Characterizing the protein-protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization.

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilizers.

Dynamic nature of BRAF or KRAS p.G12C mutations in second-line therapy for advanced colorectal cancer patients: do early and late effects exist?

INTRODUCTION: The mitogen-activated protein kinase (MAPK) signalling network aberrations in metastatic colorectal cancer (mCRC) generate intrinsic dynamic effects and temporal variations that are crucial but often overlooked in clinical trial populations. Here, we investigate the time-varying impact of MAPK pathway mutation genotype on each treatment line's contribution to the overall clinical course. METHODS: The PROMETEO study focused on mCRC patients undergoing second-line treatment at 20 hospitals. We evaluated genotypes and employed flexible models to analyse the dynamic effect of each mutation. RESULTS: We examined data derived from 1160 patients. The effects of KRAS G12C or G12V, and BRAF V600E are clearly time-varying, with unexpected consequences such as the deleterious effect of BRAF V600E vs other genotypes dissipating over time when subjects receive antiangiogenics, or KRAS G12V and G12C showing increasing aggressiveness over time. Thus, contrary to expectations, the 12-month survival rate from the second line for those who survived >6 months was 49.9% (95% CI, 32.7-67.3) for KRAS G12C and 59% (95% CI, 38.5-80.6) for BRAF V600E. CONCLUSIONS: The dynamic perspective is essential for understanding the behaviour of tumours with specific genotypes, especially from the second line onward. This may be relevant in patient monitoring and treatment decision-making, particularly in cases with distinct mutations.

Selective Aster inhibitors distinguish vesicular and nonvesicular sterol transport mechanisms.

The Aster proteins (encoded by the Gramd1a-c genes) contain a ligand-binding fold structurally similar to a START domain and mediate nonvesicular plasma membrane (PM) to endoplasmic reticulum (ER) cholesterol transport. In an effort to develop small molecule modulators of Asters, we identified 20α-hydroxycholesterol (HC) and U18666A as lead compounds. Unfortunately, both 20α-HC and U18666A target other sterol homeostatic proteins, limiting their utility. 20α-HC inhibits sterol regulatory element-binding protein 2 (SREBP2) processing, and U18666A is an inhibitor of the vesicular trafficking protein Niemann-Pick C1 (NPC1). To develop potent and selective Aster inhibitors, we synthesized a series of compounds by modifying 20α-HC and U18666A. Among these, AI (Aster inhibitor)-1l, which has a longer side chain than 20α-HC, selectively bound to Aster-C. The crystal structure of Aster-C in complex with AI-1l suggests that sequence and flexibility differences in the loop that gates the binding cavity may account for the ligand specificity for Aster C. We further identified the U18666A analog AI-3d as a potent inhibitor of all three Aster proteins. AI-3d blocks the ability of Asters to bind and transfer cholesterol in vitro and in cells. Importantly, AI-3d also inhibits the movement of low-density lipoprotein (LDL) cholesterol to the ER, although AI-3d does not block NPC1. This finding positions the nonvesicular Aster pathway downstream of NPC1-dependent vesicular transport in the movement of LDL cholesterol to the ER. Selective Aster inhibitors represent useful chemical tools to distinguish vesicular and nonvesicular sterol transport mechanisms in mammalian cells.

Anti-allodynic and anti-hyperalgesic activity of (±)-licarin A in neuropathic rats via NO-cyclic-GMP-ATP-sensitive K+ channel pathway.

The study aimed to examine the effect of intraperitoneal and intrathecal (±)-licarin A in neuropathic pain induced by L5 and L6 spinal nerve ligation (SNL) in male Wistar rats and the possible involvement of the NO-cGMP-ATP-sensitive K+ channel pathway. Neuropathic pain signs (allodynia and hyperalgesia) were evaluated on postoperative Day 14 using von Frey filaments. Single intraperitoneal (0.01, 0.1, 1, and 10 mg/kg) and intrathecal (0.01, 0.1, 1, and 10 µg/rat) administration of (±)-licarin A improved allodynia and hyperalgesia. The (±)-licarin A-induced anti-allodynic and anti-hyperalgesic activity was prevented by the intrathecal injection of  l-NAME (100 µg/rat; nonselective nitric oxide synthase inhibitor), ODQ (10 µg/rat; guanylate cyclase inhibitor), and glibenclamide (50 µg/rat; adenosine triphosphate (ATP)-sensitive K+ channel blocker). The data suggest that (±)-licarin A exerts its anti-allodynic and anti-hyperalgesic activity by activating the NO-cGMP-ATP-sensitive K+ channel pathway.

