Chemico-Physical Properties of Some 1,1'-Bis-alkyl-2,2'-hexane-1,6-diyl-bispyridinium Chlorides Hydrogenated and Partially Fluorinated for Gene Delivery.

The development of very efficient and safe non-viral vectors, constituted mainly by cationic lipids bearing multiple charges, is a landmark for in vivo gene-based medicine. To understand the effect of the hydrophobic chain's length, we here report the synthesis, and the chemico-physical and biological characterization, of a new term of the homologous series of hydrogenated gemini bispyridinium surfactants, the 1,1'-bis-dodecyl-2,2'-hexane-1,6-diyl-bispyridinium chloride (GP12_6). Moreover, we have collected and compared the thermodynamic micellization parameters (cmc, changes in enthalpy, free energy, and entropy of micellization) obtained by isothermal titration calorimetry (ITC) experiments for hydrogenated surfactants GP12_6 and GP16_6, and for the partially fluorinated ones, FGPn (where n is the spacer length). The data obtained for GP12_6 by EMSA, MTT, transient transfection assays, and AFM imaging show that in this class of compounds, the gene delivery ability strictly depends on the spacer length but barely on the hydrophobic tail length. CD spectra have been shown to be a useful tool to verify the formation of lipoplexes due to the presence of a "tail" in the 288-320 nm region attributed to a chiroptical feature named ψ-phase. Ellipsometric measurements suggest that FGP6 and FGP8 (showing a very interesting gene delivery activity, when formulated with DOPE) act in a very similar way, and dissimilar from FGP4, exactly as in the case of transfection, and confirm the hypothesis suggested by previously obtained thermodynamic data about the requirement of a proper length of the spacer to allow the molecule to form a sort of molecular tong able to intercalate DNA.

Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers.

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.

ITC study on the interaction of some bile salts with tragacanth, Arabic, and guar gums with potential cholesterol-lowering ability.

Introduction: The urge of designing new safe and natural functional foods to control blood lipids and dispensable without the need of physician supervision, has increased especially after the coming into effect of the recent EU Commission regulation 2022/860, that regulates the consumption of "red yeast rice," made by fermentation of rice with Monascus purpureus, and perceived as a natural functional food, due to a health risk for frail consumers. The results of the present work are a part of the systematic study we are carrying out of the binding ability of some soluble dietary fibers (SDF) from different natural sources toward selected bile salts (BS). Methods: Measurements were carried out by isothermal titration calorimetry (ITC) with the idea to shed light on the mechanism, if any, by which they show cholesterol-lowering activity. Results and discussion: Epidemiological studies are sometimes conflicting and offer only hypothesis about the mechanism of action, the most accredited being the reduction of reabsorption of BS in the gut. Previous measurements done on negatively charged pectin and alginate, showed specific binding interaction with monomer NaDC for pectin and no interaction at all for alginate. Chitosan, positively charged and soluble only at low pH, in 100 mM acetate buffer at pH = 3 shows strong exothermic interactions with NaTC and NaTDC. Here we considered two plant exudates (Arabic gum and tragacanth gum) and guar gum, extracted from guar beans, and their interaction with the same bile salts. ITC measurements do not evidence specific interactions between gums and the studied BS, so that their cholesterol lowering ability, if any, is due to a different mechanism very probably bound to the viscosity increase. Moreover, the addition of NaC, the most abundant BS in the bile, at very low concentration (under the cmc) causes a structural change of the solution. The obtained results seem to corroborate the hypothesis that the cholesterol lowering activity is related to the increase in viscosity of guar solution favored by NaC, the major component of the bile.

Effects of online genetics education on physician assistant interviewing skills.

OBJECTIVES: This study was designed to evaluate the impact of an Internet-based educational program on clinically relevant communication behaviors. METHODS: A randomized trial enrolled 50 physician assistants (PAs); 40 PAs completed the trial (18 in the intervention group and 22 in the control group). Participants conducted simulated visits that were coded for completeness of family history, referral for genetic services, and overall interviewing style using the Roter Interaction Analysis System (RIAS). RESULTS: Compared with PAs in the control group, PAs who took the Web course elicited more complete family histories (7.2 vs 5.0 of a 13-member pedigree; t = 2.3, P < .05) and more detailed cancer histories (3.2 vs 2.3 of five affected family members; t = 2.7, P < .001) but did not make more genetics referrals. Overall, intervention-group PAs used a less patient-centered interviewing style than controls (1.9 vs 3.2; t = 2.8, P < .01). CONCLUSIONS: More comprehensive assessment of family history was associated with lower patient-centeredness, suggesting a need for caution with regard to unintended consequences of targeted communication interventions on interviewing style.

On the mechanism of the cholesterol lowering ability of soluble dietary fibers: Interaction of some bile salts with pectin, alginate, and chitosan studied by isothermal titration calorimetry.

