The glutaminase-dependent acid resistance system. Qualitative and quantitative assays and analysis of its distribution in enteric bacteria

Neutralophilic bacteria have developed several strategies to overcome the deleterious effects of acid stress. In particular, the amino acid-dependent systems are widespread, with their activities overlapping, covering a rather large pH range, from 6 to <2. Recent reports showed that an acid resistance (AR) system relying on the amino acid glutamine (AR2_Q), the most readily available amino acid in the free form, is operative in Escherichia coli, Lactobacillus reuteri and some Brucella species. This system requires a glutaminase active at acidic pH and the antiporter GadC to import L-glutamine and export either glutamate (the glutamine deamination product) or GABA. The latter occurs when the deamination of glutamine to glutamate, via acid-glutaminase (YbaS/GlsA), is coupled to the decarboxylation of glutamate to GABA, via glutamate decarboxylase (GadB), a structural component of the glutamate-dependent AR (AR2) system, together with GadC. Taking into account that AR2_Q could be widespread in bacteria and that until now assays based on ammonium ion detection were typically employed, this work was undertaken with the aim to develop assays that allow a straightforward identification of the acid-glutaminase activity in permeabilised bacterial cells (qualitative assay) as well as a sensitive method (quantitative assay) to monitor in the pH range 2.5-4.0 the transport of the relevant amino acids in vivo. The qualitative assay is colorimetric, rapid and reliable and provides several additional information, such as co-occurrence of AR2 and AR2_Q in the same bacterial species and assessment of the growth conditions that support maximal expression of glutaminase at acidic pH. The quantitative assay is HPLC-based and allows to concomitantly measure the uptake of glutamine and the export of glutamate and/or GABA via GadC in vivo and depending on the external pH. Finally, an extensive bioinformatic genome analysis shows that the gene encoding the glutaminase involved in AR2_Q is often nearby or in operon arrangement with the genes coding for GadC and GadB. Overall, our results indicate that AR2_Q is likely to be of prominent importance in the AR of enteric bacteria and that it modulates the enzymatic as well as antiport activities depending on the imposed acidic stress

Comparing Passenger Satisfaction, Employees’ Perspective and Performance on Quality and Safety Indicators: A field study

This paper aims to analyze the impact that different attributes related to a Regional Airport service and the socio-economic factors of the passengers have on the passenger’s overall satisfaction. The study also compared passenger and employee satisfaction in relation to the service offered by the airport, to identify possible critical areas of improvement. An Ordinal Logistic Regression (OLR) approach was used to model how the attributes considered for qualifying airport services and the socio-economic variables impact the predicted variable (i.e., passenger satisfaction). Furthermore, the results were triangulated to include quality and safety performance indicators as an objective anchor point for the performance of the company. The findings indicate interesting areas of difference between the perceptions of the passengers and airport employees regarding a company’s services and its performance. The company managers in the key areas of operation were then asked to select the main areas of improvement among the ones highlighted by the survey’s results. Quality and safety indicators were also helpful in enriching the analysis and indicating good synergy with the suggestions collected from the passengers’ and the employees’ surveys, offering yet another complementary perspective

Glutaminase activity and glutamine-dependent acid resistance in enteric bacteria

Many neutralophilic bacteria have developed several strategies to overcome the life-threatening effect of the exposure to a mild-to-harsh acid stress [1]. Among these strategies, the amino acid-dependent systems were shown to be quite widespread amongst gram-positive and –negative bacteria, and their activities overlap so to cover a rather large pH range, from 6 to <2 [1]. Recently an acid resistance (AR) system relying on the amino acid glutamine, the most readily available amino acid in the free form, was shown to be operative in E. coli, L. reuteri and some Brucella species [2-4]. This system imports L-glutamine via the antiporter GadC, also a structural component of the glutamate-dependent AR system, and exports via GadC either glutamate or GABA, depending on whether only the deamination of glutamine into glutamate occurs, or glutamate is also decarboxylated to yield GABA by the enzyme GadB. We have undertaken a bioinformatic analysis to study the distribution of the glutaminase-dependent AR system in enteric bacteria and developed two assays. The first assay was developed for a rapid screening of the glutaminase activity and the growth conditions that support its maximum expression. The second assay was developed to measure the amount of glutamate/GABA exported. Our results indicate that the glutamine-dependent AR is likely to be of prominent importance in enteric bacteria. [1] Lund et al. (2014) FEMS MIcrobiol Rev 38:1091-125. [2] Lu et al. (2013) Cell Res 23:635–644. [3] Teixeira et al. (2014) Food Microbiol 42:172-180. [4] Freddi et al. (2017) Front Microbiol 8:2236

The glutamine-depend acid resistance system in neutralophilic bacteria

Background: The amino acid-dependent acid resistance (AR) systems are widespread in neutralophilic bacteria. Their activities overlap so to cover a rather large acidic pH range, from 6 to &lt;2. Recently, the AR system relying on glutamine (AR2_Q) was shown to be operative in Escherichia coli, Lactobacillus reuteri and some Brucella species. AR2_Q requires the glutaminase GlsA/YbaS and the membrane antiporter GadC, both proteins are active at acidic pH. GadC imports L-glutamine and exports either glutamate (the glutamine deamination product) or GABA, the decarboxylation product of glutamate, via glutamate decarboxylase (GadB), a structural component of the glutamate-dependent AR system, together with GadC (AR2). Objectives: This work was undertaken to study the distribution of the AR2_Q in bacteria and to develop a qualitative acid-glutaminase assay and a quantitative GadC-mediated transport assay. Methods: The qualitative assay takes advantage of the color change of the pH indicator bromocresol green. The quantitative assay is fluorometric, HPLC-based, to concomitantly measure glutamine uptake, and glutamate and GABA export. Results: The colorimetric assay is fast and reliable. It also provides information on co-occurrence of AR2 and AR2_Q in bacterial species. The HPLC-based assay provided evidence that glutamine uptake and the selective export of glutamate and/or GABA via GadC is finely modulated in the pH range 2.5-4.0. A bioinformatic genome analysis showed that the gene encoding acid-glutaminase is often nearby or in operon arrangement with the genes encoding GadC and/or GadB. Overall, our results suggest that AR2_Q is likely to be of prominent importance especially for enteric bacteria AR