Crimean-Congo Hemorrhagic Fever, Spain, 2013-2021.

Crimean-Congo hemorrhagic fever (CCHF) is a viral infectious disease for which distribution of the main vector, Hyalomma spp. ticks, is expanding. We analyzed all 10 cases of CCHF diagnosed in Spain during 2013-2021; case-patient median age was 56.5 years, and 7 were men. We identified CCHF virus genotypes III and V. Six case-patients acquired the infection in urban areas. Sixty percent of patients were infected in summer and 40% in spring. Two patients met criteria for hemophagocytic syndrome. Seven patients survived. The epidemiologic pattern of CCHF in Spain is based on occasional cases with an elevated mortality rate. Genotype III and, to a less extent also genotype V, CCHF circulates in humans in a common geographic area in Spain. Those data suggest that the expansion pathways are complex and may change over time. Physicians should remain alert to the possibility of new CCHF cases.

Sohlh1 loss of function male and female infertility model impacts overall health beyond gonadal dysfunction in mice†.

Reproductive longevity is associated with health outcomes. Early menopause, loss of ovarian function, and male infertility are linked to shorter lifespan and increased adverse health outcomes. Here we examined the extragonadal effects of whole animal loss of spermatogenesis and oogenesis specific basic helix-loop-helix 1 (Sohlh1) gene in mice, a well-described mouse model of female and male infertility. Sohlh1 encodes a transcription factor that is primarily expressed in the male and female germline and regulates germline differentiation. The Sohlh1 knockout mouse model, just like human individuals with SOHLH1 loss of function, presents with hypergonadotropic hypogonadism and loss of ovarian function in females and impaired spermatogenesis in males, with a seemingly gonad restricted phenotype in both sexes. However, extragonadal phenotyping revealed that Sohlh1 deficiency leads to abnormal immune profiles in the blood and ovarian tissues of female animals, sex-specific alterations of metabolites, and behavior and cognition changes. Altogether, these results show that Sohlh1 deficiency impacts overall health in both male and female mice.

Endotracheal tubes coated with a broad-spectrum antibacterial ceragenin reduce bacterial biofilm in an in vitro bench top model.

BACKGROUND: Ventilator-associated pneumonia is one of the most common nosocomial infections, caused mainly by bacterial/fungal biofilm. Therefore, it is necessary to develop preventive strategies to avoid biofilm formation based on new compounds. OBJECTIVES: We performed an in vitro study to compare the efficacy of endotracheal tubes (ETTs) coated with the ceragenin CSA-131 and that of uncoated ETTs against the biofilm of clinical strains of Pseudomonas aeruginosa (PA), Escherichia coli (EC) and Staphylococcus aureus (SA). METHODS: We applied an in vitro bench top model using coated and uncoated ETTs that were treated with three different clinical strains of PA, EC and SA for 5 days. After exposure to biofilm, ETTs were analysed for cfu count by culture of sonicate and total number of cells by confocal laser scanning microscopy. RESULTS: The median (IQR) cfu/mL counts of PA, EC and SA in coated and uncoated ETTs were, respectively, as follows: 1.00 × 101 (0.0-3.3 × 102) versus 3.32 × 109 (6.6 × 108-3.8 × 109), P < 0.001; 0.0 (0.0-5.4 × 103) versus 1.32 × 106 (2.3 × 103-5.0 × 107), P < 0.001; and 8.1 × 105 (8.5 × 101-1.4 × 109) versus 2.7 × 108 (8.6 × 106-1.6 × 1011), P = 0.058. The median (IQR) total number of cells of PA, EC and SA in coated and non-coated ETTs were, respectively, as follows: 11.0 [5.5-not applicable (NA)] versus 87.9 (60.5-NA), P = 0.05; 9.1 (6.7-NA) versus 62.6 (42.0-NA), P = 0.05; and 97.7 (94.6-NA) versus 187.3 (43.9-NA), P = 0.827. CONCLUSIONS: We demonstrated significantly reduced biofilm formation in coated ETTs. However, the difference for SA was not statistically significant. Future clinical studies are needed to support our findings.

