IKKß promotes metabolic adaptation to glutamine deprivation via phosphorylation and inhibition of PFKFB3.

Glutamine is an essential nutrient for cancer cell survival and proliferation. Enhanced utilization of glutamine often depletes its local supply, yet how cancer cells adapt to low glutamine conditions is largely unknown. Here, we report that IκB kinase ß (IKKß) is activated upon glutamine deprivation and is required for cell survival independently of NF-κB transcription. We demonstrate that IKKß directly interacts with and phosphorylates 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase isoform 3 (PFKFB3), a major driver of aerobic glycolysis, at Ser269 upon glutamine deprivation to inhibit its activity, thereby down-regulating aerobic glycolysis when glutamine levels are low. Thus, due to lack of inhibition of PFKFB3, IKKß-deficient cells exhibit elevated aerobic glycolysis and lactate production, leading to less glucose carbons contributing to tricarboxylic acid (TCA) cycle intermediates and the pentose phosphate pathway, which results in increased glutamine dependence for both TCA cycle intermediates and reactive oxygen species suppression. Therefore, coinhibition of IKKß and glutamine metabolism results in dramatic synergistic killing of cancer cells both in vitro and in vivo. In all, our results uncover a previously unidentified role of IKKß in regulating glycolysis, sensing low-glutamine-induced metabolic stress, and promoting cellular adaptation to nutrient availability.

The B56α subunit of PP2A is necessary for mesenchymal stem cell commitment to adipocyte.

Adipose tissue plays a major role in maintaining organismal metabolic equilibrium. Control over the fate decision from mesenchymal stem cells (MSCs) to adipocyte differentiation involves coordinated command of phosphorylation. Protein phosphatase 2A plays an important role in Wnt pathway and adipocyte development, yet how PP2A complexes actively respond to adipocyte differentiation signals and acquire specificity in the face of the promiscuous activity of its catalytic subunit remains unknown. Here, we report the PP2A phosphatase B subunit B56α is specifically induced during adipocyte differentiation and mediates PP2A to dephosphorylate GSK3ß, thereby blocking Wnt activity and driving adipocyte differentiation. Using an inducible B56α knock-out mouse, we further demonstrate that B56α is essential for gonadal adipose tissue development in vivo and required for the fate decision of adipocytes over osteoblasts. Moreover, we show B56α expression is driven by the adipocyte transcription factor PPARγ thereby establishing a novel link between PPARγ signaling and Wnt blockade. Overall, our results reveal B56α is a necessary part of the machinery dictating the transition from pre-adipocyte to mature adipocyte and provide fundamental insights into how PP2A complex specifically and actively regulates unique signaling pathway in biology.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

Micronutrient Status and Nutritional Intake in 0- to 2-Year-old Children Consuming a Cows' Milk Exclusion Diet.

OBJECTIVES: To study micronutrient status and nutritional intake from complementary feeding in children on a cows' milk exclusion (CME) diet. METHODS: Fifty-seven children with cows' milk allergy, younger than 2 years, were included in a cross-sectional study. Blood was analyzed for micronutrient status. Complementary feeding was defined as all solids and liquids except of breast milk, and assessed by 3-day food diary. The results were analyzed according to 3 feeding patterns: mainly breast-fed (mBF), partially breast-fed, and no breast milk group (nBM). RESULTS: The children had a median age of 9 months and micronutrient status was within normal range for total homocysteine (p-tHcy), s-B12, s-folate, b-Hb, s-ferritin, s-zinc, and s-25(OH)D. There were no significant differences between feedings groups, except for B12-biomarkers. The mBF had higher p-tHcy (P < 0.000) and lower s-B12 (P = 0.002) compared nBM. Vitamin B12 deficiency (p-tHcy >6.5 µmol/L combined with s-B12 <250 pmol/L) was found in 12% of participants, most frequently among the mBF (36%) and none in nBM group (P = 0.009). Vitamin B12 intake from complementary feeding was negatively correlated with p-tHcy (r = -0.479, P = 0.001) and positively with s-B12 (r = 0.410, P = 0.003). Iron deficiency anemia was found in 5%. Iron intake correlated positively with b-Hb (r = 0.324, P = 0.02). Zinc deficiency was found in 7% and low 25(OH)D in 9%. Vitamin D intake was positively correlated with the use of supplements (r = 0.456, P = 0.001). CONCLUSION: The risk of B12 deficiency was high in mBF infants on CME diet, and complementary feeding was associated with better B12 status. Iron, zinc, and vitamin D deficiencies were present in all feeding groups. Complementary feeding should be introduced at 4 to 6 months of age. Vitamin D supplement is recommended to ensure adequate intake.

