ATR-FTIR spectroscopy of plasma supported by multivariate analysis discriminates multiple sclerosis disease.

Multiple sclerosis (MS) is one of the most common neurodegenerative diseases showing various symptoms both of physical and cognitive type. In this work, we used attenuated total reflection Fourier transformed infrared (ATR-FTIR) spectroscopy to analyze plasma samples for discriminating MS patients from healthy control individuals, and identifying potential spectral biomarkers helping the diagnosis through a quick non-invasive blood test. The cohort of the study consists of 85 subjects, including 45 MS patients and 40 healthy controls. The differences in the spectral features both in the fingerprint region (1800-900 cm-1) and in the high region (3050-2800 cm-1) of the infrared spectra were highlighted also with the support of different chemometric methods, to capture the most significant wavenumbers for the differentiation. The results show an increase in the lipid/protein ratio in MS patients, indicating changes in the level (metabolism) of these molecular components in the plasma. Moreover, the multivariate tools provided a promising rate of success in the diagnosis, with 78% sensitivity and 83% specificity obtained through the random forest model in the fingerprint region. The MS diagnostic tools based on biomarkers identification on blood (and blood component, like plasma or serum) are very challenging and the specificity and sensitivity values obtained in this work are very encouraging. Overall, the results obtained suggest that ATR-FTIR spectroscopy on plasma samples, requiring minimal or no manipulation, coupled with statistical multivariate approaches, is a promising analytical tool to support MS diagnosis through the identification of spectral biomarkers.

Thermal Liquid Biopsy (TLB) of Blood Plasma as a Potential Tool to Help in the Early Diagnosis of Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) is frequently characterized by a variety of clinical signs, often exhibiting little specificity. The diagnosis requires a combination of medical observations and instrumental tests, and any support for its objective assessment is helpful. OBJECTIVE: Herein, we describe the application of thermal liquid biopsy (TLB) of blood plasma samples, a methodology for predicting the occurrence of MS with a noninvasive, quick blood test. METHODS: TLB allows one to define an index (TLB score), which provides information about overall real-time alterations in plasma proteome that may be indicative of MS. RESULTS: This pilot study, based on 85 subjects (45 MS patients and 40 controls), showed good performance indexes (sensitivity and specificity both around 70%). The diagnostic methods better discriminate between early stage and low-burden MS patients, and it is not influenced by gender, age, or assumption of therapeutic drugs. TLB is more accurate for patients having low disability level (≤ 3.0, measured by the expanded disability status scale, EDSS) and a relapsing-remitting diagnosis. CONCLUSION: Our results suggest that TLB can be applied to MS, especially in an initial phase of the disease when diagnosis is difficult and yet more important (in such cases, accuracy of prediction is close to 80%), as well as in personalized patient periodic monitoring. The next step will be determining its utility in differentiating between MS and other disorders, in particular in inflammatory diseases.

Thermo-Plasmonic Killing of Escherichia coli TG1 Bacteria.

Plasmonic photo-thermal therapy (PPTT) is a minimally invasive, drug-free, therapy based on the properties of noble metal nanoparticles, able to convert a bio-transparent electromagnetic radiation into heat. PPTT has been used against cancer and other diseases. Herein, we demonstrate an antimicrobial methodology based on the properties of gold nanorods (GNRs). Under a resonant laser irradiation GNRs become highly efficient light to heat nano-converters extremely useful for PPTT applications. The concept here is to assess the antimicrobial effect of easy to synthesize, suitably purified, water-dispersible GNRs on Escherichia coli bacteria. A control on the GNRs concentration used for the process has been demonstrated critical in order to rule out cytotoxic effects on the cells, and still to be able to generate, under a near infrared illumination, an adequate amount of heat suited to increase the temperature up to ≈50 °C in about 5 min. Viability experiments evidenced that the proposed system accomplished a killing efficiency suitable to reducing the Escherichia coli population of about 2 log CFU (colony-forming unit).

Antimicrobial Effects of Chemically Functionalized and/or Photo-Heated Nanoparticles.

