Empirical monotherapy with meropenem or combination therapy: the microbiological point of view.

The increase in the number of clinical isolates of multiresistant Enterobacteriaceae and Pseudomonas aeruginosa raises problems in decision-making on empirical treatments for severe Gram-negative bacilli-associated infections. The aim of our study is to determine the resistance of meropenem in our setting and the co-resistance of a combination of this compound with two antibiotics from different families: amikacin and ciprofloxacin. Between 2009 and 2013, a total of 81,310 clinical isolates belonging to the main species of Enterobacteriaceae and 39,191 clinical isolates of P. aeruginosa isolated in 28 hospitals in the Valencian Community on the South East Mediterranean Coast of Spain were analyzed using data provided by RedMiva (microbiological surveillance network of the Valencian Community). Meropenem resistance in Enterobacteriaceae increased from 0.16 % in 2009 to 1.25 % in 2013. Very few Enterobacteriaceae strains resistant to meropenem were sensitive to ciprofloxacin; in contrast, the combination of meropenem and amikacin led to a marked decrease in the risk of the microorganisms being resistant to both drugs (RR = 34 in 2013). In the case of P. aeruginosa, meropenem resistance also increased (from 14.32 % in 2009 to 24.52 % in 2013). Most meropenem-resistant P. aeruginosa isolates were also resistant to fluoroquinolones. However, the addition of amikacin led to a more than three-fold decrease in the risk of resistance. In our setting, empirical treatment with meropenem is adequate in enterobacterial infections, but poses difficulties when infection due to P. aeruginosa is suspected, in which case a combination of meropenem and amikacin has been shown to have a higher microbiological success rate.

Source investigation of two outbreaks of skin and soft tissue infection by Mycobacterium abscessus subsp. abscessus in Venezuela.

Outbreaks of soft tissue or skin infection due to non-tuberculous mycobacteria are reported frequently in scientific journals but in general the infection source in these outbreaks remains unknown. In Venezuela, in two distinct outbreaks, one after breast augmentation surgery and another after hydrolipoclasy therapy, 16 patients contracted a soft tissue infection due to Mycobacterium abscessus subsp. abscessus. Searching for the possible environmental infection sources in these outbreaks, initially the tap water (in the hydrolipoclasy therapy outbreak) and a surgical skin marker (in the breast implant surgery outbreak), were identified as the infection sources. Molecular typing of the strains with a variable number tandem repeat typing assay confirmed the tap water as the infection source but the molecular typing technique excluded the skin marker. We discuss the results and make a call for the implementation of stringent hygiene and disinfection guidelines for cosmetic procedures in Venezuela.

Hypoxia induces the expression of transketolase-like 1 in human colorectal cancer.

BACKGROUND AND AIMS: Transketolase-like (TKTL) 1 is one of the key enzymes for anaerobic sugar degradation even in the presence of oxygen (aerobic glycolysis). Transketolase-dependent reactions supply malignant tumors with ribose and NADPH. Therefore, TKTL1 activity could be crucial for tumor proliferation and survival. The aim of the study was to evaluate the expression of TKTL1 in colorectal cancer (CRC) and its regulation under hypoxic conditions. METHODS: We studied TKTL1 mRNA and protein expression in CRC cell lines and human CRC biopsies by quantitative real-time PCR, Western blotting and immunohistochemistry. Regulation of TKTL1 under oxygen depletion was analyzed by cultivating cells either in a three-dimensional spheroid model or in a hypoxia incubator chamber. RESULTS: TKTL1 mRNA was heterogeneously expressed in monolayers of cells with high levels in HT-29 and SW480. TKTL1 protein was also clearly detectable in HT-29 and SW480. Hypoxia-inducible factor (HIF)-1α protein expression correlated with TKTL1 protein expression in SW480 spheroids over time. On the one hand, induction of hypoxia in T84 spheroids did not induce TKTL1; on the other hand, hypoxia by incubation at 1% O2 in a hypoxia incubator chamber clearly showed an upregulation of TKTL1. In 50% of CRC patients, TKTL1 protein expression was upregulated in tumor compared to non-tumor tissue. The immunohistochemical staining of TKTL1 in CRC patient samples resulted in 14 positive and 30 negative samples. CONCLUSIONS: TKTL1 expression correlated with HIF-1α protein expression and was induced upon hypoxic conditions which could facilitate energy supply to tumors under these circumstances.