Typhoid Fever Diagnosis in Endemic Countries: A Clog in the Wheel of Progress?

Typhoid fever causes significant morbidity and mortality in developing countries, with inaccurate estimates in some countries affected, especially those situated in Sub-Saharan Africa. Disease burden assessment is limited by lack of a high degree of sensitivity and specificity by many current rapid diagnostic tests. Some of the new technologies, such as PCR and proteomics, may also be useful but are difficult for low-resource settings to apply as point-of-care diagnostics. Weak laboratory surveillance systems may also contribute to the spread of multidrug resistant Salmonella serovar Typhi across endemic areas. In addition, most typhoid-endemic countries employ serological tests that have low sensitivity and specificity making diagnosis unreliable. Here we review currently available typhoid fever diagnostics, and advances in serodiagnosis of S. Typhi.

Genomic analysis of sexual dimorphism of gene expression in the mouse adrenal gland.

A relevant gender difference exists in adrenal physiology and propensity to disease. In mice, a remarkable sexual dimorphism is present in several components of the hypothalamic-pituitary-adrenal axis, with females displaying higher adrenal weight, plasma ACTH, corticosterone, and aldosterone levels than males. The molecular bases of this sexual dimorphism are little known. We have compared global gene expression profiles in males vs. female mouse adrenal glands and also studied the effect that testosterone treatment and castration have on adrenal gene expression in female vs. male mice, respectively. Our study evidenced a set of 71 genes that are coordinately modulated according to sex and hormonal treatments and represent the core sexually dimorphic expression program in the mouse adrenal gland. Moreover, we show that some genes involved in steroid metabolism have a remarkable sexual dimorphic expression and identify new potential markers for the adrenal X-zone, a transitory cellular layer in the inner adrenal cortex, which spontaneously regresses at puberty in males and during the first pregnancy in females and has an uncertain physiological role. Finally, sexually dimorphic expression of the transcriptional regulators Nr5a1 and Nr0b1 may explain at least in part the differences in adrenal steroidogenesis between sexes.

Prognostic significance of residual tumor in patients with epithelial ovarian carcinoma stage IV in a 20 year perspective.

OBJECTIVE: We aimed to evaluate prognostic factors impacting on overall survival during a 20 year period with substantial changes in surgical approach and chemotherapy management of patients with epithelial ovarian carcinoma stage IV. DESIGN: A retrospective population-based study. SETTING: The Norwegian Radium Hospital during 1985-2005. POPULATION: Three hundred and ninety-four patients with epithelial ovarian carcinoma stage IV treated at the Norwegian Radium Hospital. METHODS: The cohort was divided into two groups (1985-1995 and 1996-2005), and clinical and pathological characteristics were compared. Univariate and multivariate analyses were performed to identify prognostic factors during 1985-1995, 1996-2005 and 1985-2005. MAIN OUTCOME MEASURES: Prognostic factors and overall survival in the three periods. RESULTS: Median overall survival improved from 1985-1995 to 1996-2005 (from 1.3 to 2.1 years). More patients had macroscopic radical surgery (28 vs. 11%), received neoadjuvant chemotherapy and were treated with platinum-taxane combination therapy from 1996-2005 compared to 1985-1995. Patients with primary surgery had improved median overall survival from 1996-2005 compared to 1985-1995. In multivariate analyses, surgical approach was not a prognostic factor for overall survival, but chemotherapy was during 1985-2005. Postoperative residual tumor was a prognostic factor for overall survival in all periods. CONCLUSIONS: Macroscopic radical surgery is a strong prognostic factor for overall survival and is achievable in a subset of patients with epithelial ovarian carcinoma stage IV. Improved selection criteria for what treatment algorithm to choose for patients with epithelial ovarian carcinoma stage IV are warranted.

