Proteomics and Integrated Techniques to Characterize Organic Residues in Funerary Findings from Italic Populations of the First Millennium BC.

Multiple analytical techniques were combined to achieve a detailed characterization of organic residues in different typologies of funerary pottery, which were found at two separate archeological sites in the Campania Region (Italy) and both dated back to the first millennium BC. Gas chromatography-mass spectrometry (GC-MS) analysis of lipids provided inconclusive results. The attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra of encrustation on two glazed bowls of the 3rd to 4th century BC were comparable to those of fresh bone, revealing the presence of hydroxyapatite and proteins, which were identified as bovine collagen chains by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS)-based proteomics. This finding confirmed that Italic populations used to inhume the dead along with votive meat offerings. Proteomics was decisive for identifying bovine milk in an unusually shaped amphora unearthed from a grave that belonged to a woman at the necropolis of the Greek colony in Cuma (7th century BC). Peptidomic analysis demonstrated that the genetic variant A1 of ß-casein was already present in the southern Mediterranean area at least 2500 years ago. Overall, these results depict an agropastoral system of Italic populations at the age of Magna Graecia based on a significant role of domesticated cattle.

Overcoming Nanoparticle-Mediated Complement Activation by Surface PEG Pairing.

Many PEGylated nanoparticles activate the complement system, which is an integral component of innate immunity. This is of concern as uncontrolled complement activation is potentially detrimental and contributes to disease pathogenesis. Here, it is demonstrated that, in contrast to carboxyPEG2000-stabilized poly(lactic-co-glycolic acid) nanoparticles, surface camouflaging with appropriate combinations and proportions of carboxyPEG2000 and methoxyPEG550 can largely suppress nanoparticle-mediated complement activation through the lectin pathway. This is attributed to the ability of the short, rigid methoxyPEG550 chains to laterally compress carboxyPEG2000 molecules to become more stretched and assume an extended, random coil configuration. As supported by coarse-grained molecular dynamics simulations, these conformational attributes minimize statistical protein binding/intercalation, thereby affecting sequential dynamic processes in complement convertase assembly. Furthermore, PEG pairing has no additional effect on nanoparticle longevity in the blood and macrophage uptake. PEG pairing significantly overcomes nanoparticle-mediated complement activation without the need for surface functionalization with complement inhibitors.

Antiproliferative effects of chalcones on T cell acute lymphoblastic leukemia-derived cells: Role of PKCß.

In this study, a series of 20 chalcone derivatives was synthesized, and their antiproliferative activity was tested against the human T cell acute lymphoblastic leukemia-derived cell line, CCRF-CEM. On the basis of the structural features of the most active compounds, a new library of chalcone derivatives, according to the structure-activity relationship design, was synthesized, and their antiproliferative activity was tested against the same cancer cell line. Furthermore, four of these derivatives (compounds 3, 4, 8, 28), based on lower IC50 values (between 6.1 and 8.9 µM), were selected for further investigation regarding the modulation of the protein expression of RACK1 (receptor for activated C kinase), protein kinase C (PKC)α and PKCß, and their action on the cell cycle level. The cell cycle analysis indicated a block in the G0/G1 phase for all four compounds, with a statistically significant decrease in the percentage of cells in the S phase, with no indication of apoptosis (sub-G0/G1 phase). Compounds 4 and 8 showed a statistically significant reduction in the expression of PKCα and an increase in PKCß, which together with the demonstration of an antiproliferative role of PKCß, as assessed by treating cells with a selective PKCß activator, indicated that the observed antiproliferative effect is likely to be mediated through PKCß induction.

Automated Cerebrospinal Fluid Cell Counts Using the New Body Fluid Mode of Sysmex UF-1000i.