Antibiotic resistance refers to when microorganisms survive and grow in the presence of specific antibiotics, a phenomenon mainly related to the indiscriminate widespread use and abuse of antibiotics. In this framework, thanks to the design and fabrication of original functional nanomaterials, nanotechnology offers a powerful weapon against several diseases such as cancer and pathogenic illness. Smart nanomaterials, such as metallic nanoparticles and semiconductor nanocrystals, enable the realization of novel drug-free medical therapies for fighting against antibiotic-resistant bacteria. In the light of the latest developments, we highlight the outstanding capabilities of several nanotechnology-inspired approaches to kill antibiotic-resistant bacteria. Chemically functionalized silver and titanium dioxide nanoparticles have been employed for their intrinsic toxicity, which enables them to exhibit an antimicrobial activity while, in a different approach, photo-thermal properties of metallic nanoparticles have been theoretically studied and experimentally tested against several temperature sensitive (mesophilic) bacteria. We also show that it is possible to combine a highly localized targeting with a plasmonic-based heating therapy by properly functionalizing nanoparticle surfaces with covalently linked antibodies. As a perspective, the utilization of properly engineered and chemically functionalized nanomaterials opens a new roads for realizing antibiotic free treatments against pathogens and related diseases.

Plasmonics Meets Biology through Optics.

Plasmonic metallic nanoparticles (NPs) represent a relevant class of nanomaterials, which is able to achieve light localization down to nanoscale by exploiting a phenomenon called Localized Plasmon Resonance. In the last few years, NPs have been proposed to trigger DNA release or enhance ablation of diseased tissues, while minimizing damage to healthy tissues. In view of the therapeutic relevance of such plasmonic NPs; a detailed characterization of the electrostatic interaction between positively charged gold nanorods (GNRs) and a negatively charged whole-genome DNA solution is reported. The preparation of the hybrid biosystem has been investigated as a function of DNA concentration by means of ζ-potential; hydrodynamic diameter and gel electrophoresis analysis. The results have pointed out the specific conditions to achieve the most promising GNRs/DNA complex and its photo-thermal properties have been investigated. The overall study allows to envisage the possibility to ingeniously combine plasmonic and biological materials and, thus, enable design and development of an original non invasive all-optical methodology for monitoring photo-induced temperature variation with high sensitivity.

Mutation analysis of the PINK1 gene in Southern Italian patients with early- and late-onset parkinsonism.

Mutations in the PINK1 gene represent the second most frequent cause of early-onset Parkinson's disease (EOPD). One or two mutated alleles were also reported in some sporadic or familial patients suffering from late-onset Parkinson's disease (LOPD). We aimed at assessing the frequency of mutations in this gene in our population. We performed a sequence analysis of PINK1 in 115 patients diagnosed with Parkinson's disease (PD) from southern Italy, including 93 sporadic cases with EOPD, 9 familial cases with EOPD, and 13 familial cases with LOPD. Three known homozygous mutations (Q456X, W437X, Q126P), corresponding to a 2.6% of all cases, were found. In particular, one mutation was detected among the sporadic cases (1.0%), one mutation among the familial early-onset patients (11.1%) and one mutation among the familial late-onset patients (7.7%). In addition, we found two heterozygous mutations (E476K, R207Q) among the sporadic patients. Only one mutation (R207Q) had not been previously described. Our results assess the role played by PINK1 in EOPD in southern Italy and illustrate the existence of mutations in this gene also in the late-onset form of the disease.

Association study between four polymorphisms in the HFE, TF and TFR genes and Parkinson's disease in southern Italy.

Iron overload may lead to neurodegenerative disorders such as Parkinson's disease (PD) and alterations of iron-related genes might be involved in the pathogenesis of this disease. The gene of haemochromatosis (HFE) encodes the HFE protein which interacts with the transferrin receptor (TFR), lowering its affinity for iron-bound transferrin (TF). We examined four known polymorphisms, C282Y and H63D in the HFE gene, G258S in the TF gene and S82G in the TFR gene, in 181 sporadic PD patients and 180 controls from Southern Italy to investigate their possible role in susceptibility to PD. No significant differences were found in genotype and allele frequencies between PD and controls for all the polymorphisms studied, suggesting that these variants do not contribute significantly to the risk of PD.

