Hosing of a Long Relativistic Particle Bunch in Plasma.

Experimental results show that hosing of a long particle bunch in plasma can be induced by wakefields driven by a short, misaligned preceding bunch. Hosing develops in the plane of misalignment, self-modulation in the perpendicular plane, at frequencies close to the plasma electron frequency, and are reproducible. Development of hosing depends on misalignment direction, its growth on misalignment extent and on proton bunch charge. Results have the main characteristics of a theoretical model, are relevant to other plasma-based accelerators and represent the first characterization of hosing.

Controlled Growth of the Self-Modulation of a Relativistic Proton Bunch in Plasma.

A long, narrow, relativistic charged particle bunch propagating in plasma is subject to the self-modulation (SM) instability. We show that SM of a proton bunch can be seeded by the wakefields driven by a preceding electron bunch. SM timing reproducibility and control are at the level of a small fraction of the modulation period. With this seeding method, we independently control the amplitude of the seed wakefields with the charge of the electron bunch and the growth rate of SM with the charge of the proton bunch. Seeding leads to larger growth of the wakefields than in the instability case.

Filamentation of a relativistic proton bunch in plasma.

We show in experiments that a long, underdense, relativistic proton bunch propagating in plasma undergoes the oblique instability, which we observe as filamentation. We determine a threshold value for the ratio between the bunch transverse size and plasma skin depth for the instability to occur. At the threshold, the outcome of the experiment alternates between filamentation and self-modulation instability (evidenced by longitudinal modulation into microbunches). Time-resolved images of the bunch density distribution reveal that filamentation grows to an observable level late along the bunch, confirming the spatiotemporal nature of the instability. We provide a rough estimate of the amplitude of the magnetic field generated in the plasma by the instability and show that the associated magnetic energy increases with plasma density.

Optimum design of family structure and allocation of resources in association mapping with lines from multiple crosses.

Family mapping is based on multiple segregating families and is becoming increasingly popular because of its advantages over population mapping. Athough much progress has been made recently, the optimum design and allocation of resources for family mapping remains unclear. Here, we addressed these issues using a simulation study, resample model averaging and cross-validation approaches. Our results show that in family mapping, the predictive power and the accuracy of quatitative trait loci (QTL) detection depend greatly on the population size and phenotyping intensity. With small population sizes or few test environments, QTL results become unreliable and are hampered by a large bias in the estimation of the proportion of genotypic variance explained by the detected QTL. In addition, we observed that even though good results can be achieved with low marker densities, no plateau is reached with our full marker complement. This suggests that higher quality results could be achieved with greater marker densities or sequence data, which will be available in the near future for many species.

A major susceptibility locus to murine lung carcinogenesis maps on chromosome 6.

Lung tumours represent a major cause of death in humans, and although smoking represents the main pathogenetic factor, inheritance also plays a part. However, the identification of possible predisposing genetic factors is difficult, because of their low penetrance. We took advantage of murine strains that are genetically susceptible or resistant to lung tumour development, to map murine genes associated with susceptibility to lung carcinogenesis. An F2 population of urethan-treated A/J x C3H/He mice was scored with 83 genetic markers. A chromosome 6 distal region, spanning mice was scored with 83 genetic markers. A chromosome 6 distal region, spanning 35 centiMorgans, contained a major lung tumour susceptibility locus. No other chromosomal region was significantly associated with lung tumour development.

Myogenic commitment of human stem cells by myoblasts Co-culture: a static vs. a dynamic approach.

