Differentiation Between Amyotrophic Lateral Sclerosis and Mimics Using Quantitative Analysis of Fasciculation with Muscle Ultrasound

Objective To determine the diagnostic accuracy of the intensity of fasciculation evaluated by muscle ultrasound in the differential diagnosis of amyotrophic lateral sclerosis(ALS).Methods We prospectively recruited patients who had ALS and neuropathy-radiculopathy attending Peking Union Medical College Hospital from 2017 to 2020.Healthy adults from a community were recruited as healthy controls.Muscle strength was assessed using the Medical Research Council(MRC)scale.At the first visit to the hospital,patients were assessed for maximal grade of fasciculations,total fasciculation score,and fasciculation grade in 16 muscle groups of bilateral upper and lower limbs using ultrasonography.The sensitivity and specificity of maximal grade of fasciculations,total fasciculation score,and fasciculation grade for the diagnosis of ALS were assessed by receiver operating characteristic analyses.Results The percentage of limb muscles with a maximal fasciculation grade higher than grade 2 in ALS patients and neuropathy-radiculopathy patients was 84.9%and 9.8%,respectively(χ^(2)=172.436,P<0.01).Of the 16 limb muscles detected,the total fasciculation score[median(interquartile range)]was 29(15,41)in ALS patients and 3(0,8)in neuropathy-radiculopathy patients(Z=9.642,P<0.001).Remarkable fasciculations were seen in ALS patients whose muscles with a MRC score ranging from 2 to 4,followed by patients with MRC score 5,and then in those with MRC score 0 and 1.The sensitivity and specificity of total fasciculation score for diagnosis of ALS were 80.6%and 93.4%,respectively(cut-off value 14).In patients with ALS,for muscles with MRC score 4 and 5,the percentage of muscles with fasciculation grades≥3 was 42.3%and 24.1%respectively,while in neuropathy-radiculopathy patients,the percentage for muscles with MRC score 4 and 5 was only 1.7%and 0,respectively.Conclusion A combined analysis of fasciculation intensity and MRC score of the limb muscles may be helpful for differential diagnosis of ALS.

Alreration of nuclear matrix-intermediate filament system and differential expression of nuclear matrix proteins during human hepatocarcinoma cell differentiation

AIM： To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells. METHODS： Cells cultured with or without 5 × 10^-3 mmol/L of hexamethylene bisacetamide （HMBA） on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin. The peptides were analyzed by matrix-assisted laser- desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS）. Data were submitted for database searching using Mascot tool （www.matrixscience.com）. RESULTS： The nuclear matrix （NM） and intermediate filament （IF） in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly. The nuclear lamina was non-uniform, and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA, the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments, including highly branched utrathin filaments could also be seen in the regular meshwork. The connection between the two kinds of filaments and the relatively thin, condensed and sharply demarcated lamina composed of intermediate- sized filaments was relatively fastened. Meanwhile, 21 NM proteins changed remarkably during SMMC-7721 cell differentiation. Four proteins, i.e. mutant Pystl, hypothetical protein, nucleophosminl, and LBP were downregulated, whereas four other proteins, eIF6, p44 subunit, 13-tubulin, and SIN3B were upregulated with the last one, SR2/ASF found only in the differentiated SMMC-7721 cells. CONCLUSION： The induced differentiation of SMMC-7721 cells by HMBA is accompanied by the configurational changes of nuclear matrix-intermediate filament （NM-IF） system and the compositional changes of nuclear matrix protein expression. These changes may be important morphological or functional indications of the cancer cell reversion.

Contribution and underlying mechanisms of CXCR4 overexpression in patients with systemic lupus erythematosus

Aberrant expression of CXCR4 has been indicated to play a role in the pathogenesis of systemic lupus erythematosus(SLE),but the mechanism of CXCR4 dysregulation in SLE is unclear.This study is aimed to explore the clinical significance and possible mechanisms of abnormal CXCR4 expression on B cells from patients with untreated SLE.Expression of CXCR4 on peripheral B cells was determined by flow cytometry and western blotting.Freshly isolated B cells were cultured with exogenous interleukin 21(IL-21)in the presence or absence of CD40 ligand(CD40L)plus anti-IgM antibody(aIgM),and changes in CXCR4 expression were detected.Involvement of phosphatidylinositol 3 kinase(PI3K)/Akt and Janus kinase/Signal transducer and activator of transcription(JAK/STAT)signaling pathways was assessed by adding blocking agents Ly294002 and AG490.Since CD63 is reported to mediate endosomal recruitment of CXCR4 and BCL6 is capable of silencing CD63 gene transcription,we also measured BCL6 and CD63 gene transcription with real-time PCR.It was shown that CXCR4 expression on B cells was significantly upregulated in SLE patients,especially in those with lupus nephritis,and was positively correlated with SLE Disease Activity Index scores and negatively with the serum complement 3 levels(Po0.05).Downregulation of CXCR4 by IL-21 was intact.In contrast,a similar effect of aIgM plus CD40L in downregulating CXCR4 expression was defective in SLE patients but was restored by co-stimulation with IL-21 in vitro.Both Ly294002 and AG490 promoted downregulation of surface CXCR4 expression on B cells from SLE patients(P=0.078 and P=0.064).Furthermore,B cells from SLE patients exhibited diminished CD63 mRNA and enhanced BCL6 mRNA expression(both Po0.05).To sum up,CXCR4 was overexpressed on SLE B cells,positively correlating with disease activity and kidney involvement.Overactivation of the PI3K/Akt and JAK/STAT pathways as well as defective CD63 synthesis may contribute to CXCR4 dysregulation in SLE.