A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast.

X-linked recessive myotubular myopathy (MTM1) is characterized by severe hypotonia and generalized muscle weakness, with impaired maturation of muscle fibres. We have restricted the candidate region to 280 kb and characterized two candidate genes using positional cloning strategies. The presence of frameshift or missense mutations (of which two are new mutations) in seven patients proved that one of these genes is indeed implicated in MTM1. The protein encoded by the MTM1 gene is highly conserved in yeast, which is surprising for a muscle specific disease. The protein contains the consensus sequence for the active site of tyrosine phosphatases, a wide class of proteins involved in signal transduction. At least three other genes, one located within 100 kb distal from the MTM1 gene, encode proteins with very high sequence similarities and define, together with the MTM1 gene, a new family of putative tyrosine phosphatases in man.

Coupling magnetic torque and force for colloidal microbot assembly and manipulation.

For targeted transport in the body, biomedical microbots (µbots) must move effectively in three-dimensional (3D) microenvironments. Swimming µbots translate via asymmetric or screw-like motions while rolling ones use friction with available surfaces to generate propulsive forces. We have previously shown that planar rotating magnetic fields assemble µm-scale superparamagnetic beads into circular µbots that roll along surfaces. In this, gravity is required to pull µbots near the surface; however, this is not necessarily practical in complex geometries. Here we show that rotating magnetic fields, in tandem with directional magnetic gradient forces, can be used to roll µbots on surfaces regardless of orientation. Simplifying implementation, we use a spinning permanent magnet to generate differing ratios of rotating and gradient fields, optimizing control for different environments. This use of a single magnetic actuator sidesteps the need for complex electromagnet or tandem field setups, removes requisite gravitational load forces, and enables µbot targeting in complex 3D biomimetic microenvironments.

Magnetically Powered Chitosan Milliwheels for Rapid Translation, Barrier Function Rescue, and Delivery of Therapeutic Proteins to the Inflamed Gut Epithelium.

Inflammatory bowel disease (IBD) is mediated by an overexpression of tumor necrosis factor-α (TNF) by mononuclear cells in the intestinal mucosa. Intravenous delivery of neutralizing anti-TNF antibodies can cause systemic immunosuppression, and up to one-third of people are non-responsive to treatment. Oral delivery of anti-TNF could reduce adverse effects; however, it is hampered by antibody degradation in the harsh gut environment during transit and poor bioavailability. To overcome these shortcomings, we demonstrate magnetically powered hydrogel particles that roll along mucosal surfaces, provide protection from degradation, and sustain the local release of anti-TNF. Iron oxide particles are embedded into a cross-linked chitosan hydrogel and sieved to produce 100-200 µm particles called milliwheels (m-wheels). Once loaded with anti-TNF, these m-wheels release 10 to 80% of their payload over 1 week at a rate that depends on the cross-linking density and pH. A rotating magnetic field induces a torque on the m-wheels that results in rolling velocities greater than 500 µm/s on glass and mucus-secreting cells. The permeability of the TNF-challenged gut epithelial cell monolayers was rescued in the presence of anti-TNF carrying m-wheels, which both neutralized the TNF and created an impermeable patch over leaky cell junctions. With the ability to translate over mucosal surfaces at high speed, provide sustained release directly to the inflamed epithelium, and provide barrier rescue, m-wheels demonstrate a potential strategy to deliver therapeutic proteins for the treatment of IBD.

Delivery and actuation of aerosolized microbots.

For disease of the lung, the physical key to effective inhalation-based therapy is size; too large (10's of µm) and the particles or droplets do not remain suspended in air to reach deep within the lungs, too small (subµm) and they are simply exhaled without deposition. µBots within this ideal low-µm size range however are challenging to fabricate and would lead to devices that lack the speed and power necessary for performing work throughout the pulmonary network. To uncouple size from structure and function, here we demonstrate an approach where individual building blocks are aerosolized and subsequently assembled in situ into µbots capable of translation, drug delivery, and mechanical work deep within lung mimics. With this strategy, a variety of pulmonary diseases previously difficult to treat may now be receptive to µbot-based therapies.

