Clonal KEAP1 mutations with loss of heterozygosity share reduced immunotherapy efficacy and low immune cell infiltration in lung adenocarcinoma.

BACKGROUND: KEAP1 mutations have been associated with reduced survival in lung adenocarcinoma (LUAD) patients treated with immune checkpoint inhibitors (ICIs), particularly in the presence of STK11/KRAS alterations. We hypothesized that, beyond co-occurring genomic events, clonality prediction may help identify deleterious KEAP1 mutations and their counterparts with retained sensitivity to ICIs. PATIENTS AND METHODS: Beta-binomial modelling of sequencing read counts was used to infer KEAP1 clonal inactivation by combined somatic mutation and loss of heterozygosity (KEAP1 C-LOH) versus partial inactivation [KEAP1 clonal diploid-subclonal (KEAP1 CD-SC)] in the Memorial Sloan Kettering Cancer Center (MSK) MetTropism cohort (N = 2550). Clonality/LOH prediction was compared to a streamlined clinical classifier that relies on variant allele frequencies (VAFs) and tumor purity (TP) (VAF/TP ratio). The impact of this classification on survival outcomes was tested in two independent cohorts of LUAD patients treated with immunotherapy (MSK/Rome N = 237; DFCI N = 461). Immune-related features were studied by exploiting RNA-sequencing data (TCGA) and multiplexed immunofluorescence (DFCI mIF cohort). RESULTS: Clonality/LOH inference in the MSK MetTropism cohort overlapped with a clinical classification model defined by the VAF/TP ratio. In the ICI-treated MSK/Rome discovery cohort, predicted KEAP1 C-LOH mutations were associated with shorter progression-free survival (PFS) and overall survival (OS) compared to KEAP1 wild-type cases (PFS log-rank P = 0.001; OS log-rank P < 0.001). Similar results were obtained in the DFCI validation cohort (PFS log-rank P = 0.006; OS log-rank P = 0.014). In both cohorts, we did not observe any significant difference in survival outcomes when comparing KEAP1 CD-SC and wild-type tumors. Immune deconvolution and multiplexed immunofluorescence revealed that KEAP1 C-LOH and KEAP1 CD-SC differed for immune-related features. CONCLUSIONS: KEAP1 C-LOH mutations are associated with an immune-excluded phenotype and worse clinical outcomes among advanced LUAD patients treated with ICIs. By contrast, survival outcomes of patients whose tumors harbored KEAP1 CD-SC mutations were similar to those with KEAP1 wild-type LUADs.

Differential properties of the two Drosophila gamma-tubulin isotypes.

The functional significance of distinct gamma-tubulins in several unrelated eukaryotes remains an enigma due to the difficulties to investigate this question experimentally. Using specific nucleotidic and immunological probes, we have demonstrated that the two divergent Drosophila gamma-tubulins, gamma-tub23C and gamma-tub37CD, are expressed in cultured cells. Gamma-tub37CD is constantly detected at the centrosome and absent in the mitotic spindle, while gamma-tub23C is extensively recruited to the centrosome during mitosis and relocalizes in the mitotic spindle. The two gamma-tubulins exhibit distinct biochemical properties. Gamma-tub23C is present in the soluble gamma-tubulin small complexes (10S) and gamma-tubulin big complexes (35S) and is loosely associated to the cytoskeleton. In contrast, gamma-tub37CD is undetectable in the soluble fraction and exhibits a tight binding to the centrosome. Syncytial embryos also contain the two gamma-tubulin isotypes, which are differentially recruited at the centrosome. Gamma-tub23C is present in the 10S soluble complexes only, while y-tub37CD is contained in the two soluble complexes and is recruited at the centrosome where it exhibits an heterogeneous binding. These results demonstrated an heterogeneity of the two Drosophila gamma-tubulin isotypes both in the cytoskeletal and the soluble fractions. They suggest the direct implication of the 35S complex in the centrosomal recruitment of gamma-tubulin and a conditional functional redundancy between the two gamma-tubulins.

Human 76p: A new member of the gamma-tubulin-associated protein family.

