GateMeClass: Gate Mining and Classification of cytometry data.

MOTIVATION: Cytometry comprises powerful techniques for analyzing the cell heterogeneity of a biological sample by examining the expression of protein markers. These technologies impact especially the field of oncoimmunology, where cell identification is essential to analyze the tumor microenvironment. Several classification tools have been developed for the annotation of cytometry datasets, which include supervised tools that require a training set as a reference (i.e. reference-based) and semisupervised tools based on the manual definition of a marker table. The latter is closer to the traditional annotation of cytometry data based on manual gating. However, they require the manual definition of a marker table that cannot be extracted automatically in a reference-based fashion. Therefore, we are lacking methods that allow both classification approaches while maintaining the high biological interpretability given by the marker table. RESULTS: We present a new tool called GateMeClass (Gate Mining and Classification) which overcomes the limitation of the current methods of classification of cytometry data allowing both semisupervised and supervised annotation based on a marker table that can be defined manually or extracted from an external annotated dataset. We measured the accuracy of GateMeClass for annotating three well-established benchmark mass cytometry datasets and one flow cytometry dataset. The performance of GateMeClass is comparable to reference-based methods and marker table-based techniques, offering greater flexibility and rapid execution times. AVAILABILITY AND IMPLEMENTATION: GateMeClass is implemented in R language and is publicly available at https://github.com/simo1c/GateMeClass.

Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells.

Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.

Molecules, Information and the Origin of Life: What Is Next?

How life did originate and what is life, in its deepest foundation? The texture of life is known to be held by molecules and their chemical-physical laws, yet a thorough elucidation of the aforementioned questions still stands as a puzzling challenge for science. Focusing solely on molecules and their laws has indirectly consolidated, in the scientific knowledge, a mechanistic (reductionist) perspective of biology and medicine. This occurred throughout the long historical path of experimental science, affecting subsequently the onset of the many theses and speculations about the origin of life and its maintenance. Actually, defining what is life, asks for a novel epistemology, a ground on which living systems' organization, whose origin is still questioned via chemistry, physics and even philosophy, may provide a new key to focus onto the complex nature of the human being. In this scenario, many issues, such as the role of information and water structure, have been long time neglected from the theoretical basis on the origin of life and marginalized as a kind of scenic backstage. On the contrary, applied science and technology went ahead on considering molecules as the sole leading components in the scenery. Water physics and information dynamics may have a role in living systems much more fundamental than ever expected. Can an organism be simply explained by a mechanistic view of its nature or we need "something else"? Probably, we can earn sound foundations about life by simply changing our prejudicial view about living systems simply as complex, highly ordered machines. In this manuscript we would like to reappraise many fundamental aspects of molecular and chemical biology and reading them through a new paradigm, which includes Prigogine's dissipative structures and informational dissipation (Shannon dissipation). This would provide readers with insightful clues about how biology and chemistry may be thoroughly revised, referring to new models, such as informational dissipation. We trust they are enabled to address a straightforward contribution in elucidating what life is for science. This overview is not simply a philosophical speculation, but it would like to affect deeply our way to conceive and describe the foundations of organisms' life, providing intriguing suggestions for readers in the field.

Peripheral blood immunophenotyping in a large cohort of patients with Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is one of the more common inherited bone marrow failure syndromes, characterized by neutropenia, occasional thrombocytopenia, and anemia. Bone marrow evaluation reveals an increased number of monocytes and mature B cells along with decreased granulocytes. However, little is known about the subpopulations of peripheral blood cells, and few previous publications have been based on a small number of patients. Here, we report a comprehensive immunophenotypic analysis from a cohort of 37 SDS patients who display impairment mostly in the myeloid compartment with a deficiency also in the number of B cells and CD4/CD8 double-negative T cells.

Tetracycline Residues in Bovine Muscle and Liver Samples from Sicily (Southern Italy) by LC-MS/MS Method: A Six-Year Study.