An in-vitro model of human bone marrow mesenchymal stem cells (hBM-MSCs) myogenic commitment by synergic effect of a differentiation media coupled with human primary skeletal myoblasts (hSkMs) co-culture was developed adopting both conventional static co-seeding and perfused culture systems. Static co-seeding provided a notable outcome in terms of gene expression with a significant increase of Desmin (141-fold) and Myosin heavy chain II (MYH2, 32-fold) at day 21, clearly detected also by semi-quantitative immunofluorescence. Under perfusion conditions, myogenic induction ability of hSkMs on hBM-MSCs was exerted by paracrine effect with an excellent gene overexpression and immunofluorescence detection of MYH2 protein; furthermore, due to the dynamic cell culture in separate wells, western blot data were acquired confirming a successful cell commitment at day 14. A significant increase of anti-inflammatory cytokine gene expression, including IL-10 and IL-4 (15-fold and 11-fold, respectively) at day 14, with respect to the pro-inflammatory cytokines IL-12A (7-fold at day 21) and IL-1ß (1.4-fold at day 7) was also detected during dynamic culture, confirming the immunomodulatory activity of hBM-MSCs along with commitment events. The present study opens interesting perspectives on the use of dynamic culture based on perfusion as a versatile tool to study myogenic events and paracrine cross-talk compared to the simple co-seeding static culture.

Lipid nano-vesicles for thyroid hormone encapsulation: A comparison between different fabrication technologies, drug loading, and an in vitro delivery to human tendon stem/progenitor cells in 2D and 3D culture.

Phosphatidylcholine (PC) vesicles loaded with Triiodothyronine (T3) were fabricated using different manufacturing methods: thin layer hydration plus sonication (TF-UF), supercritical liposome formation (SC), and microfluidic technology (MF). Vesicles obtained by MF had the lowest mean diameter (88.61 ± 44.48 nm) with a Zeta Potential of -20.1 ± 5.90 mV and loading of 10 mg/g (encapsulation efficiency: 57%). In contrast, SC vesicles showed extremely low encapsulation efficiency (<10%) probably due to T3 solubility in ethanol/carbon dioxide mixture; despite TF-UF vesicles exhibiting good size (167.7 ± 90 nm; Zp -8.50 ± 0.60 mV) and loading (10 mg/g), poor mass recovery was obtained (50% loss). MF vesicles had low cytotoxicity, and they were well enough internalized by both HeLa and human tendon stem/progenitor cells (hTSPCs). Their biological activity was also monitored in both 2D and 3D cultures of hTSPCs supplemented with therapeutical concentrations of PC/T3 nano-liposomes. 2D culture showed almost similar constitutive gene expression compared to control culture supplemented with free-T3. On the contrary, when hTPSCs 3D culture was assembled, it showed a more evident homogeneous distribution of FITC labeled vesicles within the high-density structure and a significant upregulation of cell constitutive genes, such as type I Collagen (4.8-fold; p < 0.0001) at day 7, compared to the control, suggesting that T3/PC formulation has increased T3 cytosolic concentration, thus improving cells metabolic activity. The study supported MF technology for nano-carriers fabrication and opens perspectives on the activity of PC/T3 nano-vesicles as innovative formulations for TPSCs stimulation in ECM secretion.

Quantitative and qualitative features of acute phase-adverse events following SARS-CoV-2 vaccination in a large sample of people with multiple sclerosis.

INTRODUCTION: Few data are available on adverse events (AE) associated to vaccines in persons with multiple sclerosis (pwMS). AIMS: to study the incidence of acute phase AE (AP-AE) related to SARS-CoV-2 mRNA vaccines in pwMS compared to a control group, and to analyze the association between AP-AE and disease modifying treatments (DMT). METHODS: This was a cross-sectional study on 438 PwMS and 481 age- and sex-matched subjects not affected by dysimmune diseases that underwent two doses of SARS-CoV-2 mRNA BNT162b2 vaccine (Pfizer/BioNtech). RESULTS: Two hundred and twenty five (51.4%) pwMS complained of ≥1 AP-AE after the first dose, 269 (61.4%) after the second dose. A logistic regression analysis revealed that only pwMS on Fingolimod and Ocrelizumab did not show a higher risk of developing AP-AE. The likelihood to present with ≥1 AP-AE, after correcting for age and sex, was significantly higher in pwMS than controls. CONCLUSIONS: This study reports qualitative and quantitative features of AP-AE associated with the first and second doses of SARS-CoV-2 vaccine in a large sample of pwMS. The only risk factor identified for developing AP-AE is female gender. AntiCD-20 monoclonal antibodies and S1P inhibitors are associated with a lower risk of AP-AE occurrence.

Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient.