The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.

Condensation-decondensation of the gamma-tubulin containing material in the absence of a structurally visible organelle during the cell cycle of Physarum plasmodia.

Genetic evidence has shown the presence of a common spindle pole organiser in Physarum amoebae and plasmodia. But the typical centrosome and mitosis observed in amoebae are replaced in plasmodia by an intranuclear mitosis devoid of any structurally defined organelle. The fate of gamma-tubulin and of another component (TPH17) of the centrosome of Physarum amoebae was investigated in the nuclei of synchronous plasmodia. These two amoebal centrosomal elements were present in the nuclear compartment during the entire cell cycle and exhibited similar relocalisation from metaphase to telophase. Three preparation methods showed that gamma-tubulin containing material was dispersed in the nucleoplasm during interphase. It constituted an intranuclear thread-like structure. In contrast, the TPH17 epitope exhibited a localisation close to the nucleolus. In late G2-phase, the gamma-tubulin containing elements condensed in a single organelle which further divided. Intranuclear microtubules appeared before the condensation of the gamma-tubulin material and treatment with microtubule poisons suggested that microtubules were required in this process. The TPH17 epitope relocalised in the intranuclear spindle later than the gamma-tubulin containing material suggesting a maturation process of the mitotic poles. The decondensation of the gamma-tubulin material and of the material containing the TPH17 epitope occurred immediately after telophase. Hence in the absence of a structurally defined centrosome homologue, the microtubule nucleating material undergoes a cycle of condensation and decondensation during the cell cycle.

The mammalian interphase centrosome: two independent units maintained together by the dynamics of the microtubule cytoskeleton.

In mammalian cells the centrosome or diplosome is defined by the two parental centrioles observed in electron microscopy and by the pericentriolar material immunostained with several antibodies directed against various centrosomal proteins (gamma-tubulin, pericentrin, centrin and centractin). Partial destabilization of the microtubule cytoskeleton by microtubule-disassembling substances induced a splitting and a slow migration of the two diplosome units to opposite nuclear sides during most of the interphase in several mammalian cell lines. These units relocated close together following drug removal, while microtubule stabilization by nM taxol concentrations inhibited this process. Cytochalasin slowed down diplosome splitting but did not affect its relocation after colcemid washing. These results account for the apparently opposite effects induced by microtubule poisons on centriole separation. Moreover, they provide new information concerning the centrosome cycle and stability. First, the centrosome is formed by two units, distinguished only by the number of attached stable microtubules, but not by pericentrin, gamma-tubulin, centrin and centractin and their potency to nucleate microtubules. Second, the centrosomal units are independent during most of the interphase. Third, according to the cell type, these centrosomal units are localized in close proximity because they are either linked or maintained close together by the normal dynamics of the microtubule cytoskeleton. Finally, the relocalization of the centrosomal units with their centrioles in cells possessing one or two centrosomes suggests that their relative position results from the overall tensional forces involving at least partially the microtubule arrays nucleated by each of these entities.

Protein complexes containing gamma-tubulin are present in mammalian brain microtubule protein preparations.

The presence of gamma-tubulin in microtubule preparations, obtained by disassembly/ assembly cycles at 0degreesC/37degreesC from the brain of several mammals, is demonstrated by immunoblotting with specific antibodies directed against three distinct regions of the protein. In contrast gamma-tubulin was absent from pure tubulin obtained by chromatography on phosphocellulose, but was retained on the column with the other microtubule-associated proteins. A large part of the gamma-tubulin was present in cold stable material remaining after microtubule disassembly at OdegreesC and was partially solubilized using high salt, thus preventing its purification by the usual assembly/disassembly procedure used for alpha/beta-tubulin heterodimers. Brain gamma-tubulin was purified by affinity chromatography with gamma-tubulin antibodies raised against its carboxyl terminal region. Purified gamma-tubulin consisted of at least two polypeptides present in equal quantities and exhibiting a pI of 6.5 and 6.6, respectively. It was associated with the alpha/beta-tubulin heterodimer and with at least five other polypeptides of 75, 105, 130, 195, and 250 kDa. With the exception of the 250 kDa polypeptide, all of these proteins seem to be present in gamma-tubulin complexes isolated from Xenopus eggs. But, in contrast with Xenopus egg complexes, brain complexes exhibited a considerable heterogeneity of their apparent masses and composition in sucrose gradient centrifugation, in agreement with the absence of an homogeneous structure in electron microscopy. Despite this heterogeneity, gamma-tubulin complexes bind quantitatively to microtubule extremities. The possibility to further use mammalian brain gamma-tubulin and some of its associated proteins in biochemical and pharmacological experiments is of interest since brain microtubule protein preparations have been extensively used for studying both microtubule dynamics and the activity of microtubule poisons.

