Antibiotic hyper-resistance in a class I aminoacyl-tRNA synthetase with altered active site signature motif.

Antibiotics target key biological processes that include protein synthesis. Bacteria respond by developing resistance, which increases rapidly due to antibiotics overuse. Mupirocin, a clinically used natural antibiotic, inhibits isoleucyl-tRNA synthetase (IleRS), an enzyme that links isoleucine to its tRNAIle for protein synthesis. Two IleRSs, mupirocin-sensitive IleRS1 and resistant IleRS2, coexist in bacteria. The latter may also be found in resistant Staphylococcus aureus clinical isolates. Here, we describe the structural basis of mupirocin resistance and unravel a mechanism of hyper-resistance evolved by some IleRS2 proteins. We surprisingly find that an up to 103-fold increase in resistance originates from alteration of the HIGH motif, a signature motif of the class I aminoacyl-tRNA synthetases to which IleRSs belong. The structural analysis demonstrates how an altered HIGH motif could be adopted in IleRS2 but not IleRS1, providing insight into an elegant mechanism for coevolution of the key catalytic motif and associated antibiotic resistance.

Correction: Naked and necked gold nanoparticles prepared by submerged alternating current arc discharge in water.

[This corrects the article DOI: 10.1039/D2RA06050G.].

Necked gold nanoparticles prepared by submerged alternating current arc discharge in water.

The article presents the method of producing gold nanoparticles using a high voltage arc discharge of alternating current with a frequency of 50 Hz in distilled water. The equipment necessary to carry out the process is described, including the construction of the reactor and the power source of a very simple design necessary to generate a high-voltage arc discharge between the electrodes. Arc discharge processes were carried out two times for 2 and 5 minutes, respectively, in ambient conditions without thermostating the reactor, at medium temperature varying in the range of 25-70 °C. The obtained gold nanoparticles were examined by means of various analytical techniques such as UV-vis spectroscopy, zeta potential measurement, energy dispersive X-ray analysis (EDS), X-ray diffraction (XRD). The morphology, surface, and size of the obtained nanoparticles were carried out using transmission electron microscopy (HRTEM) and dynamic light scattering (DLS). The concentration of the obtained colloids were determined using the mass spectrometry ICP-MS technique. The results show that high-voltage AC arc discharge is a simple and effective way to obtain stable gold nanoparticles under environmentally friendly conditions at relatively low production costs, and can be considered as an alternative to arc discharge nanoparticles synthesis by means of direct current (DC) methods.

[Neutrophils in the tumor microenvironment-foes or friends?] / Neutrophile Granulozyten im Tumormikromilieu ­ Feind oder Freund?

Neutrophils are the most abundant white blood cells in the human circulation and are usually associated with inflammation and with fighting infections. In recent years, the role of these cells during cancer progression has been a matter of increasing interest. Tumor-associated neutrophils (TANs) accumulate in cancer patients and represent an important negative prognostic marker in a broad variety of neoplasms. Accordingly, TANs represent a highly attractive therapeutic target. TAN may exhibit tumor-promoting or -inhibiting functions. Pro-tumor neutrophils support tumor angiogenesis and growth and promote metastatic dissemination of tumors via establishment of the premetastatic niche. Studies in animal models have already shown that the depletion of TANs or the inhibition of their migration bears therapeutic potential. Multiple pathways and mediators that induce pro-tumoral functions in neutrophils have been identified. In this review, we provide an up-to-date overview of the pro- and anti-tumor properties of neutrophils as well as the environmental cues that regulate these distinct functions. We also report on our own work that comprehensively investigated the role of neutrophils in head and neck cancer.

[The microbiome in head and neck tumors-initial findings and outlook]. / Das Mikrobiom bei Kopf-Hals-Tumoren ­ erste Erkenntnisse und Ausblick.

