Current-Controlled Nanospray Ionization Mass Spectrometry

The hypothesis that direct determination of electrospray current would provide a viable method for maintaining spray stability to enable optimal nanospray analysis was tested by building a feedback apparatus capable of reading the current and readjusting the emitter voltage in real time. The apparatus consists of a current-sensing circuit that reads the voltage drop across a resistor located between the high-voltage power supply and the nanospray emitter. A low voltage proportional to the observed current is generated and sent to a data acquisition card. The information is used by a proportional-derivative-integral (PID) algorithm to calculate the magnitude of a low-voltage signal that is used to control the power supply output. Any variation of current across the sensing resistor is thus counteracted by an opposite-direction variation of the high voltage applied to the nanospray emitter. In this way, the apparatus adjusts the emitter voltage to achieve a preset value of current, which it strives to maintain over time in spite of any possible variation of the parameters influencing the spray regime. Preliminary results have shown that the feedback apparatus is capable of establishing and maintaining stable spray for samples that are usually considered challenging in traditional voltage-controlled analysis, such as those consisting of nucleic acid solutions with high salt loads. For these types of samples, the total ion count recorded in current-controlled mode was significantly more stable than that observed in voltage-controlled mode. At the same time, overall signal intensities and signal-to-noise ratios were also significantly improved. Setting the target nanospray current to a predefined value and letting the apparatus reach the target without operator intervention enabled the acquisition of viable data from solutions containing up to 2.5 M ammonium acetate, which are ordinarily difficult by traditional manual tuning. A deeper understanding of the current–voltage relationships for samples of very different compositions is expected to enable one not only to predict the target current that should be used for a certain analysis, but also to devise algorithms to change such target as a function of predictable variations of sample properties and analytical conditions. This will allow for optimal performance to be maintained during on-line gradient chromatography in which the nature of the sprayed solution may vary very widely during the course of the analysis

Inhibitory effects of archetypical nucleic acid ligands on the interactions of HIV-1 nucleocapsid protein with elements of Ψ-RNA

Disrupting the interactions between human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein and structural elements of the packaging signal (Ψ-RNA) could constitute an ideal strategy to inhibit the functions of this region of the genome leader in the virus life cycle. We have employed electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS) to assess the ability of a series of nucleic acid ligands to bind selected structures of Ψ-RNA and inhibit their specific interactions with NC in vitro. We found that the majority of the ligands included in the study were able to form stable non-covalent complexes with stem–loop 2, 3 and 4 (SL2–4), consistent with their characteristic nucleic acid binding modes. However, only aminoglycosidic antibiotics were capable of dissociating preformed NC•SL3 and NC•SL4 complexes, but not NC•SL2. The apparent specificity of these inhibitory effects is closely dependent on distinctive structural features of the different NC•RNA complexes. The trends observed for the IC(50) values correlate very well with those provided by the ligand binding affinities and the dissociation constants of target NC•RNA complexes. This systematic investigation of archetypical nucleic acid ligands provides a valid framework to support the design of novel ligand inhibitors for HIV-1 treatment

Revisiting Plus-Strand DNA Synthesis in Retroviruses and Long Terminal Repeat Retrotransposons: Dynamics of Enzyme: Substrate Interactions

Although polypurine tract (PPT)-primed initiation of plus-strand DNA synthesis in retroviruses and LTR-containing retrotransposons can be accurately duplicated, the molecular details underlying this concerted series of events remain largely unknown. Importantly, the PPT 3′ terminus must be accommodated by ribonuclease H (RNase H) and DNA polymerase catalytic centers situated at either terminus of the cognate reverse transcriptase (RT), and in the case of the HIV-1 enzyme, ∼70Å apart. Communication between RT and the RNA/DNA hybrid therefore appears necessary to promote these events. The crystal structure of the HIV-1 RT/PPT complex, while informative, positions the RNase H active site several bases pairs from the PPT/U3 junction, and thus provides limited information on cleavage specificity. To fill the gap between biochemical and crystallographic approaches, we review a multidisciplinary approach combining chemical probing, mass spectrometry, NMR spectroscopy and single molecule spectroscopy. Our studies also indicate that nonnucleoside RT inhibitors affect enzyme orientation, suggesting initiation of plus-strand DNA synthesis as a potential therapeutic target

Behind the Mirror: Chirality Tunes the Reactivity and Cytotoxicity of Chloropiperidines as Potential Anticancer Agents

The pressing demand for sustainable antitumor drugs prompted us to investigate 3-chloropiperidines as potential mustard-based anticancer agents. In this study, an explorative set of variously decorated monofunctional 3-chloropiperidines (M-CePs) was efficiently synthesized through a fast and affordable route providing high yields of pure racemates and enantiomers. Consistently with their reactivity, M-CePs were demonstrated to alkylate DNA in vitro. On a panel of carcinoma cell lines, M-CePs exhibited low nanomolar cytotoxicity indexes, which showed their remarkable activity against pancreatic cancer cells and in all cases performed strikingly better than the chlorambucil control. Very interestingly, stereochemistry modulated the activity of M-CePs in unexpected ways, pointing to additional molecular mechanisms of action beyond the direct damage of genomic DNA. This encouraging combination of efficacy and sustainability suggests they are valid candidates for anticancer agent development