We examined a total of 369 bovine liver and muscle samples for the detection of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), and doxycycline (DOX) residues by implementation and validation of a LC-MS/MS method. The method showed good recovery values between 86% and 92% at three levels of concentrations. The linearity tests revealed r² > 0.996 for all the tetracyclines examined. Furthermore, the Youden test revealed that the method was robust. Only 14.4% of the samples showed OTC and TC residues in a concentration range of 10.4⁻40.2 µg kg-1. No CTC and DOX residues were found in all the samples analyzed. Liver samples showed the highest average values (31.5 ± 20.6 and 21.8 ± 18.9 for OTC and TC, respectively). The results showed a low incidence of TCs in all the samples examined, in comparison with other studies reported in the literature. A significant decrease in TC residues frequency was found from 2013 (p < 0.05). This work reports for the first time epidemiological data on the presence of TC residues in liver and muscle samples of cattle farmed in Sicily (Southern Italy). The very low incidence of TC residues indicates a continuous improvement in farming techniques in Southern Italy, which is essential to ensure consumers' protection.

Ataluren-driven restoration of Shwachman-Bodian-Diamond syndrome protein function in Shwachman-Diamond syndrome bone marrow cells.

Shwachman-Diamond syndrome (SDS) is a rare inherited recessive disease mainly caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene, which encodes for the homonymous protein SBDS, whose function still remains to be fully established. SDS affects several organs causing bone marrow failure, exocrine pancreatic insufficiency, skeletal malformations, and cognitive disorders. About 15% of SDS patients develop myelodysplastic syndrome (MDS) and are at higher risk of developing acute myeloid leukemia (AML). Deficiency in SBDS expression has been associated with increased apoptosis and lack of myeloid differentiation in bone marrow hematopoietic progenitors. Importantly, most SDS patients carry nonsense mutations in SBDS. Since ataluren is a well-characterized small molecule inhibitor that can suppress nonsense mutations, here, we have assessed the efficacy of this drug in restoring SBDS expression in hematopoietic cells obtained from a cohort of SDS patients. Remarkably, we show that ataluren treatment readily restores SBDS protein expression in different cell types, particularly bone marrow stem cells. Furthermore, ataluren promotes myeloid differentiation in hematopoietic progenitors, reduces apoptotic rate in primary PBMCs, and brings mammalian target of rapamycin phosphorylation levels back to normal in both lymphoblasts and bone marrow mesenchymal stromal cells (BM-MSCs). Since a specific therapy against SDS is currently lacking, these results provide the rationale for ataluren repurposing clinical trials.

Targeting Cancer with Phytochemicals via Their Fine Tuning of the Cell Survival Signaling Pathways.

The role of phytochemicals as potential prodrugs or therapeutic substances against tumors has come in the spotlight in the very recent years, thanks to the huge mass of encouraging and promising results of the in vitro activity of many phenolic compounds from plant raw extracts against many cancer cell lines. Little but important evidence can be retrieved from the clinical and nutritional scientific literature, where flavonoids are investigated as major pro-apoptotic and anti-metastatic compounds. However, the actual role of these compounds in cancer is still far to be fully elucidated. Many of these phytochemicals act in a pleiotropic and poorly specific manner, but, more importantly, they are able to tune the reactive oxygen species (ROS) signaling to activate a survival or a pro-autophagic and pro-apoptosis mechanism, depending on the oxidative stress-responsive endowment of the targeted cell. This review will try to focus on this issue.

Assessed and Emerging Biomarkers in Stroke and Training-Mediated Stroke Recovery: State of the Art.

Since the increasing update of the biomolecular scientific literature, biomarkers in stroke have reached an outstanding and remarkable revision in the very recent years. Besides the diagnostic and prognostic role of some inflammatory markers, many further molecules and biological factors have been added to the list, including tissue derived cytokines, growth factor-like molecules, hormones, and microRNAs. The literatures on brain derived growth factor and other neuroimmune mediators, bone-skeletal muscle biomarkers, cellular and immunity biomarkers, and the role of microRNAs in stroke recovery were reviewed. To date, biomarkers represent a possible challenge in the diagnostic and prognostic evaluation of stroke onset, pathogenesis, and recovery. Many molecules are still under investigation and may become promising and encouraging biomarkers. Experimental and clinical research should increase this list and promote new discoveries in this field, to improve stroke diagnosis and treatment.

Toxic Metals in Pelagic, Benthic and Demersal Fish Species from Mediterranean FAO Zone 37.