Epidemiological of cat scratch disease among inpatients in the Spanish health system (1997-2015).

Cat scratch disease, whose causative agent is Bartonella henselae, is an anthropozoonosis with a worldwide distribution that causes significant public health problems. Although it is an endemic disease in Spain, the available data are very limited. The aim of our study was to describe cat scratch disease inpatients in the National Health System (NHS) of Spain. This was a retrospective descriptive study using the minimum basic data set (CMBD in Spanish) in patients admitted to hospitals of the NHS between 1997 and 2015 with a diagnosis of cat scratch disease (ICD-9: 078.3). We found 781 hospitalized patients diagnosed with cat scratch disease. The mean age (± SD) was 30.7 ± 25.3 years old. The male/female ratio was 1.1:1. The incidence rate over the study period was 0.93 (95% CI, 0.86-0.99) cases per million person-years. The incidence rate in men was 0.98 cases per million person-years and that in women was 0.88 cases per million person-years. The cases were more frequent from September to January. A total of 652 (83.5%) cases were urgent hospital admissions. The average hospital stay was 8.4 ± 8.9 days. The overall lethality rate of the cohort was 1.3%. We have demonstrated that CSD causes a substantial burden of disease in Spain, affecting both adult and pediatric patients with a stable incidence rate. Our data suggest that CSD is benign and self-limited, with low mortality, and its incidence is possibly underestimated. Finally, there is a need for a common national strategy for data collection, monitoring, and reporting, which would facilitate a more accurate picture and the design of more strategic control measures. Hospital discharge records (HDRs) could be a good database for the epidemiological analysis of the hospital management of CSD.

Epidemiological assessment of 5598 brucellosis inpatients in Spain (1997-2015).

Brucellosis remains one of the main zoonoses worldwide. Epidemiological data on human brucellosis in Spain are scarce. The objective of this study was to assess the epidemiological characteristics of inpatient brucellosis in Spain between 1997 and 2015. A retrospective longitudinal descriptive study was performed. Data were requested from the Health Information Institute of the Ministry of Health and Equality, which provided us with the Minimum Basic Data Set of patients admitted to the National Health System. We also obtained data published in the System of Obligatory Notifiable Diseases. A total of 5598 cases were registered. The period incidence rate was 0.67 (95% CI 0.65-0.68) cases per 100 000 person-years. We observed a progressive decrease in the number of cases and annual incidence rates. A total of 3187 cases (56.9%) came from urban areas. The group most at risk comprised men around the fifth decade of life. The average (±s.d.) hospital stay was 12.6 days (±13.1). The overall lethality rate of the cohort was 1.5%. The number of inpatients diagnosed with brucellosis decreased exponentially. The group of patients with the highest risk of brucellosis in our study was males under 45 years of age and of urban origin. The lethality rate has reduced to minimum values. It is probable that hospital discharge records could be a good database for the epidemiological analysis of the hospital management of brucellosis and offer a better information collection system than the notifiable diseases system (EDO in Spanish).

Corticosteroid Pulses for Hospitalized Patients with COVID-19: Effects on Mortality.

OBJECTIVES: To assess the influence of corticosteroid pulses on 60-day mortality in hospitalized patients with severe COVID-19. METHODS: We designed a multicenter retrospective cohort study in three teaching hospitals of Castilla y León, Spain (865,096 people). We selected patients with confirmed COVID-19 and lung involvement with a pO2/FiO2<300, excluding those exposed to immunosuppressors before or during hospitalization, patients terminally ill at admission, or those who died in the first 24 hours. We performed a propensity score matching (PSM) adjusting covariates that modify the probability of being treated. Then, we used a Cox regression model in the PSM group to consider factors affecting mortality. RESULTS: From 2933 patients, 257 fulfilled the inclusion and exclusion criteria. 124 patients were on corticosteroid pulses (250 mg of methylprednisolone for three days), and 133 were not. 30.3% (37/122) of patients died in the corticosteroid pulse group and 42.9% (57/133) in the nonexposed cohort. These differences (12.6%, 95% CI [8·54-16.65]) were statically significant (log-rank 4.72, p = 0, 03). We performed PSM using the exact method. Mortality differences remained in the PSM group (log-rank 5.31, p = 0.021) and were still significant after a Cox regression model (HR for corticosteroid pulses 0.561; p = 0.039). CONCLUSIONS: This study provides evidence about treatment with corticosteroid pulses in severe COVID-19 that might significantly reduce mortality. Strict inclusion and exclusion criteria with that selection process set a reliable frame to compare mortality in both the exposed and nonexposed groups.