BACKGROUND: We evaluated the new body fluid module on Sysmex UF1000-i (UF1000i-BF) for analysis of white blood cell (WBC) and red blood cell (RBC) in cerebrospinal fluid (CSF). METHODS: WBC and RBC counting were compared between UF1000i-BF and Fuchs-Rosenthal counting chamber in 67 CSF samples. This study also included the evaluation of between-day precision, limit of blank (LoB), limit of detection (LoD), functional sensitivity (limit of quantitation, LoQ), carryover and linearity. Diagnostic agreement for differentiation between normal and increased WBC counts (≥5.0 × 10(6) /L) was also assessed. RESULTS: The agreement between UF1000i-BF and manual WBC counts was otpiaml in all CSF samples (r = 0.99; y = 1.05x + 0.09). A modest overestimation was noticed in samples with WBC < 30 × 10(6) /L (r = 0.95; y = 1.21x - 0.15). A good agreement was observed for RBC counts (r = 0.98; y = 1.15x + 0.55), particularly in samples with RBC ≥ 18 × 10(6) /L (r = 0.98; y = 1.01x + 8.90). Between-day precision was good, with coefficient of variations (CVs) lower than 7.2% for both WBC and RBC. The LoBs were 0.1 × 10(6) WBC/L and 1.2 × 10(6) RBC/L, the LoDs were 0.7 × 10(6) WBC/L and 5.5 × 10(6) RBC/L, the LoQs were 2.4 × 10(6) WBC/L and 18.0 × 10(6) RBC/L, respectively. Linearity was excellent (r = 1.00 for both WBC and RBC). Carryover was negligible. Excellent diagnostic agreement was obtained at 4.5 × 10(6) WBC/L cut-off (sensitivity, 100%; specificity, 97.4%). CONCLUSION: The UF1000i-BF provides rapid and accurate WBC and RBC counts in clinically relevant values of CSF cells. The use of UF1000i-BF may hence allow to replace routine optical counting, except for samples displaying abnormal WBC counts or abnormal scattergram distribution, for which differential cell counts may still be required.

Infection control strategy and primary care assistance in Campania region during the national lockdown due to COVID-19 outbreak: the experience of two tertiary emergency centers.

BACKGROUND: COVID-19 pandemic has markedly affected emergency care, due to sudden limitation of health care capacity by general practitioners (GP) and urgent need for infection control strategies. We evaluated the activity of the Emergency Department (ED) during the national lockdown (March 8-April 30), as well as the outcomes of our infection control strategy. RESULTS: Despite a reduction in access by one fifth, a proportion of febrile patients comparable to 2019 was seen (829/2492, 33.3% vs 4580/13.342, 34.3%, p = 0.3). Diagnostic swab for COVID-19 was performed in 25% of patients, especially in subjects with co-morbidities or multiple access. Six infected cases were identified, all presenting with febrile disease. Only two positive patients fulfilled the criteria for diagnostic swab provided by the Italian Health Authorities, because of close contact with suspected or confirmed cases. The rate of admission for febrile or respiratory conditions was higher than the same period of 2019 (33.4% vs 25.9%, p < 0.0001). None of the 105 health-care professionals working during the study time lapse exhibited anti-SARS-CoV-2 seroconversion. Among the 589 patients with information available, 54.9% declared no medical consultation at all prior to coming to ED, while only 40 (of which 27 with fever) had been examined by their GP before coming to ED. Nevertheless, 35.6% of the cases were already taking medications. None of the 9 patients requiring intensive care reported recent pediatric consultation, despite symptoms duration up to 30 days. CONCLUSION: Our results provide evidence that the reduced capacity of primary care facilities during the national lockdown may have caused a high rate of self-medication as well as a delayed provision of care in some patients. Identification of pediatric patients affected with SARS-CoV-2 infection remains a challenge because of the absence of reliable predictive factors. Finally, the use of specific triage centers, with dedicated pathways to diagnose SARS-CoV-2 infection, trace contacts and allow adequate care after swabs, is effective in preventing spreading of the infection.

Complete Blood Count as point of care testing QBC STAR™: Preliminary evaluation.