A single genetic alteration, a guanine-to-cytosine transversion, is responsible for the acquisition of malignant properties by K-ras genes of two human tumor cell lines established from carcinomas of the bladder (A1698) and lung (A2182). As a consequence, arginine instead of the normal glycine is incorporated into the K-ras-coded p21 proteins at amino acid position 12. This mutation creates a restriction enzyme polymorphism that can be used to screen human cells for transforming K-ras genes. This approach was used to identify the mutational event responsible for the malignant activation of a K-ras oncogene in a squamous cell lung carcinoma of a 66-year-old man; this point mutation was not present in either the normal bronchial or parenchymal tissue or in the blood lymphocytes. Hence, malignant activation of a ras oncogene appears to be specifically associated with the development of a human neoplasm.

A bioavailability study on microbeads and nanoliposomes fabricated by dense carbon dioxide technologies using human-primary monocytes and flow cytometry assay.

Supercritical Emulsion Extraction (SEE) and Supercritical assisted Liposome formation (SuperLip), use dense gases such as carbon dioxide (dCO2) to fabricate advanced micro/nanocarriers. SEE uses dCO2 to extract solvent from the oily phase of an emulsion and obtain biopolymer microbead; For this study, poly-Lactic Acid (PLA) microbeads of 1 ±â€¯0.2 µm in mean size loaded at 1 µg/mgPLA with Rhodamine B (ROD) were prepared by SEE; the beads showed a solvent residue lower than 10 ppm and encapsulated the fluorochrome with an efficiency of 90%. SuperLip uses dCO2 to enhance lipid/ethanol/water mixing and to promote the ethanol extraction from liposome suspension. In this case, phosphatidyl-choline (PC) vesicles with a mean size of 0.2 ±â€¯0.05 µm and loaded with Fluorescein Iso-ThioCyanate (FITC) at 8 µg/mgPC were prepared; small unilamellar structure was observed for all the vesicles with FITC encapsulation efficiency of 80%. Ethanol residue of 50 ppm was measured in all the liposome suspensions. The bioavailability of microbeads and nanoliposomes was assessed through incubation with human monocytes previously isolated from healthy donors' blood. A specifically optimized protocol that allowed their quenching on the cell surface was developed to monitor by flow cytometer assay only the cell population that effectively internalized the carriers. When microbeads were tested, the percentage of alive internalizing monocytes was of about 30%. An internalization of 96.1 ±â€¯21% was, instead, obtained at dosage of 0.1 mg/mL for nanoliposomes. In this last case, monocytes showed a vitality of almost 100% after vesicles internalization at all the concentrations studied; on the other hand, cell apoptosis progressively increased in a dose/response manner, after polymer microbeads phagocytosis. The proposed data suggested that dCO2 technologies can be reliably used to fabricate intracellular carriers.

Gene p53 mutations are restricted to poorly differentiated and undifferentiated carcinomas of the thyroid gland.

The p53 gene was analyzed in tumor specimens obtained from 52 patients with various types of carcinoma of the thyroid gland by a combined molecular and immunocytochemical approach. The histologic types included 37 well-differentiated papillary and follicular carcinomas, 8 poorly differentiated, and 7 undifferentiated carcinomas. The p53 gene was shown to be unaffected in all differentiated tumors by single-strand conformation polymorphism analysis. However, in two out of eight (25%) of poorly differentiated carcinomas and five out of seven (71%) undifferentiated carcinomas, p53 mutations were identified and subsequently characterized by DNA sequencing. One undifferentiated carcinoma displayed two areas with varying degrees of differentiation. The comparative analysis of the p53 gene, in both the more and the less differentiated area of this tumor, clearly showed that the p53 mutation was confined to the latter component of the tumor specimen. These results indicate that mutations of the p53 gene are associated with the most aggressive histologic types of thyroid tumors, such as the undifferentiated carcinoma and, to a certain extent, the poorly differentiated carcinoma, and that the alterations of this gene represent a late genetic event in human thyroid carcinogenesis.

Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype.