Characterization of the virulence of Pseudomonas aeruginosa strains causing ventilator-associated pneumonia.

BACKGROUND: The objective of this study was to evaluate the virulence of P. aeruginosa ventilator-associated pneumonia (VAP) strains (cases) in terms of biofilm production and other phenotypic and genotypic virulence factors compared to P. aeruginosa strains isolated from other infections (controls). METHODS: Biofilm production was tested to assess biomass production and metabolic activity using crystal violet binding assay and XTT assay, respectively. Pigment production (pyocyanin and pyoverdine) was evaluated using cetrimide agar. Virulence genes were detected by conventional multiplex PCR and virulence was tested in an in vivo model in Galleria mellonella larvae. RESULTS: We did not find statistically significant differences between VAP and no-VAP strains (p > 0.05) regarding biofilm production. VAP strains had no production of pyocyanin after 24 h of incubation (p = 0.023). The distribution of virulence genes between both groups were similar (p > 0.05). VAP strains were less virulent than non-VAP strains in an in vivo model of G. mellonella (p < 0.001). CONCLUSION: The virulence of VAP-Pseudomonas aeruginosa does not depend on biofilm formation, production of pyoverdine or the presence of some virulence genes compared to P. aeruginosa isolated from non-invasive locations. However, VAP strains showed attenuated virulence compared to non-VAP strains in an in vivo model of G. mellonella.

Selective digestive decontamination solution used as "lock therapy" prevents and eradicates bacterial biofilm in an in vitro bench-top model.

BACKGROUND: Most preventing measures for reducing ventilator-associated pneumonia (VAP) are based mainly on the decolonization of the internal surface of the endotracheal tubes (ETTs). However, it has been demonstrated that bacterial biofilm can also be formed on the external surface of ETTs. Our objective was to test in vitro the efficacy of selective digestive decontamination solution (SDDs) onto ETT to prevent biofilm formation and eradicate preformed biofilms of three different microorganisms of VAP. METHODS: We used an in vitro model in which we applied, at the subglottic space of ETT, biofilms of either P. aeruginosa ATCC 15442, or E. coli ATCC 25922, or S. aureus ATCC 29213, and the SDDs at the same time (prophylaxis) or after 72 h of biofilm forming (treatment). ETT were incubated during 5 days with a regimen of 2 h-locks. ETT fragments were analyzed by sonication and confocal laser scanning microscopy to calculate the percentage reduction of cfu and viable cells, respectively. RESULTS: Median (IQR) percentage reduction of live cells and cfu/ml counts after treatment were, respectively, 53.2% (39.4%-64.1%) and 100% (100%-100.0%) for P. aeruginosa, and 67.9% (46.7%-78.7%) and 100% (100%-100.0%) for E. coli. S. aureus presented a complete eradication by both methods. After prophylaxis, there were absence of live cells and cfu/ml counts for all microorganisms. CONCLUSIONS: SDDs used as "lock therapy" in the subglottic space is a promising prophylactic approach that could be used in combination with the oro-digestive decontamination procedure in the prevention of VAP.

Asynchrony between the early embryo and the reproductive tract affects subsequent embryo development in cattle.