INTRODUCTION: Point of care testing (POCT) represents a valuable option when laboratory data shall be urgently available for timely clinical management, with a turnaround time (TAT) that is unfeasible using conventional laboratory instrumentation. This study was aimed to compare the performance of QBC STAR™ compared to Sysmex XN-module and the reference optical microscopy (OM) assessment. MATERIAL AND METHODS: One hundred peripheral blood samples, collected in K3 EDTA tubes, and 50 capillary blood samples obtained by finger stick were analyzed with QBC STAR™, Sysmex XN-module, and OM. Data were compared with Passing-Bablok regression and Bland-Altman plots. RESULTS: The Passing-Bablok regression analysis (QBC STAR™ capillary sample vs XN-module) yielded slopes comprised between 0.30 and 1.37, while the intercepts ranged between -17.57 and 232.6. Bland-Altman plots yielded relative bias comprised between -4.87% (for MN QBC STAR™ capillary sample vs XN-module) and 27% (PLT QBC STAR™ capillary sample vs XN-module). A significant bias was found for all parameters except MN and WBC, RBC in all and pediatric samples, and HB in adults samples. CONCLUSION: The results of this analytical evaluation suggest that QBC STAR™ may not be the ideal tool for performing complete blood count analysis for diagnostic purposes, while it could be more useful in urgent/emergent conditions, such as for rapid monitoring of some hematological parameters (eg, WBC and HB).

Standardization and harmonization in hematology: Instrument alignment, quality control materials, and commutability issue.

INTRODUCTION: In the hub and spoke laboratory network, the number of hematology analyzers (HAs) within each core center has increased, and the control of HAs alignment is becoming necessary requirement to ensure analytical quality. In this scenario, HA alignment can be assessed by analyzing the same control material used for internal quality control on multiple HAs, assuming its commutability. The aim of the study was to verify the applicability of a protocol for the alignment of HAs based on control material rather than on fresh whole-blood samples. METHODS: The alignment of five HAs was evaluated for red (RBC, Hb, MCV, RET), white (WBC, NE, LY, MO, EO, BA, IG), and platelet (PLT) series parameters, following a protocol by SIBioC, using human sample (HS) and quality control material (QC), after the verification of commutability, according to the IFCC protocol. Maximum bias was derived from biological variation data. RESULTS: A complete alignment between instruments was confirmed for the majority of the parameters investigated both for HS and QC material. Partial misalignments or inconcludent results were instead evident for MCV, MO, EO, BA, and IG. Interestingly, QC material was found to be not commutable for LY, MO, and BA. CONCLUSION: The alignment of hematologic analyzers for main cell population parameters may be verified with both QC and HS, displaying consistent results and interpretation. The evaluation for some white series parameters (EO, BA, and IG) is critical, and particular attention must be paid to the values of the material used for the alignment.

Clinical significance of cell population data (CPD) on Sysmex XN-9000 in septic patients with our without liver impairment.

BACKGROUND: This study evaluated the clinical significance of cell population data (CPD) parameters obtained on Sysmex XN-9000 in septic patients admitted to intensive care unit (ICU) and stratified according to liver function. METHODS: The study population consisted in 84 patients, 44 of whom did not develop sepsis (NS), whereas the remaining 40 developed sepsis (SE) (n=24) or septic shock (SS) (n=16). Two hundred ostensibly healthy blood donors [healthy subjects (HS)], undergoing routine blood testing before a regular blood donation, were studied. RESULTS: Except for neutrophils and lymphocytes cell size (NE-FCS and LY-Z), all other CPD values were significantly different in ICU patients compared to HS. Neutrophils and monocytes fluorescence intensity (NE-SFL and MO-X) values were significantly higher in SS compared to sepsis and not develop sepsis patients. The value of many parameters was also different according to liver function. Overall, MO-X and neutrophils fluorescence intensity (NE-SFL) exhibited the best performance for diagnosing sepsis in all patients (AUC, 0.75 and 0.72), as well as in those with (AUC, 0.95 and 0.89) or without (AUC, 0.72 for both) liver impairment. These parameters were also significantly correlated with Sequential Organ Failure Assessment (SOFA) score. CONCLUSIONS: This study suggested that some novel CPD parameters (namely NE-SFL and MO-X) may provide useful information for diagnosis and management of sepsis.