The role of trastuzumab in patients with her2 positive small (Pt1mi/a) breast cancers, a multicenter retrospective study

SIMPLE SUMMARY: Treatment of small HER2+ breast cancers with adjuvant Trastuzumab is still controversial. This study aims to measure the effect of Trastuzumab in early stages of breast cancer (pT1mic/a pN0/1mi) in terms of disease recurrence and to identify which factors most affect the prognosis of small HER2+ tumors. We retrospectively selected and reviewed 100 HER2+ pT1mic-pT1a breast cancer patients with a median follow-up of 86 months. In our study the primary outcome was the disease recurrence rate, which appeared to be significantly lower among patients who received adjuvant Trastuzumab. Among the patients who did not receive adjuvant Trastuzumab, HR− HER2+ tumors showed a risk seven times higher of relapse. The results of our study indicate that adjuvant Trastuzumab reduces the risk of developing a disease recurrence even in small HER2+ tumors. Adjuvant targeted therapy should be considered in patients with HR− HER2+ tumors, the category with the highest risk of recurrence. ABSTRACT: The treatment with adjuvant Trastuzumab in patients diagnosed with HER2+ small breast cancers is controversial: limited prospective data from randomized trials is available. This study aims to measure the effect of Trastuzumab in the early stages of breast cancer (pT1mic/a pN0/1mi) in terms of disease recurrence and to identify which are the factors that most affect the prognosis of small HER2+ tumors. One hundred HER2+ pT1mic-pT1a breast cancer patients who were treated in three Turin Breast Units between January 1998 and December 2018 were retrospectively selected and reviewed. Trastuzumab was administered to 23 patients. Clinicopathological features and disease-free survival (DFS) were compared between different subgroups. The primary outcome was the disease recurrence rate. Median follow-up time was 86 months. Compared to pT1a tumors, pT1mic lesions had a higher tumor grade (84% of pT1mic vs. 55% of pT1a; p = 0.003), a higher Ki-67 index (81% vs. 46%; p = 0.007) and were more frequently hormone receptor (HR) negative (69% vs. 36%, p = 0.001). Disease recurrence rate was significantly lower among patients who received adjuvant Trastuzumab (p = 0.02), with this therapy conferring an 85% reduction in the risk of relapse (HR 0.15; p = 0.02). Among the patients who did not receive adjuvant Trastuzumab, the only factor significantly associated with an increased risk of developing a recurrence was the immunohistochemical (IHC) subtype: in fact, HR− HER2+ tumors showed a risk seven times higher of relapsing (HR 7.29; p = 0.003). Adjuvant Trastuzumab appears to reduce the risk of disease recurrence even in small HER2+ tumors. The adjuvant targeted therapy should be considered in patients with HR− HER2+ tumors since they have the highest risk of recurrence, independently from size and grade

Patterns of care at the end of life: a retrospective study of Italian patients with advanced breast cancer

Abstract Objectives To better understand the type of care offered to Italian patients with advanced breast cancer at the End-of-Life (EoL), we conducted a retrospective observational study. EoL was defined as the period of six months before death. Methods One hundred and twenty-one patients with advanced breast cancer (ABC) treated at IRCCS San Martino Policlinic Hospital who died between 2017 and 2021 were included. Data about patient, disease, and treatment characteristics from breast cancer diagnosis to death, along with information about comorbidities, medications, imaging, specialist evaluations, hospitalization, palliative care and home care, hospice admissions, and site of death were collected. Results 98.3% of the patients received at least one line of active treatment at EoL; 52.8% were hospitalized during the selected period. Palliative (13.9%), psychological (7.4%), and nutritional evaluations (8.2%) were underutilized. Palliative home care was provided to 52% of the patients. Most of the patients died at home (66.1%) and fewer than one out of five (18.2%) died at the hospital. Among the patients who died at home, 27.3% had no palliative support. Conclusions Our findings indicate that palliative care in EoL breast cancer patients is still inadequate. Only a minority of patients had psychological and nutritional support While low nutritional support may be explained by the fact that typical symptoms of ABC do not involve the gastrointestinal tract, the lack of psychological support suggests that significant barriers still exist. Data on the site of death are encouraging, indicating that EoL management is increasingly home centered in Italy

Geomorphological analysis of the San Domino Island (Tremiti Islands, Southern Adriatic Sea). Results from the 2019 Geomorphological Field Camp of the MSc in Geological Science and Technology (University of Chieti-Pescara)

The 2019 Geomorphological Field Camp at San Domino Island (Tremiti Islands, Southern Adriatic Sea) is the result of geological and geomorphological field work activities carried out by a group of students attending the Geomorphological field mapping course of the Master’s Degree in Geological Science and Technology (University of Chieti-Pescara). The main map (1:5000 scale) was obtained through an integrated approach that incorporates morphometric analysis, geological and geomorphological field mapping, and geomorphological profiles drawing. Activities were carried out by all students, divided into six working groups of three to four persons each. The field camp and field work activities made it possible to produce a detailed thematic map, as a scientific tool to depict the San Domino Island landscape, and to outline some geomorphological issues in terms of possible constraints to landscape evolution, geomorphological processes distribution, and natural hazard assessment