Vacuolated PAS-Positive Lymphocytes on Blood Smear: An Easy Screening Tool and a Possible Biomarker for Monitoring Therapeutic Responses in Late Onset Pompe Disease (LOPD).

Background: Primary aim was to investigate the diagnostic value of PAS-positive vacuolated lymphocytes on blood smear in Late Onset Pompe Disease (LOPD) patients and, secondly, to evaluate its potential utility in monitoring treatment effects. Methods: We examined blood smear of 26 LOPD patients. We evaluated 10 treated and 16 untreated LOPD patients. Among the latter group, 7 patients later initiated ERT and were tested again 6 months after start. Blood smear was also sampled from 82 controls and 19 patients with other muscle glycogenoses (MGSDs). PAS staining was used to evaluate: (1) presence of lymphocytes with glycogen-filled vacuoles, (2) quantification of vacuolated lymphocytes. Results: We found that PAS-positive lymphocytes were significantly higher in LOPD patients than in controls or other MGSDs (p < 0.05 and p < 0.001, respectively). ROC curve for discriminating between untreated LOPD patients and controls yielded an AUC of 1.00 (95%CI 1.00-1.00; p < 0.0001). PAS-positive lymphocyte cutoff level of >10 yielded sensitivity of 100% (95%CI 78-100%), specificity of 100% (95%CI 96-100%), and positive predictive value of 100%. Patients studied before and after ERT showed a dramatic decrease of PAS-positive vacuolated lymphocytes number (p = 0.016). In other MGSDs, PAS-positive lymphocytes were significantly lower that untreated LOPD patients but higher than controls. Conclusions: Our data suggest that the Blood Smear Examination (BSE) for PAS-positive lymphocytes quantification could be used as a simple and sensitive test for a quick screening of suspected Pompe disease. The quantification of vacuolated lymphocytes appears to be also a valuable tool for monitoring the efficacy of treatment in LOPD patients.

Myasthenia Gravis: Unusual Presentations and Diagnostic Pitfalls.

BACKGROUND: Myasthenia gravis (MG) is an autoimmune disorder presenting with fluctuating, fatigable muscle weakness. Initial symptoms classically involve ocular and proximal limb muscles. Rarely, MG may onset with unusual features, so it can be misdiagnosed with other neuromuscular diseases. OBJECTIVE: To describe unusual and atypical presentations of MG in a large cohort of patients, considering and discussing diagnostic difficulties and pitfalls. METHODS: We report on 21 out of 508 MG patients, coming to our department in the last 27 years and presenting with atypical or unusual features. The diagnosis was achieved performing a careful clinical examination, a proper neurophysiological assessment, the neostigmine test, the AChR and MuSK antibodies assay and chest CT-scan. RESULTS: Patients with atypical/unusual MG onset were the 4.4% of all MG patients population. We describe seven different clinical categories: asymmetric distal upper limbs weakness, foot drop, isolated triceps brachii weakness and foot drop, post exertional axial weakness with dropped head, acute facial dyplegia, limb-girdle MG and MG with sudden lower limbs weakness and recurrent falls. CONCLUSIONS: Atypical and unusual presentations may increase the risk to misdiagnose or delay MG diagnosis. Isolated limb-girdle presentation is the most frequent atypical form in our series.

Impaired Muscle Mitochondrial Biogenesis and Myogenesis in Spinal Muscular Atrophy.

IMPORTANCE: The important depletion of mitochondrial DNA (mtDNA) and the general depression of mitochondrial respiratory chain complex levels (including complex II) have been confirmed, implying an increasing paucity of mitochondria in the muscle from patients with types I, II, and III spinal muscular atrophy (SMA-I, -II, and -III, respectively). OBJECTIVE: To investigate mitochondrial dysfunction in a large series of muscle biopsy samples from patients with SMA. DESIGN, SETTING, AND PARTICIPANTS: We studied quadriceps muscle samples from 24 patients with genetically documented SMA and paraspinal muscle samples from 3 patients with SMA-II undergoing surgery for scoliosis correction. Postmortem muscle samples were obtained from 1 additional patient. Age-matched controls consisted of muscle biopsy specimens from healthy children aged 1 to 3 years who had undergone analysis for suspected myopathy. Analyses were performed at the Neuromuscular Unit, Istituto di Ricovero e Cura a Carattere Scientifico Foundation Ca' Granda Ospedale Maggiore Policlinico-Milano, from April 2011 through January 2015. EXPOSURES: We used histochemical, biochemical, and molecular techniques to examine the muscle samples. MAIN OUTCOMES AND MEASURES: Respiratory chain activity and mitochondrial content. RESULTS: Results of histochemical analysis revealed that cytochrome-c oxidase (COX) deficiency was more evident in muscle samples from patients with SMA-I and SMA-II. Residual activities for complexes I, II, and IV in muscles from patients with SMA-I were 41%, 27%, and 30%, respectively, compared with control samples (P < .005). Muscle mtDNA content and cytrate synthase activity were also reduced in all 3 SMA types (P < .05). We linked these alterations to downregulation of peroxisome proliferator-activated receptor coactivator 1α, the transcriptional activators nuclear respiratory factor 1 and nuclear respiratory factor 2, mitochondrial transcription factor A, and their downstream targets, implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P < .001), COX4 (26.6%; P < .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P < .001). Conversely, the levels of expression of 3 myogenic regulatory factors-muscle-specific myogenic factor 5, myoblast determination 1, and myogenin-were higher in muscles from patients with SMA compared with muscles from age-matched controls (P < .05). CONCLUSIONS AND RELEVANCE: Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes.