Reducing high blood cholesterol is an important strategy to decrease the chances of a cardiovascular disease occurrence, the main cause of mortality in western developed countries. Therefore, the search for an alternative therapeutic or preventive approach being natural, biocompatible, and not toxic is still more relevant than ever. This need is particularly felt in Pediatrics for treating childhood hypercholesterolemia, due to statins interference in the production of steroid hormones in prepuberal children. Notwithstanding the general acceptance of the healthy role of the fibers in the diet, the mechanism underlying the cholesterol-lowering ability of soluble fibers is still under discussion. Therefore, we started a systematic study of the binding ability of some soluble dietary fibers (SDF) originated from different natural sources toward selected bile salts (BS) by isothermal titration calorimetry (ITC). Here we report the results of our ITC studies on the interaction of alginate, pectin and chitosan with sodium cholate (NaC), sodium deoxycholate (NaDC), sodium taurocholate (NaTC) and sodium taurodeoxycholate (NaTDC). Thermodynamic data on the micelle formation process of the above bile salts, as a premise to the study of their binding ability to the SDF, are also reported. Alginate does not show specific binding interaction with BS, while pectin shows a strong exothermic bond with NaDC in monomeric form. Chitosan, positively charged and soluble only at low pH, shows strong exothermic interactions with NaTC and NaTDC (soluble at pH = 3 in acetate buffer) with precipitate formation. For NaTC, the exothermic peak starts at about 5 mM. At this concentration NaTC bound on the fiber reaches locally the cmc value and micelles start forming on the fiber inducing its conformational change. For NaTDC the same process occurs at much lower concentrations, due to lower cmc, and with a greater quantity of heat involved. The first set of results here presented shows that for some SDF the binding of BS could be an important mechanism in cholesterol lowering but not the only one. The information here presented could be a starting point for the design of optimized functional foods with high cholesterol lowering ability.

The psychophysical impact of COVID-19 lockdown on pediatric patients with rheumatological disease.

BACKGROUND: COVID-19 was declared a pandemic on 11 March 2020 and unprecedented containment measures were taken to limit its spreading. These exceptional measures may have an impact on the mental health of the population. OBJECTIVES: We hypothesize that children with the rheumatological chronic disease might be a population at higher risk of psychological distress. DESIGN: This study evaluated the psychological effect of social distancing in pediatric rheumatological patients and its possible correlation with disease relapse. SETTING: Pediatric patients diagnosed with juvenile idiopathic arthritis (JIA) were included in the study. SUBJECTS AND METHODS: They completed the Multidimensional Anxiety Scale for Children (MASC-2) to assess anxious symptoms, the Children Depression Inventory Self Report (CDI-2 SR) for depression symptoms and a semi-structured questionnaire to collect their activities. Their parents completed the Children Depression Inventory 2 Parent (CDI-2 P). RESULTS: The MASC-2 test showed high values in anxiety factors. The CDI-2 SR test showed high values for Ineffectiveness and Functional Problems. The CDI-2 P showed high values for Emotional Problems factor. All the patients with high level of depression were females. The result is that those who take therapy were younger, have reduced values in Functional Problems and in Social Anxiety. 47% of the sample had disease relapse. CONCLUSIONS: During the COVID-19 period of isolation, in patients with JIA, there was an increase in anxious and depressive symptoms in female adolescents, a tendency to disease relapse, despite generally good compliance to therapy. Those under treatment were younger and showed reduced values in Functional Problems, Social Anxiety, and Humiliation/Rejection. LIMITATIONS: Limited number of patients to whom questionnaires could be distributed.

MicroRNA profiling of paediatric AML with FLT-ITD or MLL-rearrangements: Expression signatures and in vitro modulation of miR-221-3p and miR-222-3p with BRD4/HATs inhibitors.

Novel therapeutic strategies are needed for paediatric patients affected by Acute Myeloid Leukaemia (AML), particularly for those at high-risk for relapse. MicroRNAs (miRs) have been extensively studied as biomarkers in cancer and haematological disorders, and their expression has been correlated to the presence of recurrent molecular abnormalities, expression of oncogenes, as well as to prognosis/clinical outcome. In the present study, expression signatures of different miRs related both to presence of myeloid/lymphoid or mixed-lineage leukaemia 1 and Fms like tyrosine kinase 3 internal tandem duplications rearrangements and to the clinical outcome of paediatric patients with AML were identified. Notably, miR-221-3p and miR-222-3p resulted as a possible relapse-risk related miR. Thus, miR-221-3p and miR-222-3p expression modulation was investigated by using a Bromodomain­containing protein 4 (BRD4) inhibitor (JQ1) and a natural compound that acts as histone acetyl transferase inhibitor (curcumin), alone or in association, in order to decrease acetylation of histone tails and potentiate the effect of BRD4 inhibition. JQ1 modulates miR-221-3p and miR-222-3p expression in AML with a synergic effect when associated with curcumin. Moreover, changes were observed in the expression of CDKN1B, a known target of miR-221-3p and miR-222-3p, increase in apoptosis and downregulation of miR-221-3p and miR-222-3p expression in CD34+ AML primary cells. Altogether, these findings suggested that several miRs expression signatures at diagnosis may be used for risk stratification and as relapse prediction biomarkers in paediatric AML outlining that epigenetic drugs, could represent a novel therapeutic strategy for high-risk paediatric patients with AML. For these epigenetic drugs, additional research for enhancing activity, bioavailability and safety is needed.