The aim of this study was to determine the effect of maternal-embryonic asynchrony in the reproductive tract (oviduct and uterus) on subsequent embryo development in cattle. Fifty Day 1invitro-produced zygotes were transferred endoscopically into the oviduct ipsilateral to the corpus luteum of heifers (n=40) that were either synchronous with the embryos (Day 1 after ovulation) or asynchronous and ahead of the embryo (Day 3 after ovulation). A subset of heifers was killed in a commercial abattoir 3, 6 or 14 days after embryo transfer. Location within the reproductive tract, developmental stage and the quality of embryos were recorded. Transfer of embryos to an advanced (asynchronous) oviduct resulted, on Day 4, in fewer embryos at the expected location (oviduct), and a greater number of degenerated and retarded embryos with a lower total cell number than for embryos in the synchronous group. Similarly, on Day 7, asynchrony led to a greater number of degenerated and retarded embryos compared with the synchronous group. Total embryo cell number was similar among groups. Although Day 15 conceptuses were longer following asynchronous transfer, only 50% of the asynchronous heifers yielded conceptuses, compared with 100% in the synchronous group. In conclusion, asynchrony between the developing embryo and the reproductive tract has a negative effect on embryo development.

A tick cell line as a powerful tool to screen the antimicrobial susceptibility of the tick-borne pathogen Anaplasma marginale.

Anaplasma marginale is the causative agent of the severe bovine anaplasmosis. The tick Rhipicephalus microplus is one of the main vectors of A. marginale in tropical and subtropical regions of the world. After the tick bite, the bacterium invades and proliferates within the bovine erythrocytes leading to anemia, impairment of milk production and weight loss. In addition, infection can cause abortion and high mortality in areas of enzootic instability. Immunization with live and inactivated vaccines are employed to control acute bovine anaplasmosis. However, they do not prevent persistent infection. Consequently, infected animals, even if immunized, are still reservoirs of the bacterium and contribute to its dissemination. Antimicrobials are largely employed for the prophylaxis of bovine anaplasmosis. However, they are often used in sublethal doses which may select pre-existing resistant bacteria and induce genetic or phenotypic variations. Therefore, we propose a new standardized in vitro assay to evaluate the susceptibility of A. marginale strains to different antimicrobials. This tool will help health professionals to choose the more adequate treatment for each herd which will prevent the selection and spread of resistant strains. For that, we initially evaluated the antimicrobial susceptibility of two field isolates of A. marginale (Jaboticabal and Palmeira) infecting bovines. The least susceptible strain (Jaboticabal) was used for the standardization of an antimicrobial assay using a culture of Ixodes scapularis-derived tick cell line, ISE6. Results showed that enrofloxacin (ENRO) at 0.25, 1 or 4 µg/mL and oxytetracycline (OTC) at 4 or 16 µg/mL are the most efficient treatments, followed by OTC at 1 µg/mL and imidocarb dipropionate (IMD) at 1 or 4 µg/mL. In addition, this proposed tool has technical advantages compared to the previously established bovine erythrocyte culture. Thereby, it may be used to guide cattle farmers to the correct use of antimicrobials. The choice of the most suitable antimicrobial is essential to eliminate persistent infections, prevent the spread of resistant strains and help controlling of bovine anaplasmosis.

Spatial and Pregnancy-Related Changes in the Protein, Amino Acid, and Carbohydrate Composition of Bovine Oviduct Fluid.

Knowledge of how the biochemical composition of the bovine oviduct is altered due to the oviduct anatomy or the presence of an embryo is lacking. Thus, the aim of this study was to assess the effect of (І) oviduct anatomy and (ІІ) embryo presence on oviductal fluid (OF) protein, amino acid, and carbohydrate composition. Cross-bred beef heifers (n = 19) were synchronized and those in standing estrus were randomly allocated to a cyclic (non-bred) or pregnant (artificially inseminated) group. All heifers were slaughtered on Day 3 after estrus. The oviducts ipsilateral to the corpus luteum from each animal were isolated, straightened and cut, separating ampulla and isthmus. Each portion was flushed with 500 µl of PBS enabling recovery of the oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (8-cell embryos; pregnant group) were located in the isthmus of the oviduct. Samples of flushing medium from the isthmus and ampulla were used for proteomic (n = 2 per group), amino acid (n = 5), and carbohydrate (n = 5) analysis. For proteomic analysis, total protein from cyclic and pregnant samples were labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3-10, 17 cm, Protean® IEF cell system, Bio Rad). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). Images were obtained with a Typhoon 9410 scanner and analyzed with Progenesis SameSpots software v 4.0. Amino acid content in the OF was determined by high performance liquid chromatography (HPLC). Glucose, lactate, and pyruvate were quantified using microfluorometric enzyme-linked assays. For the proteomic assessment, the results of the image analysis were compared by ANOVA. For both amino acid and carbohydrate analyses, statistical analysis was carried out by 2-way ANOVA with the Holm-Sidak nonparametric post hoc analysis. On Day 3 post-estrus, OF composition varied based on (І) anatomical region, where isthmic metabolites were present in lower (i.e., lactate, glycine, and alanine) or higher (i.e., arginine) concentrations compared to the ampulla; and (ІІ) embryo presence, which was correlated with greater, arginine, phosphoglycerate kinase 1, serum albumin, α-1-antiproteinase and IGL@ protein concentrations. In conclusion, data indicate that the composition of bovine OF is anatomically dynamic and influenced by the presence of an early embryo.

