The Complex Dance of the Two Conformers of Bis(trifluoromethanesulfonyl)imide as a Function of Pressure and Temperature.

Absorbance spectra of two ionic liquids, the short alkyl chain N-trimethyl-N-propylammonium bis(trifluoromethanesulfonyl)imide (TMPA-TFSI) and the longer chain N-trimethyl-N-hexylammonium bis(trifluoromethanesulfonyl)imide (TMHA-TFSI) are reported as a function of pressure and temperature. The occurrence of various phase transitions is evidenced by the changes in the relative concentration of the cisoid and transoid conformers of their common TFSI anion. The infrared spectrum of TMPA-TFSI was measured at 300 K with an applied pressure varying over the 0-5 GPa range. Above 0.2 GPa only the trans conformer is detected, suggesting the occurrence of a pressure induced crystallization. When pressure is applied to TMHA-TFSI at T = 310 K, both TFSI conformers subsist up to ∼11 GPa. However, the clear change of their intensity ratio observed around 2 GPa, suggests the onset of a glass phase as supported by measurements carried out at 4.2 GPa along a cooling/heating cycle. A careful analysis of the spectra collected along different p-T thermodynamic paths shows the occurrence of a cold crystallization at 295 K on heating from 139 K along the p = 0.5 GPa isobar. The rich phase diagrams of the two ionic liquids is the result of the competition among the anion-cation intermolecular interactions, the lower energy of trans-TFSI with respect to cis-TFSI and the smaller volume of cis-TFSI with respect to trans-TFSI.

Genome-Wide DNA Methylation Analysis Identifies Novel Hypomethylated Non-Pericentromeric Genes with Potential Clinical Implications in ICF Syndrome.

INTRODUCTION AND RESULTS: Immunodeficiency, centromeric instability and facial anomalies syndrome (ICF) is a rare autosomal recessive disease, characterized by severe hypomethylation in pericentromeric regions of chromosomes (1, 16 and 9), marked immunodeficiency and facial anomalies. The majority of ICF patients present mutations in the DNMT3B gene, affecting the DNA methyltransferase activity of the protein. In the present study, we have used the Infinium 450K DNA methylation array to evaluate the methylation level of 450,000 CpGs in lymphoblastoid cell lines and untrasformed fibroblasts derived from ICF patients and healthy donors. Our results demonstrate that ICF-specific DNMT3B variants A603T/STP807ins and V699G/R54X cause global DNA hypomethylation compared to wild-type protein. We identified 181 novel differentially methylated positions (DMPs) including subtelomeric and intrachromosomic regions, outside the classical ICF-related pericentromeric hypomethylated positions. Interestingly, these sites were mainly located in intergenic regions and inside the CpG islands. Among the identified hypomethylated CpG-island associated genes, we confirmed the overexpression of three selected genes, BOLL, SYCP2 and NCRNA00221, in ICF compared to healthy controls, which are supposed to be expressed in germ line and silenced in somatic tissues. CONCLUSIONS: In conclusion, this study contributes in clarifying the direct relationship between DNA methylation defect and gene expression impairment in ICF syndrome, identifying novel direct target genes of DNMT3B. A high percentage of the DMPs are located in the subtelomeric regions, indicating a specific role of DNMT3B in methylating these chromosomal sites. Therefore, we provide further evidence that hypomethylation in specific non-pericentromeric regions of chromosomes might be involved in the molecular pathogenesis of ICF syndrome. The detection of DNA hypomethylation at BOLL, SYCP2 and NCRNA00221 may pave the way for the development of specific clinical biomarkers with the aim to facilitate the identification of ICF patients.

A field survey on the presence of prednisolone and prednisone in urine samples from untreated cows.

Prednisolone is a synthetic glucocorticoid widely employed in bovine clinical practice that may also be used illegally as a growth promoter. Recent in vitro and in vivo studies lend support to the hypothesis that prednisolone could be synthesised from cortisol in untreated cattle subjected to stressful events. To verify such a hypothesis, a field survey was conducted on urine samples collected from 131 guaranteed untreated cows and analysed for the presence of prednisolone and prednisone - in some instances also for cortisol and cortisone - with a validated LC/MS-MS method. None of the examined samples exhibited either prednisolone levels higher than the CCα limit (around 0.70 µg l⁻¹) or prednisone, being therefore officially compliant for both analytes. Trace amounts of prednisolone, approximately estimated in the range 0.1-0.3 µg l⁻¹ were found in only seven samples from cows also showing urinary cortisol and cortisone levels higher than those detected in negative specimens, as the result of a probable stress condition.