Perceptions and Likelihood of Medical Students to Pursue a Career in Neurosurgery: Findings from the Philippines.

BACKGROUND: The number of neurosurgeons in the Philippines is less than ideal for its population, so there is a need to recruit and train more. This study aimed to determine medical students' perception of neurosurgery and their likelihood of pursuing a career in this specialty. METHODS: A cross-sectional survey was conducted of medical students in the Philippines to assess their perceptions and likelihood of pursuing a career in neurosurgery. Data obtained were analyzed using descriptive statistics and a χ2 test with a significance level of 0.05. RESULTS: A total of 627 medical students completed the survey, with a response rate of 72.4%. The mean age was 23.8 years, and almost half (49.1%) were women. They had mostly negative perceptions of neurosurgery, and only 18.7% were likely to pursue a career in this specialty. Reasons included poor work-life balance, poor understanding of neuroscience and neurosurgery, and the self-perceived lack of manual dexterity. The most common sources of their perceptions included lectures, movies, and neurosurgery residents and consultants whom they met in hospital. Previous exposure to neurosurgery lectures or rotations, going back to their hometown to work, and having a physician parent were associated with a higher likelihood of pursuing neurosurgery. CONCLUSIONS: Our study showed that medical students' perceptions of neurosurgery were generally negative and that only 18.7% were likely to pursue it as a career. Major changes would have to be made to improve students' perceptions to attract more students to the field and increase the neurosurgical workforce.

Hydranencephaly: Clinical Features and Survivorship in a Retrospective Cohort.

OBJECTIVE: Hydranencephaly is a congenital central nervous system disorder characterized by the complete or near-complete absence of the cerebral cortex and basal ganglia. Because of its rarity, data on the clinical features and survivorship remain sparse. We aim to determine the clinical features and survivorship of a cohort of patients with hydranencephaly. METHODS: We performed a retrospective cohort study of all patients diagnosed with hydranencephaly at our institution from 2008 to 2018. Data on demographics, clinical features, presence of comorbidities, surgical operations performed, and status on last follow-up were collected. Survival curves were generated using Kaplan-Meier analysis. RESULTS: Fifty patients were included in the cohort, who had a median age at diagnosis of 4 months and a female predilection. The most common clinical manifestations were macrocephaly (92%) and seizures or myoclonic movements. Infection was present in 36% of cases, endocrinopathies in 22%, dysmorphisms in 20%, and cardiac disease in 8%. Twenty patients underwent shunt insertion, with half developing a postoperative complication at a mean follow-up of 14.9 months. The median survival of the cohort was not reached at 7.5 years. Among the patients with follow-up, characteristics were similar between the surgical and nonsurgical groups, except for the greater incidence of infections in the nonsurgical group. The survival curves among the groups were significantly different, with a hazard ratio of 3.731 in the nonsurgical group. CONCLUSIONS: In this large single-center retrospective cohort of patients with hydranencephaly, novel findings are presented regarding the clinical manifestations and survivorship of this condition.

Dietary glutamine supplementation suppresses epigenetically-activated oncogenic pathways to inhibit melanoma tumour growth.