Impact of Sepsis Flow Chip, a novelty fast microbiology method, in the treatment of bacteremia caused by Gram-negative bacilli.

OBJECTIVE: The aim of this study was to assess the impact of the information provided by the new Sepsis Chip Flow system (SFC) and other fast microbiological techniques on the selection of the appropriate antimicrobial treatment by the clinical researchers of an antimicrobial stewardship team. METHODS: Two experienced clinical researchers performed the theoretical exercise of independently selecting the treatment for patients diagnosed by bacteremia due to bacilli gram negative (BGN). At first, the clinicians had only available the clinical characteristics of 74 real patients. Sequentially, information regarding the Gram stain, MALDI-TOF, and SFC from Vitro were provided. Initially, the researchers prescribed an antimicrobial therapy based on the clinical data, later these data were complementing with information from microbiological techniques, and the clinicians made their decisions again. RESULTS: The data provided by the Gram stain reduced the number of patients prescribed with combined treatments (for clinician 1, from 23 to 7, and for clinician 2, from 28 to 12), but the use of carbapenems remained constant. In line with this, the data obtained by the MALDI-TOF also decreased the combined treatment, and the use of carbapenems remained unchanged. By contrast, the data on antimicrobial resistance provided by the SFC reduced the carbapenems treatment. CONCLUSIONS: From the theoretical model the Gram stain and the MALDI-TOF results achieved a reduction in the combined treatment. However, the new system tested (SFC), due to the resistance mechanism data provided, not only reduced the combined treatment, it also decreased the prescription of the carbapenems.

Transcutaneous electrical nerve stimulation for spasticity: A systematic review. / Estimulación eléctrica nerviosa transcutánea como tratamiento de la espasticidad: una revisión sistemática.

INTRODUCTION: Although transcutaneous electrical nerve stimulation (TENS) has traditionally been used to treat pain, some studies have observed decreased spasticity after use of this technique. However, its use in clinical practice is still limited. Our purpose was twofold: to determine whether TENS is effective for treating spasticity or associated symptoms in patients with neurological involvement, and to determine which stimulation parameters exert the greatest effect on variables associated with spasticity. DEVELOPMENT: Two independent reviewers used PubMed, PEDro, and Cochrane databases to search for randomised clinical trials addressing TENS and spasticity published before 12 May 2015, and selected the articles that met the inclusion criteria. Of the initial 96 articles, 86 were excluded. The remaining 10 articles present results from 207 patients with a cerebrovascular accident, 84 with multiple sclerosis, and 39 with spinal cord lesions. CONCLUSIONS: In light of our results, we recommend TENS as a treatment for spasticity due to its low cost, ease of use, and absence of adverse reactions. However, the great variability in the types of stimulation used in the studies, and the differences in parameters and variables, make it difficult to assess and compare any results that might objectively determine the effectiveness of this technique and show how to optimise parameters.

Prospective evaluation of toceranib phosphate in metastatic canine osteosarcoma.

Efficacious therapies for measurable metastatic canine osteosarcoma (OSA) are generally lacking. Preliminary retrospective studies suggested that approximately 50% of dogs with measurable metastatic OSA experienced clinical benefit (objective response or clinically meaningful disease stabilisation) following toceranib (TOC) treatment. The purpose of this clinical trial was to prospectively evaluate the clinical outcome following TOC treatment in dogs with measurable pulmonary metastatic OSA. A secondary goal was to identify potential biomarkers of clinical benefit by measuring changes in plasma vascular endothelial growth factor (VEGF) and circulating regulatory T-cell (Treg) percentage. Twenty-two dogs with pulmonary metastasis from appendicular OSA having undergone previous amputation were treated prospectively with TOC. Adverse events (AEs) were common but predominantly low grade. Nine patients were withdrawn from the study prior to the week 8 assessment of response either due to progressive disease (PD), decreased quality of life or owner perceived unacceptable AEs. Of the patients evaluable for disease progression at week 8 (or earlier), 3/17 (17.6 %) had stable disease with the remainder having PD. The median progression-free survival time for all patients was 57 days (range 7-176 days) with a median overall survival time of 89 days (range 7-574 days). Plasma VEGF concentrations were significantly elevated in patients after 4 weeks of TOC treatment, but no changes were observed in percentage of Treg in peripheral blood. Overall, the results of this clinical trial do not support the use of TOC as single agent therapy for canine metastatic OSA.