Lack of association between G-protein coupled receptor kinase 5 gene and Parkinson's disease.

The major component of Lewy Bodies (LB), the pathological hallmark of Parkinson's disease (PD) is α-synuclein, most prominently phosphorylated at serine 129. G-protein coupled receptor kinase 5 (GRK5) has been reported to phosphorylate α-synuclein in vitro, enhancing the α-synuclein toxicity to dopaminergic neurons in Drosophila model. Moreover, GRK5 was found in LBs from brain of PD patients. A genetic association study performed in the Japanese population revealed haplotypic association of the GRK5 gene with susceptibility to sporadic PD. We aimed at investigating whether four polymorphisms within the GRK5 gene (rs871196, rs2420616, rs7069375, rs4752293) could represent a risk factor for sporadic PD in Southern Italy. We genotyped 446 patients with PD and 450 controls for these markers and did not find any significant association with the disease at allelic, genotypic and haplotypic level. Our results indicate that the GRK5 gene does not confer risk to sporadic PD in our sample from Southern Italy.

Compound heterozygosity in DJ-1 gene non-coding portion related to parkinsonism.

In this study we analysed the DJ-1 gene in 40 sporadic patients with early onset Parkinson's disease and 100 appropriate controls, originated from southern Italy. We identified a single patient with age at onset of 38 years carrying two previously undescribed heterozygous mutations, both located in non-coding regions. The first mutation was a nucleotide change in the promoter region of the gene (g.159C>G) and the second one was an insertion in the intron 4 splice site (IVS4+3insA). In the same patient, genomic rearrangements were excluded. No DJ-1 mutations were found in the remaining parkinsonian patients. Our results support the growing importance of mutations in non-coding portion of human genome, and confirm that alterations in DJ-1 are a cause, even if rare, of early-onset Parkinson's disease.

Identification of the novel D297fsX318 PINK1 mutation and phenotype variation in a family with early-onset Parkinson's disease.

Herein we first describe a novel homozygous single nucleotide deletion in PINK1 exon 4 (889delG) which results in a loss of kinase domain on the PINK1 protein (D297fsX318). This mutation was identified in two brothers with early-onset Parkinson disease (EOPD) from a Sicilian consanguineous family. Of note, while one of the two patients developed mental deterioration and psychiatric problems, the other showed no cognitive decline. The present study supports the view that PINK1 is a pathogenic gene in some Italian families with EOPD and contributes to define the PINK1-associated phenotype.

Glucocerebrosidase gene mutations are associated with Parkinson's disease in southern Italy.

Recent studies have reported an association between the glucocerebrosidase (GBA) gene and Parkinson's disease (PD). To elucidate the role of this gene in our population, we screened 395 PD patients and 483 controls from southern Italy for the N370S and the L444P mutations. We found 11 patients (2.8%) carrying a heterozygous mutant GBA allele, whereas only one control subject (0.2%) had a heterozygous substitution (P = 0.0018). These results strongly suggest that Italian carriers of a GBA mutation have an increased risk of developing PD.

Myocardial 123metaiodobenzylguanidine uptake in genetic Parkinson's disease.

Myocardial (123)Metaiodobenzylguanidine (MIBG) enables the assessment of postganglionic sympathetic cardiac innervation. MIBG uptake is decreased in nearly all patients with Parkinson's disease (PD). Our objective was to evaluate MIBG uptake in patients with genetic PD. We investigated MIBG uptake in 14 patients with PD associated with mutations in different genes (Parkin, DJ-1, PINK1, and leucine-rich repeat kinase 2 -LRRK2), in 15 patients with idiopathic PD, and 10 control subjects. The myocardial MIGB uptake was preserved in 3 of the 4 Parkin-associated Parkinsonisms, in 1 of the 2 patients with DJ-1 mutations, in 1 of the 2 brothers with PINK1 mutations, in 3 of the 6 unrelated patients with Gly2019Ser mutation in the LRRK2 gene, whereas it was impaired in all patients with idiopathic PD. MIBG was preserved in all control subjects. Our study shows that myocardial MIGB uptake was normal in 8 of 14 patients with genetic PD, suggesting that cardiac sympathetic denervation occurs less frequently in genetic PD than in idiopathic PD. Our findings also demonstrate that MIGB uptake has a heterogeneous pattern in genetic PD, because it was differently impaired in patients with different mutations in the same gene or with the same gene mutation.