Hematologic improvement and response in elderly AML/RAEB patients treated with valproic acid and low-dose Ara-C.

The histone deacetylase inhibitor (HDACi) valproic acid (VPA) has been shown to be active on acute myeloid leukemia (AML) and refractory anemia with excess of blasts (RAEB). Thirty-one elderly AML/RAEB patients (AML n=25; RAEB n=6) with a high rate of comorbidity were entered in a phase II study with low-dose cytarabine (Ara-C) and VPA. Fitness was evaluated by means of the Comprehensive Geriatric Assessment (CGA), including the Cumulative Illness Rating Scale (CIRS) score, the self-sufficiency scores of Activity of Daily Living (ADL) and Instrumental Activity of Daily Living (IADL). Eight patients obtained a lasting complete remission and 3 other patients obtained hematologic improvement for a total response rate of 35%. Five of 11 responding patients were relapsed or resistant after a previous treatment with Ara-C. Seven of 11 responding patients were assessed as frail at enrollment and/or had IADL impairment. Grades 3 and 4 toxicities were mainly hematological. Low-dose Ara-C and VPA is a relatively non-toxic combination with good therapeutic activity in elderly patients with AML/RAEB. This therapeutic approach represents an alternative treatment for patients who cannot undergo standard induction therapy.

Hip fractures in Italy: 2000-2005 extension study.

SUMMARY: A total of 507,671 people > or =65 experienced hip fractures between 2000 and 2005. In 2005, 94,471 people > or =65 were hospitalized due to hip fractures, corresponding to a 28.5% increase over 6 years. Most fractures occurred in patients > or =75 (82.9%; n = 420,890; +16% across 6 years), particularly in women (78.2%; n = 396,967). INTRODUCTION: We aimed to analyze incidence and costs of hip fractures in Italy over the last 6 years. METHODS: We analyzed the national hospitalization and DRG databases concerning fractures occurred in people > or =65 between 2000 and 2005. RESULTS: A total of 507,671 people > or =65 experienced hip fractures across 6 years, resulting in about 120,000 deaths. In year 2005 94,471 people aged > or =65 were hospitalized due to hip fractures, corresponding to a 28.5% increase over 6 years. The majority of hip fractures occurred in patients > or =75 (82.9%; n = 420,890; +16% across 6 years) and particularly in women (78.2%; n = 396,967). Among women, 84.2% of fractures (n = 334,223; +28.0% over 6 years) were experienced by patients > or =75, which is known to be the age group with the highest prevalence of osteoporosis, accounting for 68.6% of the overall observed increase in the total number of fractures. Hip fractures in men > or =75 increased by 33.1% (up to 16,540). Hospitalization costs increased across the six examined years (+36.1%) reaching 467 million euros in 2005, while rehabilitation costs rose up to 531 million in the same year. CONCLUSIONS: Hip fractures of the elderly are increasing and represent a major health problem in industrialized countries such as Italy.

In vivo lamellar keratoplasty using platelet-rich plasma as a bioadhesive.

PURPOSE: The use of standard sutures has been replaced by platelet-rich plasma (PRP), a bioadhesive agent, in several surgical procedures. This prompted us to test PRP efficacy in experimental lamellar keratoplasty. METHODS: After lamellar anterior keratoplasty, PRP with a mean concentration of 807,564 platelets/mm(3) was used to attach the corneal flap to the stromal surface in 12 New Zealand white rabbits. 10-0 nylon sutures were used in one control group of 12 animals and no suture was used in a second control group of six rabbits. Animals were killed at days 2, 7, 30, and 90 for histological and smooth muscle actin (SMA) immunohistochemical analysis. RESULTS: The PRP group showed a tight corneal graft from the first postoperative hours until they were killed. A transparent cornea was seen at 30 days and remained clear until the end of the protocol. Histological specimens showed no signs of ocular inflammation in any animal within the PRP group. Electron microscopy showed normal morphological features on the flap and stromal bed, and a clear interface zone without cells or debris. The number of stromal myofibroblasts was lower than that seen in the suture group at 3 months postsurgery. The sutured group showed an attached cornea with signs of inflammation around the knots. All flaps without PRP or sutures were completely detached in the immediate postoperative period. CONCLUSIONS: PRP was useful for attaching the corneal flap and it was well tolerated by the rabbit corneal tissue. Corneal healing was satisfactory. Further studies on PRP adhesiveness in grafts with donor corneas should be performed before considering its use in patients.