Conversion of Sugars and Biomass to Furans Using Heterogeneous Catalysts in Biphasic Solvent Systems.

Within the last decade, interest in using biphasic systems for producing furans from biomass has grown significantly. Biphasic systems continuously extract furans into the organic phase, which prevents degradation reactions and potentially allows for easier separations of the products. Several heterogeneous catalyst types, including zeolites, ion exchange resins, niobium-based, and others, have been used with various organic solvents to increase furan yields from sugar dehydration reactions. In this minireview, we summarized the use of heterogeneous catalysts in biphasic systems for furfural and 5-hydroxymethylfurfural production from the past five years, highlighting trends in chemical and physical properties that effect catalytic activity. Additionally, the selection of an organic solvent for a biphasic system is extremely important and we review and discuss properties of the most commonly used organic solvents.

PD-1 expression by canine T cells and functional effects of PD-1 blockade.

The co-inhibitory checkpoint molecule programmed death receptor 1 (PD-1) can trigger T cell functional exhaustion upon binding to its ligand PD-L1 expressed on tumour cells or macrophages. PD-1 blocking antibodies have generated remarkable results in human cancer patients, including inducing durable responses in a number of advanced cancers. Therefore, monoclonal antibodies specific for canine PD-1 were assessed for T cell binding and induction of functional activation. A total of 5-10% of CD4 T cells and 20-25% of CD8 T cells from healthy dogs expressed PD-1, and PD-1 expression was upregulated on T cells from dogs with cancer. Functionally, PD-1 antibodies significantly enhanced T-cell activation, as assessed by proliferation and interferon-gamma (IFN-γ) production. PD-1 antibodies also reversed T-cell suppression induced by canine soluble PD-L1 and by tumour cells and tumour explant fragments. These findings indicate that PD-1 antibodies have potential for use in cancer immunotherapy in dogs.

Role of monocyte recruitment in hemangiosarcoma metastasis in dogs.

Canine hemangiosarcoma (HSA) is a highly malignant tumour associated with short survival times because of early and widespread metastasis. In humans and rodents, monocytes play key roles in promoting tumour metastasis through stimulating tumour cell extravasation, seeding, growth and angiogenesis. Therefore, we investigated the potential association between monocyte infiltration and tumour metastasis in HSA and other common canine tumours. Immunohistochemistry was used to quantify CD18+ monocytes within metastases. We found that HSA metastases had significantly greater numbers of CD18+ monocytes compared with metastases from other tumour types. HSA cells were the highest producers of the monocyte chemokine CCL2, and stimulated canine monocyte migration in a CCL2 dependent manner. These results are consistent with the hypothesis that overexpression of CCL2 and recruitment of large numbers of monocytes may explain in part the aggressive metastatic nature of canine HSA. Thus, therapies designed to block monocyte recruitment may be an effective adjuvant strategy for suppressing HSA metastasis in dogs.

Immune regulation of canine tumour and macrophage PD-L1 expression.

Expression of programmed cell death receptor ligand 1 (PD-L1) on tumor cells has been associated with immune escape in human and murine cancers, but little is known regarding the immune regulation of PD-L1 expression by tumor cells and tumor-infiltrating macrophages in dogs. Therefore, 14 canine tumor cell lines, as well as primary cultures of canine monocytes and macrophages, were evaluated for constitutive PD-L1 expression and for responsiveness to immune stimuli. We found that PD-L1 was expressed constitutively on all canine tumor cell lines evaluated, although the levels of basal expression were very variable. Significant upregulation of PD-L1 expression by all tumor cell lines was observed following IFN-γ exposure and by exposure to a TLR3 ligand. Canine monocytes and monocyte-derived macrophages did not express PD-L1 constitutively, but did significantly upregulate expression following treatment with IFN-γ. These findings suggest that most canine tumors express PD-L1 constitutively and that both innate and adaptive immune stimuli can further upregulate PD-L1 expression. Therefore the upregulation of PD-L1 expression by tumor cells and by tumor-infiltrating macrophages in response to cytokines such as IFN-γ may represent an important mechanism of tumor-mediated T-cell suppression in dogs as well as in humans.