Yellow laser performance of Dy³âº in co-doped Dy,Tb:LiLuF4.

We present laser results obtained from a Dy³âº-Tb³âº co-doped LiLuF4 crystal, pumped by a blue emitting InGaN laser diode, aiming for generation of a compact 578 nm source. We exploit the yellow Dy³âº transition 4F(9/2)⇒6H(13/2) to generate yellow laser emission. The lifetime of the lower laser level is quenched, via energy transfer, to co-doped Tb³âº ions in the fluoride crystal. We report the growth technique, spectroscopic study, and room temperature continuous wave laser results in a hemispherical cavity at 574 nm, and with a highly reflective output coupler at 578 nm. A yellow laser at 578 nm is very relevant for metrological applications, in particular for pumping of the forbidden ¹S0-³P0 ytterbium clock transition, which is recommended as a secondary representation of the second in the international system of units.

Multiple polarization orange and red laser emissions with Pr:BaY2F8.

We investigated the polarization of continuous-wave laser emission in the orange region, at 607 nm, and in the red region, at 639 nm and 643 nm, from a Pr:BaY2F8 (Pr:BYF) crystal, pumped by a 445 nm laser diode. We achieved linearly polarized emission along two optic axes of the crystal by changing its orientation with respect to the pump. Simultaneous emission of two orthogonal linear polarizations was observed in the orange region, at the same wavelength, and in the red region, with concurrent emission from the two separate lines.

Continuous-wave and Q-switched operation of a resonantly pumped Ho³âº:KY3F10 laser.

We report continuous-wave and repetitively Q-switched operation of a resonantly pumped Ho3+:KY3F10 laser at room temperature. End pumped by a Tm3+-doped silica fiber laser operating at 1938 nm, a maximum laser power of 7.8 W was obtained at a wavelength of ∼2041 nm for 21 W of absorbed pump power, corresponding to a slope efficiency of 60.7% with respect to absorbed power. At a repetition rate of 10 kHz up to 0.78 mJ, energy per pulse was demonstrated with pulse widths of 100 ns. The beam propagation factor (M2) was measured to be <1.26 at the maximum output power.

In-band pumped Ho3+:KY3F10 2 µm laser.

We report the first observation to our knowledge of room-temperature continuous-wave laser operation on the (5)I(7)→(5)I(8) transition of Ho(3+) ions in a KY(3)F(10) single crystal. Using a Tm-doped silica fiber laser operating at 1938 nm as a pump source, a maximum laser power of 1.8 W was obtained at a wavelength of ~2040 nm for 27 W of absorbed pump power with a slope efficiency of 19.1% with respect to absorbed power. At low cavity output coupling, the lasing wavelength shifted to 2060.5 nm. The beam propagation factor (M(2)) was measured to be <1.06 at the maximum output power, confirming fundamental transverse-mode (TEM(00)) operation. Performing a Caird analysis, we determined resonator round-trip losses and intrinsic slope efficiency of 30% and 43.8%, respectively.

Compact passively Q-switched diode-pumped Tm:LiLuF4 laser with 1.26 mJ output energy.

We demonstrate efficient continuous-wave (CW) and passively Q-switched Tm:LiLuF(4) laser operation near 1.9 µm. The CW slope efficiency reached 54.8% with respect to absorbed power. Stable passive Q-switching with Cr(2+):ZnS saturable absorbers resulted in minimum pulse duration of 7.6 ns and maximum pulse energy and peak power of 1.26 mJ and 166 kW, respectively.

Passively Q-switched Tm:YLF laser.

Stable passive Q-switching of a Tm: LiYF4 laser is obtained using polycrystalline Cr2+: ZnS as a saturable absorber. The achieved maximum pulse energy of 0.9 mJ and peak power of 65 kW for a pulse duration of ∼14 ns represent substantial improvement and highest values for a passively Q-switched diode-pumped Tm laser operating at ∼1.9 µm.

High-efficiency 1.9 µm Tm3+:LiLuF4 thin-disk laser.

