ABSTRACT
The virus is the smallest known replicative unit, usually in nanometer-range sizes. The most simple and sensitive detection assay involves molecular amplification of nucleic acids. This work shows a novel, straightforward detection based on the interaction of viral particles with fluorescent nanoconstructs without using enzymatic amplification, washing or separation steps. Fluorescent nanoconstructs are prepared with individual quantum dots of different emitting green and red fluorescence as a core. They are decorated with aptamers developed to recognise the receptor-binding region of the SARS-CoV-2 spike protein. Nanoconstructs can recognise SARS-CoV-2 viral particles fixed onto a coverglass generating aggregates. Meanwhile, SARS-CoV-2 viral particles/nanoconstructs complexes in solution yield aggregates and complexes, which a fluorescence microscope can visualise. The multiple molecular recognition allowed the detection of SARS-CoV-2 viral particles from a few microliters of patient swabs. This specific SARS-CoV-2/nanoconstructs interaction generates insoluble and precipitating aggregates. By using a mixture of green and red fluorescent nanoconstructs, upon the viral particle interaction, they yield heterochromatic green, red and yellow spectral fluorescence, easily identifiable by a fluorescence microscope. Washing and separation steps are not required, and aggregates allow one to easily recognise them, offering a sensitive, simple, and cheap alternative for viral detection.
ABSTRACT
In the last few years, polymerase chain reaction analysis is frequently required to improve the detection of pathogen infections in central nervous system as a potential cause of neurological disorders and neuropsychiatric symptoms. The goal of this paper is to set up a fast, cheap and reliable molecular approach for qualitative detection of six neurotropic pathogens. A method based on PCR has been designed and implemented to guarantee the qualitative DNA detection of herpes simplex virus types 1 and 2 (HSVI/II), Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella-zoster virus (VZV), rubella virus (RUBV) and Toxoplasma gondii in the cerebrospinal fluid, where otherwise they are barely detectable. Each PCR assay was tested using dilutions of positive controls, which demonstrated a sensitivity allowing to detect up to 102 copies/ml in PCR and 10 copies/ml in real-time PCR for each pathogen. Once been set up, the protocol was applied to evaluate the cerebrospinal fluid from 100 patients with suspected infectious diseases of the central nervous system and 50 patients without any infection. The method allowed to identify 17 positive cerebrospinal fluid with polymerase chain reaction and 22 with real-time PCR (RT-PCR), respectively. Therefore, application of RT PCR allows a fast and sensitive evaluation of neurotropic DNA pathogens in the course of diagnostic routine within neurological units.
Subject(s)
Central Nervous System Infections/virology , Central Nervous System/virology , Virus Diseases/virology , Evaluation Studies as Topic , Humans , Real-Time Polymerase Chain Reaction/methods , Viruses/geneticsABSTRACT
Nonalcoholic fatty liver disease (NAFLD) is becoming an increasing cause of chronic liver damage. The decision of start a medical treatment is based on the documented risk of progression to cirrhosis and liver cancer, when steatohepatitis (NASH) occurs. The therapy of this syndrome requires, as obviously, some considerations on the natural history of the condition, on the efficacy and safety of various therapeutic options, as well as on the costs. Treatment of patients with NAFLD has typically been focused on the management of associated conditions such as obesity, diabetes mellitus and hyperlipemia. Weight loss improves insulin sensitivity, and NASH may resolve with weight reduction. Insulin resistance seems to be the common denominator in many cases of NAFLD. Two classes of drugs have been shown to correct insulin resistance: biguanides (e.g., metformin) and thiazolidinediones (e.g., rosiglitazone and pioglitazone). The last two decades have witnessed a considerable progress in the understanding of the mechanisms respon-sible for the fibrogenic progression of chronic liver diseases. Several drugs believed to be hepatoprotective or antifibrotic agent as UDCA, betaine, vitamin E, lecithin, beta-carotene and selenium have been used in patients with NASH. Silybin is the main component of silymarin that is absorbed when linked whith a phytosome. This substance reduces in rats the lipid-peroxidation and the activaction of hepatic stellate cells. In humans, some non controlled data show that silybin is able to reduce insulin resistance, liver steatosis and plasma markers of liver fibrosis.