A single gamma-tubulin gene and mRNA, but two gamma-tubulin polypeptides differing by their binding to the spindle pole organizing centres.

Cells of eukaryotic organisms exhibit microtubules with various functions during the different developmental stages. The identification of multiple forms of alpha- and beta-tubulins had raised the question of their possible physiological roles. In the myxomycete Physarum polycephalum a complex polymorphism for alpha- and beta-tubulins has been correlated with a specific developmental expression pattern. Here, we have investigated the potential heterogeneity of gamma-tubulin in this organism. A single gene, with 3 introns and 4 exons, and a single mRNA coding for gamma-tubulin were detected. They coded for a polypeptide of 454 amino acids, with a predicted molecular mass of 50,674, which presented 64-76% identity with other gamma-tubulins. However, immunological studies identified two gamma-tubulin polypeptides, both present in the two developmental stages of the organism, uninucleate amoebae and multinucleate plasmodia. The two gamma-tubulins, called gamma s- and gamma f-tubulin for slow and fast electrophoretic mobility, exhibited apparent molecular masses of 52,000 and 50,000, respectively. They were recognized by two antibodies (R70 and JH46) raised against two distinct conserved sequences of gamma-tubulins. They were present both in the preparations of amoebal centrosomes possessing two centrioles and in the preparations of plasmodial nuclear metaphases devoid of structurally distinct polar structures. These two gamma-tubulins exhibited different sedimentation properties as shown by ultracentrifugation and sedimentation in sucrose gradients. Moreover, gamma s-tubulin was tightly bound to microtubule organizing centers (MTOCs) while gamma f-tubulin was loosely associated with these structures. This first demonstration of the presence of two gamma-tubulins with distinct properties in the same MTOC suggests a more complex physiological role than previously assumed.

Oligomerization state in solution of the cell cycle regulators p13suc1 from the fission yeast and p9cksphy from the myxomycete Physarum, two members of the cks family.

The cks proteins (for cdc2 kinase subunit) are essential cell cycle regulators. They interact strongly with the mitotic cdc2 kinase, but the mechanism and the biological function of this association still await understanding. The oligomerization state in solution of two members of this ubiquitous protein family, the suc1 gene product from the fission yeast and the newly cloned cksphy gene product from the myxomycete Physarum, was investigated by small-angle X-ray scattering (SAXS) and biochemical methods. We found that the major molecular species are monodispersed monomeric proteins. Minor amounts of dimeric suc1 proteins were also found, but no equilibrium between the two forms was observed and surprisingly, the hexameric assemblies observed in the crystal structure of the human ckshs2 homolog were not detected. These apparent discrepancies between proteins that display cross-complementation address the question of the control of the cks oligomerization process and its link to the biological function.

Pulse oximetry: assuring accuracy.

The principles of pulse oximetry are explored in this article. It describes the accuracy of the machine as well as its limitations in practice. Specific disease entities, external factors, and laboratory values that interfere with the pulse oximeter's accuracy are described. Practical approaches, proper probe applications, and alternative methods of measuring the patient's oxygenation status are also discussed.

[Alkalizing activity of a calcium-bicarbonate-containing water, evaluated for pH, in patients with gastroesophageal reflux]. / Attività alcalinizzante di un'acqua bicarbonato-calcica valutata con pHmetria in soggetti con reflusso gastro-esofageo.

