IS1R-mediated plasticity of IncL/M plasmids leads to the insertion of bla OXA-48 into the Escherichia coli Chromosome.

The OXA-48 carbapenemase is mainly encoded by ∼ 62-kb IncL/M plasmids. However, chromosome-mediated genes have been observed in Escherichia coli isolates. In this work, we investigated the genetic environment of OXA-48 in members of the family Enterobacteriaceae (n = 22) to understand how the OXA-48-encoding gene is transferred into the E. coli chromosome. The OXA-48-encoding gene was located within intact Tn1999.2 transposons in the ∼ 62-kb plasmids or within a truncated variant of Tn1999.2 for the OXA-48-encoding genes located in the chromosomes of E. coli bacteria. The analysis of the Tn1999.2 genetic environment revealed an inverted orientation of the transposon in five ∼ 62-kb plasmids (5/14 [35%]) and in all chromosome inserts (n = 8). The sequencing of pRA35 plasmid showed that this orientation of Tn1999.2 and the acquisition of an IS1R insertion sequence generated a 21.9-kb IS1R-based composite transposon encoding OXA-48 and designated Tn6237. The sequencing of a chromosomal insert encoding OXA-48 also revealed this new transposon in the E. coli chromosome. PCR mapping showed the presence of this element in all strains harboring an OXA-48-encoding chromosomal insert. However, different insertion sites of this transposon were observed in the E. coli chromosome. Overall, these findings indicate a plasticity of the OXA-48 genetic environment mediated by IS1R insertion sequences. The insertion sequences can induce the transfer of the OXA-encoding gene into E. coli chromosomes and thereby promote its persistence and expression at low levels.

Cloning and characterization of a new intestinal inflammation-associated colonic epithelial Ste20-related protein kinase isoform.

Intestinal epithelial cells respond to inflammatory extracellular stimuli by activating mitogen activated protein kinase (MAPK) signaling, which mediates numerous pathophysiological effects, including intestinal inflammation. Here, we show that a novel isoform of SPS1-related proline alanine-rich kinase (SPAK/STE20) is involved in this inflammatory signaling cascade. We cloned and characterized a SPAK isoform from inflamed colon tissue, and found that this SPAK isoform lacked the characteristic PAPA box and alphaF loop found in SPAK. Based on genomic sequence analysis the lack of PAPA box and alphaF loop in colonic SPAK isoform was the result of specific splicing that affect exon 1 and exon 7 of the SPAK gene. The SPAK isoform was found in inflamed and non-inflamed colon tissues as well as Caco2-BBE cells, but not in other tissues, such as liver, spleen, brain, prostate and kidney. In vitro analyses demonstrated that the SPAK isoform possessed serine/threonine kinase activity, which could be abolished by a substitution of isoleucine for the lysine at position 34 in the ATP-binding site of the catalytic domain. Treatment of Caco2-BBE cells with the pro-inflammatory cytokine, interferon gamma, induced expression of the SPAK isoform. Over-expression of the SPAK isoform in Caco2-BBE cells led to nuclear translocation of an N-terminal fragment of the SPAK isoform, as well as activation of p38 MAP kinase signaling cascades and increased intestinal barrier permeability. These findings collectively suggest that pro-inflammatory cytokine signaling may induce expression of this novel SPAK isoform in intestinal epithelia, triggering the signaling cascades that govern intestinal inflammation.

Targeting colorectal cancer-associated bacteria: A new area of research for personalized treatments.

Most cases of colorectal cancer (CRC) are sporadic, and numerous studies have suggested that gut microbiota may play a crucial role in CRC development. Escherichia coli is a member of the gut microbiota frequently associated with colorectal tumors. CRC-associated E. coli strains frequently harbor the pks genomic island. This genomic island is responsible for the synthesis of colibactin genotoxin, which increases tumor numbers in CRC mouse models. We recently showed that targeting ClbP, a key enzyme involved in colibactin synthesis, blocks the deleterious effect of this toxin in vitro and leads to a significant decrease in tumor numbers in vivo. Altogether, our results suggest that the personalized treatment of CRC should also take into consideration the bacteria associated with the tumor in order to limit their deleterious effects.

First experience in Italy with a new transcutaneous bone conduction implant.

Since 2011, transcutaneous bone-anchored auditory implants have been an alternative to the classic percutaneous implant (Baha) for bilateral conductive/mixed hearing loss that cannot be corrected by surgery. Recently, a new transcutaneous device has been approved for clinical use. Its internal component is made of the classic titanium Baha fixture, coupled to a 27 mm diameter subcutaneous circular magnet. The external component includes a second circular magnet 29 mm in diameter and a digital sound processor. To date, there are no reports describing the results of the application of this device. The aim of the present study is to report on the anatomical and functional results of transcutaneous Baha implantation in three patients: two adults, one with syndromic aural atresia and one with bilateral conductive hearing loss due to bilateral tympanomastoidectomy, and an 8-year-old child with non-syndromic aural atresia. No major intraoperative or postoperative complications were observed. The three patients tolerated the external magnet, with no signs of skin irritation. Functional results were good: median unaided free-field PTA (0.5-3 kHz) was 50 dB HL (range = 41-66 dB HL); with the transcutaneous Baha median PTA (0.5-3 kHz) was 27 dB HL (range = 25-30 dB HL) and median gain was 25 dB HL (range = 11-39 dB HL). Preliminary results encourage use of the device as a valuable alternative to other implantable devices in these patients. To ensure the success of treatment, several precautions are suggested including gradually increasing use during the first post-operative months to favour skin adaptation to magnet pressure. In addition to skin reactions, in a paediatric age most concerns are related to the curvature of the skull, which may induce tenting of the skin over the internal magnet.