Subject(s)
Fatty Liver/drug therapy , Fatty Liver/therapy , Anti-Obesity Agents/therapeutic use , Cytokines/antagonists & inhibitors , Cytokines/metabolism , Humans , Hypoglycemic Agents/therapeutic use , Insulin Resistance , Lipid Peroxidation , Liver Cirrhosis , Obesity/drug therapy , Weight LossSubject(s)
Central Nervous System Vascular Malformations/complications , Central Nervous System Vascular Malformations/drug therapy , Indomethacin/therapeutic use , Migraine Disorders/complications , Migraine Disorders/drug therapy , Adult , Central Nervous System Vascular Malformations/pathology , Female , Humans , Male , Migraine Disorders/pathologyABSTRACT
Huntington's disease (HD) and dominant ataxias (SCA) represent neurodegenerative hereditary diseases dominantly transmitted for which a direct and accurate genetic test is now available for molecular confirmation and presymptomatic test. Predictive testing programs, according to published international guidelines, are available worldwide. A large number of subjects (n=165) required a predictive HD diagnosis, although only 36% completed the program flow-chart and received the final genetic result (26 had a positive, 34 negative result for mutation). In 4 cases, an allele of intermediate range (33-34 CAGs) was found. Two of these shared the intermediate allele with an expanded repeat. In this case, we estimated the patient's risk to have affected children over the usually reported 50%. In 4 cases, the presymptomatic diagnosis was requested by persons at-risk for SCA1 and SCA3/Machado-Joseph disease. There were no adverse events to results of both HD and SCA presymptomatic diagnoses.
Subject(s)
DNA Mutational Analysis/psychology , Genetic Counseling/psychology , Genetic Testing/psychology , Huntington Disease/genetics , Neuropsychological Tests/standards , Patient Education as Topic/standards , Spinocerebellar Ataxias/genetics , Age of Onset , Female , Humans , Huntington Disease/psychology , Male , Predictive Value of Tests , Spinocerebellar Ataxias/psychology , Trinucleotide Repeat Expansion/geneticsABSTRACT
OBJECTIVE: The authors investigated programming (Bereitschaftspotential or BP) and control activity (Skilled Performance Positivity or SPP) of a bimanual, sequential, skilled motor act in off-therapy Parkinson's disease (PD) patients. METHODS: We recorded Movement Related Potentials (MRPs) in 12, non-demented, off-therapy parkinsonian patients and in 17 control subjects who were performing a skilled, time-locked motor act, which was not routine in their everyday life but had to be learned: the Skilled Performance Task (SPT). BP, SPP and correct performances were evaluated in grand average waveforms and in sequential blocks. RESULTS: The analysis of correct performances showed that accuracy in PD patients was significantly lower than in the control group and this accuracy did not improve throughout the blocks. A significantly low level of performances was associated with an increased BP amplitude (P<0.05) and decreased SPP amplitude (P<0.05) in PD patients. CONCLUSION: Our findings suggest that skill motor learning is impaired in non-demented unmedicated PD patients. We discuss the view that PD patients may allocate more attentional resources, as suggested by the increased BP amplitude, the decreased SPP amplitude and the low correct performances, in order to perform a new skilled motor act.
Subject(s)
Brain Mapping , Brain/physiopathology , Evoked Potentials, Somatosensory/physiology , Parkinson Disease/physiopathology , Psychomotor Performance/physiology , Aged , Attention , Brain/physiology , Electroencephalography , Electromyography , Electrooculography , Female , Hand/innervation , Humans , Male , Middle Aged , Motor Skills/physiology , Reference Values , Video RecordingABSTRACT
It has been proposed that in rat and murine tissues glucosidase II (GII) is formed by two subunits, GIIalpha and GIIbeta, respectively, responsible for the catalytic activity and the retention of the enzyme in the endoplasmic reticulum (ER). To test this proposal we disrupted genes (gls2alpha(+) and gls2beta(+)) encoding GIIalpha and GIIbeta homologs in Schizosaccharomyces pombe. Both mutant cells (gls2alpha and gls2beta) were completely devoid of GII activity in cell-free assays. Nevertheless, N-oligosaccharides formed in intact gls2alpha cells were identified as Glc(2)Man(9)GlcNAc(2) and Glc(2)Man(8)GlcNAc(2), whereas gls2beta cells formed, in addition, small amounts of Glc(1)Man(9)GlcNAc(2). It is suggested that this last compound was formed by GIIalpha transiently present in the ER. Monoglucosylated oligosaccharides facilitated glycoprotein folding in S. pombe as mutants, in which formation of monoglucosylated glycoproteins was completely (gls2alpha) or severely (gls2beta and UDP-Glc:glycoprotein:glucosyltransferase null) diminished, showed ER accumulation of misfolded glycoproteins when grown in the absence of exogenous stress as revealed by (a) induction of binding protein-encoding mRNA and (b) accumulation of glycoproteins bearing ER-specific oligosaccharides. Moreover, the same as in mammalian cell systems, formation of monoglucosylated oligosaccharides decreased the folding rate and increased the folding efficiency of glycoproteins as pulse-chase experiments revealed that carboxypeptidase Y arrived at a higher rate but in decreased amounts to the vacuoles of gls2alpha than to those of wild type cells.