Technical progress in molecular biology has allowed for a more detailed analysis of the composition of the human microbiome in recent years. Inter- and intraindividual differences in microbiome composition have been demonstrated, which in part correlate with the occurrence of certain diseases. For some of the so-called oncomicrobes, a direct relationship between their effect on the host organism and carcinogenesis has been demonstrated, predominantly for gastrointestinal cancers. Initial results for head and neck cancer show inter- and intraindividual differences in the local microbiota of the tumor environment, with certain bacterial strains over- or underrepresented. Our results confirm these findings, e.g., by showing a relative abundance of fusobacteria in tumor tissue while streptococci were relatively reduced. Currently available results show a high degree of inter- and intraindividual variation, thus requiring larger patient cohorts for functional analyses.

[Exosomes as immune regulators in head and neck cancer]. / Exosomen als Immunregulatoren in Kopf-Hals-Karzinomen.

Exosomes, virus-sized nanovesicles, are utilized as messenger systems of our body to communicate with other body cells and regulate immune functions. Almost all cells produce exosomes and are able to interact with immune cells in the blood stream and peripheral body areas. Different markers on the surface of exosomes are necessary for immune cell adhesion and interaction. Furthermore, many types of exosome-immune cell interaction, such as surface receptor contact and phagocytosis, are known. As carriers of different cargos, exosomes affect different immune cell types in head and neck cancers: So far, T cells, natural killer cells, macrophages, and dendritic cells have been described in this context. For diagnostic purposes, a combined analysis of different parameters including protein amount, nucleic acid/protein expression, and the immunosuppressive impact of exosomes could empower exosomes as useful tools for evaluation of tumor promotion and progression in the future.

Absolute quantification reveals the stable transmission of a high copy number variant linked to autoinflammatory disease.

BACKGROUND: Dissecting the role copy number variants (CNVs) play in disease pathogenesis is directly reliant on accurate methods for quantification. The Shar-Pei dog breed is predisposed to a complex autoinflammatory disease with numerous clinical manifestations. One such sign, recurrent fever, was previously shown to be significantly associated with a novel, but unstable CNV (CNV_16.1). Droplet digital PCR (ddPCR) offers a new mechanism for CNV detection via absolute quantification with the promise of added precision and reliability. The aim of this study was to evaluate ddPCR in relation to quantitative PCR (qPCR) and to assess the suitability of the favoured method as a genetic test for Shar-Pei Autoinflammatory Disease (SPAID). RESULTS: One hundred and ninety-six individuals were assayed using both PCR methods at two CNV positions (CNV_14.3 and CNV_16.1). The digital method revealed a striking result. The CNVs did not follow a continuum of alleles as previously reported, rather the alleles were stable and pedigree analysis showed they adhered to Mendelian segregation. Subsequent analysis of ddPCR case/control data confirmed that both CNVs remained significantly associated with the subphenotype of fever, but also to the encompassing SPAID complex (p < 0.001). In addition, harbouring CNV_16.1 allele five (CNV_16.1|5) resulted in a four-fold increase in the odds for SPAID (p < 0.001). The inclusion of a genetic marker for CNV_16.1 in a genome-wide association test revealed that this variant explained 9.7 % of genetic variance and 25.8 % of the additive genetic heritability of this autoinflammatory disease. CONCLUSIONS: This data shows the utility of the ddPCR method to resolve cryptic copy number inheritance patterns and so open avenues of genetic testing. In its current form, the ddPCR test presented here could be used in canine breeding to reduce the number of homozygote CNV_16.1|5 individuals and thereby to reduce the prevalence of disease in this breed.

Comparison between fine needle aspiration cytology (FNAC) and core needle biopsy (CNB) in the diagnosis of breast lesions.

AIM: To compare the diagnostic accuracy of fine-needle aspiration cytology (FNAC) and core needle biopsy (CNB) in patients with USdetected breast lesions. PATIENTS AND METHODS: Between September 2011 and May 2013, 3469 consecutive breast US examinations were performed. 400 breast nodules were detected in 398 patients. 210 FNACs and 190 CNBs were performed. 183 out of 400 (46%) lesions were surgically removed within 30 days form diagnosis; in the remaining cases, a six month follow up US examination was performed. Sensitivity, specificity, diagnostic accuracy, positive predictive (PPV) and negative predictive (NPV) values were calculated for FNAC and CNB. RESULTS: 174 out of 400 (43%) malignant lesions were found while the remaining 226 resulted to be benign lesions. 166 out of 210 (79%) FNACs and 154 out of 190 (81%) CNBs provided diagnostic specimens. Sensitivity, specificity, diagnostic accuracy, PPV and NPV of 97%, 94%, 95%, 91% and 98% were found for FNAC, and values of 92%, 82%, 89%, 92% and 82% were obtained for CNB. Sensitivity, specificity, diagnostic accuracy, PPV and NPV of 97%, 96%, 96%, 97% and 96% were found for FNAC, and values of 97%, 96%, 96%, 97% and 96% were obtained for CNB. CONCLUSION: FNAC and CNB provide similar values of diagnostic accuracy.

