LncRNAs expression profile in a family household cluster of COVID-19 patients.

More than 3 years after the start of SARS-CoV-2 pandemic, the molecular mechanisms behind the viral pathogenesis are still not completely understood. Long non-coding RNAs (lncRNAs), well-known players in viral infections, can represent prime candidates for patients' risk stratification. The purpose of the current study was to investigate the lncRNA profile in a family cluster of COVID-19 cases with different disease progression, during the initial wave of the pandemic and to evaluate their potential as biomarkers for COVID-19 evolution. LncRNA expression was investigated in nasopharyngeal swabs routinely collected for diagnosis. Distinct expression patterns of five lncRNAs (HOTAIR, HOTAIRM1, TMEVPG1, NDM29 and snaR) were identified in all the investigated cases, and they were associated with disease severity. Additionally, a significant increase in the expression of GAS5-family and ZFAS1 lncRNAs, which target factors involved in the inflammatory response, was observed in the sample collected from the patient with the most severe disease progression. An lncRNA prognostic signature was defined, opening up novel research avenues in understanding the interactions between lncRNAs and SARS-CoV-2.

Leukemic conversion involving RAS mutations of type 1 CALR-mutated primary myelofibrosis in a patient treated for HCV cirrhosis: a case report.

Somatic frameshift mutations in exon 9 of calreticulin (CALR) gene are recognized as disease drivers in primary myelofibrosis (PMF), one of the three classical Philadelphia-negative myeloproliferative neoplasms (MPNs). Type 1/type 1-like CALR mutations particularly confer a favorable prognostic and survival advantage in PMF patients. We report an unusual case of PMF incidentally diagnosed in a 68-year-old woman known with hepatitis C virus (HCV) cirrhosis who developed a progressive painful splenomegaly, without anomalies in blood cell counts. While harboring a type 1 CALR mutation, the patient underwent a leukemic transformation in less than 1 year from diagnosis, with a lethal outcome. Analysis of paired DNA samples from chronic and leukemic phases by a targeted next-generation sequencing (NGS) panel and single-nucleotide polymorphism (SNP) microarray revealed that the leukemic clone developed from the CALR-mutated clone through the acquisition of genetic events in the RAS signaling pathway: an increased variant allele frequency of the germline NRAS Y64D mutation present in the chronic phase (via an acquired uniparental disomy of chromosome 1) and gaining NRAS G12D in the blast phase. SNP microarray analysis showed five clinically significant copy number losses at regions 7q22.1, 8q11.1-q11.21, 10p12.1-p11.22, 11p14.1-p11.2, and Xp11.4, revealing a complex karyotype already in the chronic phase. We discuss how additional mutations, detected by NGS, as well as HCV infection and antiviral therapy, might have negatively impacted this type 1 CALR-mutated PMF. We suggest that larger studies are required to determine if more careful monitoring would be needed in MPN patients also carrying HCV and receiving anti-HCV treatment.

Kinetics and persistence of cellular and humoral immune responses to SARS-CoV-2 vaccine in healthcare workers with or without prior COVID-19.

SARS-CoV-2 vaccines are highly efficient against severe forms of the disease, hospitalization and death. Nevertheless, insufficient protection against several circulating viral variants might suggest waning immunity and the need for an additional vaccine dose. We conducted a longitudinal study on the kinetics and persistence of immune responses in healthcare workers vaccinated with two doses of BNT162b2 mRNA vaccine with or without prior SARS-CoV-2 infection. No new infections were diagnosed during follow-up. At 6 months, post-vaccination or post-infection, despite a downward trend in the level of anti-S IgG antibodies, the neutralizing activity does not decrease significantly, remaining higher than 75% (85.14% for subjects with natural infection, 88.82% for vaccinated after prior infection and 78.37% for vaccinated only). In a live-virus neutralization assay, the highest neutralization titres were present at baseline and at 6 months follow-up in persons vaccinated after prior infection. Anti-S IgA levels showed a significant descending trend in vaccinated subjects (p < 0.05) after 14 weeks. Cellular immune responses are present even in vaccinated participants with declining antibody levels (index ratio 1.1-3) or low neutralizing activity (30%-40%) at 6 months, although with lower T-cell stimulation index (p = 0.046) and IFN-γ secretion (p = 0.0007) compared to those with preserved humoral responses.

Recent advances in gastric cancer early diagnosis.