Tumour cells adapt to nutrient deprivation in vivo, yet strategies targeting the nutrient poor microenvironment remain unexplored. In melanoma, tumour cells often experience low glutamine levels, which promote cell dedifferentiation. Here, we show that dietary glutamine supplementation significantly inhibits melanoma tumour growth, prolongs survival in a transgenic melanoma mouse model, and increases sensitivity to a BRAF inhibitor. Metabolomic analysis reveals that dietary uptake of glutamine effectively increases the concentration of glutamine in tumours and its downstream metabolite, αKG, without increasing biosynthetic intermediates necessary for cell proliferation. Mechanistically, we find that glutamine supplementation uniformly alters the transcriptome in tumours. Our data further demonstrate that increase in intra-tumoural αKG concentration drives hypomethylation of H3K4me3, thereby suppressing epigenetically-activated oncogenic pathways in melanoma. Therefore, our findings provide evidence that glutamine supplementation can serve as a potential dietary intervention to block melanoma tumour growth and sensitize tumours to targeted therapy via epigenetic reprogramming.

α-Ketoglutarate attenuates Wnt signaling and drives differentiation in colorectal cancer.

Genetic-driven deregulation of the Wnt pathway is crucial but not sufficient for colorectal cancer (CRC) tumourigenesis. Here, we show that environmental glutamine restriction further augments Wnt signaling in APC mutant intestinal organoids to promote stemness and leads to adenocarcinoma formation in vivo via decreasing intracellular alpha-ketoglutarate (aKG) levels. aKG supplementation is sufficient to rescue low-glutamine induced stemness and Wnt hyperactivation. Mechanistically, we found that aKG promotes hypomethylation of DNA and histone H3K4me3, leading to an upregulation of differentiation-associated genes and downregulation of Wnt target genes, respectively. Using CRC patient-derived organoids and several in vivo CRC tumour models, we show that aKG supplementation suppresses Wnt signaling and promotes cellular differentiation, thereby significantly restricting tumour growth and extending survival. Together, our results reveal how metabolic microenvironment impacts Wnt signaling and identify aKG as a potent antineoplastic metabolite for potential differentiation therapy for CRC patients.

Transcriptional diversity and bioenergetic shift in human breast cancer metastasis revealed by single-cell RNA sequencing.

Although metastasis remains the cause of most cancer-related mortality, mechanisms governing seeding in distal tissues are poorly understood. Here, we establish a robust method for the identification of global transcriptomic changes in rare metastatic cells during seeding using single-cell RNA sequencing and patient-derived-xenograft models of breast cancer. We find that both primary tumours and micrometastases display transcriptional heterogeneity but micrometastases harbour a distinct transcriptome program conserved across patient-derived-xenograft models that is highly predictive of poor survival of patients. Pathway analysis revealed mitochondrial oxidative phosphorylation as the top pathway upregulated in micrometastases, in contrast to higher levels of glycolytic enzymes in primary tumour cells, which we corroborated by flow cytometric and metabolomic analyses. Pharmacological inhibition of oxidative phosphorylation dramatically attenuated metastatic seeding in the lungs, which demonstrates the functional importance of oxidative phosphorylation in metastasis and highlights its potential as a therapeutic target to prevent metastatic spread in patients with breast cancer.

Correction: The anti-metastatic activity of collagenase-2 in breast cancer cells is mediated by a signaling pathway involving decorin and miR-21.

The original microRNA hybridization data for this article, which has been available for the scientific community upon request, has now been deposited in the GEO repository under accession number GSE124432.

p53 Promotes Cancer Cell Adaptation to Glutamine Deprivation by Upregulating Slc7a3 to Increase Arginine Uptake.

Cancer cells heavily depend on the amino acid glutamine to meet the demands associated with growth and proliferation. Due to the rapid consumption of glutamine, cancer cells frequently undergo glutamine starvation in vivo. We and others have shown that p53 is a critical regulator in metabolic stress resistance. To better understand the molecular mechanisms by which p53 activation promotes cancer cell adaptation to glutamine deprivation, we identified p53-dependent genes that are induced upon glutamine deprivation by using RNA-seq analysis. We show that Slc7a3, an arginine transporter, is significantly induced by p53. We also show that increased intracellular arginine levels following glutamine deprivation are dependent on p53. The influx of arginine has minimal effects on known metabolic pathways upon glutamine deprivation. Instead, we found arginine serves as an effector for mTORC1 activation to promote cell growth in response to glutamine starvation. Therefore, we identify a p53-inducible gene that contributes to the metabolic stress response.