CXCR2-mediated tumor-associated neutrophil recruitment is regulated by IFN-ß.

The chemokine receptor CXCR2 and its ligands CXCL1, CXCL2 and CXCL5 play an important role in homing of tumor-associated neutrophils (TANs) into developing tumors. TANs are known to support the development of blood vessels in growing solid tumors, hence contributing to tumor growth. Here, we show that the migration of neutrophils is influenced by endogenous interferon-beta (IFN-ß) via regulation of such chemokines and their receptor. We could demonstrate that CXCL1 and CXCL2 gradients are formed in tumor-bearing mice, i.e., low chemokine level in bone marrow (BM) and high level in the tumor. This supports migration of neutrophils into the tumor. Moreover, expression of CXCR2 was highest on neutrophils from BM and lowest in TANs. Importantly, although IFN-ß appears to have only a minor influence on the expression of CXCR2, it strongly regulates the CXCR2 ligands. In the absence of endogenous IFN-ß, they were expressed significantly higher in tumor-infiltrating neutrophils. Treatment of such neutrophils from tumor-bearing Ifnb1(-/-) mice with recombinant IFN-ß downregulated CXCR2 ligand expression to wild-type levels. This explains the reduced migration of neutrophils into tumors and the diminished tumor angiogenesis in IFN-ß-sufficient mice. Our results add a novel functional aspect of the type I IFN system as effector molecules of natural cancer surveillance and open interesting possibilities for antineutrophil therapies against cancer.

Too fast, too soon to call it "probiotic".

Probiotics (bacteria or yeasts) were defined by the Food Agricultural Organization (FAO) and the World Health Organization (WHO) joint report as live microorganisms which when administered in adequate amounts (in food or as a dietary supplement) confer a health benefit on the host. The best-demonstrated potential clinical benefits of probiotic agents, specifically in the pediatric population, are in the prevention and management of acute diarrhea, antibiotic associated diarrhea, and evidence is mounting on their potential benefits in atopic disease, inflammatory bowel conditions, and necrotizing enterocolitis. Their beneficial effects seem to be strain specific, thus, pooling data from different strains may result in misleading conclusions. Because there was no international consensus on methodology to assess efficiency and safety of probiotics, in 2001 the FAO/WHO undertook work to compile and evaluate the scientific evidence on functional and safety aspects of probiotics. International criteria have been developed to formulate unequivocal criteria for probiotic bacterial strains and products that contain them. More recently, the European Food Safety Authority (EFSA) highlighted as critical factors for probiotic health claim submissions genetic typing, internationally recognised naming protocols and evidence of consistency in the final product.

The infrequent simultaneous genetic alterations in glioblastoma multiforme (LOH 10, 17, 19q, TP53 mutation and EGFR amplification) with short clinical course.

We described the case of an unusual, complex genetic alteration in 57 year-old male patient with glioblastoma multiforme (GBM) with short survival (6 and half months). Alterations consisted of p53 mutation, LOH 10, LOH 17, LOH 19q and EGFR amplification. LOH1p, LOH 9 and LOH 13 were negative. Immunohistochemical study did not correlate with molecular results. The overexpression of TP53 protein and RB protein was detected only in small percentage of cells and interestingly the overexpression of EGFR was present only focally. Immnunostainings for PTEN, P16, PI3-K were negative. Additionally, we observed an overexpression of IGFB2 protein. This case indicates the accumulation of molecular changes in glioblastoma multiforme in patient with short survival.

