A New Method to Quantify Ifosfamide Blood Levels Using Dried Blood Spots and UPLC-MS/MS in Paediatric Patients with Embryonic Solid Tumours

Ifosfamide blood concentrations are necessary to monitor its therapeutic response, avoiding any adverse effect. We developed and validated an analytical method by UPLC-MS/MS to quantify ifosfamide in dried blood spots (DBS). Blood samples were collected on Whatman 903® filter paper cards. Five 3 mm disks were punched out from each dried blood spot. Acetonitrile and ethyl acetate were used for drug extraction. Chromatographic separation was carried out in an Acquity UPLC equipment with a BEH-C18 column, 2.1 x 100 mm, 1.7 μm (Waters®). The mobile phase consisted in 5 mM ammonium formate and methanol:acetonitrile (40:48:12 v/v/v) at 0.2 mL/min. LC-MS/MS detection was done by ESI+ and multiple reaction mode monitoring, ionic transitions were m/z¹⁺ 260.99 > 91.63 for ifosfamide and 261.00 > 139.90 for cyclophosphamide (internal standard). This method was linear within a 100–10000 ng/mL range and it was accurate, precise and selective. Ifosfamide samples in DBS were stable for up to 52 days at -80°C. The procedure was tested in 14 patients, ages 1 month to 17 years (9 males and 5 females), with embryonic tumours treated with ifosfamide, alone or combined, at a public tertiary referral hospital. Ifosfamide blood levels ranged from 11.1 to 39.7 μmol/L at 12 hours after the last infusion, while 24-hour levels ranged from 0.7–19.7 μmol/L. The median at 12 hours was 19.5 μmol/L (Q₂₅ 14.4–Q₇₅ 29.0) and 3.8 μmol/L (Q₂₅ 1.5–Q₇₅ 9.9) at 24 hours, p<0.001. This method is feasible to determine ifosfamide plasma levels in paediatric patients.     

Sawfishes and Other Elasmobranch Assemblages from the Mio-Pliocene of the South Caribbean (Urumaco Sequence,Northwestern Venezuela)

The Urumaco stratigraphic sequence, western Venezuela, preserves a variety of paleoenvironments that include terrestrial, riverine, lacustrine and marine facies. A wide range of fossil vertebrates associated with these facies supports the hypothesis of an estuary in that geographic area connected with a hydrographic system that flowed from western Amazonia up to the Proto-Caribbean Sea during the Miocene. Here the elasmobranch assemblages of the middle Miocene to middle Pliocene section of the Urumaco sequence (Socorro, Urumaco and Codore formations) are described. Based on new findings, we document at least 21 taxa of the Lamniformes, Carcharhiniformes, Myliobatiformes and Rajiformes, and describe a new carcharhiniform species (†Carcharhinus caquetius sp. nov.). Moreover, the Urumaco Formation has a high number of well-preserved fossil Pristis rostra, for which we provide a detailed taxonomic revision, and referral in the context of the global Miocene record of Pristis as well as extant species. Using the habitat preference of the living representatives, we hypothesize that the fossil chondrichthyan assemblages from the Urumaco sequence are evidence for marine shallow waters and estuarine habitats.     

Hospital Admission following Acute Kidney Injury in Kidney Transplant Recipients Is Associated with a Negative Impact on Graft Function after 1-Year