Chromosomal localization of telomeric sequences in three species of Akodon (Rodentia, Sigmodontinae).

The distribution of the vertebrate telomeric sequence T2AG3 in three species of the rodent genus Akodon was examined by FISH with a peptide nucleic acid probe. In addition to the expected telomeric hybridization, non-telomeric signals were observed in the three species. In A. dolores, centromeric signals were visible in two of the four biarmed autosome pairs featuring Robertsonian polymorphism, indicating the retention of at least part of the telomeric sequences during the fusion process, and an interstitial signal of lower intensity was observed in the short arm of another. In A. boliviensis, a strong signal was observed near the centromeric end of the first chromosome pair. The first pair of A. azarae (homologous to the first pair of A. boliviensis) showed a similar but markedly amplified signal, and a subcentromeric signal in the X chromosome corresponding to a heterochromatic region; additionally, interstitial signals of lower intensity were present in one to four chromosomes in the majority of cells examined.

Specific heterochromatic banding of metaphase chromosomes using nuclear yellow.

The bis-benzimidazole compound nuclear yellow (NY) belongs to the same chemical family as the DNA binding fluorochromes Hoechst 33258 and Hoechst 33342. Spectroscopic studies of NY alone and in the presence of calf thymus DNA show high DNA binding affinity and behavior similar to the Hoechst fluorochromes above. Mitotic metaphase chromosomes from Balb/c mice stained with NY show C-banding and weak G/Q-banding, both of them disappearing after distamycin A (DA) or methyl green (MG) counterstaining. The same staining of human metaphase chromosomes from lymphocyte cultures, however, reveal only faint G/Q-banding (NY) and a characteristic DA-DAPI-like banding (NY-DA, NY-MG). Image analysis of NY stained human chromosomes, confirms that NY is suitable for studying polymorphisms affecting size in the pericentromeric heterochromatin of pairs 1, 9 and 16, and shows significant enhancement of NY fluorescence induced by DA in DA-DAPI heterochromatin. Our spectroscopic and cytological results show that NY, either alone or counterstained with DA or MG, can be used for DNA cytochemistry and chromosome banding. Possible mechanisms for the banding patterns induced by NY are discussed.

[Evaluation of an immunoenzymatic method for determining the tumor marker CA 15-3)]. / Valutazione di una metodologia immunoenzimatica per la determinazione del marcatore tumorale CA 15-3.

CA 15-3 determination has graved to be of clinical utility for diagnosis, evaluation and monitoring patients suffering from tumor. The aim of our study was to compare Sorin (kit ETI CA 15-3 K) and BMI (Enzymun-test CA 15.3) screening between two methodologies' capability. The results obtained were evaluated by using a Passing and Bablok correlation, Spearman rank correlation coefficient. The results obtained shown an over-valuation of the Sorin method vs. BMI.

[Evaluation of an immunoenzymatic method to determine the tumor marker CA 125]. / Valutazione di una metodologia immunoenzimatica per la determinazione del marcatore tumorale CA 125.

CA 125 determination has proved to be of clinical utility for diagnosis, evaluation and monitoring patients suffering from tumor. The aim of our study was to compare Sorin (kit ETI CA 125 K) and BMI (Enzymun-Test CA 125) screening between two methodologies' capability. The result obtained was evaluated by using a Passing and Bablok correlation, Spearman rank correlation coefficient. The results obtained show a low correlation between the two methods.

[295 cases of brucellosis treated with minocycline]. / Su 295 casi di brucellosi trattati con minociclina.

295 cases of acute and subacute cases of brucellosis were treated with minocycline. It is concluded that because of its in vitro activity, its penetrability in macrophages and granulomas and its tolerance, minocycline is the antibiotic of choice for brucellosis.

[Our experience with a combination of rifampicin and minocycline in the therapy of brucellosis]. / La nostra esperienza con l'associazione rifampicina-minociclina nella terapia della brucellosi.

After a brief review of the literature on the treatment of brucellosis the results obtained with a new protocol (Rifampicin + Minocycline for 20 days) are reported. The combination was well-tolerated and undeniably effective producing a 94% cure rate.

BDNF/ TrkB interaction regulates migration of SVZ precursor cells via PI3-K and MAP-K signalling pathways.

Neuroblasts born in the subventricular zone (SVZ) migrate along the rostral migratory stream, reaching the olfactory bulb (OB) where they differentiate into local interneurons. Several extracellular factors have been suggested to control specific steps of this process. The brain-derived neurotrophic factor (BDNF) has been demonstrated to promote morphological differentiation and survival of OB interneurons. Here we show that BDNF and its receptor TrkB are expressed in vivo throughout the migratory pathway, implying that BDNF might also mediate migratory signals. By using in vitro models we demonstrate that BDNF promotes migration of SVZ neuroblasts, acting both as inducer and attractant through TrkB activation. We show that BDNF induces cAMP response element-binding protein (CREB) activation in migrating neuroblasts via phosphatidylinositol 3-kinase (PI3-K) and mitogen-activated protein kinase (MAP-K) signalling. Pharmacological blockade of these pathways on SVZ explants significantly reduces CREB activation and impairs neuronal migration. This study identifies a function of BDNF in the SVZ system, which involves multiple protein kinase pathways leading to neuroblast migration.