Gastric cancer (GC) remains an important cause of cancer death worldwide with a high mortality rate due to the fact that the majority of GC cases are diagnosed at an advanced stage when the prognosis is poor and the treatment options are limited. Unfortunately, the existing circulating biomarkers for GC diagnosis and prognosis display low sensitivity and specificity and the GC diagnosis is based only on the invasive procedures such as upper digestive endoscopy. There is a huge need for less invasive or non-invasive tests but also highly specific biomarkers in case of GC. Body fluids such as peripheral blood, urine or saliva, stomach wash/gastric juice could be a source of specific biomarkers, providing important data for screening and diagnosis in GC. This review summarized the recently discovered circulating molecules such as microRNAs, long non-coding RNAs, circular RNAs, which hold the promise to develop new strategies for early diagnosis of GC.

Inefficient spin trapping of superoxide in the presence of nitric-oxide: implications for studies on nitric-oxide synthase uncoupling.

Uncoupling of nitric-oxide synthase (NOS) by deficiency of the substrate L-arginine or the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) is known to generate the reactive oxygen species H2O2 and superoxide. Discrimination between these two compounds is usually achieved by spin trapping of superoxide. We measured superoxide formation by uncoupled rat neuronal NOS, which contained one equivalent of tightly bound BH4 per dimer, using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap. As expected, the Ca2+-stimulated enzyme exhibited reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity that was accompanied by generation of superoxide and H2O2 in the absence of added L-arginine and BH4. Addition of BH4 (10 microM) did not significantly affect the rate of H2O2 formation but almost completely inhibited the apparent formation of superoxide, suggesting direct formation of H2O2. Although L-arginine (0.1 mM) increased the rate of NADPH oxidation about two-fold, the substrate largely attenuated apparent formation of both superoxide and H2O2, indicating that the spin trap did not efficiently outcompete the reaction between NO and superoxide. The efficiency of DEPMPO to scavenge superoxide in the presence of NO was studied by measuring free NO with a Clark-type electrode under conditions of NO/superoxide cogeneration. Neuronal NOS half-saturated with BH4 and the donor compound 3-morpholinosydnonimine (SIN-1) were used as enzymatic and nonenzymatic sources of NO/superoxide, respectively. Neither of the two systems gave rise to considerable NO signals in the presence of 50-100 mM DEPMPO, and even at 400 mM the spin trap uncovered less than 50% of the NO release that was detectable in the presence of 5000 U/ml superoxide dismutase. These results indicate that DEPMPO and all other currently available superoxide spin traps do not efficiently outcompete the reaction with NO. In addition, the similar behavior of nNOS and SIN-1 provides further evidence for NO as initial product of the NOS reaction.

Membrane penetration of nitric oxide and its donor S-nitroso-N-acetylpenicillamine: a spin-label electron paramagnetic resonance spectroscopic study.

S-nitroso-N-acetylpenicillamine (SNAP) is a pharmacological agent with diverse biological effects that are mainly attributable to its favorable characteristics as a nitric oxide (NO)-evolving agent. It is found that SNAP incorporates readily into dimyristoyl phosphatidylcholine (DMPC) bilayer membranes; and an approximate penetration profile was obtained from the depth dependence of the perturbation that it exerts on spin-labeled lipid chains. The profile of SNAP locates it deep in the hydrophobic core of both fluid- and gel-phase membranes. The spin relaxation enhancement of spin-labeled phospholipids with nitroxide group located at different depths in DMPC membranes was determined for nitric oxide (NO) and molecular oxygen (O(2)), at close to atomic spatial resolution. The relaxation enhancement, which is proportional to the corresponding vertical membrane profile of the concentration-diffusion product, was measured in the gel and fluid phases of the lipid bilayer. No significant membrane penetration was observed in the gel phase for the two water-dissolved gases. In the fluid phase, the transmembrane profiles of NO and O(2) are similar and could be well described by a sigmoidal function with a maximum in the center of the bilayer, but that of NO is less steep and is shifted toward the center of the membrane, relative to that of O(2). These differences can be attributed mainly to the difference in hydrophobicity between the two gases and the presence of the donor in the NO experiments. The biological implications of the above results are discussed.

EPR spectroscopy of common nitric oxide - spin trap complexes.

Two commonly used hydrophobic and hydrophilic spin traps for NO, namely Fe2+(DETC)(2)and Fe2+(MGD)(2), respectively, were analyzed via EPR spectroscopy. EPR spectra of trapped NO, together with field position standards, were recorded both in the frozen state and at room temperature. We present a detailed characterization of the EPR spectra of the above paramagnetic NO complexes, concerning g-value, hyperfine splitting and linewidths. This study also provides spectroscopic data required to develop a quantitative and sensitive detection system for nitric oxide both in hydrophobic and hydrophilic aqueous media.