Inhibiting glutamine utilization creates a synthetic lethality for suppression of ATP citrate lyase in KRas-driven cancer cells.

Metabolic reprogramming is now considered a hallmark of cancer cells. KRas-driven cancer cells use glutaminolysis to generate the tricarboxylic acid cycle intermediate α-ketoglutarate via a transamination reaction between glutamate and oxaloacetate. We reported previously that exogenously supplied unsaturated fatty acids could be used to synthesize phosphatidic acid-a lipid second messenger that activates both mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTOR complex 2 (mTORC2). A key target of mTORC2 is Akt-a kinase that promotes survival and regulates cell metabolism. We report here that mono-unsaturated oleic acid stimulates the phosphorylation of ATP citrate lyase (ACLY) at the Akt phosphorylation site at S455 in an mTORC2 dependent manner. Inhibition of ACLY in KRas-driven cancer cells in the absence of serum resulted in loss of cell viability. We examined the impact of glutamine (Gln) deprivation in combination with inhibition of ACLY on the viability of KRas-driven cancer cells. While Gln deprivation was somewhat toxic to KRas-driven cancer cells by itself, addition of the ACLY inhibitor SB-204990 increased the loss of cell viability. However, the transaminase inhibitor aminooxyacetate was minimally toxic and the combination of SB-204990 and aminooxtacetate led to significant loss of cell viability and strong cleavage of poly-ADP ribose polymerase-indicating apoptotic cell death. This effect was not observed in MCF7 breast cancer cells that do not have a KRas mutation or in BJ-hTERT human fibroblasts which have no oncogenic mutation. These data reveal a synthetic lethality between inhibition of glutamate oxaloacetate transaminase and ACLY inhibition that is specific for KRas-driven cancer cells and the apparent metabolic reprogramming induced by activating mutations to KRas.

Lessons Learned From Five Years of Newborn Screening for Severe Combined Immunodeficiency in Israel.

BACKGROUND: Implementation of newborn screening (NBS) programs for severe combined immunodeficiency (SCID) have advanced the diagnosis and management of affected infants and undoubtedly improved their outcomes. Reporting long-term follow-up of such programs is of great importance. OBJECTIVE: We report a 5-year summary of the NBS program for SCID in Israel. METHODS: Immunologic and genetic assessments, clinical analyses, and outcome data from all infants who screened positive were evaluated and summarized. RESULTS: A total of 937,953 Guthrie cards were screened for SCID. A second Guthrie card was requested on 1,169 occasions (0.12%), which resulted in 142 referrals (0.015%) for further validation tests. Flow cytometry immune-phenotyping, T cell receptor excision circle measurement in peripheral blood, and expression of TCRVß repertoire for the validation of positive cases revealed a specificity and sensitivity of 93.7% and 75.9%, respectively, in detecting true cases of SCID. Altogether, 32 SCID and 110 non-SCID newborns were diagnosed, making the incidence of SCID in Israel as high as 1:29,000 births. The most common genetic defects in this group were associated with mutations in DNA cross-link repair protein 1C and IL-7 receptor α (IL-7Rα) genes. No infant with SCID was missed during the study time. Twenty-two SCID patients underwent hematopoietic stem cell transplantation, which resulted in a 91% survival rate. CONCLUSIONS: Newborn screening for SCID should ultimately be applied globally, specifically to areas with high rates of consanguineous marriages. Accumulating data from follow-up studies on NBS for SCID will improve diagnosis and treatment and enrich our understanding of immune development in health and disease.

Algorithmic prediction of failure modes in healthcare.

BACKGROUND: Preventing medical errors is crucial, especially during crises like the COVID-19 pandemic. Failure Modes and Effects Analysis (FMEA) is the most widely used prospective hazard analysis in healthcare. FMEA relies on brainstorming by multi-disciplinary teams to identify hazards. This approach has two major weaknesses: significant time and human resource investments, and lack of complete and error-free results. OBJECTIVES: To introduce the algorithmic prediction of failure modes in healthcare (APFMH) and to examine whether APFMH is leaner in resource allocation in comparison to the traditional FMEA and whether it ensures the complete identification of hazards. METHODS: The patient identification during imaging process at the emergency department of Sheba Medical Center was analyzed by FMEA and APFMH, independently and separately. We compared between the hazards predicted by APFMH method and the hazards predicted by FMEA method; the total participants' working hours invested in each process and the adverse events, categorized as 'patient identification', before and after the recommendations resulted from the above processes were implemented. RESULTS: APFMH is more effective in identifying hazards (P < 0.0001) and is leaner in resources than the traditional FMEA: the former used 21 h whereas the latter required 63 h. Following the implementation of the recommendations, the adverse events decreased by 44% annually (P = 0.0026). Most adverse events were preventable, had all recommendations been fully implemented. CONCLUSION: In light of our initial and limited-size study, APFMH is more effective in identifying hazards (P < 0.0001) and is leaner in resources than the traditional FMEA. APFMH is suggested as an alternative to FMEA since it is leaner in time and human resources, ensures more complete hazard identification and is especially valuable during crisis time, when new protocols are often adopted, such as in the current days of the COVID-19 pandemic.