Subject(s)
Protein Folding , alpha-Glucosidases/chemistry , Animals , Gene Deletion , Gene Expression Regulation, Enzymologic , Mice , Rats , Schizosaccharomyces , alpha-Glucosidases/geneticsABSTRACT
Interaction of monoglucosylated oligosaccharides with ER lectins (calnexin and/or calreticulin) facilitates glycoprotein folding but this interaction is not essential for cell viability under normal conditions. We obtained two distinct single Schizosaccharomyces pombe mutants deficient in either one of the two pathways leading to the formation of monoglucosylated oligosaccharides. The alg6 mutant does not glucosy- late lipid-linked oligosaccharides and transfers Man9GlcNAc2 to nascent polypeptide chains and the gpt1 mutant lacks UDP-Glc:glycoprotein glucosyltransferase (GT). Both single mutants grew normally at 28 degreesC. On the other hand, gpt1/alg6 double-mutant cells grew very slowly and with a rounded morphology at 28 degreesC and did not grow at 37 degreesC. The wild-type phenotype was restored by transfection of the double mutant with a GT-encoding expression vector or by addition of 1 M sorbitol to the medium, indicating that the double mutant is affected in cell wall formation. It is suggested that facilitation of glycoprotein folding mediated by the interaction of monoglucosylated oligosaccharides with calnexin is essential for cell viability under conditions of extreme ER stress such as underglycosylation of proteins caused by the alg6 mutation and high temperature. In contrast, gls2/alg6 double-mutant cells that transfer Man9GlcNAc2 and that are unable to remove the glucose units added by GT as they lack glucosidase II (GII), grew at 37 degreesC and had, when grown at 28 degreesC, a phenotype of growth and morphology almost identical to that of wild-type cells. These results indicate that facilitation of glycoprotein folding mediated by the interaction of calnexin and monoglucosylated oligosaccharides does not necessarily require cycles of reglucosylation-deglucosylation catalyzed by GT and GII.
Subject(s)
Endoplasmic Reticulum/metabolism , Glucosyltransferases/metabolism , Schizosaccharomyces/growth & development , Schizosaccharomyces/metabolism , Carbohydrate Sequence , Glucosyltransferases/genetics , Glycosylation , Molecular Sequence Data , Mutagenesis , Oligosaccharides/metabolism , Phenotype , Protein Folding , Schizosaccharomyces/genetics , TemperatureABSTRACT
A key element in the quality control of glycoprotein folding is the UDP-Glc:glycoprotein glucosyltransferase (GT), which in cell-free assays exclusively glucosylates misfolded glycoproteins. In order to test if such a protein conformation is a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT, a Schizosaccharomyces pombe double mutant (gls2/alg6) was constructed. With this mutant, Man9GlcNAc2 is transferred to proteins and no removal of glucose units added by GT occurs as it lacks glucosidase II. The same proportion of glucosylated (Glc1Man9GlcNAc2) and unglucosylated (Man9GlcNAc2 and Man8GlcNAc2) endoplasmic reticulum (ER)-specific compounds was produced when cells were pre-incubated for 10, 20 or 30 min and further incubated with [14C]glucose for 10 min at 28 degrees C with or without 5 mM dithiothreitol (DTT), thus indicating not only that DTT did not affect protein glucosylation but also that no increased glucosylation of glycoproteins occurred in the presence of the drug. Monitoring Golgi-specific modifications of oligosaccharides after pulse-chase experiments performed in the presence or absence of 5 mM DTT showed that exit of the bulk of glycoproteins synthesized from the ER and thence their proper folding had been prevented by the drug. Cells pulse-chase labeled at 37 degrees C in the absence of DTT also yielded glucosylated and unglucosylated protein-linked oligosaccharides without Golgi-specific modifications. It was concluded that a misfolded protein conformation is not a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT.
