Objective To develop an isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method for determining the concentration of 5-methyltertrahydrofolate (5MT) in human serum.Methods After qualitative analysis of 5MT reference,its purity was identified by two methods with different principle,i.e.,mass balance and quantitative nuclear magnetic resonance.The definite value and uncertainty of 5MT concentrations in 6 serum samples containing same level of 5MT were assessed by ultra performance liquid chromatography-triple quadrupole tandem mass spectrometer equipped with electrospray ionization in monitoring mode of multiple reactions,in which 13C5-5MT was used as the internal standard.The precision,accuracy,limit of detection (LOD) and limit of quantification (LOQ) of the method were also evaluated.Results After qualitative analysis for confirming the principal component in the sample,the purity of 5MT reference was measured as 76.65％ by mass balance and quantitative nuclear magnetic resonance method.The definite value of 5MT in the serum sample was 8.22 ng/mL,the extended uncertainty was 0.54 ng/mL,LOD was 0.05 ng/mL,LOQ was 0.50 ng/mL and the repeatability and inter-batch precision CV were within 5.0％.The accuracy of measured results was verified by using NIST SRM 1955 since all the values of 3 levels were within the range of certified value with the extended uncertainty.Conclusion A accurately quantitative determination for the concentration of 5 MT in human serum with high specificity and sensitivity was established by using ID-LC-MS/MS method.

OBJECTIVETo clarify the natural course and explore impact factors of distant metastasis in rectal cancer patients who received total mesorectal excision(TME) following neoadjuvant chemoradiotherapy (CRT).

METHODSBetween Januray 2008 and December 2013, 317 patients with locally advanced rectal cancer who underwent radical surgical resection following neoadjuvant CRT (pre- and postoperative simple fluorouracil or fluorouracil combined with oxaliplatin plus preoperative three dimensional conformal radiotherapy) at Department of Colorectal Surgery in Fujian Medical University Union Hospital were included. Univariate analysis and Cox regression were performed to evaluate the clinicopathological parameters that may be associated with distant metastasis.

RESULTSDuring a median follow-up of 39 months(range 15 - 89 months), 72 patients(22.7%) had disease recurrence, including local recurrence in 8 patients, and distant metastasis in 67 patients (among whom 3 patients had both). Distant metastasis occurred in 86.5%(58/67) patients during the first three years after surgery. The 3-year cumulative distant metastatic rate in all the patients was 22.4%. The 5-year overall survival rate in distant metastatic patient was significantly lower than that of non-distant metastatic patients following neoadjuvant CRT (36.2% vs. 81.2%, P=0.000). Univariate analysis showed that ypT stage (χ(2)=13.304, P=0.010), ypN stage(χ(2)=23.416, P=0.000), ypTNM stage (χ(2)=31.765, P=0.000) and RCRG(χ(2)=16.246, P=0.000) were associated with distant metastasis. Cox regression revealed that ypTNM stage(HR=1.959, 95% CI:1.171 ~ 3.277, P=0.010) was the only independent risk factor of distant metastasis.

CONCLUSIONSDistant metastasis is the early event during the progression in rectal cancer. ypTNM stage is the only independent risk factor of distant metastasis in locally advanced rectal cancer patients who undergo TME following neoadjuvant CRT.

Chemoradiotherapy ; Digestive System Surgical Procedures ; Fluorouracil ; therapeutic use ; Humans ; Neoadjuvant Therapy ; Neoplasm Metastasis ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Organoplatinum Compounds ; therapeutic use ; Rectal Neoplasms ; drug therapy ; pathology ; surgery ; Risk Factors ; Survival Rate