Styloid Process Elongation on Cervical Spine Computed Tomography is Associated with the Enthesopathy-Related Diseases of Ankylosing Spondylitis and Diffuse Idiopathic Skeletal Hyperostosis.

BACKGROUND: Enthesopathy may lead to calcification of the stylohyoid ligament and can cause elongation of the styloid process (SP). OBJECTIVES: To evaluate whether SP elongation is associated with two common enthesitis-related diseases: ankylosing spondylitis (AS) and diffuse idiopathic skeletal hyperostosis (DISH). METHODS: Cervical spine computed tomography (CT) examinations of patients with DISH (n=64, Resnick criteria), AS (n=24, New York criteria) and a controls (no radiological signs of DISH or AS, n=54) were retrospectively evaluated. The DISH group was further divided into patients with and without cervical DISH. The length of right and left SP was measured independently by two readers on coronal and sagittal curved reformats. The average right and left styloid length and average length per person were compared among the groups. RESULTS: Demographic characteristics were similar between the DISH and control groups (average age 68.2 ± 15.7, 69.2 ± 12.7 years, male:female ratio 48:16 and 35:19, respectively, P > 0.05), whereas age was significantly lower (average age: 53 ± 15 years, P < 0.0001) in the AS group, which was also composed mainly of men. The AS and DISH groups had significantly longer SP compared to controls (AS 37.9 ± 9.6 mm, DISH 34.4 ± 9 mm, control 30.3 ± 10.1 mm, P < 0.05). There was no correlation between age and SP length. Inter-reader reliability of SP measurements was excellent in all groups (ICC = 0.998, P < 0.0001). CONCLUSIONS: SP elongation is associated with both AS and DISH substantiating the enthesopathy-related pathophysiology of this finding.

Antagonistic effects of doublecortin and MARK2/Par-1 in the developing cerebral cortex.

Abnormal neuronal migration is manifested in brain malformations such as lissencephaly. The impairment in coordinated cell motility likely reflects a faulty mechanism of cell polarization or coupling between polarization and movement. Here we report on the relationship between the polarity kinase MARK2/Par-1 and its substrate, the well-known lissencephaly-associated gene doublecortin (DCX), during cortical radial migration. We have previously shown using in utero electroporation that reduced MARK2 levels resulted in multipolar neurons stalled at the intermediate zone border, similar to the phenotype observed in the case of DCX silencing. However, whereas reduced MARK2 stabilized microtubules, we show here that knock-down of DCX increased microtubule dynamics. This led to the hypothesis that simultaneous reduction may alleviate the phenotype. Coreduction of MARK2 and DCX resulted in a partial restoration of the normal neuronal migration phenotype in vivo. The kinetic behavior of the centrosomes reflected the different molecular mechanisms activated when either protein was reduced. In the case of reducing MARK2 processive motility of the centrosome was hindered, whereas when DCX was reduced, centrosomes moved quickly but bidirectionally. Our results stress the necessity for successful coupling between the polarity pathway and cytoplasmic dynein-dependent activities for proper neuronal migration.

Involvement of Na(+), K(+)-ATPase and endogenous digitalis-like compounds in depressive disorders.

BACKGROUND: Sodium and potassium-activated adenosine triphosphatase (Na(+), K(+)-ATPase) and endogenous digitalis-like compounds (DLC) in the brain have been implicated in the pathogenesis of mood disorders. This hypothesis was examined by the determination of Na(+), K(+)-ATPase/DLC system in parietal cortex of patients with different mood disorders and two animal models of depression. METHODS: Na(+), K(+)-ATPase concentrations in human brain synaptosomal fractions, from patients with mood disorders, schizophrenia, and normal individuals, were determined by (3)H-ouabain binding assay. Alpha isoforms were quantified by Western blotting. Brain DLC were measured using sensitive enzyme linked immunosorbant assay (ELISA). The effects of ouabain and ouabain-antibodies on behavior were determined in two animal models of depression. RESULTS: (3)H-ouabain binding in bipolar patients was significantly lower than in major depressed and schizophrenic patients. Na(+), K(+)-ATPase alpha isoforms in synaptosomal fractions were not different among the groups. DLC levels in the parietal cortex of bipolar patients were significantly higher than in normal individuals and depressed patients. Injection of lipopolysaccharide (intraperitoneally) to rats elicited depression-like symptoms, which were significantly attenuated by pre-injection of ouabain-antibodies. Injection of ouabain and ouabain-antibodies (intracerebroventricular) reduced depression-like symptoms in the forced swimming test in rats. CONCLUSIONS: The results support the possibility that Na(+), K(+)-ATPase and endogenous DLC participate in the pathogenesis of depressive disorders.