Subject(s)
Glucosyltransferases/metabolism , Protein Folding , Dithiothreitol/pharmacology , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Glucose/metabolism , Glucosyltransferases/genetics , Heat-Shock Response , Mutation , Oligosaccharides/metabolism , Protein Conformation/drug effects , Protein Processing, Post-Translational/drug effects , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolismABSTRACT
Previous work indicated that diacylglycerol (DG) molecules translocate across the cytoplasm of mammalian cells, a process relevant to the signalling role of this lipid as protein kinase C activator. Here we investigated the possible mechanism underlying DG translocation. We examined the interaction between 1,2-di-[1-14C]oleoyl-sn-glycerol and rat liver cytosol (rlc) using assays based on Lipidex-1000 and on coelution on Sepharose CL 6B. We measured high DG binding activity and found that it resides in cytosolic proteins and not in cytosolic lipids. Chromatography of rlc proteins on Sepharose CL 6B showed profiles in which the activity measured by either method coincided. Further, we showed that the DG-rlc protein interaction results in the stabilization of DG in a micellar form, eluting in the void volume of Sepharose CL 6B. Such stabilized micelles are reminiscent of insect lipophorins and may represent a new, thus far unrecognized, mode of lipid transport within living cells.
Subject(s)
Diglycerides/metabolism , Lipoproteins/metabolism , Micelles , Animals , Biological Transport , Chromatography, Agarose , Cytosol/metabolism , Dextrans/metabolism , Female , Liver/ultrastructure , Oleic Acid/metabolism , RatsABSTRACT
To investigate the effects of spontaneous chronic hypoglycemia on the peripheral and central nervous system, a multimodal neurophysiological evaluation [median somatosensory (mSEP), brain stem auditory (BAEP), and visual (VEP) evoked potentials recordings] was performed in seven insulinoma patients before and 3 and 6 months after surgical removal of tumor. Before surgery, mSEP findings showed abnormal reduction in peripheral wrist-Erb conduction velocity in three patients as well as a pathological increase in Erb-N13, N13-N20, and Erb-N20 conduction times in five cases. BAEP and VEP recordings gave pathological results in two patients. Moreover, during hypoglycemia, the III-V and I-V interpeak latencies of BAEPs were significantly prolonged (P < 0.01 and P < 0.005, respectively) compared to recordings in euglycemia. After 6 months, a mSEP recovery, even if partial was noted in four patients, BAEPs were normalized in one case, and VEPs were unmodified. Compared to presurgery data, these recordings showed a significant (P < 0.05), but incomplete, shortening of BAEPs (III-V and I-V interpeak latencies) and mSEPs (Erb-N13 and Erb-N20 conduction times). Our findings demonstrate that multiple and selective neurophysiological abnormalities are present in insulinoma patients, confirm that hypoglycemia impairs suddenly brain stem function, and show that after tumor removal, long recovery times for improvement of some neurophysiological anomalies are requested.
Subject(s)
Brain/physiopathology , Hypoglycemia/physiopathology , Insulinoma/complications , Pancreatic Neoplasms/complications , Peripheral Nerves/physiopathology , Adolescent , Adult , Evoked Potentials, Auditory, Brain Stem , Evoked Potentials, Somatosensory , Evoked Potentials, Visual , Female , Humans , Hypoglycemia/etiology , Insulinoma/surgery , Male , Middle Aged , Pancreatic Neoplasms/surgeryABSTRACT
Cortical functions concerned with the execution of skilled movements can be studied through complex interactive tasks. Skilled performance task (SPT) offers the greatest deal of information about the electrophysiological components reflecting pre-programming, execution of the movement and control of the results. Overall, these components are indicated as "movement-related brain macropotentials' (MRBMs). Among them, Bereitschaftspotential (BP) reflects cerebral processes related to the preparation of movement and skilled performance positivity (SPP) reflects control processes on the result of performance. There is some evidence supporting a training effect on MRBMs, but less clear is whether long-term practice of a skilled activity could modify learning strategies of a new skilled task. We recorded MRBMs in subjects trained for a long time to perform a highly skillful athletic activity, i.e. gun shooting, and in a group of control subjects without any former experience in skilled motor activities. Our findings demonstrated the existence of a relationship between pre-programming and performance control, as suggested by decrease of BP amplitude and increase of SPP amplitude in presence of high levels of performance. Long-term practice seems to develop better control models on performance, that reduce the need of a high mental effort in pre-programming a skilled action.
