[Comparison of etoposide combined with G-CSF and cyclophosphamide combined with G-CSF in the mobilization of autologous peripheral blood stem cells in patients with multiple myeloma].

Objective: The effect and safety of etoposide combined with G-CSF were compared with those of cyclophosphamide combined with G-CSF in autologous peripheral blood mobilization in patients with multiple myeloma (MM) . Methods: Patients with MM who received autologous peripheral blood stem cell mobilization and collection in the Department of Hematology, Beijing Chaoyang Hospital Affiliated to Capital Medical University from January 1, 2020 to July 31, 2023 were included. A total of 134 patients were screened by propensity score matching technology according to a 1∶1 ratio. A total of 67 cases were each treated with ETO combined with G-CSF mobilization scheme (ETO group) and CTX combined with G-CSF mobilization scheme (CTX group). Their clinical data were retrospectively analyzed. Results: ①Collection results: the ETO and CTX groups [2 (1-3) d vs 2 (1-5) d; P<0.001] and CD34(+) cells [7.62×10(6) (2.26×10(6)-37.20×10(6)) /kg vs 2.73×10(6) (0.53×10(6)-9.85×10(6)) /kg; P<0.001] were collected. The success rate of collection was 100.0% (67/67) versus 76.1% (51/67) (P<0.001). Excellent rate of collection was 82.1% (55/67) versus 20.9% (14/67; P<0.001). Two patients in the ETO group switched protocols after 1 day of collection, and 11 patients in the CTX group switched protocols after 1-2 days of collection. ②Adverse reactions: granular deficiency with fever (21.5%[14/65] vs. 10.7%[6/56]; P=0.110), requiring platelet transfusion [10.7% (7/65) vs 1.8% (1/56) ; P=0.047]. ③Until the end of follow-up, 63 cases in the ETO group and 54 cases in the CTX group have undergone autologous transplantation. The median number of CD34(+) cells infused in the two groups was 4.62×10(6) (2.14×10(6)-19.89×10(6)) /kg versus 2.62×10(6) (1.12×10(6)-5.31×10(6)) /kg (P<0.001), neutrophil implantation time was 11 (9-14) d versus 11 (10-14) d (P=0.049), and platelet implantation time was 11 (0-19) d vs. 12 (0-34) d (P=0.035). One case in the CTX group experienced delayed platelet implantation. Conclusion: The mobilization scheme of etoposide combined with G-CSF requires relatively platelet transfusion, but the collection days are shortened. The collection success rate, excellent rate, and the number of CD34(+) cells obtained are high, and the neutrophil and platelet engraftment is accelerated after transplantation.

[Prognostic value of the Second Revision of the International Staging System in patients with newly diagnosed transplant-eligible multiple myeloma].