RECQL5: Another DNA helicase potentially involved in hereditary breast cancer susceptibility.

There is still around 50% of the familial breast cancer (BC) cases with an undefined genetic cause, here we have used next-generation sequencing (NGS) technology to identify new BC susceptibility genes. This approach has led to the identification of RECQL5, a member of RECQL-helicases family, as a new BC susceptibility candidate, which deserves further study. We have used a combination of whole exome sequencing in a family negative for mutations in BRCA1/2 throughout (BRCAX), in which we found a probably deleterious variant in RECQL5, and targeted NGS of the complete coding regions and exon-intron boundaries of the candidate gene in 699 BC Spanish BRCAX families and 665 controls. Functional characterization and in silico inference of pathogenicity were performed to evaluate the deleterious effect of detected variants. We found at least seven deleterious or likely deleterious variants among the cases and only one in controls. These results prompt us to propose RECQL5 as a gene that would be worth to analyze in larger studies to explore its possible implication in BC susceptibility.

A phase 2 study of panitumumab with irinotecan as salvage therapy in chemorefractory KRAS exon 2 wild-type metastatic colorectal cancer patients.

BACKGROUND: Targeted agents are standard treatment for RAS wild-type metastatic colorectal cancer in the first- and second-line settings. This phase 2 study determined the benefit of targeting the epidermal growth factor receptor (EGFR) with panitumumab plus irinotecan in irinotecan-refractory patients. METHODS: KRAS exon-2 wild-type patients failing prior irinotecan received panitumumab (6 mg/kg) and irinotecan (180 mg/m²) every 2 weeks. The primary endpoint was the overall response rate (ORR). Secondary endpoints included safety, progression-free survival (PFS) and overall survival (OS). KRAS exon-2 status was evaluated centrally, along with NRAS, BRAF mutations, epiregulin, amphiregulin, PTEN and EGFR copy number status, and correlated with efficacy. RESULTS: Sixty-one patients were treated. Among the 46 wild-type RAS patients, the ORR was 15.2% (seven partial responses), with median PFS of 3.8 months (95% CI 2.7-4.3) and median OS of 12.5 months (95% CI 6.7-15.9). Wild-type BRAF patients showed a 13.0% response rate. No significant correlations between response and baseline biomarker expression were identified. Common grade 3-4 adverse events were diarrhoea and rash (18.0% each), hypomagnesaemia and asthenia (8.2% each). CONCLUSIONS: The addition of panitumumab to irinotecan as salvage therapy is feasible but has limited activity in irinotecan-refractory metastatic colorectal cancer. No biomarkers predictive of response were identified.

Genotypes and population genetics of cryptococcus neoformans and cryptococcus gattii species complexes in Europe and the mediterranean area.