Bicarbonate-calcic water Ferrarelle has been administered both in the fasting state and during meals to patients suffering from gastro-esophageal reflux disease submitted to computerized pHmetry. Marked and lasting increase of esophageal and gastric pH was observed with significant differences from the effect of tap water. In addition, patients reported improvement of heart burn and acidity after the administration of the bicarbonate-calcic water. The alkalizing effect of the mineral water employed is therefore fully confirmed.

Mineral waters in treatment of metabolic changes from fatigue in sportsmen.

The authors list the principal metabolic consequences of fatigue in athletes with special reference to alterations of fluid-electrolyte balance, acid-base balance, macroelements and trace elements. They then go on to stress the role which mineral waters, especially the bicarbonate ones, can play in the compensation of these disorders thus preventing or curing the fatigue syndrome in athletes.

[A case of intestinal giardiasis associated with immunoglobulin deficiency and intestinal lymph node hyperplasia]. / Su di un caso di giardiasi intestinale associata a deficit immunoglobulinico e iperplasia nodulare linfatica intestinale.

The authors recall the main etiopathogenetic, immunological and clinical features of Giardiasis; they report on a patient suffering from intestinal Giardiasis associated with immunoglobulin deficiency and nodular lymphatic hyperplasia of the gut. They report the results of medical therapy.

[Hydrological therapy of renal lithiasis]. / Terapia idrologica della litiasi renale.

The spa treatment most widely used in the management of renal calculosis is the drinking of a certain amount of mineral water under certain predetermined conditions of temperature, time and rhythm. These treatments are always indicated unless there are obstacles to the passage of urine or general contraindications. Chances for success are increased if the diagnosis is exact and the stone has been located. The results obtained by various authors are reported and their statistical validity is discussed. Favorable effects consist in: increased diuresis with urine dilution and correspondingly reduced concentration of lithogenic salts and hence supersaturation of urine; reduced concentration of microorganisms; changes in the physiological condition of the renal medulla; changes in inhibitors of crystallization, in urinary pH; mechanical effect on the urinary passages; increased ureter motility; expulsion of small stones and sand; preventive action on recurrences after surgery and after extra-corporeal shock-wave treatment, percutaneous therapy and uretero-nephroscopy.

Drug-membrane interactions: silymarin, silibyn and microsomal membranes.

Silymarin and silibyn are extracted from the seeds of Silybum marianum and used as a liver protectant because of their free radical scavenging. When incorporated into rabbit liver microsomes they cause a small decrease in the flourescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) but not of 1-anilinononaphthalene-8-sulphonic acid (ANS), incorporated into the membranes. They do, however, reduce the fluorescence intensity of incorporated ANS without changing the wavelength of maximum intensity. These observations suggest that the drugs are incorporated into the hydrophobic-hydrophilic interface of the microsomal bilayer and perturb the structure by influencing the packing of the acyl chains.

[The effects of Levissima oligomineral water on diuresis and water exchange]. / Considerazioni sulle influenze esercitate dall'acqua oligominerale Levissima sulla diuresi e sul ricambio idrico.

Research on healthy and diseased subjects and laboratory animals have shown that Levissima oligomineral water: a) has no unpleasant or harmful subjective and/or objective side-effects even when taken at high doses for long periods. b) Encourages diuresis. Comparison with saline solution of the same osmolality showed: 1) mean increase in diuresis: 16%; 2) more rapid elimination of water; 3) significant increase in free water clearance; 4) no change in the stock of electrolytes during protracted administration. c) Influences purine exchange: 1) by increasing uric acid clearance (same comparison); 2) by reducing the hyperuricaemising effect of a rapid i.v. fructose load; 3) by opposing hyperuricaemia due to depression of mechanisms responsible for the increase of uric acid owing to enhancement of serum lactic acid after the administration of alcohol. d) Results in a characteristic change in certain coagulation parameters when compared with saline solution and tap water. It is suggested that this method be used to recognise the persistence over time of the biological activities of bottled mineral waters.