Based on the biomechanical mechanism of human upper limb, the disadvantages of traditional rehabilitation training and the current status of upper limb rehabilitation robot, a six degree of freedom, flexible adjustment, wearable upper limb rehabilitation exoskeleton design scheme is proposed. Firstly, the mechanics of each joint of the upper limb is analyzed, and the virtual prototype design of the whole mechanical structure of the upper limb rehabilitation wearable exoskeleton is carried out by using CATIA three-dimensional software. The tooth transmission of the forearm and the upper arm single row four point contact ball bearing with internal/external rotation and the shoulder flexible passive adjustment mechanism (viscoelastic damper) are innovatively designed. Then, the joints of the upper limb rehabilitation exoskeleton are analyzed, theoretical analysis and calculation of the driving torque, the selection of the motor and gearbox of each driving joint are carried out. Finally, the whole finite element analysis of the upper limb exoskeleton is carried out. The research and experimental results showed that the design scheme of the upper limb exoskeleton assist structure is highly feasible, which can help the patients with upper limb paralysis and motor dysfunction self-rehabilitation.
Biomechanical Phenomena ; Exoskeleton Device ; Humans ; Robotics ; Stroke Rehabilitation ; Torque ; Upper Extremity ; Wearable Electronic Devices

Since the discovery of the C9ORF72 gene in 2011, great advances have been achieved in its genetics and in identifying its role in disease models and pathological mechanisms; it is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS patients with C9ORF72 expansion show heterogeneous symptoms. Those who are C9ORF72 expansion carriers have shorter survival after disease onset than non-C9ORF72 expansion patients. Pathological and clinical features of C9ORF72 patients have been well mimicked via several models, including induced pluripotent stem cell-derived neurons and transgenic mice that were embedded with bacterial artificial chromosome construct and that overexpressing dipeptide repeat proteins. The mechanisms implicated in C9ORF72 pathology include DNA damage, changes of RNA metabolism, alteration of phase separation, and impairment of nucleocytoplasmic transport, which may underlie C9ORF72 expansion-related ALS/FTD and provide insight into non-C9ORF72 expansion-related ALS, FTD, and other neurodegenerative diseases.

Autophagy is an evolutionarily-conserved self-degradative process that maintains cellular homeostasis by eliminating protein aggregates and damaged organelles. Recently, vesicle-associated membrane protein-associated protein B (VAPB), which is associated with the familial form of amyotrophic lateral sclerosis, has been shown to regulate autophagy. In the present study, we demonstrated that knockdown of VAPB induced the up-regulation of beclin 1 expression, which promoted LC3 (microtubule-associated protein light chain 3) conversion and the formation of LC3 puncta, whereas overexpression of VAPB inhibited these processes. The regulation of beclin 1 by VAPB was at the transcriptional level. Moreover, knockdown of VAPB increased autophagic flux, which promoted the degradation of the autophagy substrate p62 and neurodegenerative disease proteins. Our study provides evidence that the regulation of autophagy by VAPB is associated with the autophagy-initiating factor beclin 1.
Autophagy ; drug effects ; physiology ; Beclin-1 ; genetics ; metabolism ; Cell Line, Transformed ; Gene Expression Regulation ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microtubule-Associated Proteins ; genetics ; metabolism ; R-SNARE Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; RNA-Binding Proteins ; genetics ; metabolism ; Transfection

GGGGCC repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). It has been reported that hexanucleotide repeat expansions in C9ORF72 produce five dipeptide repeat (DPR) proteins by an unconventional repeat-associated non-ATG (RAN) translation. Within the five DPR proteins, poly-PR and poly-GR that contain arginine are more toxic than the other DPRs (poly-GA, poly-GP, and poly-PA). Here, we demonstrated that poly-PR peptides transferred into cells by endocytosis in a clathrin-dependent manner, leading to endoplasmic reticulum stress and cell death. In SH-SY5Y cells and primary cortical neurons, poly-PR activated JUN amino-terminal kinase (JNK) and increased the levels of p53 and Bax. The uptake of poly-PR peptides by cells was significantly inhibited by knockdown of clathrin or by chlorpromazine, an inhibitor that blocks clathrin-mediated endocytosis. Inhibition of clathrin-dependent endocytosis by chlorpromazine significantly blocked the transfer of poly-PR peptides into cells, and attenuated poly-PR-induced JNK activation and cell death. Our data revealed that the uptake of poly-PR undergoes clathrin-dependent endocytosis and blockade of this process prevents the toxic effects of synthetic poly-PR peptides.