[Chronic lumbago after unstable intertrochanteric femoral fracture: a new syndrome or sporadic feature of hip biomechanics after surgery? A case report]. / Lombalgia cronica invalidante dopo frattura intertrocanterica instabile di femore: nuova sindrome o sporadica alterazione biomeccanica dell'anca dopo la chirurgia? Case report.

OBJECTIVE: Only in 2002, more than 86,000 hip fractures were registered in Italy in male and female patients over 45 years old, with 9% progression compared to 1999. In this paper, we report a clinic case of a patient experiencing working problems for a chronic back pain after an unstable extracapsular hip fracture. SUBJECT AND METHOD: The patient, a 54 years old woman, factory worker, complaining of low back pain weeks after the hip fracture, was evaluated and scheduled for a computer tomography examination of the lumbar spine. The C.T. scans were evaluated to assess eventual evidence of disc problems and/or cross sectional changes in density and fibro-adipose degeneration of muscular body of the psoas muscle. RESULT: Because back pain matched with the evidence of cross sectional changes in psoas density without any evidence of disc herniation, the patient was scheduled for aspecific back pain training. After a specific rehabilitation protocol the patient had 75% pain relief after four weeks. CONCLUSION: Age and diseases related changes are commonly seen in the lumbar spine, but the relationship between these changes and symptoms of back pain is not straightforward. Our case report suggest that back pain following extra capsular hip fracture may involve the psoas muscle. The prognosis might be favourable following a specific back training that is actually object of an ongoing controlled trial.

Low back pain after unstable extracapsular hip fractures: randomized control trial on a specific training.

AIM: This study compares the efficacy of a walking recovery rehabilitation program with or without specific back exercises in patients affected by unstable extracapsular hip fractures and secondary back pain. Further, it reports data and images about analysed tomographic changes of the psoas muscle ipsilateral to fractures. METHODS: A prospective, randomized, parallel treatment trial was carried out. Patients admitted for rehabilitation after fractures were evaluated for secondary back pain. If clinically indicated, patients were further scheduled for a computed tomography (CT) scan of the lumbar spine by which we evaluated cross-sectional changes in density and fibroadipose degeneration of the body of the psoas muscle. When back pain matched cross-sectional changes in psoas density, the patient was eligible for our study, enrolled and randomly assigned to 1 of 2 study groups. Both groups were scheduled for hip rehabilitation and walking training plus a back protocol for the study group only. Pain was evaluated subjectively with a visual analog scale to calculate treatment effectiveness and Harris hip score was used to assess the outcome after surgery. RESULTS: Thirty-seven patients out of about hundred were enrolled. In all cases CT scans showed age-related changes commonly seen in the lumbar spine and significant fibroadipose degeneration and altered density in the ipsilateral psoas muscles. With back exercises added to the standard rehabilitation protocol, all study patients recovered significantly better than control group patients (P<0.04) in only 4 weeks of treatment. CONCLUSION: Results show how hip fractures may cause psoas changes that can be of relevance for a more rational choice of physical exercises. Causes of back pain may be more complex than described by the textbooks and prognosis is more favourable following specific back training.

Incidence and costs of hip fractures compared to acute myocardial infarction in the Italian population: a 4-year survey.