We present the first demonstration of a 792 nm diode-pumped Tm3+:LiLuF4 thin-disk laser operation around 1.9 µm. In multimode configuration, up to 21 W of output power and a maximal slope efficiency of 49% with an optical-to-optical efficiency of 40% was demonstrated. A tuning range from 1899 nm to 1927 nm could be achieved by inserting an etalon into the cavity.

Diode-pumped Pr:BaY2F8 continuous-wave orange laser.

We report the realization of the continuous wave laser emission in the orange at 607 nm from a Pr:BaY(2)F(8) (Pr:BYF) crystal pumped by a blue GaN laser diode. A maximal output power of 78 mW is obtained in a quasi-single transverse mode beam. The effect of reabsorption losses at the laser wavelength is also evidenced.

Anti-Stokes luminescence cooling of Tm3+ doped BaY2F8.

We report laser-induced cooling with thulium-doped BaY2F8 single crystals grown using the Czochralski technique. The spectroscopic characterization of the crystals has been used to evaluate the laser cooling performance of the samples. Cooling by 3 degrees below ambient temperature is obtained in a single-pass geometry with 4.4 Watts of pump laser power at lambda = 1855 nm.

Analysis and classification of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and a chemometric approach.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a useful technique for the identification of bacteria on the basis of their characteristic protein mass spectrum fingerprint. Highly standardized instrumental analytical performance and bacterial culture conditions are required to achieve useful information. A chemometric approach based on multivariate analysis techniques was developed for the analysis of MALDI data of different bacteria to allow their identification from their fingerprint. Principal component analysis, linear discriminant analysis (LDA) and soft independent modelling of class analogy (SIMCA) were applied to the analysis of the MALDI MS mass spectra of two pathogenic bacteria, Escherichia coli O157:H7 and Yersinia enterocolitica, and the non-pathogenic E. coli MC1061. Spectra variability was assessed by growing bacteria in different media and analysing them at different culture growth times. After selection of the relevant variables, which allows the evaluation of an m/z value pattern with high discriminant power, the identification of bacteria by LDA and SIMCA was performed independently of the experimental conditions used. In order to better evaluate the analytical performance of the approach used, the ability to correctly classify different bacteria, six wild-type strains of E. coli O157:H7, was also studied and a combination of different chemometric techniques with a severe validation was developed. The analysis of spiked bovine meat samples and the agreement with an independent chemiluminescent enzyme immunoassay demonstrated the applicability of the method developed for the detection of bacteria in real samples. The easy automation of the MALDI method and the ability of multivariate techniques to reduce interlaboratory variability associated with bacterial growth time and conditions suggest the usefulness of the proposed MALDI MS approach for rapid routine food safety checks.

Hollow-fiber flow field-flow fractionation of whole blood serum.

Hollow-fiber flow field-flow fractionation is here applied to untreated, whole human blood serum. Matrix-assisted, laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) of serum fractions shows mass signals in the <30,000 M(r) range where low-abundance, serum protein components are known to be present, though a membrane of nominal 30,000 Da cutoff was employed for the fractionation device. Using diluted sera spiked with low amounts (0.06-0.1%, w/w) of an artificial mixture constituted the human adrenocorticotropic hormone fragments 18-39 (M(r)=2465.7) and 7-38 (M(r)=3659.2), and of bovine insulin (M(r)=5734), horse cytochrome c (M(r)=12384) and chicken lysozyme (M(r)=14388), a hybrid fractionation/microfiltration mechanism shows to govern the separation of the low-M(r) components.

Serum protein profiling in patients with inflammatory bowel diseases using selective solid-phase bulk extraction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemometric data analysis.

The identification of new biomarkers or a disease-related protein fingerprint for inflammatory bowel diseases (IBDs) represents a major task in the diagnosis, prognosis and pharmacological therapy. To address these issues, a simple and rapid analytical proteomic method for serum protein profiling based on selective beads-based solid-phase bulk extraction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and chemometric data analysis was developed. Serum proteins from healthy subjects (22) and patients with Crohn's disease (15) and ulcerative colitis (26) were selectively extracted according to reversed-phase (C18), strong anion-exchange (SAX) and metal ion affinity (IDA-Cu(II)) principles. This approach allowed enrichment of serum proteins/peptides due to the high interaction surface between analytes and the solid phase and high recovery due to the elution step performed directly on the MALDI-target plate. The MALDI-TOF MS serum protein profiles were acquired and, after a data pre-processing step, analyzed by linear discriminant analysis (LDA), a chemometric classification technique, in order to classify serum samples among healthy subjects and patients with inflammatory bowel diseases (IBDs). Since the high number of variables in the MALDI spectra (more than 16000 m/z values) prevents the use of LDA, the variables were reduced to 10-20 by features selection, thus allowing the evaluation of a pattern of m/z values with high discriminant power. Serum protein profiles obtained by reversed-phase extraction and the selection of 20 m/z values gave the best overall prediction ability (96.9%). The recognition of these m/z values may allow the identification of protein biomarkers involved in IBDs.