A human homologue of the Drosophila tumour suppressor gene l(2)gl maps to 17p11.2-12 and codes for a cytoskeletal protein that associates with nonmuscle myosin II heavy chain.

Inactivation of the tumour suppressor gene lethal(2) giant larvae (D-lgl) of Drosophila leads to malignant transformation of the presumptive adult optic centers in the larval brain and tumours of the imaginal discs. These malignancies result from the disorganization of a cytoskeletal network in which the D-LGL protein participates. Here we describe the isolation of a cDNA encoding the human homologue to the D-lgl gene designated as hugl. The hugl cDNA detects a locus spanning at least 25 kilobases (kb) in human chromosome band 17p11.2-12, which is centromeric to the p53 gene and recognizes a 4.5 kb RNA transcript. The hugl gene is expressed in brain, kidney and muscle but is barely seen in heart and placenta. Sequence analysis of the hugl cDNA demonstrates a long open reading frame, which has the potential to encode a protein of 1057 amino acids with a predicted molecular weight of 115 kDaltons (kD). To further substantiate and identify the HUGL protein, we have prepared polyclonal rabbit antibodies against synthetic peptides corresponding to the amino and carboxyl termini of the conceptual translation product of the hugl gene. The affinity-purified anti-HUGL antibodies recognize a single protein with an apparent molecular weight of approximately 115 kD. Similar to the Drosophila protein, HUGL is part of a cytoskeletal network and, is associated with nonmuscle myosin II heavy chain and a kinase that specifically phosphorylates HUGL at serine residues.

The genomic structure of the DMBT1 gene: evidence for a region with susceptibility to genomic instability.

Increasing evidence has accumulated for an involvement of the inactivation of tumour suppressor genes at chromosome 10q in the carcinogenesis of brain tumours, melanomas, and carcinomas of the lung, the prostate, the pancreas, and the endometrium. The gene DMBT1 (Deleted in Malignant Brain Tumours 1) is located at chromosome 10q25.3-q26.1, within one of the putative intervals for tumour suppressor genes. DMBT1 is a member of the scavenger-receptor cysteine-rich (SRCR) superfamily and displays homozygous deletions or lack of expression in glioblastoma multiforme, medulloblastoma, and in gastrointestinal and lung cancers. Based on these properties, DMBT1 has been proposed to be a candidate tumour suppressor gene. We have determined the genomic sequence of DMBT1 to allow analyses of mutations. The gene has at least 54 exons that span a genomic region of about 80 kb. We have identified a putative exon with coding potential for a transmembrane domain. Our data further suggest that alternative splicing gives rise to isoforms of DMBT1 with a differential utilization of SRCR domains and SRCR interspersed domains. The major part of the gene harbours locus specific repeats. These repeats may point to the DMBT1 locus as a region susceptible to chromosomal instability.

Differential transcriptional regulation of the monocyte-chemoattractant protein-1 (MCP-1) gene in tumorigenic and non-tumorigenic HPV 18 positive cells: the role of the chromatin structure and AP-1 composition.