Objective: To verify the predictive value of the Second Revision of the International Staging System (R2-ISS) in newly diagnosed patients with multiple myeloma (MM) who underwent first-line autologous hematopoietic stem cell transplantation (ASCT) in a new drug era in China. Methods: This multicenter retrospective cohort study enrolled patients with newly diagnosed MM from three centers in China (Beijing Chao-Yang Hospital, Capital Medical University; the First Affiliated Hospital, Sun Yat-Sen University, and the Second Affiliated Hospital of Naval Medical University) from June 2008 to June 2018. A total of 401 newly diagnosed patients with MM who were candidates for ASCT were enrolled in this cohort, all received proteasome inhibitor and/or immunomodulator-based induction chemotherapy followed by ASCT. Baseline and follow-up data were collected. The patients were regrouped using R2-ISS. Progression-free survival (PFS) and overall survival (OS) were analyzed. The Kaplan-Meier method was used to analyze the survival curve and two survival curves were compared using the log-rank test. Cox regression analysis were performed to analyze the relationship between risk factors and survival. Results: The median age of the patients was 53 years (range 25-69 years) and 59.5% (240 cases) were men. Newly diagnosed patients with renal impairment accounted for 11.5% (46 cases). According to Revised-International Staging System (R-ISS), 74 patients (18.5 %) were diagnosed with stage Ⅰ, 259 patients (64.6%) with stage Ⅱ, and 68 patients (17.0%) with stage Ⅲ. According to the R2-ISS, the distribution of patients in each group was as follows: 50 patients (12.5%) in stage Ⅰ, 95 patients (23.7%) in stage Ⅱ, 206 patients (51.4%) in stage Ⅲ, and 50 patients (12.5%) in stage Ⅳ. The median follow-up time was 35.9 months (range, 6-119 months). According to the R2-ISS stage, the median PFS in each group was: 75.3 months for stage Ⅰ; 62.0 months for stage Ⅱ, 39.2 months for stage Ⅲ, and 30.3 months for stage Ⅳ; and the median OS was not reached, 86.6 months, 71.6 months, and 38.5 months, respectively. There were statistically significant differences in PFS and OS between different groups (both P<0.001). Multivariate Cox regression analysis showed that stages Ⅲ and Ⅳ of the R2-ISS were independent prognostic factors for PFS (HR=2.37, 95%CI 1.30-4.30; HR=4.50, 95%CI 2.35-9.01) and OS (HR=4.20, 95%CI 1.50-11.80; HR=9.53, 95%CI 3.21-28.29). Conclusions: The R2-ISS has significant predictive value for PFS and OS for transplant-eligible patients with MM in the new drug era. However, the universality of the R2-ISS still needs to be further verified in different populations.

[The prognostic value of dynamic minimal residual disease after autologous hematopoietic stem cell transplantation in patients with multiple myeloma in novel-agent era].

Objective: To investigate the clinical prognostic value of dynamic minimal residual disease (MRD) after autologous hematopoietic stem cell transplantation (AHSCT) in patients with multiple myeloma (MM). Methods: Patients with MM who underwent AHSCT in Beijing Chao-Yang Hospital from February 2016 to December 2019 were enrolled in this study. All the patients in the study had complete baseline data at the diagnosis. AHSCT was performed after induction chemotherapy. Response evaluation was performed after induction therapy. All the patients were assessed at approximately 100 days after AHSCT. Bone marrow MRD by NGF was performed every three months and dynamically monitored for at least 12 months. All the patients were divided into different groups according to cytogenetics and MRD status. Survivals in different groups were analyzed by IBM SPSS 22.0 statistical software. Results: A total of 150 patients with MM were enrolled in this study at last, including 66 patients in the cytogenetic standard risk group and 84 patients in the cytogenetic high-risk group. The median age was 54 years (range 30-68 years) and 87 male patients (58.0%) was in the study. The median follow-up was 36 months (range 16-72 months). Patients in the standard-risk group had better clinical prognosis than those in the high-risk group [median PFS in the standard-risk group was not achieved, and median PFS in the high-risk group was 45 months (P<0.001); median OS of both groups was not reached, and the estimated 3-year OS rate of the standard-risk group and the high-risk group was 95.2% and 78.9%, respectively (P=0.001)]. According to MRD status of patients, patients in each group were divided into three subgroups: persistent positive (Ppos), transient negative (Tneg) and persistent negative (Pneg). The median OS and median PFS of all subgroups in the standard-risk group was not reached (P=0.324 and P=0.086). In high-risk group, the median OS of MRD Pneg subgroup was not reached, and the estimated 3-year OS rate was 100%; The median OS of MRD Ppos subgroup was 52 months, and MRD Tneg subgroup only 31 months (P=0.002); the median PFS of MRD Pneg group was not reached, and the estimated 3-year PFS rate was 85.4%; median PFS of MRD Ppos subgroup was 40 months, and MRD Tneg subgroup only 17 months (P=0.001). Conclusions: MRD Pneg might overcome the adverse prognosis of MM patients with high-risk cytogenetics. However, MRD Tneg might be a poor prognostic factor for the patients with cytogenetic high-risk MM.

[Autologous stem cell transplantation improve the survival of newly diagnosed multiple myeloma patients].