INTRODUCTION: Few data are available about the incidence and costs of hip fractures in Italy. We aimed to determine the impact of hip fractures vs. acute myocardial infarction (AMI). METHODS: We studied the national hospitalization database to calculate their incidence and costs in adults aged >or=45 between 1999 and 2002. RESULTS: In 2002, there were 86,719 hip fractures with a 10.0% increase over 4 years. We observed a predominance of women (77.1%) and a strong age effect: 92.7% of patients were >or=65 years old and 80% of fractures occurred in women aged >or=75, showing a clear relationship with the incidence of osteoporosis. Hospitalizations due to AMI after 45 years of age in 1999 were only 9% higher than those for hip fracture, although this difference increased over the 4 examined years up to 24%. Considering the DRGs costs, hip fractures resulted in being more expensive than AMI overall and concerning elderly people. CONCLUSIONS: This study shows that in the Italian population aged >or=45, hospitalizations following hip fracture and AMI between 1999 and 2002 were comparable, while hip fractures' direct costs were higher and grew faster than costs for AMI. Hip fractures in Italy are a serious medical problem and a leading health-cost driver.

[Methodology assessment of the first-aid in a company]. / Metodologia di valutazione dell'adeguatezza del primo soccorso aziendale.

The Ministerial Decree 388/03 establishes the first aid duties and organisation. It classifies the health firms into three groups, according to the types of activities carried out, to the number of the employees involved, and to the risks linked to it. The membership of a firm to a group reflects upon its standard equipment of facilities, the training required for the employees and the duties of communication to carry out to the Health Service. During the activity of vigilance we have verified that a lot of firms, belonging to our territory, have not applied correctly the body of rules regarding the first aid. For this reason a project aiming at providing information about the application of this decree has been started.

An optic micro-switch for an eyelid response to foster environmental control in children with minimal motor behaviour.

This study assessed whether two children (11.9- and 9.7-years-old) with profound multiple disabilities and minimal motor behaviour could learn to control environmental stimulation using an eyelid response with a newly developed micro-switch. The response consisted of raising the eyelid markedly (i.e. by looking upward or opening the eyes widely). The micro-switch developed for this target response consisted of an electronically regulated optic sensor mounted on an eyeglasses' frame. Data showed that the children learned the target eyelid response to activate the micro-switch and to increase their level of environmental stimulation. Responding was largely maintained at a 2-month post-intervention check. These results indicate that continued work in this area has positive implications for the rehabilitation of children with most serious disabilities.

Contribution of the copper ions in the dinuclear active site to the stability of Carcinus aestuarii hemocyanin.

We have investigated the effect of copper binding on the structural properties of hemocyanin (Hc). To this aim, we have studied the holo- and apo-form of the protein, both in the hexameric and in the monomeric state (CaeSS2 subunit), with experimental approaches that report on the protein aggregation and conformational stability. The results of gel-filtration chromatography and small angle X-ray scattering (SAXS) provide evidence that the hydrodynamic and gyration radius (R(g)) of Hc in the hexameric form only slightly increase upon copper removal, whereas a remarkable enhancement in the R(g) value is observed for the CaeSS2 monomer. CD measurements in the far- and near-UV region indicate that removal of copper only marginally affects the conformation of the hexameric Hc. Instead, copper depletion in the CaeSS2 strongly alters the tertiary structure of the monomer (near-UV CD), even though it is almost inconsequential on the secondary structure content (far-UV CD). These findings are fully consistent with the results of limited proteolysis experiments showing that the hexameric Hc is similarly resistant to proteolysis by trypsin both in the holo- and apo-form. Conversely, the apo-form of CaeSS2 monomer is much more susceptible to proteolytic attack by trypsin than the holo-form. Based on SAXS measurements, the concentration-dependent oligomerization process for apo-CaeSS2 has been analyzed on the basis of a thermodynamic model involving a concentration-dependent equilibrium between a monomer in a native-like and an hexameric aggregate of monomers.

Structural role of the copper ions in the dinuclear active site of Carcinus aestuarii hemocyanin.

In this work we show, by a combination of biochemical and biophysical approaches, that the copper ions bound in the binuclear active site of Carcinus aestuarii hemocyanin play a stabilizing role on the tertiary structure of the protein. Upon removal of copper, the monomeric hemocyanin, but not the hexameric oligomer, undergoes changes at the level of tertiary structure while the secondary structure is almost unaffected. By Small-Angle X-Ray Scattering, supported by gel chromatography measurements, it can be concluded that the apo-monomer, but not the holo form or the hexameric form, undergoes a slow time-dependent oligomerization process.

Allogeneic bone marrow transplantation (BMT) for adults with acute lymphoblastic leukemia (ALL): predictive role of minimal residual disease monitoring on relapse.