The expression of the monocyte-chemoattractant-protein-1 (MCP-1) is closely linked with a non-tumorigenic phenotype in somatic cell hybrids made between the human papillomavirus type 18 (HPV 18) positive cervical carcinoma cell line HeLa and normal human fibroblasts. In contrast, MCP-1 transcription is absent in tumorigenic segregants derived from the same hybrids or in parental HeLa cells. Selectivity of MCP-1 transcription, which is regulated at the level of initiation of transcription, is mainly based on differences in the location and extension of DNAse I-hypersensitive regions (DHSR) at both ends of the gene. While TNF-alpha only moderately increases the sensitivity of pre-existing 5'-DHSRs, a 3'-end DHSR became strongly induced exclusively in non-malignant hybrids. DNA sequencing showed that the 3'-DHSR coincides with an additional AP-1 site located approximately 600 bp downstream of the polyadenylation site. Analyses of AP-1 composition revealed that MCP-1 is only expressed in those cells where jun-family members were mainly heterodimerized with the fos-related protein fra-1. In contrast, in tumorigenic cells the 1: 1 ratio between jun and fra-1 is disturbed and the MCP-1 gene is no longer expressed. Hence, alterations in the heterodimerization pattern of AP-1 and its selective accessibility to opened chromatin may represent a novel regulatory pathway in the regulation of chemokines in malignant and non-malignant HPV-positive cells.

Isolation, differential splicing and protein expression of a DNase on the human X chromosome.

A systematic search for genes differentially expressed in human tissues resulted in the isolation of a gene encoding a protein with high homology to DNase I. In addition to the recently described cDNA sequence (Parrish et al., 1995) we have isolated a transcript, alternatively spliced in the 5' noncoding region. The gene is located between the QM and the XAP-2 gene in Xq28 and encodes a 302 amino acid protein with 39% identity to human DNase I. Besides a high homology at the nucleotide and amino acid level, most exon-intron boundaries of DNase I and DNase X are identical, indicating that both genes may have evolved from a common ancestor. The predicted function was verified by expression of a recombinant protein in an inducible bacterial system and detection of DNase activity. In contrast to DNase I a 18 kdal amino terminal fragment of the full length 35 kdal protein exhibited DNase activity.

Mapping of X chromosome inversion breakpoints [inv(X)(q11q28)] associated with FG syndrome: a second FG locus [FGS2]?

FG syndrome is an X-linked condition comprising mental retardation, congenital hypotonia, macrocephaly, distinctive facial changes, and constipation or anal malformations. In a linkage analysis, we mapped a major FG syndrome locus [FGS1] to Xq13, between loci DXS135 and DXS1066. The same data, however, clearly demonstrated genetic heterogeneity. Recently, we studied a French family in which an inversion [inv(X)(q12q28)] segregates with clinical symptoms of FG syndrome. This suggests that one of the breakpoints corresponds to a second FG syndrome locus [FGS2]. We report the results of fluorescence in situ hybridization analysis performed in this family using YACs and cosmids encompassing the Xq11q12 and Xq28 regions. Two YACs, one positive for the DXS1 locus at Xq11.2 and one positive for the color vision pigment genes and G6PD loci at Xq28, were found to cross the breakpoints, respectively. We postulate that a gene might be disrupted by one of the breakpoints.

The relationship between allelic imbalance on 17p, p53 mutation and p53 overexpression in head and neck cancer.

The huge majority of head and neck squamous cell carcinoma (HNSCC) show alterations of p53 either on the genetic level or on the protein level. Allelic imbalance (AI)/loss of heterozygosity (LOH) on 17p at the p53 locus is frequent in HNSCC. However, the complex relationship between these phenomena is poorly understood in HNSCC. We investigated one group of 39 HNSCC for: a) allelic imbalance on 17p using 4 microsatellite markers located throughout this chromosomal arm; b) mutations of p53 in exons 5-9; and c) overexpression of p53 using two antibodies located on opposite ends of the protein. AI/LOH was detected in 44% at the locus TP53, rising to 69% when regarding all 4 markers on 17p. Therefore, our data are in line with the assumption of additional tumour suppressor genes on 17p in HNSCC. A nuclear accumulation of p53 (51%) was independent from the antibody and the recognised epitope. At the first glance there was no correlation between overall p53 mutation (36%) and overexpression. However, it appeared that, with very few exceptions, only nonsense mutations did not lead to p53 overexpression, while missense mutations did. As overexpression of p53 was 15% more frequent than p53 mutations and only 35% of the tumours with p53 overexpression carried a p53 mutation, our data support the hypothesis of additional mechanisms of p53 overexpression. AI/LOH at the p53 locus in 83% of all tumours with a p53 mutation is in line with Knudson's theory of inactivation of tumour suppressor genes.