A total of 476 European isolates (310 Cryptococcus neoformans var. grubii, 150 C. neoformans var. neoformans, and 16 C. gattii species complex) from both clinical and environmental sources were analyzed by multi-locus sequence typing. Phylogenetic and population genetic analyses were performed. Sequence analysis identified 74 sequence types among C. neoformans var. neoformans (VNIV), 65 among C. neoformans var. grubii (56 VNI, 8 VNII, 1 VNB), and 5 among the C. gattii species complex (4 VGI and 1 VGIV) isolates. ST23 was the most frequent genotype (22%) among VNI isolates which were mostly grouped in a large clonal cluster including 50% of isolates. Among VNIV isolates, a predominant genotype was not identified. A high percentage of autochthonous STs were identified in both VNI (71%) and VNIV (96%) group of isolates. The 16 European C. gattii species complex isolates analyzed in the present study originated all from the environment and all belonged to a large cluster endemic in the Mediterranean area. Population genetic analysis confirmed that VNI group of isolates were characterized by low variability and clonal expansion while VNIV by a higher variability and a number of recombination events. However, when VNI and VNIV environmental isolates were compared, they showed a similar population structure with a high percentage of shared mutations and the absence of fixed mutations. Also linkage disequilibrium analysis reveals differences between clinical and environmental isolates showing a key role of PLB1 allele combinations in host infection as well as the key role of LAC1 allele combinations for survival of the fungus in the environment. The present study shows that genetic comparison of clinical and environmental isolates represents a first step to understand the genetic characteristics that cause the shift of some genotypes from a saprophytic to a parasitic life style.

Gene expression and metabolic response of bovine oviduct epithelial cells to the early embryo.

During its journey through the oviduct, the bovine embryo may induce transcriptomic and metabolic responses, via direct or indirect contact, from bovine oviduct epithelial cells (BOECs). An in vitro model using polyester mesh was established, allowing the study of the local contact during 48 h between a BOEC monolayer and early embryos (2- or 8-cell stage) or their respective conditioned media (CM). The transcriptomic response of BOEC to early embryos was assessed by analyzing the transcript abundance of SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP and AGR3 selected from previous in vivo studies and GPX4, NFE2L2, SCN9A, EPSTI1 and IGFBP3 selected from in vitro studies. Moreover, metabolic analyses were performed on the media obtained from the co-culture. Results revealed that presence of early embryos or their CM altered the BOEC expression of NFE2L2, GPX4, SMAD6, IGFBP3, ROCK2 and SCN9A. However, the response of BOEC to two-cell embryos or their CM was different from that observed to eight-cell embryos or their CM. Analysis of energy substrates and amino acids revealed that BOEC metabolism was not affected by the presence of early embryos or by their CM. Interestingly, embryo metabolism before embryo genome activation (EGA) seems to be independent of exogenous sources of energy. In conclusion, this study confirms that early embryos affect BOEC transcriptome and BOEC response was embryo stage specific. Moreover, embryo affects BOEC via a direct contact or via its secretions. However transcriptomic response of BOEC to the embryo did not manifest as an observable metabolic response.

Evaluation of the Alfred™ turbidity monitoring system (Alifax®) following sonication in the diagnosis of central venous catheter colonization.

The conventional diagnostic techniques for catheter colonization (CC) take at least 48 h to yield results. Therefore, new diagnostic procedures that speed up the time necessary for results are needed. Our main objective was to assess the efficacy of the combination of sonication, turbidity monitoring, and MALDI-TOF to detect CC and catheter-related bloodstream infection (C-RBSI). For 1 year, we assessed central venous catheter (CVC) tips that arrived at the microbiology laboratory from adult patients admitted to our institution. CVC tips were cut, inoculated into 2.5 ml of BHI, and sonicated for 1 min. The suspension was then processed using Gram stain, quantitative culture (gold standard), and preincubation on the Alfred™ system. We analyzed the validity values of our new diagnostic approach for prediction of CC and C-RBSI and compared them with those of the gold standard. We collected a total of 167 catheters, 33 (19.8%) of which were colonized. We confirmed 21 episodes of C-RBSI. The distribution of microorganisms in colonized CVCs was as follows: Gram-positive, 68.4%; Gram-negative, 5.3%; and yeasts, 26.3%. The validity values for CC and C-RBSI using the new procedure were as follows: S, 39.4%/61.9%; Sp, 100%/100%; PPV, 100%/100%; and NPV, 87.0%/94.8%. The combination of sonication with a pre-incubation period based on turbidity monitoring using the Alfred™ system followed by MALDI-TOF proved to be a useful tool that was faster than conventional culture for ruling out C-RBSI. Future studies are needed to assess the clinical and economic impact of this diagnostic approach.