Tm-doped LiLuF4 crystal for efficient laser action in the wavelength range from 1.82 to 2.06 microm.

Room-temperature continuous-wave laser action in a novel Tm-doped LiLuF(4) crystal emitting at around 1.9 microm is demonstrated. The crystal growth process, spectroscopic measurements, and laser performance are presented for different Tm concentrations. An overall tunability extending from 1817 to 2056 nm, a slope efficiency of 46%, and a maximum output power in excess of 1.1 W are reported.

Visible laser emission of solid state pumped LiLuF(4):Pr(3+).

In the present work we report on the growth, spectroscopy and laser results of LiLuF(4):Pr(3+) 1.25% in the melt. Room temperature polarized absorption and emission spectra have been recorded and the decay time of the (3)P0 manifold has been measured. Finally efficient room temperature laser emission have been obtained at 522.8 nm, 607.25 nm, 640.17 nm and 721.5 nm under 480 nm pumping by means of an optically pumped semiconductor laser.

High efficiency room temperature laser emission in heavily doped Yb:YLF.

We report the tunable, CW and quasi CW laser operation at room temperature of an highly doped (30% at.) Yb:YLF crystal longitudinally pumped by a fiber coupled laser diode array. The CW output power is 1.15 W vs. an absorbed pump power of 6 W, with a slope efficiency of 31%. In quasi-CW operation (20% duty factor @10 Hz) an output power of 4 W with an absorbed power of 9.5 W, and a slope efficiency of 62.8% were obtained. The tuning range spans from 1022 to 1075 nm. To our knowledge, these are among the best experimental results obtained at room temperature with Yb doped YLF.

Proteomic-based analysis of nuclear signaling: PLCbeta1 affects the expression of the splicing factor SRp20 in Friend erythroleukemia cells.

An extensive body of evidence links inositide-specific phospholipase C (PLC) to the nucleus and the main isoform located in the nucleus is PLCbeta(1). Constitutive overexpression of nuclear PLCbeta(1) has been previously shown to inhibit Friend erythroleukemia cells differentiation and to induce cell cycle progression targeting cyclin D3. The aim of this study was to identify new proteins regulated by PLCbeta(1) overexpression, given the role exerted by its signaling in the nucleus during cell growth and differentiation. To identify novel downstream effectors of nuclear PLCbeta(1)-dependent signaling in Friend erythroleukemia cells, we performed the high-resolution 2-DE-based proteomic analysis. Using a proteomic approach we found that SRp20, a member of the highly conserved SR family of splicing regulators, was down-regulated in cells overexpressing nuclear PLCbeta(1) as compared with wild-type cells. Reduction in SRp20 was confirmed by 2-D Western blotting. Moreover, we have shown that nuclear PLCbeta(1) is bound to the SRp20 splicing factor. Indeed, by immunoprecipitation and subcellular fractioning, we have demonstrated that endogenous PLCbeta(1) and SRp20 physically interact in the nucleus. Here we show the existence of a PLCbeta(1)-specific target, the splicing factor SRp20, whose expression is specifically down-regulated by the nuclear signaling evoked by PLCbeta(1).

Hollow-fiber flow field-flow fractionation: a gentle separation method for mass spectrometry of native proteins.

Low-impact ionization sources like electrospray ionization (ESI) and matrix-assisted, laser desorption/ionization (MALDI) equipped with time-of-flight (TOF) mass analyzers provide intact protein analysis over a very wide molar mass range. ESI/TOFMS provides also indications on the higher-order structure of intact proteins and non-covalent protein complexes. However, direct analysis of intact proteins mixtures in real samples shows limited success, mainly because spectra become very complex to interpret. This is also due to sample contaminants, and to the mechanism of competitive ionization in ESI or MALDI. Rapid and efficient sample clean-up and separation methods can significantly enhance the power of TOFMS for intact protein analysis. However, if protein native conditions want to be maintained, the methods should affect neither the three-dimensional structure nor the non-covalent chemistry of the proteins. Reversed-phase (RP) HPLC, size-exclusion chromatography (SEC), and capillary zone electrophoresis (CZE) are on-line or off-line coupled to ESI/TOFMS or MALDI/TOFMS. In fact, these separation methods often show limitations when applied to the analysis of native proteins. Organic modifiers and saline buffers are required in the case of RP HPLC or CZE. They can induce protein degradation or affect ionization when MS is performed after separation. High voltages used in CZE can contribute to alter proteins from their native form. In the case of high molar mass proteins, SEC is scarcely selective, and barely able to detect protein aggregates. Sample entanglement/adsorption on the stationary phase can also occur.