a satellite focus as luminal-B and luminal-A subtypes, respectively. Whole-genome copy number profiles were highly similar in both tumour components. Shared copy number alterations (CNAs) included gains of chromosome 1q21.1-q43 and losses of chromosome 16q11.2-q24.3, the locus of the CDH1/E-cadherin tumour suppressor gene. CNAs detected only in the main tumour included a gain of chromosome 20q12-q13.33 and a loss of chromosome 1p36.33-p34.3, which has recently been associated with the solid variant of ILBC. Next generation sequencing revealed an identical, truncating CDH1 mutation (p.G169fs*5) in both tumour components confirming a common clonal ancestry. In conclusion, we confirm the recently described variant of ILBC with solid-papillary-like growth pattern and provide evidence that it evolves from classical ILBC by subclonal evolution.

Synergistic anti-cancer effects of epigenetic drugs on medulloblastoma cells.

PURPOSE: Medulloblastomas are aggressive brain malignancies. While considerable progress has been made in the treatment of medulloblastoma patients with respect to overall survival, these patients are still at risk of developing neurologic and cognitive deficits as a result of anti-cancer therapies. It is hypothesized that targeted molecular therapies represent a better treatment option for medulloblastoma patients. Therefore, the aim of the present study was to test a panel of epigenetic drugs for their effect on medulloblastoma cells under mild hypoxic conditions that reflect the physiological concentrations of oxygen in the brain. METHODS: Protein levels of histone deacetylase 1 (HDAC1) and DNA methyltransferase 1 (DNMT1) in medulloblastoma-derived cells (Daoy and D283 Med), as well as in developing and differentiated brain cells, were determined and compared. Class I and II histone deacetylase inhibitors (HDACi) and a DNMT inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), were applied to Daoy and D283 Med cells, and their effects were studied using viability, apoptosis and cancer sphere assays. RESULTS: We found that in HDAC1 and DNMT1 overexpressing medulloblastoma-derived cells, cell death was induced under various epigenetic drug conditions tested. At low HDACi concentrations, however, a pro-proliferative effect was observed. Parthenolide, a drug that affects cancer stem cells, was found to be efficient in inducing cell death in both cell lines tested. In contrast, we found that Daoy cells were more resistant to 5-aza-dC than D283 Med cells. When suberoylanilide hydroxamic acid (SAHA) and parthenolide were individually applied to both cell lines in combination with 5-aza-dC, a synergistic effect on cell survival was observed. CONCLUSIONS: Our current results suggest that the application of HDACi in combination with drugs that target DNMT may represent a promising option for the treatment of medulloblastoma.

Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target.

In human cells, thousands of predominantly neuronal genes are regulated by the repressor element 1 (RE1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). REST/NRSF represses transcription of these genes in stem cells and non-neuronal cells by tethering corepressor complexes. Aberrant REST/NRSF expression and intracellular localization are associated with cancer and neurodegeneration in humans. To date, detailed molecular analyses of REST/NRSF and its C-terminal repressor complex have been hampered largely by the lack of sufficient amounts of purified REST/NRSF and its complexes. Therefore, the aim of this study was to express and purify human REST/NRSF and its C-terminal interactors in a baculovirus multiprotein expression system as individual proteins and coexpressed complexes. All proteins were enriched in the nucleus, and REST/NRSF was isolated as a slower migrating form, characteristic of nuclear REST/NRSF in mammalian cells. Both REST/NRSF alone and its C-terminal repressor complex were functionally active in histone deacetylation and histone demethylation and bound to RE1/neuron-restrictive silencer element (NRSE) sites. Additionally, the mechanisms of inhibition of the small-molecule drugs 4SC-202 and SP2509 were analyzed. These drugs interfered with the viability of medulloblastoma cells, where REST/NRSF has been implicated in cancer pathogenesis. Thus, a resource for molecular REST/NRSF studies and drug development has been established.

Optimized preoperative fasting times decrease ketone body concentration and stabilize mean arterial blood pressure during induction of anesthesia in children younger than 36 months: a prospective observational cohort study.

BACKGROUND: In pediatric anesthesia, preoperative fasting guidelines are still often exceeded. OBJECTIVE: The objective of this noninterventional clinical observational cohort study was to evaluate the effect of an optimized preoperative fasting management (OPT) on glucose concentration, ketone bodies, acid-base balance, and change in mean arterial blood pressure (MAP) during induction of anesthesia in children. METHODS: Children aged 0-36 months scheduled for elective surgery with OPT (n = 50) were compared with peers studied before optimizing preoperative fasting time (OLD) (n = 50) who were matched for weight, age, and height. RESULTS: In children with OPT (n = 50), mean fasting time (6.0 ± 1.9 h vs 8.5 ± 3.5 h, P < 0.001), deviation from guideline (ΔGL) (1.2 ± 1.4 h vs 3.7 ± 3.1 h, P < 0.001, ΔGL>2 h 8% vs 70%), ketone bodies (0.2 ± 0.2 mmol·l(-1) vs 0.6 ± 0.6 mmol·l(-1) , P < 0.001), and incidence of hypotension (MAP <40 mmHg, 0 vs 5, P = 0.022) were statistically significantly lower and MAP after induction was statistically significantly higher (55.2 ± 9.5 mmHg vs 50.3 ± 9.8 mmHg, P = 0.015) as compared to children in the OLD (n = 50) group. Glucose, lactate, bicarbonate, base excess, and anion gap did not significantly differ. CONCLUSION: Optimized fasting times improve the metabolic and hemodynamic condition during induction of anesthesia in children younger than 36 months of age.