We have recently reported the activation of a new oncogene in human papillary thyroid carcinomas. This oncogene, papillary thyroid carcinoma (PTC), is a novel rearranged version of the ret tyrosine-kinase protooncogene. Thyroid neoplasms include a broad spectrum of malignant tumors, ranging from well-differentiated tumors to undifferentiated anaplastic carcinomas. To determine the frequency of ret oncogene activation, we analyzed 286 cases of human thyroid tumors of diverse histologic types. We found the presence of an activated form of the ret oncogene in 33 (19%) of 177 papillary carcinomas. By contrast, none of the other 109 thyroid tumors, which included 37 follicular, 15 anaplastic, and 18 medullary carcinomas, and 34 benign lesions, showed ret activation.

Molecular characterization of a thyroid tumor-specific transforming sequence formed by the fusion of ret tyrosine kinase and the regulatory subunit RI alpha of cyclic AMP-dependent protein kinase A.

The ret oncogene frequently has been found activated in papillary thyroid carcinomas. A previous characterization of ret activation revealed recombination of its tyrosine kinase domain and sequences derived from an uncharacterized locus (D10S170). The mechanism leading to this recombination was identified as a paracentric inversion of the long arm of chromosome 10, inv(10)(q11.2q21), with the breakpoints occurring where ret and D10S170 were mapped. To further characterize the activation of ret in papillary thyroid carcinomas, we have now isolated and sequenced a second type of ret oncogenic rearrangement not involving the D10S170 locus. The nucleotide sequence indicated that the transforming activity was created by the fusion of the ret tyrosine kinase domain with part of the RI alpha regulatory subunit of protein kinase A (PKA). This is the first example of an oncogenic activity involving a PKA gene. PKA is the main intracellular cyclic AMP receptor, and its RI alpha subunit gene is located on chromosome 17q. RI alpha-ret transcripts encode two isoforms of the chimeric protein (p76 and p81), which display constitutive tyrosine phosphorylation as well as a tyrosine kinase enzymatic activity. Under nonreducing conditions, both isoforms are found in a dimeric configuration because of both homo- and heterodimer formation. Thus, the in vivo activation of ret in human papillary thyroid carcinomas is provided by the fusion of its tyrosine kinase domain with different genes and can be mediated by different mechanisms of gene rearrangement.

Corticosteroid microparticles produced by supercritical-assisted atomization: process optimization, product characterization, and "in vitro" performance.

In this work, the production of dexametasone and dexametasone acetate microparticles is proposed using supercritical-assisted atomization (SAA). This process is based on the solubilization of supercritical carbon dioxide in a liquid solution containing the drug; then, the ternary mixture is sprayed through a nozzle and submicroparticles are formed as a consequence of the enhanced atomization. Several process parameters such as different organic solvent (methanol and acetone), solute concentration and flow rate ratio between the liquid solution and carbon dioxide are investigated; their influence is evaluated on the morphology and size of precipitated particles. Spherical corticosteroid particles with mean diameters ranging from 0.5 to 1.2 microm are produced at the optimum operating conditions and narrow particle size distributions (PSDs) have also been obtained. No drug degradation was observed after SAA processing and solvent residues of 300 and 500 ppm for acetone and methanol, respectively, were measured. Drug microparticles produced by SAA can be semi-crystalline or amorphous depending on the process condition; a micronized drug surface area ranging from about 4 to 5 m2/g was also observed. The "in vitro" activity of both untreated and SAA processed glucocorticoids was tested on the release of pro-inflammatory cytokines from stimulated cells. The results shown that SAA-glucocorticoids have retained the activity of the parent untreated compounds and, in the case of dexamethasone, SAA processing improves drug performance.

Cellular immune reaction to human malignant melanoma and breast carcinoma cells.