The incidence and outcomes of acute kidney injury (AKI) in kidney transplantation are poorly known. Retrospective cohort analysis was performed on the data of all patients (≥3 months after transplantation and ≥16 years of age) admitted to the hospital due to medical or surgical complications from 2007 to 2010. We analyzed 458 kidney transplant recipients, 55.2% men, median age 49 (IQR, 36–58) years, median of 12.5 (IQR, 3–35) months after kidney transplantation; admitted to the hospital due to medical or surgical complications. Most of the patients received a kidney from a deceased donor (62.2%), the primary cause for hospital admission was infection (60.7%) and 57 (12.4%) individuals were diagnosed with acute rejection (AR). The incidence of AKI was 82.3%: 31.9% stage 1, 29.3% stage 2 and 21.2% stage 3. Intensive care unit (ICU) admission (OR 8.90, 95% CI: 1.77–44.56 p = 0.008), infection (OR 5.73, 95% CI: 2.61–12.56, p<0.001) and the use of contrast media (OR 9.34, 95% CI: 2.04–42.70, p = 0.004) were the independent risk factors for AKI development. The mortality rate was 2.1% and all patients who died were diagnosed with AKI. Even after the exclusion of AR cases, at the end of 12 months, the individuals with AKI exhibited higher percent changes in creatinine values when compared with individuals without AKI (9.1% vs. -4.3%; p<0.001). According to KDIGO system, we found a high incidence of AKI among the complications of renal transplantation. As in other scenarios, AKI was associated with renal function loss at 1-year after the hospital discharge.     

A Pilot Study Identifying a Set of microRNAs As Precise Diagnostic Biomarkers of Acute Kidney Injury

In the last decade, Acute Kidney Injury (AKI) diagnosis and therapy have not notably improved probably due to delay in the diagnosis, among other issues. Precocity and accuracy should be critical parameters in novel AKI biomarker discovery. microRNAs are key regulators of cell responses to many stimuli and they can be secreted to the extracellular environment. Therefore, they can be detected in body fluids and are emerging as novel disease biomarkers. We aimed to identify and validate serum miRNAs useful for AKI diagnosis and management. Using qRT-PCR arrays in serum samples, we determined miRNAs differentially expressed between AKI patients and healthy controls. Statistical and target prediction analysis allowed us to identify a panel of 10 serum miRNAs. This set was further validated, by qRT-PCR, in two independent cohorts of patients with relevant morbi-mortality related to AKI: Intensive Care Units (ICU) and Cardiac Surgery (CS). Statistical correlations with patient clinical parameter were performed. Our results demonstrated that the 10 selected miRNAs (miR-101-3p, miR-127-3p, miR-210-3p, miR-126-3p, miR-26b-5p, miR-29a-3p, miR-146a-5p, miR-27a-3p, miR-93-3p and miR-10a-5p) were diagnostic biomarkers of AKI in ICU patients, exhibiting areas under the curve close to 1 in ROC analysis. Outstandingly, serum miRNAs estimated before CS predicted AKI development later on, thus becoming biomarkers to predict AKI predisposition. Moreover, after surgery, the expression of the miRNAs was modulated days before serum creatinine increased, demonstrating early diagnostic value. In summary, we have identified a set of serum miRNAs as AKI biomarkers useful in clinical practice, since they demonstrate early detection and high diagnostic value and they recognize patients at risk.     

Trisomy 8, a Cytogenetic Abnormality in Myelodysplastic Syndromes,Is Constitutional or Not?

Isolated trisomy 8 is not considered presumptive evidence of myelodysplastic syndrome (MDS) in cases without minimal morphological criteria. One reason given is that trisomy 8 (+8) can be found as a constitutional mosaicism (cT8M). We tried to clarify the incidence of cT8M in myeloid neoplasms, specifically in MDS, and the diagnostic value of isolated +8 in MDS. Twenty-two MDS and 10 other myeloid neoplasms carrying +8 were studied. Trisomy 8 was determined in peripheral blood by conventional cytogenetics (CC) and on granulocytes, CD3+ lymphocytes and oral mucosa cells by fluorescence in situ hybridization (FISH). In peripheral blood CC, +8 was seen in 4/32 patients. By FISH, only one patient with chronic myelomonocytic leukemia showed +8 in all cell samples and was interpreted as a cT8M. In our series +8 was acquired in all MDS. Probably, once discarded cT8M by FISH from CD3+ lymphocytes and non-hematological cells, +8 should be considered with enough evidence to MDS.     