Oxidative stress in blood platelets from schizophrenic patients.

Oxidative stress in blood platelets is observed in various diseases, including neuropsychiatric disorders. The aim of our study was to evaluate oxidative stress in blood platelets from patients with schizophrenic disorders by measuring the activity of the platelet antioxidative enzyme, superoxide dismutase (SOD), concomitant with the level of thiobarbituric acid reactive species (TBARS). In blood platelets obtained from schizophrenic patients (with paranoid schizophrenia according to DSM-IV criteria) and from healthy volunteers the level of reactive oxygen species was also measured via chemiluminescence. In resting blood platelets from schizophrenic patients the chemiluminescence was higher than in platelets from control subjects (P < 0.05), but in thrombin-activated platelets an increase (about 53%) of chemiluminescence was observed, however this increase was lower than in thrombin-stimulated platelets from healthy subjects (101.5%). The results indicate that in platelets from schizophrenic patients generation of reactive oxygen species is enhanced. Moreover, we observed that SOD activity in blood platelets from schizophrenic patients was significantly lower than in control platelets and that a correlation exists between increased lipid peroxidation and inhibition of the activity of this antioxidative enzyme in schizophrenic platelets.

Influence of composition of diluent on populations of yeasts and moulds recovered from raw fruits.

AIMS: The aims of this study were (i) to determine the retention of viability of mycoflora removed from raw fruits, and how this affected diluents used to prepare samples for enumeration of propagules, and (ii) to evaluate the performance of recovery media for supporting colony development. METHODS AND RESULTS: Yeasts and moulds removed from seven types of raw fruit were held in seven diluents for 1 h before plating on dichloran rose bengal chloramphenicol (DRBC) agar and plate count agar supplemented with chloramphenicol (100 micro g ml-1) (PCAC). Significant reductions (P=0.05) in populations of yeasts, moulds, and yeasts plus moulds occurred within the 1 h holding period, regardless of diluent composition. Overall, retention of viability was not influenced by diluent composition, and neither DRBC agar nor PCAC were superior in supporting colony development. CONCLUSIONS: The composition of diluents used to prepare food samples for mycological analysis has little affect on the number of yeasts and moulds recovered from seven types of naturally contaminated raw fruit. Both DRBC agar and PCAC are suitable as enumeration media. SIGNIFICANCE AND IMPACT OF THE STUDY: Diluents and media most often recommended for enumerating yeasts and moulds in foods are appropriate for raw fruits.

Obstetric complications and Apgar score in early-onset schizophrenic patients with prominent positive and prominent negative symptoms.

UNLABELLED: The objective of the study was to find associations between obstetric complications (OCs) history and schizophrenia course and symptoms. We analysed the obstetric and psychiatric history of 50 DSM IV schizophrenic subjects who experienced their first schizophrenia episode in adolescence, and 30 healthy controls. Obstetrical data and Apgar scores were obtained from medical records and evaluated with the Lewis and Murray Scale. Based on patients' documentation [including longitudinal evaluation with Positive and Negative Syndrome Scale (PANSS)] the symptom profile and the course of schizophrenia were determined. RESULTS: we distinguished two major groups of patients: with prominent negative and prominent positive symptoms. Schizophrenics with prominent negative symptoms and a chronic schizophrenia course had significantly more definite OCs and lower Apgar scores than patients with prominent positive symptoms and controls. Subjects who had a positive OCs history were more than four times likely to develop schizophrenia in adolescence than those without such a history (OR=4.64; 95% CI=1.29-17.51) with the likelihood of developing schizophrenia with prominent negative symptoms especially high (OR=7.31; 95% CI=1.80-29.65). An Apgar score of between 0 and 3 after birth was associated with an increased risk for developing schizophrenia (OR=2.25; 95% CI=0.56-9.12), especially with prominent negative symptoms (OR=3.71; 95% CI=0.84-16.32). The findings support the hypothesis of a role of OCs in developing early-onset schizophrenia and suggest the associations of the OCs history with a specific symptoms profile (prominent negative symptoms) and a chronic course of schizophrenia.