Fish represents a nutrient-rich food but, at the same time, is one of the most important contributor to the dietary intake of heavy metals. The aim of this study was to assess residual levels of Pb, Cd and Hg in different species, caught from FAO zones 37 1.3 and 37 2.2, particularly small pelagic, benthic and demersal fishes. The results obtained showed the absence of toxic metal in fishes from FAO zone 37 1.3. Relating to FAO zone 37 2.2, instead, in all samples we observed the absence of Pb, small concentrations of Cd (0.081±0.022 mg/kg) and higher Hg residual levels (0.252±0.033 mg/kg). Particularly, the trend of Cd contamination was similar in all species whereas Hg showed high levels in demersal, intermediate in pelagic and low in benthic species. However, only Cd concentrations exceed the MRL in mackerel, mullet, sea-bream fishes, according to Regulation CE n. 629/2008 and n. 488/2014.

Essential and non-essential elements in white lupin (Lupinus albus L.) cultivated in Southern Italy.

We assessed the presence of V, Cr, Ni, Cu, Zn, As, Se, Sb, Cd, and Pb in white lupin samples cultivated in Southern Italy by the validation of an Inductively Coupled Plasma Mass Spectrometry (ICP-MS) method. The ICP-MS method validation showed satisfactory values of linearity (r2 > 0.999), recovery (87.4-100.7%), repeatability, and reproducibility values. Zinc was the most abundant element; showing mean concentrations of 0.778 ± 0.09 mg/Kg wet weight (w.w.) and a maximum of 1.013 mg/Kg w.w., followed by copper (0.191 ± 0.05 mg/Kg w.w.). Among the non-essential elements, important levels of cadmium were found (0.017 ± 0.004 mg/Kg w.w.), with 28% exceeding the limits set by the EU Regulation. The results of this work confirm the role of white lupins and other legumes in reducing the pH of the soil, increasing the exchangeable forms of Cd. This work also provides the first data on the nutritional and antinutritional properties of white lupins cultivated in Italy.

Acute phase response after zoledronic acid is associated with long-term effects on white blood cells.

We have recently reported a long-lasting decrease in circulating γδ T cells in osteoporotic patients on oral amino-bisphosphonates (N-BPs). Here we verify whether these changes are associated with the occurrence of acute phase response (APR) to intravenous (IV) zoledronic acid (ZOL) or changes of other circulating white blood cells (WBC). WBC count was obtained before and 1 year after a single IV administration of 5 mg ZOL in 36 osteoporotic patients (mean age 72 ± 9, range 45-86 years) without other relevant diseases; 12 of 36 patients developed the classical APR. After 1 year in the patients who experienced an APR, but not in the others, a significant decrease not only of γδ T cells (-30 %), but also of total lymphocytes (-11 %) and eosinophils (-27 %), was observed. The mechanism leading to the observed decrease of circulating lymphocytes and eosinophils remains unclear, but our observation opens a new frontier for the understanding of the immunoeffects of N-BPs.

Toxic metals in Loggerhead sea turtles (Caretta caretta) stranded freshly dead along Sicilian coasts.

BACKGROUND: The Loggerhead sea turtle (Caretta caretta) is a marine reptile belonging to a monophyletic group of chelonians. As these animals are long-lived, they have the ability to accumulate pollutants. AIM: To collect epidemiological data on toxic metals in marine Loggerhead sea turtles. MATERIALS AND METHODS: Forty Loggerhead sea turtles comprising 25 males and 15 females stranded freshly dead between 2013 and 2018 along the coasts of Sicily, Southern Italy, were examined for arsenic, cadmium, and lead accumulation in muscle and adipose tissues by means of a validated ICP-MS method. A modified K index as a growth condition factor, namely Fulton's K index, was used. Samples were tested in duplicate. A Wilcoxon rank sum test was carried out to evaluate metal contents differences between muscle and adipose tissues and between genders. RESULTS: The Fulton's K index suggested a good body condition of the C. caretta recovered with mean values of 5.34 ± 3.40 (n = 40; ±SD). Detectable concentrations of lead were found in 70% of the samples analysed with mean values of 0.65 ± 1.67 mg/kg wet weight and 0.51 ± 1.29 mg/kg wet weight in muscle and adipose tissues, respectively. No significant differences in arsenic, cadmium, and lead were detected between genders. In addition, no significant correlation was found between modified K index and concentrations of arsenic, cadmium, and lead. CLINICAL RELEVANCE: Findings on muscle and adipose tissues suggest chronic exposure of Caretta caretta to high concentrations of especially lead which might negatively affect health and welfare of these marine turtles although body condition was good.