Immature B Cell Egress from Bone Marrow Is SOCS3 Independent

Suppressor of cytokine signaling (SOCS)-3 has been suggested to regulate CXCR4 signaling in a variety of human cell lines. In mice, conditional SOCS3 inactivation in hematopoietic cells including B-lineage lymphocytes has been reported to exacerbate CXCR4-signaling and focal adhesion kinase phosphorylation, which resulted in altered immature B cell distribution in bone marrow (BM) due to sustained α4β1 integrin-mediated adhesion to the extracellular matrix. However, a recent study examining conditional SOCS3 deletion specifically in B-lineage cells failed to detect significant roles in B-lineage cell retention in BM. In this study we carefully examined the role played by SOCS3 in CXCR4 signaling in developing B cell subsets. We show that in mice conditionally deficient in SOCS3 exclusively in B cells (Socs3 ^fl/fl Mb1 ^cre/+) there was no detectable difference in B cell development in BM and in periphery. We show that SOCS3 deficient and sufficient immature B cell subsets are similarly distributed between BM parenchyma and sinusoids, and are equally competent at exiting BM into peripheral blood. Furthermore, we found no significant differences in CXCR4 desensitization upon ligand exposure in developing B lymphocyte subsets. Consequently, SOCS3-deficient and sufficient B-lineage cell migration towards CXCL12 in vitro was undistinguishable, and B-lineage cell amoeboid motility within BM parenchyma was also unaffected by SOCS3-deficiency. Thus we conclude that SOCS3 has no detectable influence on biological processes known to be controlled by CXCR4 signaling.     

The Role of Polymerase Chain Reaction of High-Risk Human Papilloma Virus in the Screening of High-Grade Squamous Intraepithelial Lesions in the Anal Mucosa of Human Immunodeficiency Virus-Positive Males Having Sex with Males

Objectives

To evaluate the advantages of cytology and PCR of high-risk human papilloma virus (PCR HR-HPV) infection in biopsy-derived diagnosis of high-grade squamous intraepithelial lesions (HSIL = AIN2/AIN3) in HIV-positive men having sex with men (MSM).

Methods

This is a single-centered study conducted between May 2010 and May 2014 in patients (n = 201, mean age 37 years) recruited from our outpatient clinic. Samples of anal canal mucosa were taken into liquid medium for PCR HPV analysis and for cytology. Anoscopy was performed for histology evaluation.

Results

Anoscopy showed 33.8% were normal, 47.8% low-grade squamous intraepithelial lesions (LSIL), and 18.4% HSIL; 80.2% had HR-HPV. PCR of HR-HPV had greater sensitivity than did cytology (88.8% vs. 75.7%) in HSIL screening, with similar positive (PPV) and negative predictive value (NPV) of 20.3 vs. 22.9 and 89.7 vs. 88.1, respectively. Combining both tests increased the sensitivity and NPV of HSIL diagnosis to 100%. Correlation of cytology vs. histology was, generally, very low and PCR of HR-HPV vs. histology was non-existent (<0.2) or low (<0.4). Area under the receiver operating characteristics (AUROC) curve analysis of cytology and PCR HR-HPV for the diagnosis of HSIL was poor (<0.6). Multivariate regression analysis showed protective factors against HSIL were: viral suppression (OR: 0.312; 95%CI: 0.099-0.984), and/or syphilis infection (OR: 0.193; 95%CI: 0.045-0.827). HSIL risk was associated with HPV-68 genotype (OR: 20.1; 95%CI: 2.04-197.82).

Conclusions

When cytology and PCR HR-HPV findings are normal, the diagnosis of pre-malignant HSIL can be reliably ruled-out in HIV-positive patients. HPV suppression with treatment protects against the appearance of HSIL.     

GSE4, a Small Dyskerin- and GSE24.2-Related Peptide,Induces Telomerase Activity,Cell Proliferation and Reduces DNA Damage,Oxidative Stress and Cell Senescence in Dyskerin Mutant Cells

Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients.     

NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103

Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103.