Diurnal profile of melatonin secretion in the acute phase of major depression and in remission.

BACKGROUND: The aim of this study was to determine whether there are differences in the 24-hour melatonin secretion profile between patients with major depression (MD) before and after clomipramine treatment, and a control group, and also whether or not there exist statistically significant differences in the melatonin levels depending on the severity of depression. MATERIAL AND METHODS: 20 patients with MD and 14 healthy volunteers took part in the study. Before and after clomipramine treatment all the patients had an 8 ml blood sample collected 7 times, at fixed hours, for measurement of the melatonin level. The severity of depression was assessed (HAMDS, BDI, POMS) before and after treatment. RESULTS: The mean melatonin concentration at each of the chosen hours was higher in persons with MD than in the control, but the differences reached statistical significance only at certain night hours. In both groups the nocturnal rise in melatonin secretion began at 12 midnight, reaching its peak at 2 am, to reach low values by 8 am, similar to the values from the previous morning. After 8 weeks of clomipramine treatment, the melatonin concentrations at 12 midnight, 2 am, and 4 am were lowered, and were elevated at 8 am, 2 pm, and 8 pm. The difference was statistically significant only at 8 pm. Melatonin secretion during both night time hours and daytime hours is higher in individuals with MD than in healthy subjects, and remains elevated after 8 weeks of effective clomipramine treatment. Those patients whose state changed the least after 8 weeks of clomipramine treatment differed in a statistically significant way in terms of melatonin secretion level at 2 pm from those patients who improved the most. CONCLUSIONS: Some patients with MD have elevated melatonin concentration levels during daytime and nighttime hours, and this melatonin secretion pattern is independent of the severity of depression.

Role of IL-18 in the secretion of Il-1beta, sIL-1RII, and IL-1Ra by human neutrophils.

In the present study we investigated the effect of IL- 18 on the production of IL-1beta, IL-1Ra and sIL-1RII by human neutrophils. Our observations indicate that rhIL-18 induces IL-1beta and, to a lesser extend, IL-1Ra and sIL-1RII production by human neutrophils isolated form peripheral blood. However, this effect was less important in comparison with LPS-stimulation. Moreover, the results obtained suggest that IL-18 can induce priming of neutrophils for IL-1beta and, to a lesser extend, IL- Ra and sIL-1RII production by LPS-stimulated cells. The capacity of IL-18 to serve as an effective modulator for IL-1beta and its regulatory proteins may have significance in the inflammatory and immune reactions mediated by IL-1beta.

Diurnal profile of melatonin concentrations in patients with major depression: relationship to the clinical manifestation and antidepressant treatment.

OBJECTIVES: The aim of the study was to establish if there are differences in the 24-hour melatonin secretion profile between patients with major depression (before, and after treatment with clomipramine) compared to those in healthy subjects. Additionally, we determined if there are differences in melatonin concentrations, depending on the severity of depression, and the presence of 24-hour rhythm disturbances. MATERIAL AND METHODS: Twenty patients with major depression and 24-hour rhythm disturbances, and 14 healthy volunteers took part in the study. Before, and after treatment with clomipramine all subjects had blood samples collected at 08:00, 14:00, 20:00, 24:00, 02:00, 04:00, and 08:00 h, for estimation of melatonin concentrations. Before and after treatment, the severity of depression was evaluated using the following scales: the Hamilton Depression Rating Scale (HADRS), Beck Depression Inventory (BDI), and clinical observation, as well as presence, and if so, severity of 24-hour rhythm disturbances were assessed. RESULTS: In individuals with major depression with marked disturbances of their diurnal rhythms, melatonin secretion is also disturbed, shown by the higher melatonin concentrations at night as compared to those in healthy individuals. However, melatonin levels were independent of the severity or the clinical manifestation of depression. Moreover, no correlation between the disturbances in their diurnal rhythms (sleep-watchfulness, diurnal mood shifts) and disturbed melatonin pattern was observed. CONCLUSIONS: Melatonin nocturnal concentrations in patients with major depression were higher than those in healthy individuals. However, the melatonin concentration values do not differentiate the patients in terms of the severity of the depressive symptoms.