Reflex Testing Rules for Cell Count and Differentiation of Nucleated Elements in Pleural and Ascitic Fluids on Sysmex XE-5000.

Flow cytometry is widely used in many laboratories for automated nucleated cell counts and their differentiation in body fluids. The implementation of new reflex testing rules on these automated instruments could open new frontiers in laboratory workflow, improving characterization of body fluids and clinical diagnosis and decreasing costs. Ascitic (150) and pleural (33) fluids were collected and assessed by XE-5000 and optical microscopy. Cell counts performed with the methods showed a Pearson's correlation of 0.98 (p < 0.0001), Passing-Bablok regression y = 0.99x + 2.44, and bias of 32.3. In ascitic fluids, the best diagnostic performance was found for polymorphonuclear and neutrophil counts on XE-5000, which exhibited areas under the curve (AUCs) 0.98 (p < 0.0001) and 0.99 (p < 0.0001), respectively. In pleural fluids the best diagnostic performance was found for polymorphonuclear percent parameter, which displayed 0.97 (p < 0.0001). Specific reflex test rules based on these parameters were characterized by 92% diagnostic concordance, 1.00 sensitivity, and 0.84 specificity with optical microscopy. The application of a set of reflex testing rules may improve the diagnostic performance of XE-5000, increasing its reliability for routine automated cell count in body fluids. We acknowledge that further studies should be planned to validate our findings according to clinical data.

Extended leukocyte differential count and C-reactive protein in septic patients with liver impairment: diagnostic approach to evaluate sepsis in intensive care unit.

BACKGROUND: Sepsis is still a major cause of death in intensive care units (ICUs) worldwide. Patients with liver impairment express an imbalanced cytokine response which alters common sepsis biphasic nature. Cytokines measurement is expensive, often unavailable, whereas leukocytes (WBC) evaluation performed through hematology analyzers can provide a practical strategy for monitoring inflammatory response. METHODS: A total of 200 healthy subjects (HS) and 84 patients (18 with, 66 without liver impairment) admitted to ICU, were assessed for International Sepsis Definitions, Sequential Organ Failure Assessment (SOFA) and Model for End-Stage Liver Disease (MELD) scores. We tested 1,022 peripheral blood samples using Sysmex XN-9000, estimating diagnostic accuracy of leukocyte differential count and nontraditional parameters through receiver operating characteristics (ROC) curves analysis compared to clinical classification. RESULTS: Median value of all-leukocyte parameters was different in ICU patients compared to HS. Leukocytes, neutrophils (NE) and immature granulocytes (IGs) in sepsis and septic shock (SS) were higher than no sepsis (NS), with an area under the curve: 0.81, 0.82 and 0.78 respectively. Lymphocytes (LY) and monocytes (MO) were significantly associated with liver impairment. CONCLUSIONS: Diagnostic accuracy of all-leukocyte parameters may provide valuable information for diagnosis and follow-up of sepsis in ICU patients, especially those with liver impairment.

Niosomes from 80s to present: the state of the art.

Efficient and safe drug delivery has always been a challenge in medicine. The use of nanotechnology, such as the development of nanocarriers for drug delivery, has received great attention owing to the potential that nanocarriers can theoretically act as "magic bullets" and selectively target affected organs and cells while sparing normal tissues. During the last decades the formulation of surfactant vesicles, as a tool to improve drug delivery, brought an ever increasing interest among the scientists working in the area of drug delivery systems. Niosomes are self assembled vesicular nanocarriers obtained by hydration of synthetic surfactants and appropriate amounts of cholesterol or other amphiphilic molecules. Just like liposomes, niosomes can be unilamellar or multilamellar, are suitable as carriers of both hydrophilic and lipophilic drugs and are able to deliver drugs to the target site. Furthermore, niosomal vesicles, that are usually non-toxic, require less production costs and are stable over a longer period of time in different conditions, so overcoming some drawbacks of liposomes. The niosome properties are specifically dictated by size, shape, and surface chemistry which are able to modify the drug's intrinsic pharmacokinetics and eventual drug targeting to the areas of pathology. This up-to-date review deals with composition, preparation, characterization/evaluation, advantages, disadvantages and application of niosomes.