Subject(s)
Contingent Negative Variation/physiology , Learning/physiology , Adult , Electroencephalography , Humans , Male , Reaction Time/physiology , Time FactorsABSTRACT
Several distinct types of voltage-gated and second-messenger-operated K+, Ca2+, Na+ and Cl- channels exist in electrically non excitable cells such as those of the hematopoietic lineage. In these cells ion channels mediate cellular functions involving intracellular biochemical responses, rather than rapid electrical signaling. The presence of the channels is required for several basic functions, such as activation, secretion of lymphokines, mitogenesis, the regulation of cell volume and the mechanisms of resistance to chemotherapeutic agents. Here IN we review the patch-clamp method for studying many characteristics of these ionic channels, particularly in blood cells.
Subject(s)
Hematopoietic Stem Cells/physiology , Ion Channels/physiology , Patch-Clamp Techniques , Arachidonic Acid/pharmacology , Cell Differentiation , Drug Resistance, Neoplasm , Macrophage Colony-Stimulating Factor/pharmacologyABSTRACT
P300 was recorded, using an 'odd ball' paradigm, in 18 parkinsonian patients before and during dopaminergic monotherapy. The data were compared with those of a homogeneous standard group of 20 subjects. The main finding was an increase in the P300 latency of parkinsonian patients before therapy, which recovered during dopaminergic monotherapy. In 11 voluntary healthy subjects the same therapy did not produce a reduction of the P300 latency. The data are discussed in relation to a possible dopaminergic component in P300 origin.
Subject(s)
Brain/physiopathology , Electroencephalography , Parkinson Disease/physiopathology , Reaction Time , Aged , Female , Humans , Levodopa/therapeutic use , Male , Middle Aged , Parkinson Disease/drug therapyABSTRACT
The Authors introduce some questions concerning cognitive impairment in Parkinsonian patients and they focus the attention on 'bradyphrenia'. They briefly discuss the methodological difficulties in studying this kind of disturbances in Parkinson's disease (PD) and the approach to this problem with the help of the Event-Related Potentials or ERPs. Finally, they review literature and their data on Contingent Negative Variation (CNV) and P300 in PD and conclude that interesting and useful informations can be obtained by means of the electrophysiological methods.
Subject(s)
Cognition Disorders/physiopathology , Parkinson Disease/physiopathology , Cognition Disorders/etiology , Electroencephalography , Electrophysiology , Evoked Potentials/physiology , Humans , Parkinson Disease/complicationsABSTRACT
Retinal and occipital visual evoked potentials and event-related potentials (P300) have been recorded in normal human subjects before and after the administration of the dopaminergic receptor antagonist, haloperidol, and/or the dopaminergic precursor L-DOPA. The data show that either retinal or occipital visual potentials and P300 are delayed by haloperidol. These findings are consistent with the hypothesis that haloperidol in healthy subjects mimicks the electrophysiological abnormalities observed in Parkinson's disease. On the other hand, L-DOPA does not generally modify these latencies in normals, while it is known to decrease the same parameters in parkinsonian patients. This is in accord with the involvement of a specific mechanism in the recovery observed in parkinsonian patients after L-DOPA therapy. Our data confirm that the alterations of visual and cognitive potentials observed in Parkinson's disease are closely related to the impairment of dopaminergic transmission.
Subject(s)
Cognition/physiology , Dopamine/administration & dosage , Electroencephalography , Evoked Potentials, Visual/physiology , Parkinson Disease/physiopathology , Acoustic Stimulation , Evoked Potentials, Auditory/drug effects , Evoked Potentials, Auditory/physiology , Evoked Potentials, Visual/drug effects , Haloperidol/administration & dosage , Humans , Reaction Time , Reference ValuesSubject(s)
Nervous System Diseases/etiology , Avitaminosis/complications , Diabetic Neuropathies/etiology , Drug-Related Side Effects and Adverse Reactions , Humans , Kidney Failure, Chronic/complications , Metals/adverse effects , Nervous System Diseases/chemically induced , Nervous System Diseases/metabolism , Paraneoplastic Syndromes/etiology , Porphyrias/complications , Solvents/adverse effectsABSTRACT
The authors consider the possible relationship between the autonomic functions and the contingent Negative Variation (CNV). This relationship has been found both in psychopathological conditions and after the administration of psychotropic drugs. Some preliminary data are reported on the correlations observed between CNV and the autonomic activation (R-R interval) during a sodium lactate infusion.