TiO2-Ag-NP adhesive photocatalytic films able to disinfect living indoor spaces with a straightforward approach.

TiO2-Ag doped nanoparticulate (TiO2-Ag-NP) adhesive photocatalytic films were used to assess the ability in dropping down the burden of indoor microbial particles. The application of an easy-to use photocatalytic adhesive film to cleanse indoor living spaces from microbial pollution, represents a novelty in the field of photocatalytic devices. Reduction was attained by photocatalysis in selected spaces, usually with overcrowding (≥ 3 individuals) in the common working daily hours, and upon indoor microclimate monitoring. TiO2-Ag doped nanoparticulate (TiO2-Ag-NP) adhesive photocatalytic films were applied within five types of living spaces, including schools and job places. The microbial pollution was assessed at time 0 (far from routine clean, ≥ 9 h) and throughout 2-4 weeks following the photocatalyst application by relative light unit (RLU) luminometry and microbial indirect assessment (colony forming units per cubic meter, CFU/m3). TiO2-Ag-NP photocatalyst reduced RLU and CFU/m3 by rates higher than 70% leading to RLU ≤ 20 and microbial presence ≤ 35 CFU/m3. The described TiO2-Ag-NP is able to reduce microbial pollution to the lowest RLU threshold (≤ 20) within 60 min in open daylight in a standardized test room of 100 m2. The correlation between RLU and CFU/m3 was positive (r = 0.5545, p < 0.05), assessing that the microbial reduction of indoor areas by the TiO2-Ag-NP adhesive film was real. Titania photocatalysts represent promising tools to ensure air cleaning and sanitization in living indoor microclimates with a low cost, feasible and straightforward approach. This approach represents an easy to handle, cost effective, feasible and efficacious approach to reduce microbial pollution in indoor spaces, by simply attaching a TiO2-Ag-NP adhesive film on the wall.

Interleukin-6: A New Marker of Advanced-Sarcopenic HCC Cirrhotic Patients.

Hepatocellular carcinoma (HCC) is the major cause of liver-related death worldwide. Interleukin 6 (IL-6) promotes the growth of the HCC microenvironment. The correlation between Child-Pugh (CP) and HCC stage and between HCC stage and sarcopenia is still not clear. Our aim was to investigate whether IL-6 is correlated with HCC stage and could represent a diagnostic marker for sarcopenia. Ninety-three HCC cirrhotic patients in different stages, according to BCLC-2022 (stages A, B, and C), were enrolled. Anthropometric and biochemical parameters, comprehensive of IL-6, were collected. The skeletal muscle index (SMI) was measured using dedicated software on computer tomography (CT) images. IL-6 level was higher in advanced (BCLC C) compared to the early-intermediate (BCLC A-B) stages (21.4 vs. 7.7 pg/mL, p < 0.005). On multivariate analysis, IL-6 levels were statistically dependent on the degree of liver disease severity (CP score) and HCC stages (p = 0.001 and p = 0.044, respectively). Sarcopenic patients presented lower BMI (24.7 ± 5.3 vs. 28.5 ± 7.0), higher PMN/lymphocyte ratio (2.9 ± 2.4 vs. 2.3 ± 1.2) and increased values of log (IL-6) (1.3 ± 0.6 vs. 1.1 ± 0.3). Univariate logistic regression between sarcopenia and log (IL-6) showed a significant odds ratio (OR 14.88, p = 0.044) with an AUC of 0.72. IL-6 appears to be an effective biomarker for the diagnosis of advanced cirrhotic HCC. In addition, IL-6 could be considered a marker of cirrhotic HCC-related sarcopenia, suggesting further investigation with BIA- or CT-dedicated software.

Immune checkpoint blockade therapy mitigates systemic inflammation and affects cellular FLIP-expressing monocytic myeloid-derived suppressor cells in non-progressor non-small cell lung cancer patients.