Polysorbate 20 vesicles as oral delivery system: in vitro characterization.

Non-phospholipid vesicles made with non-ionic surfactants represent a promising alternative to the more widely studied liposomes. The main aim of the present work is to evaluate if vesicles of polysorbate 20 may be used as delivery systems for oral administration of drugs. Then in vitro stability and mucoadhesion studies in simulated gastrointestinal fluids were carried out. The colloidal stability of the surfactant vesicles was determined by size and fluorescence-dequenching assay, while their mucoadhesive properties were evaluated by light-scattering and protein assay. The results of in vitro stability demonstrated that the pHs and enzymes (pepsin and/or pancreatin) of the gastrointestinal fluids had not influence on surfactant vesicle stability. However, in presence of bile salts the nanosize vesicles showed a release of fluorescent marker (about 11% at 2 h and 28% at 4 h), whereas they were stable in size as confirmed by the light scattering experiments. Finally, the in vitro mucoadhesive experiments showed that the capacity of nanovesicles to adsorb mucin was higher at neutral pH than at acidic pH. As a conclusion of these preliminary studies, the surfactant vesicles could be considered a versatile tool for the oral delivery of drugs with poor stability in gastrointestinal tract and low permeability. Nevertheless, further work is required in order to examine the interaction with and/or the transport route through the epithelial cells of the gastrointestinal wall.

Niosomes encapsulating Ibuprofen-cyclodextrin complexes: preparation and characterization.

A new delivery system based on ibuprofen-ß-cyclodextrin (ßCd) complexation and its loading into non-ionic surfactant vesicles (NSVs) was developed to improve ibuprofen therapeutic efficacy in topical formulations. The proposed strategy exploits the well known solubilizing and stabilizing properties of cyclodextrins together with the high tolerability and percutaneous absorption enhancing properties of NSVs. The complexing capacity of Cds in the presence of Ibuprofen in aqueous solution was evaluated by means of phase solubility studies. The technique used to obtain solid ibuprofen-ßCd complexes was the co-lyophilization method. The influence of the preparation method on the physicochemical properties of the final product was evaluated by means of Fourier Transform Infrared Spectroscopy and Differential scanning calorimetry studies. Ibuprofen-ßCd complexes were included in Tween 20/Cholesterol vesicles and characterized in terms of size, zeta (ζ)-potential, stability, drug entrapment efficiency and drug release. The best ibuprofen-ßCd-NSV system exhibited in vitro drug permeation properties significantly improved with respect to those of the plain drug suspension.

Starch-to-sugar conversion in wood parenchyma of field-growing Laurus nobilis plants: a component of the signal pathway for embolism repair?

The ability of stems of Laurus nobilis (L.) to refill embolised xylem conduits was studied in plants both at optimal water supply (W) and under conditions of soil drought inducing xylem pressures (Px) of -1.54 (S1) and -2.35 MPa (S2). Starch depolymerisation in wood parenchyma was measured as percentage of cells 'with high starch content' (HSC-cells) counted under a microscope. HSC-cells decreased during embolism and increased again in refilled stems. A direct relationship was found between percentage of HSC-cells and Px, with HSC-cells between 65 and 75% of the total at Px ≥ -0.6 MPa, at which recovery from PLC was recorded. At low transpiration, starch re-appeared in wood parenchyma cells but only in plants that showed diurnal stomatal opening (W- and S1-plants). In S2-plants showing diurnal stomatal closure and nocturnal opening with Px between -1.2 to -2.4 MPa, HSC-cells were only 25% and plants did not recover from PLC. This finding suggests that (i) the Px threshold for embolism repair was ≥ -0.6 MPa, and (ii) impeded phloem loading limits starch content in wood parenchyma and embolism repair. We conclude that starch depolymerisation acts as a signal to phloem unloading sugars to embolised conduits thus generating the necessary osmotic gradients driving refilling.