Mutational analysis of EFHC1 gene in Italian families with juvenile myoclonic epilepsy.

OBJECTIVES: Mutations in the EFHC1 gene have been reported in six juvenile myoclonic epilepsy (JME) families from Mexico and Belize. In this study, we screened 27 unrelated JME Italian families for mutations in the EFHC1 gene. MATERIALS AND METHODS: Twenty-seven families (86 affected individuals, 52 women) with at least two affected members with JME were selected. DNA was isolated from peripheral blood lymphocytes by standard methods and each exon of the EFHC1 gene was amplified and sequenced using intronic primers. RESULTS: Two heterozygous mutations were identified in three unrelated families. One (R353 W) was a novel missense mutation, while the F229 L mutation was previously described (say which on of the two occurred in two families). Both mutations cosegregated with the disease. In a fourth family, the variant 545G-->A (resulting in the amino acid substitution R182 H) cosegregated with JME. CONCLUSIONS: The results of our study extend the distribution of EFHC1 mutations to the white population and confirm the high level of genetic heterogeneity associated with JME.

Electroclinical features of a family with simple febrile seizures and temporal lobe epilepsy associated with SCN1A loss-of-function mutation.

PURPOSE: To report in detail the electroclinical features of a large family in which we recently identified a missense mutation (M145T) of a well-conserved amino acid in the first transmembrane segment of domain I of the human SCN1A. We showed that the mutation is associated with a loss of SCN1A function. METHODS: The family originates from southern Italy and contains 35 members spread over four generations. Of the 14 affected individuals, the 13 still living members (7 males, mean age 36.6 +/- 20.4) underwent a complete electroclinical evaluation. RESULTS: All 13 affected family members had febrile seizures (FS) up to the age of 6 years. Age at onset of FS ranged from 5 to 45 months with a mean age of 12.8 +/- 12.9 months. One of the 13 was affected by post-traumatic epilepsy. Three of the 13 later developed temporal lobe epilepsy (TLE) with both simple focal seizures, and also very rare focal complex or nocturnal secondary generalized tonic-clonic seizures. In two of the three patients who later developed TLE, the MRI studies revealed mesial temporal sclerosis. CONCLUSIONS: Our findings illustrate that SCN1A mutations can cause simple FS associated with TLE, which differ from the characteristic clinical spectrum of GEFS+. It is open to conjecture if this unusual phenotype might at least in part be related to the fact that M145T is the first missense mutation found in DIS1 of SCN1A.

Further evidence of genetic heterogeneity in families with autosomal dominant nocturnal frontal lobe epilepsy.

PURPOSE: Mutations in the genes encoding the alfa(2), alfa(4) and beta(2) subunits of the neuronal nicotinic acetylcholine receptor (nAChR) play a causative role in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Moreover, variations in the promoter of the corticotropic-releasing hormone gene (CRH) were also associated with ADNFLE. Here, we investigated whether nine brain-expressed genes (CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNB2, CHRNB3, CHRNB4), encoding distinct nAChR subunits, and CRH are associated with the disease in three distinct ADNFLE families from Southern Italy. METHODS: There were 14 living affected individuals (9 women), ranging in age from 14 to 57 years, pertaining to three unrelated families. Age at onset of seizures clustered around 9 years of age (range from 7 and 16 years, mean: 9.1 years+/-3.8). All affected individuals manifested nocturnal partial seizures of frontal lobe origin, which were well controlled by medications. Exon 5 of CHRNA4 and CHRNB2 genes, harboring all the known mutations, was sequenced in the probands. Then, we performed a linkage study on 13 affected and 26 non-affected individuals belonging to the three families with microsatellite markers and an intragenic polymorphisms encompassing the chromosome localization of the nAChR subunit genes and of the CRH gene. RESULTS: Mutational and linkage analyses allowed us to exclude the involvement of all known nAChR subunit genes and of the CRH gene in ADNFLE in our families. CONCLUSION: Our results further illustrate the considerable genetic heterogeneity for such a syndrome, despite the quite homogeneous clinical picture. It is therefore reasonable to hypothesize that at least another gene not belonging to the nAChR gene family, in addition to CRH, is involved in the pathogenesis of ADNFLE.