We developed a PCR-based method to monitor clonogenic IgH VDJ rearrangement as a possible predictor of relapse in patients with acute B-ALL after allogeneic bone marrow transplantation (BMT). We studied 23 patients at diagnosis, before and after BMT. At the time of BMT, 13 patients were in first complete remission, eight in second complete remission and two in relapse. Four patients were PCR negative before BMT and remained PCR negative also after BMT (-/- pattern). They are still in remission after a median follow-up of 41 months. Nineteen patients were MRD-positive before BMT: three were PCR negative at first determination after BMT (+/- pattern) and maintain remission. Sixteen patients were PCR-positive at first determination after BMT (+/+ pattern): five became PCR negative (+/+/- pattern) (four with chronic graft-versus-host disease (GVHD) and two after donor lymphocyte infusions (DLI)). Nine patients remained PCR-positive (+/+/+ pattern) (four remain in remission, and six relapsed); two patients died before transplant. In conclusion, PCR negative patients before BMT remained negative post-BMT; many pre-BMT positive patients had initial MRD positivity after BMT: 37% of them achieved a molecular remission with cGVHD or DLI.

Fludarabine, ARA-C, idarubicin and G-CSF (FLAG-Ida), high dose ARA-C and early stem cell transplant. A feasable and effective therapeutic strategy for de novo AML patients.

Forty-three consecutive patients with de novo and untreated non M3 AML aged 60 or less entered the study. The mean age of patients was 50 (range 15-60). The induction regimen (FLAG-Ida) included fludarabine (30 mg/sqm), Ara-C (2 g/sqm) on days 1-5, and idarubicin (10 mg/sqm) on days 1, 3, 5. G-CSF (300 mcg/day) was administered s.c. 12 hours before starting fludarabine and was continued for five days. HDT with stem cell rescue was planned for all patients in first CR after one course of high dose Ara-C (HDAC) consolidation and in good clinical conditions. Forty-two (98%) patients were evaluable for response. One patient died during induction (2%). CR was achieved in 35 patients (82%). Twenty-three patients, 66% of those achieving CR, underwent autologous (N = 17) or allogeneic (N = 6) transplantation. With a median follow up of 24 months, the average median duration of CR is 17 months (range 3-66) and the median survival is 20 months (range 1-83). Overall the 5 year projected disease free survival (DFS) and overall survival (OS) were 37% and 43%, respectively. Among patients who underwent stem cell transplantation DFS and OS were 53% and 69%, respectively. The median time to PMN recovery (> 0.5 x 10(9)/l) was 17 days (range 10-28) and 50 x 10(9)/l platelets were reached at a median of 17 days (12-38). In conclusion FLAG-Ida regimen is effective, low toxic and improves feasibility of stem cell transplant.

Lymphoplasmacytic lymphoma/immunocytoma: towards a disease-targeted treatment?

Lymphoplasmacytic-lymphoplasmacytoid lymphoma (LPL)/Waldenstrom's macroglobulinemia (WM) or immunocytoma (IMC) consists of diffuse proliferation of small mature B lymphocytes, plasmacytoid lymphocytes, and plasma-cells. The nosographic definition includes the lack of histological, immunophenotypic, cytogenetic, and molecular markers considered specific of other types of lymphoma. The cells show surface Ig (usually IgM), B-cell-associated antigens and display the CD5-, CD23- and CD10- phenotype, which allows for differential diagnosis from B-CLL and mantle cell lymphoma. t(9;14)(p13;q32) chromosomal translocation has been found in 50% of all LPL cases. The cytogenetic rearrangement juxtaposes the PAX-5 gene, which encodes for an essential transcription factor for B-cell proliferation and differention, to the Ig heavy chain gene. The combination of chlorambucil and prednisone holds as the standard treatment and seems to guarantee good control of the disease in most patients. Similar therapeutic results have been described with the combination of cyclophosphamide, vincristine, prednisone with (CHOP) or without doxorubicin (CVP), or with a combination of other alkylating agents and prednisone. Nucleoside analogues, alone or in combination with alkylating agents and anthracyclines, provide good salvage therapy for IMC and being increasingly employed as first line therapy. In a multicentric European trial Foran et al. administered the chimeric anti-CD20-monoclonal antibody (Rituximab) to 28 patients with previously treated IMC. Seven out of 25 evaluable patients (28%) achieved a partial response. Byrd et al. examined the outcome of 7 previously treated WM patients who received weekly infusions of rituximab (375 mg/m2). Therapy was well tolerated by all patients, and there was no decrease in cellular immune function, or significant infectious morbidity. Partial responses were noted in three of these patients, including two with fludarabine-refractory disease. These data suggest that rituximab exerts clinical activity on heavily pre-treated patients with WM. Furthermore, Weide et al. first reported that WM-associated polyneuropathy can be treated effectively with a combination of chemotherapy and the anti-CD20 monoclonal antibody rituximab. Most published trials exploring the efficacy of high dose treatment as salvage therapy for relapsed or refractory low grade non Hodgkin's lymphoma have included prevalently follicular or lymphocytic lymphomas. In selected high risk patients radioimmunotherapy with autologous stem-cell rescue, and myeloablative therapy followed either by autologous stem cell transplantation (SCT) or allogeneic SCT might represent an alternative strategy.