A video-intervention to improve clinician attitudes toward patients with sickle cell disease: the results of a randomized experiment.

BACKGROUND: Clinician attitudes toward patients are associated with variability in the quality of health care. Attitudes are typically considered difficult to change, and few interventions have attempted to do so. Negative attitudes toward adults with sickle cell disease have been identified as an important barrier to the receipt of appropriate pain management for this patient population. OBJECTIVE: To test the effect of a video-intervention designed to improve clinician attitudes toward adults with sickle cell disease. INTERVENTIONS: An 8-minute video depicting a clinician expert and patients discussing challenges in seeking treatment for sickle cell pain. DESIGN AND PARTICIPANTS: A randomized post-test only control group design was used to assess the impact of the intervention on the attitudes of 276 nurses and housestaff working at a large, urban, academic medical center. MAIN MEASURES: Attitudes toward adult sickle cell patients assessed using 5- and 6-point Likert-scale items. Exploratory factor analysis was used to identify underlying attitudinal domains and develop scales. Examples of the negative and positive attitudes assessed include clinician estimates of the percentage of SCD patients that exaggerate pain (negative) or make clinicians glad they went into medicine (positive). KEY RESULTS: Compared to the control group, the intervention group exhibited decreased negative attitudes (Difference in means = -8.9, 95%CI [-14.2, -3.6]; Cohen's d = 0.41), decreased endorsement of certain patient behaviors as "concern-raising" (Difference in means = -7.8, 95%CI [-13.1, -2.5]; Cohen's d = 0.36), and increased positive attitudes toward sickle cell patients (Difference in means = 6.6, 95% CI [0.6, 12.6]; Cohen's d = 0.27). CONCLUSIONS: Our results suggest that the attitudes of clinicians toward sickle cell patients may be improved through a short and relatively easy to implement intervention. Whether the attitudinal differences associated with our intervention are sustainable or are linked to clinical outcomes remains to be seen.

How does decision complexity affect shared decision making? An analysis of patient-provider antiretroviral initiation dialogue.

OBJECTIVES: This study analyzed patient-provider dialogue regarding anti-retroviral therapy (ART) initiation, assessing the degree to which shared decision making (SDM) occurred. METHODS: We analyzed 24 audio-recorded dialogues between 14 HIV providers and their patients regarding ART initiation. We coded transcribed dialogues for seven SDM elements. We stratified dialogues into three levels of decision complexity (basic, intermediate, complex) based on patient CD4 counts and evaluated SDM criteria fulfillment at each level of decision complexity. RESULTS: There were five basic, twelve intermediate, and seven complex decisions in our sample. While only two met the defined criteria for SDM, the mean number of SDM elements present increased with each level of decision complexity. Discussion of the clinical issue requiring the decision occurred most frequently (88%), while discussion of pros/cons (13%), patient's understanding (21%), and decision alternatives (29%) occurred least frequently. CONCLUSION/PRACTICE IMPLICATIONS: While few dialogues met the defined SDM criteria, providers are having conversations that respond to decision complexity. Clinicians should be aware that discussion of pros/cons, alternatives, and uncertainties are frequently skipped, even when these elements are clearly relevant, as in complex decisions. In addition, rhetorical questions to assess patient preferences and understanding are insufficient to fully engage patients in SDM.

Motivational interviewing by HIV care providers is associated with patient intentions to reduce unsafe sexual behavior.

OBJECTIVE: Motivational interviewing (MI) can promote behavior change, but HIV care providers rarely have training in MI. Little is known about the use of MI-consistent behavior among untrained providers. This study examines the prevalence of such behaviors and their association with patient intentions to reduce high-risk sexual behavior. METHODS: Audio-recorded visits between HIV-infected patients and their healthcare providers were searched for counseling dialog regarding sexual behavior. The association of providers' MI-consistence with patients' statements about behavior change was assessed. RESULTS: Of 417 total encounters, 27 met inclusion criteria. The odds of patient commitment to change were higher when providers used more reflections (p=0.017), used more MI consistent utterances (p=0.044), demonstrated more empathy (p=0.049), and spent more time discussing sexual behavior (p=0.023). Patients gave more statements in favor of change (change talk) when providers used more reflections (p<0.001) and more empathy (p<0.001), even after adjusting for length of relevant dialog. CONCLUSION: Untrained HIV providers do not consistently use MI techniques when counseling patients about sexual risk reduction. However, when they do, their patients are more likely to express intentions to reduce sexual risk behavior. PRACTICE IMPLICATIONS: MI holds promise as one strategy to reduce transmission of HIV and other sexually transmitted infections.