Parkinsonism and essential tremor in a family with pseudo-dominant inheritance of PARK2: an FP-CIT SPECT study.

We report a family with 5 affected individuals manifesting either essential tremor (ET), Parkinsonism, or both, consistent with pseudo-dominant inheritance of PARK2. Two homozygotes presented postural and kinetic tremor several years before the onset of Parkinsonism. Postural and kinetic tremor mimicking ET was the only feature in 1 homozygous and 2 heterozygous carriers of the mutation. Striatal dopamine transporter density was reduced in accordance with phenotype and number of mutated alleles. In 3 homozygotes and 1 heterozygote, a 2-year follow-up single photon emission computed tomography suggested no progression of nigrostriatal deficit.

Genetic heterogeneity in patients with pantothenate kinase-associated neurodegeneration and classic magnetic resonance imaging eye-of-the-tiger pattern.

We performed a detailed molecular study in two unrelated families with pantothenate kinase-associated neurodegeneration (PKAN) and the specific magnetic resonance imaging (MRI) eye-of-the-tiger pattern. In the first family with classic PKAN, linkage analysis using polymorphic markers from the PANK2 region ruled out linkage with this locus, and no mutation of the PANK2 gene was found. In the second family with atypical PKAN, we identified a novel homozygous C-to-T transition at nucleotide 1069 of the PANK2 gene, which resulted in an arginine to tryptophane substitution at codon 357. As far as we are aware, this is the first case of classic PKAN with the specific MRI eye-of-the-tiger pattern not carrying a PANK2 mutation. Therefore, the present observation reinforces the notion of the phenotypic and genetic heterogeneity in PKAN.

Identification of an Nav1.1 sodium channel (SCN1A) loss-of-function mutation associated with familial simple febrile seizures.

Febrile seizures (FS) affect 5-12% of infants and children up to 6 years of age. There is now epidemiological evidence that FS are associated with subsequent afebrile and unprovoked seizures in approximately 7% of patients, which is 10 times more than in the general population. Extensive genetic studies have demonstrated that various loci are responsible for familial FS, and the FEB3 autosomal-dominant locus has been identified on chromosome 2q23-24, where the SCN1A gene is mapped. However, gene mutations causing simple FS have not been found yet. Here we show that the M145T mutation of a well conserved amino acid in the first transmembrane segment of domain I of the human Na(v)1.1 channel alpha-subunit cosegregates in all 12 individuals of a large Italian family affected by simple FS. Functional studies in mammalian cells demonstrate that the mutation causes a 60% reduction of current density and a 10-mV positive shift of the activation curve. Thus, M145T is a loss-of-function mutant. These results show that monogenic FS should also be considered a channelopathy.

DJ-1 mutations and parkinsonism-dementia-amyotrophic lateral sclerosis complex.

DJ-1 gene mutations have been found to cause early-onset Parkinson's disease. We report a family from southern Italy with three brothers affected by early-onset parkinsonism, dementia, and amyotrophic lateral sclerosis. Molecular analysis of the DJ-1 gene in two living patients showed a novel homozygous mutation in exon 7 (E163K) and a new homozygous mutation (g.168_185dup) in the promoter region of the gene. Both mutations cosegregated with the disease and were detected in a heterozygous state in the patients' mother and their healthy siblings. Our findings expand the spectrum of clinical presentations associated with mutations in DJ-1 gene.