First line therapy with fludarabine combinations in 42 patients with either post myelodysplastic syndrome or therapy related acute myeloid leukaemia.

Acute myeloid leukaemias (AML) evolving from a myelodysplastic syndrome (MDS) or secondary to chemoradiotherapy frequently display unfavorable biologic characteristics. This may explain the lower remission rate obtained with conventional chemotherapy. Recently, the association of Fludarabine with intermediate dose Ara-C has produced interesting results particularly in high risk AML patients. Here, we report on 42 secondary AML patients treated with a combination of Fludarabine, intermediate dose Ara-C, G-CSF with or without an antracycline (FLANG, FLAG-IDA or FLAG). Overall, complete remissions (CR) were documented in 14 patients (33%) and partial responses (PR) in 12 (29%), while 10 patients proved resistant (24%). Six patients (14%) died early. The presence of a prognostically unfavorable karyotype had a negative impact on the CR rate (20% compared to 50% for patients with an intermediate prognosis karyotype, p 0.05). Patients treated with FLAG, FLANG and FLAG-IDA had similar CR rates. At the time of this analysis, after a mean follow-up of 12 months, the mean duration of CR is 16 months (range 3-66) and the mean survival is 11 months (range 1-67). The median time to granulocyte recovery (neutrophils > 0.5 x 10(9)/l) was 20 days (range 12-39) and 50 x 10(9)/l platelets were reached at a median of 26 days (range 9-56). Taken together, these Fludarabine containing regimens proved to be an effective and tolerable treatment for patients with secondary AML. Patients above 70 years of age may also benefit from this therapy, however the problem of treating patients with adverse chromosomal abnormalities still remains unresolved.

GM-CSF, ARA-C, VP-16 and idarubicin (GM-IVA), a short, and effective induction treatment for de novo AML, suitable for the elderly.

GM-IVA is a short and effective induction therapy of non M3 de novo AML including GM-CSF (300 mcg 12 hrs before starting therapy), Ara-C (250 mg/sqm c.i. x 3 days), VP16 (100 mg/sqm x 3 days) and idarubicin (12 mg/sqm x 3 days); it was followed by a fludarabine containing salvage protocol (FLANG). Patients <60 years of age achieving CR received 2 courses of FLANG and autologous or allogeneic BMT when possible. Patients >60 years of age in CR received a second course of GM-IVA. Twenty-one consecutive patients (mean age 64, range 29-85) entered the study. Three patients (14%) died during induction therapy. After one course of GM-IVA, CR was achieved in 12 patients (57%). Two further patients were salvaged with FLANG therapy so that the final CR rate was 14/21 (67%). In elderly patients the final CR rate (62%) is noteworthy, considering that 6 patients were >70 years of age and 3 were >80. All three patients >80 achieved CR (lasting 5 to 7 months). The median time of granulocyte and platelet recovery was 15 days. Our scheme was well tolerated. In the group of elderly patients 3 out of 14 died during induction (21%) and 4 life-threatening infections were observed (28%). The short duration of cytotoxic therapy and perhaps the use of G-CSF contributed to a reduction of the hospitalization period (median of 22 days), thus providing major savings on induction costs and allowing for better utilization of beds as well as significantly improving patients' quality of life.