Urogenital schistosomiasis is associated with signatures of microbiome dysbiosis in Nigerian adolescents.

Urogenital schistosomiasis is a neglected tropical disease caused by the parasite Schistosoma haematobium, which resides in the vasculature surrounding the urogenital system. Previous work has suggested that helminthic infections can affect the intestinal microbiome, and we hypothesized that S. haematobium infection could result in an alteration of immune system-microbiota homeostasis and impact the composition of the gut microbiota. To address this question, we compared the fecal microbiomes of infected and uninfected schoolchildren from the Argungu Local Government Area of Kebbi State, Nigeria, detecting significant differences in community composition between the two groups. Most remarkably, we observed a decreased abundance of Firmicutes and increased abundance of Proteobacteria - a shift in community structure which has been previously associated with dysbiosis. More specifically, we detected a number of changes in lower taxa reminiscent of inflammation-associated dysbiosis, including decreases in Clostridiales and increases in Moraxellaceae, Veillonellaceae, Pasteurellaceae, and Desulfovibrionaceae. Functional potential analysis also revealed an enrichment in orthologs of urease, which has been linked to dysbiosis and inflammation. Overall, our analysis indicates that S. haematobium infection is associated with perturbations in the gut microbiota and may point to microbiome disruption as an additional consequence of schistosome infection.

MiR-135 suppresses glycolysis and promotes pancreatic cancer cell adaptation to metabolic stress by targeting phosphofructokinase-1.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. It thrives in a nutrient-poor environment; however, the mechanisms by which PDAC cells undergo metabolic reprogramming to adapt to metabolic stress are still poorly understood. Here, we show that microRNA-135 is significantly increased in PDAC patient samples compared to adjacent normal tissue. Mechanistically, miR-135 accumulates specifically in response to glutamine deprivation and requires ROS-dependent activation of mutant p53, which directly promotes miR-135 expression. Functionally, we found miR-135 targets phosphofructokinase-1 (PFK1) and inhibits aerobic glycolysis, thereby promoting the utilization of glucose to support the tricarboxylic acid (TCA) cycle. Consistently, miR-135 silencing sensitizes PDAC cells to glutamine deprivation and represses tumor growth in vivo. Together, these results identify a mechanism used by PDAC cells to survive the nutrient-poor tumor microenvironment, and also provide insight regarding the role of mutant p53 and miRNA in pancreatic cancer cell adaptation to metabolic stresses.

Gene expression profiling of human liver transplants identifies an early transcriptional signature associated with initial poor graft function.

Liver ischemia-reperfusion injury occurring in orthotopic liver transplantation (OLT) may be responsible for early graft failure. Molecular mechanisms underlying initial poor graft function (IPGF) have been poorly documented in human. The purpose of this study was to identify the major transcriptional alterations occurring in human livers during OLT. Twenty-one RNA extracts derived from liver transplant biopsies taken after graft reperfusion were compared with 7 RNA derived from normal control livers. Three hundred seventy-one genes were significantly modulated and classified in molecular pathways relevant to liver metabolism, inflammatory response, cell proliferation and liver protection. Grafts were then subdivided into two groups based on their peak levels of serum aspartate amino transferase within 72 h after OLT (group 1, non-IPGF: 14 patients; group 2, IPGF: 7 patients). The two corresponding data sets were compared using a supervised prediction method. A new set of genes able to correctly classify 71% of the patients was defined. These genes were functionally associated with oxidative stress, inflammation and inhibition of cell proliferation. This study provides a comprehensive picture of the transcriptional events associated with human OLT and IPGF. We anticipate that such alterations provide a framework for the elucidation of the molecular mechanisms leading to IPGF.

IKKß activates p53 to promote cancer cell adaptation to glutamine deprivation.