Use of human stem cells in Huntington disease modeling and translational research.

Huntington disease (HD) is a devastating neurological disorder caused by an extended CAG repeat in exon 1 of the gene that encodes the huntingtin (HTT) protein. HD pathology involves a loss of striatal medium spiny neurons (MSNs) and progressive neurodegeneration affects the striatum and other brain regions. Because HTT is involved in multiple cellular processes, the molecular mechanisms of HD pathogenesis should be investigated on multiple levels. On the cellular level, in vitro stem cell models, such as induced pluripotent stem cells (iPSCs) derived from HD patients and HD embryonic stem cells (ESCs), have yielded progress. Approaches to differentiate functional MSNs from ESCs, iPSCs, and neural stem/progenitor cells (NSCs/NPCs) have been established, enabling MSN differentiation to be studied and disease phenotypes to be recapitulated. Isolation of target stem cells and precursor cells may also provide a resource for grafting. In animal models, transplantation of striatal precursors differentiated in vitro to the striatum has been reported to improve disease phenotype. Initial clinical trials examining intrastriatal transplantation of fetal neural tissue suggest a more favorable clinical course in a subset of HD patients, though shortcomings persist. Here, we review recent advances in the development of cellular HD models and approaches aimed at cell regeneration with human stem cells. We also describe how genome editing tools could be used to correct the HTT mutation in patient-specific stem cells. Finally, we discuss the potential and the remaining challenges of stem cell-based approaches in HD research and therapy development.

Predictive chromosomal clusters of synchronous and metachronous brain metastases in clear cell renal cell carcinoma.

Synchronous (early) and metachronous (late) brain metastasis (BM) events of sporadic clear cell renal cell carcinoma (ccRCC) (n = 148) were retrospectively analyzed using comparative genomic hybridization (CGH). Using oncogenetic tree models and cluster analyses, chromosomal imbalances related to recurrence-free survival until BM (RFS-BM) were analyzed. Losses at 9p and 9q appeared to be hallmarks of metachronous BM events, whereas an absence of detectable chromosomal changes at 3p was often associated with synchronous BM events. Correspondingly, k-means clustering showed that cluster 1 cases generally exhibited low copy number chromosomal changes that did not involve 3p. Cluster 2 cases had a high occurrence of -9p/-9q (94-98%) deletions, whereas cluster 3 cases had a higher frequency of copy number changes, including loss at chromosome 14 (80%). The higher number of synchronous cases in cluster 1 was also associated with a significantly shorter RFS-BM compared with clusters 2 and 3 (P = 0.02). Conversely, a significantly longer RFS-BM was observed for cluster 2 versus clusters 1 and 3 (P = 0.02). Taken together, these data suggest that metachronous BM events of ccRCC are characterized by loss of chromosome 9, whereas synchronous BM events may form independently of detectable genetic changes at chromosomes 9 and 3p.

Etiologic influence on chromosomal aberrations in European hepatocellular carcinoma identified by CGH.

Previous studies suggest different pathways in the molecular development of hepatocellular carcinoma (HCC). We investigated the pattern of chromosomal imbalances in HCC depending on the type of underlying liver disease as detected by comparative genomic hybridization in 67 cases of primary HCC occurring in non-cirrhotic livers (n=30), in liver cirrhosis (LC) related to alcohol intake (n=9), cryptogenic or metabolic changes (n=11), and chronic viral hepatitis B or C (n=17). HCC were treated by liver resection in 48 patients and transplantation in 19 patients. The 10-year disease-free and overall survival rates were 51% and 68%, respectively. The copy number changes occurring in more than 10% of cases were gains at 8q (55%), 1q (49%), 7q (15%), 7p (13%), 6p (12%), and 20q (12%), as well as losses at 8p (55%), 4q (33%), 6q (33%), 13q (25%), 14q (24%), 17p (22%), 16q (19%), 1p (18%), 18q (16%), 9p (13%), 10q (13%), 4p (12%), and 9q (12%). HCC arising in alcoholic LC showed a different pattern with significantly fewer net changes (p=0.008), particularly fewer chromosomal gains (p=0.008) and fewer breakpoints (p=0.003) compared to the other investigated HCC subgroups. Future clinical studies should evaluate the prognostic relevance of these findings.

Three-dimensional reconstruction of Trypanosoma brucei editosomes using single-particle electron microscopy.

RNA editing within the mitochondria of kinetoplastid protozoa is performed by a multicomponent -macromolecular machine known as the editosome. Editosomes are high molecular mass protein assemblies that consist of about 15-25 individual polypeptides. They bind pre-edited transcripts and convert them into translation-competent mRNAs through a biochemical reaction cycle of enzyme-catalyzed steps. At steady-state conditions, several distinct complexes can be purified from mitochondrial detergent lysates. They likely represent RNA editing complexes at different assembly stages or at different functional stages of the processing reaction. Due to their low cellular abundance, single-particle electron microscopy (EM) represents the method of choice for their structural characterization. This chapter describes a set of techniques suitable for the purification and structural characterization of RNA editing complexes by single-particle EM. The RNA editing complexes are isolated from the endogenous pool of mitochondrial complexes by tandem-affinity purification (TAP). Since the TAP procedure results in the isolation of a mixture of different RNA editing complexes, the isolates are further subjected to an isokinetic ultracentrifugation step to separate the complexes based on their sedimentation behavior. The use of the "GraFix" protocol is presented that combines mild chemical cross-linking with ultracentrifugation. Different sample preparation protocols including negative staining, cryo-negative staining, and unstained cryotechniques as well as the single-particle image processing of electron microscopical images are described.