An in vitro microassay was used to study cytotoxic reactivity of lymphocytes of 19 patients with malignant melanoma, 8 patients with breast carcinoma, and 18 normal subjects on cell cultures of malignant melanoma and breast carcinoma. In each of the twelve experiments, peripheral blood lymphocytes from individuals with and without cancer were tested simultaneously on two or three different target cells. Cytotoxic reactivity, evaluated by a comparison of the number of target cells remaining after incubation with lymphocytes with those incubated with medium only, was found in 20 cancer patients (74%) and 13 individuals without cancer (72%). The strength of lymphocyte reactivity of the cancer and of the non-cancer group did not differ significantly. Of the 27 cancer patients, 8 were positive only on the homologous target cells, 7 only on the opposite cells, and 5 on both types; 7 were negative. Short-term melanoma cell cultures were more lysable than were established cell lines; however, no direct correlation between the growth rate during the test period and susceptibility to lysis was seen. The blood group of the lymphocyte donors had no influence on cytotoxic reactivity.

Quantitative analysis of genetic susceptibility to liver and lung carcinogenesis in mice.

One-week-old male and female mice of the A, BALB/c (C), and C3H/He (C3) strains and of the AC3 and CC3 F1 hybrids were treated with a single dose (300 mg/kg s.c.) of urethan and then kept without further treatment until 30 and 40 weeks (males) or 30 and 65 weeks (females). The degree of difference in susceptibility to hepatocarcinogenesis between the susceptible C3 and the resistant A and C mice, in the different age and sex groups, was 4-12-fold by the analysis of the number of nodules/cm3 (N/cm3), which represents an estimation of tumor frequency and was more than 400-fold as indicated by the percentage of liver volume occupied by nodules (V%), an estimation of tumor size. With regard to lung carcinogenesis, mice of the A strain proved 5-10- and 20-70-fold more susceptible than the C and the C3 mice, respectively, as indicated by the number of microscopically identified lung tumors/mouse (N), an estimation of tumor frequency. The lung tumor size, as estimated by the mean tumor volume (V), was similar in A and C mice but much higher in the A than in the C3 groups (13- approximately 1000-fold difference). The AC3 hybrid was highly susceptible to both liver and lung carcinogenesis. The CC3 hybrid was as susceptible to lung carcinogenesis as its C parent and had an intermediate susceptibility to hepatocarcinogenesis. Our results indicate that a major determinant in the genetic susceptibility to hepatocarcinogenesis, and perhaps to lung carcinogenesis too, is tumor growth, as evidenced by a much greater tumor size in genetically susceptible than resistant mice.

Factors influencing the expression of endogenous retrovirus-related sequences in the liver of B6C3 mice.

The expression of RNA transcripts from three families of endogenous retrovirus-related sequences was investigated during liver cell proliferation in B6C3 mice. Treatment with a single dose of the liver mitogen and promoter of mouse hepatocarcinogenesis 1, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), or with carbon tetrachloride (CCl4), induced liver cell proliferation at days 2 and 3 after treatment. Both of these treatments led to a marked increase in Moloney murine leukemia virus-related 6 kilobase RNAs, which were most abundant at day 1 after TCPOBOP treatment and at day 2 after CCl4. Intracisternal A particle-related 6 kilobase RNAs were markedly increased at days 1 and 2 after TCPOBOP and at days 1, 2, and 3 after CCl4. VL30-related transcripts were slightly decreased after TCPOBOP, but they were markedly increased at days 1 and 2 following CCl4. The livers of 15-day-old untreated mice contained about a 3-fold higher level of Moloney murine leukemia virus-related RNAs than adult liver. Intracisternal A particle-related 6-kilobase transcripts were present at 3-fold higher abundance in 7-day-old than in 15-day-old or adult liver. RNAs homologous to VL30 were detected at about the same levels in infant as well as adult livers. Inhibition of protein synthesis by the administration of cycloheximide to adult mice caused a marked increase in the amount of Moloney murine leukemia virus-, intracisternal A particle-, and VL30-related RNAs in the livers of the treated mice, suggesting the existence of labile proteins that normally regulate the abundance of these transcripts. We postulate that the amounts of these putative regulatory proteins vary during both normal development and carcinogenesis and also in response to specific agents that induce liver cell proliferation.

Expression of retroviral sequences and oncogenes in murine hepatocellular tumors.