One of the hallmarks of cancer is the ability to reprogram cellular metabolism to increase the uptake of necessary nutrients such as glucose and glutamine. Driven by oncogenes, cancer cells have increased glutamine uptake to support their highly proliferative nature. However, as cancer cells continue to replicate and grow, they lose access to vascular tissues and deplete local supply of nutrients and oxygen. We previously showed that many tumor cells situate in a low glutamine microenvironment in vivo, yet the mechanisms of how they are able to adapt to this metabolic stress are still not fully understood. Here, we report that IκB-kinase ß (IKKß) is needed to promote survival and its activation is accompanied by phosphorylation of the metabolic sensor, p53, in response to glutamine deprivation. Knockdown of IKKß decreases the level of wild-type and mutant p53 phosphorylation and its transcriptional activity, indicating a novel relationship between IKKß and p53 in mediating cancer cell survival in response to glutamine withdrawal. Phosphopeptide mass spectrometry analysis further reveals that IKKß phosphorylates p53 on Ser392 to facilitate its activation upon glutamine deprivation, independent of the NF-κB pathway. The results of this study offer an insight into the metabolic reprogramming in cancer cells that is dependent on a previously unidentified IKKß-p53 signaling axis in response to glutamine depletion. More importantly, this study highlights a new therapeutic strategy for cancer treatment and advances our understanding of adaptive mechanisms that could lead to resistance to current glutamine targeting therapies.

First clinical symptom as a prognostic factor in systemic sclerosis: results of a retrospective nationwide cohort study.

The objective of the study is to determine the importance of the mode of onset as prognostic factor in systemic sclerosis (SSc). Data were collected from the Spanish Scleroderma Registry (RESCLE), a nationwide retrospective multicenter database created in 2006. As first symptom, we included Raynaud's phenomenon (RP), cutaneous sclerosis, arthralgia/arthritis, puffy hands, interstitial lung disease (ILD), pulmonary arterial hypertension (PAH), and digestive hypomotility. A total of 1625 patients were recruited. One thousand three hundred forty-two patients (83%) presented with RP as first symptom and 283 patients (17%) did not. Survival from first symptom in those patients with RP mode of onset was higher at any time than those with onset as non-Raynaud's phenomenon: 97 vs. 90% at 5 years, 93 vs. 82% at 10 years, 83 vs. 62% at 20 years, and 71 vs. 50% at 30 years (p < 0.001). In multivariate analysis, factors related to mortality were older age at onset, male gender, dcSSc subset, ILD, PAH, scleroderma renal crisis (SRC), heart involvement, and the mode of onset with non-Raynaud's phenomenon, especially in the form of puffy hands or pulmonary involvement. The mode of onset should be considered an independent prognostic factor in systemic sclerosis and, in particular, patients who initially present with non-Raynaud's phenomenon may be considered of poor prognosis.

A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis.

The K562 cell line serves as a model to study the molecular mechanisms associated with leukemia differentiation. We show here that cotreatment of K562 cells with PMA and low doses of SB202190 (SB), an inhibitor of the p38 MAPK pathway, induced a majority of cells to differentiate towards the megakaryocytic lineage. Electronic microscopy analysis showed that K562 cells treated with PMA+SB exhibited characteristic features of physiological megakaryocytic differentiation including the presence of vacuoles and demarcation membranes. Differentiation was also accompanied by a net increase in megakaryocytic markers and a reduction of erythroid markers, especially when both effectors were present. PMA effect was selectively mediated by new PKC isoforms. Differentiation of K562 cells by the combination of PMA and SB required Erk1/2 activation, a threshold of JNK activation and p38 MAPK inhibition. Interestingly, higher concentrations of SB, which drastically activated JNK, blocked megakaryocytic differentiation, and considerably increased cell death in the presence of PMA. c-DNA microarray membranes and PCR analysis allow us to identify a set of genes modulated during PMA-induced K562 cell differentiation. Several gene families identified in our screening, including ephrins receptors and some angiogenic factors, had never been reported so far to be regulated during megakaryocytic differentiation.