Palatini approach to 1/R gravity and its implications to the late Universe

By applying the Palatini approach to the 1/R-gravity model it is possible to explain the present accelerated expansion of the Universe. Investigation of the late Universe limiting case shows that: (i) due to the curvature effects the energy-momentum tensor of the matter field is not covariantly conserved; (ii) however, it is possible to reinterpret the curvature corrections as sources of the gravitational field, by defining a modified energy-momentum tensor; (iii) with the adoption of this modified energy-momentum tensor the Einstein's field equations are recovered with two main modifications: the first one is the weakening of the gravitational effects of matter whereas the second is the emergence of an effective varying "cosmological constant"; (iv) there is a transition in the evolution of the cosmic scale factor from a power-law scaling $a\propto t^{11/18}$ to an asymptotically exponential scaling $a\propto \exp(t)$; (v) the energy density of the matter field scales as $\rho_m\propto (1/a)^{36/11}$; (vi) the present age of the Universe and the decelerated-accelerated transition redshift are smaller than the corresponding ones in the $\Lambda$CDM model.Comment: 5 pages and 2 figures. Accepted in PR

Is there a place for nutritional supplements in the treatment of idiopathic male infertility?

Objective: Infertility affects 15% of couples in fertile age. Male factor is a cause of infertility in almost half of cases, mainly due to oligoasthenoteratozoospermia (OAT). The purpose of this study is to review the effects of nutritional supplements as medical treatment for idiopathic male infertility. Material and methods: A Pub Med and Medline review of the published studies utilizing nutritional supplements for the treatment of male infertility has been performed. Results: Clinical trials on Vitamin E, Vitamin A, Vitamin C. Arginine, Carnitine, N-Acetyl-Carnitine, Glutathione, Coenzyme Q10, Selenium and Zinc were reviewed. Although there is a wide variability in selected population, dose regimen and final outcomes, nutritional supplements both alone and in combination seems to be able to improve semen parameters (sperm count, sperm motility and morphology) and pregnancy rate in infertile men. Conclusions: There are rising evidences from published randomized trials and systematic review suggesting that nutritional supplementation may improve semen parameters and the likelihood of pregnancy in men affected by OAT. This improvement, however, is not consistent and there is a wide variation in the treatment regimens used. Well designed and adequately powered RCTs are needed to better clarify the role of nutritional supplements as treatment for male infertility

Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands

The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5′(rA)4:(dT)4 tract and the 3′ PPT-U3 junction. Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin. These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure

Efficacy and Safety of High-Dose Immunoglobulin-Based Regimen in Statin-Associated Autoimmune Myopathy: A Multi-Center and Multi-Disciplinary Retrospective Study

Statin-associated autoimmune myopathy is a rare muscle disorder, characterized by autoantibodies against HMGCR. The anti-HMGCR myopathy persists after statin, and often requires immunosuppressive therapy. However, there is not a standardized therapeutic approach. The purpose of this study is to report the effectiveness of the immunosuppressive treatment employed in a multi-center and multi-disciplinary cohort of patients affected by anti-HMGCR myopathy, in which an immunoglobulin (IVIG)-based treatment strategy was applied. We collected 16 consecutive patients with a diagnosis of anti-HMGCR myopathy, between 2012 and 2019, and recorded data on clinical and laboratory presentation (i.e., muscle strength, serum CK levels, and anti-HMGCR antibody titer) and treatment strategies. Our results highlight the safety and efficacy of an induction therapy combining IVIG with GCs and/or methotrexate to achieve persistent remission of the disease and steroid-free maintenance. Under IVIG-based regimens, clinical improvement and CK normalization occurred in more than two thirds of patients by six months. Relapse rate was low (3/16) and 2/3 relapses occurred after treatment suspension. Nearly 90% of the patients who successfully discontinued GCs were treated with a triple immunosuppressive regimen. In conclusion, an IVIG-based regimen, which particularly includes high-dose immunoglobulin, GCs and methotrexate, can provide a fast remission achievement with GC saving

New investigations on the 32S(3He,d)33Cl reaction at 9.6 MeV bombarding energy

The 32S(3He,d)33Cl one-proton transfer reaction is a powerful tool to investigate the spectroscopy of low-lying states in the proton-rich 33Cl nucleus. However, the extraction of firm differential cross-section data at various angles to benchmark and constrain theoretical models is made challenging by the presence of competitive reactions on target contaminants. In this paper we report on arecent measurement using a new generation hodoscope of silicon detectors, capable to detect and identify emitted deuterons down to energies of the order of 2 MeV. The high angular segmentation of our hodoscope combined with a suitable target to control possible contaminants, allowed to unambiguously disentangle the contribution of various states in 33Cl, in particular the 2.352 MeV state lying just few tens of keV above the proton separation energy