Cancer cells favor the generation of myeloid cells with immunosuppressive and inflammatory features, including myeloid-derived suppressor cells (MDSCs), which support tumor progression. The anti-apoptotic molecule, cellular FLICE (FADD-like interleukin-1ß-converting enzyme)-inhibitory protein (c-FLIP), which acts as an important modulator of caspase-8, is required for the development and function of monocytic (M)-MDSCs. Here, we assessed the effect of immune checkpoint inhibitor (ICI) therapy on systemic immunological landscape, including FLIP-expressing MDSCs, in non-small cell lung cancer (NSCLC) patients. Longitudinal changes in peripheral immunological parameters were correlated with patients' outcome. In detail, 34 NSCLC patients were enrolled and classified as progressors (P) or non-progressors (NP), according to the RECIST evaluation. We demonstrated a reduction in pro-inflammatory cytokines such as IL-8, IL-6, and IL-1ß in only NP patients after ICI treatment. Moreover, using t-distributed stochastic neighbor embedding (t-SNE) and cluster analysis, we characterized in NP patients a significant increase in the amount of lymphocytes and a slight contraction of myeloid cells such as neutrophils and monocytes. Despite this moderate ICI-associated alteration in myeloid cells, we identified a distinctive reduction of c-FLIP expression in M-MDSCs from NP patients concurrently with the first clinical evaluation (T1), even though NP and P patients showed the same level of expression at baseline (T0). In agreement with the c-FLIP expression, monocytes isolated from both P and NP patients displayed similar immunosuppressive functions at T0; however, this pro-tumor activity was negatively influenced at T1 in the NP patient cohort exclusively. Hence, ICI therapy can mitigate systemic inflammation and impair MDSC-dependent immunosuppression.

Long-term effects of amino-bisphosphonates on circulating γδ T cells.

The aim of this study was to explore whether desensitization to the occurrence of the acute-phase response (APR) in patients previously treated with amino-bisphosphonates (N-BPs) is due to a long-lasting reduction in the number of circulating γδ T cells. Circulating lymphocyte subpopulation counts were obtained from 63 patients with postmenopausal or senile osteoporosis at baseline and after 2 days and 12 months of the first intravenous (IV) 5 mg zoledronic acid (ZOL) infusion. At baseline both the proportion and absolute number of circulating γδ T cells were significantly higher in patients who had never used N-BPs vs. previous users, either oral or IV. A typical APR was observed in none of the patients given IV ZOL a year earlier, in 6 (22 %) of the patients previously treated with oral N-BPs, and in 13 (57 %) of the patients naive to any N-BP treatment. In patients naive to N-BPs, a significant reduction in both total lymphocytes and their subsets was observed 2 days after ZOL infusion; all these changes returned to baseline values 1 year later with the exception of γδ T cells, which remained significantly lower in terms of both proportion and absolute number. These results indicate for the first time that both IV and oral N-BP treatments are associated with a long-lasting decrease in circulating γδ T cells, and this may explain the lower incidence of APR in patients previously exposed to N-BPs. Other clinical implications of this sustained effect of N-BPs on immune-regulatory cells might be important.

First Report on the Presence of Toxic Metals and Metalloids in East Asian Bullfrog (Hoplobatrachus rugulosus) Legs.

We examined the presence of As, Cr, Cd, and Pb in 42 samples of farmed East Asian bullfrog (Hoplobatrachus rugulosus) from Vietnam and Thailand by inductively coupled plasma-mass spectrometry (ICP-MS). An estimation of the dietary intake and exposure to the toxic elements analysed was also carried out. The results showed very high As levels, with mean values of 0.094 ± 0.085 mg/Kg w.w. and a maximum of 0.22 mg/Kg. No significant differences were found for As contents between areas of production (p > 0.05). No detectable Cd contents were found in all the samples examined. The Pb concentrations of the East Asian bullfrog legs samples were below the European Commission's permitted levels. The Cr and Pb contents of the East Asian bullfrog produced in Vietnam were significantly higher than that produced in Thailand (p < 0.05). The target hazard quotient (THQ) ratio for Cr was not exceeded for all the samples analysed. In contrast, the benchmark dose lower confidence limit (BMDL) and THQ ratios for As were exceeded, indicating carcinogenic and non-carcinogenic risks for those who consume this type of food. The results of this work confirm the role of As-contaminated water absorption as an important source of arsenic for these adult organisms.