Single-fiber Electromyography in the Extensor Digitorum Communis for the Predictive Prognosis of Ocular Myasthenia Gravis： A Retrospective Study of 102 Cases

Background： Single-fiber electromyography （SFEMG） abnormality in the extensor digitorum communis （EDC） was reported in ocular myasthenia gravis （OMG）, which indicated subclinical involvement beyond extraocular muscles in OMG patients. The relationship between the abnormal findings of SFEMG in EDC and the probability for OMG to develop generalized myasthenia gravis （GMG） is unknown, This retrospective study aimed to determine the predictive value of abnormality of SFEMG in EDC of OMG patients. Methods： One-hundred and two OMG patients underwent standard clinical diagnosis process and SFEMG test in EDC muscle when diagnosed and were clinically followed up for 5 years. The SFEMG data were compared between different clinical groups according to thymus status, onset age, and different outcome of OMG developing. Chances of progressing to GMG were compared between two different groups according to SFEMG and repetitive nerve stimulation （RNS） results, acetylcholine receptor antibody （AchRAb） titer, thymus status, and onset age. Results： Abnormal SFEMG results were observed in 84 （82.4%） patients. The mean jitter, percentage of jitter 〉55 Its （%）, and blocking were higher in OMG patients than in healthy volunteers. There were no statistical differences in jitter analysis between thymoma group and non-thymoma group （P = 0.65）, or between the later OMG group and the later GMG group （P = 0.31）, including mean jitter, percentage of jitter 〉55 Its （%）, and blocking. Elderly group （〉45 years old） had a higher mean jitter than younger group （t = 2.235, P = 0.028）. Total 55 OMG developed GMG, including 47 in abnormal SFEMG group while 8 in normal SFEMG group. There was no statistical difference in the conversion rates between the two groups （x^2 = 0.790, P = 0.140）. RNS abnormality, AchRab titer, or onset age had no correlation with OMG prognosis （P = 0.150, 0.070, 0.120, respectively） while thymoma did （x^2 = 0.510, P = 0.020）. Conclusion： SFEMG test in the EDC showed high abnormality in OMG, suggesting subclinical involvement other than extraocular muscles. Nevertheless, the abnormal jitter analysis did not predict the prognosis of OMG according to clinical follow-up.

Changes of Nuclear Matrix Proteins Following the Differentiation of Human Osteosarcoma MG-63 Cells

Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide （HMBA）. Their nuclear matrix proteins （NMPs） were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOF-MS analysis, and were submitted for NCBI database searches by Mascot tool. There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.

Quantitating Changes in Jitter and Spike Number Using Concentric Needle Electrodes in Amyotrophic Lateral Sclerosis Patients

Background： Single-fiber electromyography （SFEMG） has been suggested as a quantitative method for supporting chronic partial denervation in amyotrophic lateral sclerosis （ALS） by the revised El Escorial criteria. Although concentric needle （CN） electrodes have been used to assess jitter in myasthenia gravis patients and healthy controls, there are few reports using CN electrodes to assess motor unit instability and denervation in neurogenic diseases. The aim of this study was to determine whether quantitative changes in jitter and spike number using CN electrodes could be used for ALS studies. Methods： Twenty-seven healthy controls and 23 ALS patients were studied using both CN and single-fiber needle （SFN） electrodes on the extensor digitorum communis muscle with an SFEMG program. The SFN-jitter and SFN-fiber density data were measured using SFN electrodes. The CN-jitter and spike number were measured using CN electrodes. Results： The mean CN-j itter was significantly increased in ALS patients （47.3 ±17.0 μs） than in healthy controls （27.4 ± 3.3 las） （P 〈 0.001 ）. Besides, the mean spike number was significantly increased in ALS patients （2.5 ± 0.5） than in healthy controls （1.7 ± 0.3） （P 〈 0.001）. The sensitivity and specificity in the diagnosis of ALS were 82.6% and 92.6% for CN-jitter （cut-offvalue： 32 gs）, and 91.3% and 96.3% for the spike number （cut-off value： 2.0）, respectively. There was no significant difference between the SFN-jitter and CN-jitter in ALS patients; meanwhile, there was no significant difference between the SFN-jitter and CN-jitter in healthy controls. Conclusion： CN-jitter and spike number could be used to quantitatively evaluate changes due to denervation-reinnervation in ALS.

A report on paraneoplastic motor neuron disease

To the Editor: Motor neuron disease (MND) is marked by upper (UMN) or lower (LMN) motor neuron signs and symptoms,[1] which in various combinations form the basis for MND classification. In this regard, amyotrophic lateral sclerosis (ALS) is the most common subtype.[2] Although otherwise deemed an untreatable degenerative disease, there is now increasing evidence of more heterogeneous pathogenesis, signaling a link between MND and paraneoplastic syndromes.[3].