The expression of endogenous retroviral sequences and of three cellular oncogenes was examined in three hepatocellular adenomas and in four carcinomas induced in male C57BL/6JDp X C3Hf/Dp F1 (hereafter called B6C3F1) mice by a single dose of nitrosodiethylamine, in five carcinomas that arose spontaneously in male C3Hf mice, and in the livers of normal age-matched control mice. In all of these adenomas and carcinomas, there was increased expression of Moloney murine leukemia virus- and intracisternal A particle-related sequences. The retrovirus-like VL30 sequence was expressed at significant levels in the normal liver of these mice and increased expression of this sequence was found in only 4 of the 12 tumors examined. Expression of endogenous mouse mammary tumor virus-related sequences was not detected in the normal livers or in any of the liver tumors. With respect to cellular oncogenes, increased expression of c-myc was seen in all of the B6C3F1 tumors. Two of five normal liver samples and all of the tumors of the C3Hf mice displayed significant expression of c-myc. There was a slight increase in expression of c-Ha-ras in some of the tumors. Increased expression of c-fos was found in only 1 of the 12 tumors. Taken together, these studies indicate that both carcinogen-induced and spontaneous liver tumor formation in mice is associated with abnormalities in the expression of endogenous retrovirus-related DNA sequences and also specific cellular oncogenes.

DNA methylation affecting the transforming activity of the human Ha-ras oncogene.

A plasmid containing the transforming Ha-ras gene and designated pT24-C3 was methylated in vitro using the sequence-specific bacterial methyltransferases HpaII and HhaI. Aliquots of the plasmid were methylated by the single enzymes or by the two enzymes simultaneously (double methylation). The transforming activity of the treated plasmids was assayed in the standard transfection assay on NIH-3T3 cells. Double methylation reduced the transforming activity of pT24-C3 about 80%, whereas treatment with the single methylating enzymes did not significantly affect the oncogene activity. Southern blot analysis of the transformants obtained with the methylated or mock-methylated pT24-C3 plasmids indicated in all the examined DNAs the presence of human Ha-ras sequences with methylation degrees consistent with the treatment of the plasmids. The Mr 21,000 oncogene protein p21 was also detected in several examined transformants. The DNA-demethylating agent 5-azacytidine restored the transforming activity of the double-methylated pT24-C3 upon 24 h incubation of transfected NIH-3T3 cells. Southern blot analysis showed integration of human Ha-ras with a methylation profile intermediate between the double-methylated and mock-methylated plasmids. It is suggested that DNA methylation of specific CG-containing target sites can affect the transforming activity of a human oncogene.

Clonal immunoglobulin gene rearrangements and normal T-cell receptor, bcl-2, and c-myc genes in primary cutaneous B-cell lymphomas.

Fourteen cases of primary cutaneous B-cell lymphomas were investigated at the immunohistochemical and molecular level to further characterize this newly defined entity. Neoplastic cells from all cases, phenotyped with a panel of monoclonal antibodies, were positive for HLA-DR, for the B-cell markers CD19, CD22, but not CD23 (except one case), and negative for the T-cell marker CD2. Monoclonal immunoglobulin light chains were demonstrated in six cases. The reactivity with the Ki-67 monoclonal antibody indicated that the neoplastic cells are proliferating. In five biopsies the presence of dendritic cells infiltrating the neoplastic areas was revealed using the monoclonal antibody Kim4b. By Southern blot analysis, clonal rearrangement of the immunoglobulin heavy chain gene (involving one or both alleles) was shown in 12 of 14 cases and of the light chain genes in 13 cases. The bcl-2 oncogene, normally involved in nodal follicular lymphomas, was in germ-line configuration. The c-myc and the beta and gamma chain genes of the T-cell receptor were also in the germ-line configuration. None of the cases presented Epstein-Barr virus sequences. These data indicate that primary cutaneous lymphomas of B-cell origin share morphological and phenotypic similarities with the nodal B-cell lymphomas of follicular histotype, are proliferating, and express in 45% of cases clear monoclonal immunoglobulin light chain; the molecular analysis confirms the B-cell derivation and the monoclonal nature of this neoplasia; it also shows that neither bcl-2 nor c-myc oncogenes are involved and that no inappropriate rearrangements of the T-cell receptor genes are found in this lymphoma.