Objective: To evaluate the effect of autologous stem cell transplantation (auto-HSCT) on treatment remission and survival of newly diagnosed multiple myeloma (MM) patients. Methods: A total of 243 new diagnosed MM patients (age ≤65 years) who had received auto-HSCT were selected, and 176 MM patients (age ≤65 years) who had not received auto-HSCT were selected as the control group to evaluate the effect of auto-HSCT on the remission and survival. To balance the distribution of prognostic factors between auto-HSCT and non-auto-HSCT patients, the propensity score matching technique was used to reduce the bias between groups in a 1∶1 scale, 64 in each group, and correlation analysis was performed. Results: A total of 128 patients (64 cases in each group) were screened by propensity score matching analysis. 64 patients received auto-HSCT after induction therapy. After auto-HSCT, 24 patients (37.5%) obtained sCR, 16 patients (25.0%) obtained CR, 15 patients (23.4%) obtained VGPR, and 9 patients (14.1%) obtained PR. The efficacy of patients with auto-HSCT was significantly better than that of non-auto-HSCT patients (P=0.032) . Progression-free survival (PFS) and overall survival (OS) were significantly longer in auto-HSCT patients compared with non-auto-HSCT patients[PFS: 42.2 (95% CI 29.9-54.5) months vs 22.4 (95% CI 17.1-27.7) months, P=0.007; OS: 87.6 (95% CI 57.3-117.9) months vs 53.9 (95% CI 36.1-71.7) months, P=0.011]. Multivariate analysis confirmed that auto-HSCT had a favorable effect on OS (HR=0.448, 95%CI 0.260-0.771, P=0.004) and PFS (HR=0.446, 95%CI 0.280-0.778, P=0.003) . Conclusion: These results demonstrated that auto-HSCT was a favorable prognostic factor for newly diagnosed MM patients.

Neuronal architecture of the second-order CO2 pathway in the brain of a noctuid moth.

Many insects possess the ability to detect fine fluctuations in the environmental CO2 concentration. In herbivorous species, plant-emitted CO2, in combination with other sensory cues, affect many behaviors including foraging and oviposition. In contrast to the comprehensive knowledge obtained on the insect olfactory pathway in recent years, we still know little about the central CO2 system. By utilizing intracellular labeling and mass staining, we report the neuroanatomy of projection neurons connected with the CO2 sensitive antennal-lobe glomerulus, the labial pit organ glomerulus (LPOG), in the noctuid moth, Helicoverpa armigera. We identified 15 individual LPOG projection neurons passing along different tracts. Most of these uniglomerular neurons terminated in the lateral horn, a previously well-described target area of plant-odor projection neurons originating from the numerous ordinary antennal-lobe glomeruli. The other higher-order processing area for odor information, the calyces, on the other hand, was weakly innervated by the LPOG neurons. The overlapping LPOG terminals in the lateral horn, which is considered important for innate behavior in insects, suggests the biological importance of integrating the CO2 input with plant odor information while the weak innervation of the calyces indicates the insignificance of this ubiquitous cue for learning mechanisms.

[Incremental cost-effectiveness of the second Xpert MTB/RIF assay for detection of Mycobacterium tuberculosis].

Objective: To study the incremental cost-effectiveness of the second Xpert assay in detection of Mycobacterium tuberculosis (Mtb) and rifampicin (RIF) resistance. Methods: We continuously collected 2 896 specimens from suspected tuberculosis patients who had undergone 2 Xpert tests in a week from March 2015 to March 2018, including 2 402 suspected tuberculosis patients with 1 523 males and 879 females, with an average age of 50 years. Among them, 2 144 specimens of sputum and 258 cases of bronchoalveolar lavage fluid were collected. We also enrolled 494 patients with suspected extrapulmonary tuberculosis, 318 males and 176 females, with an average age of 42 years. Among them, 157 pleural effusion specimens, 106 cerebrospinal fluid specimens, 34 urine specimens and 197 pus specimens were collected. All specimens were subjected to two Xpert tests, smear microscopy, liquid rapid culture (BACTEC MGIT 960), and positively cultured bacteria were tested for drug susceptibility. Results: Among the 2 896 specimens from suspected tuberculosis patients, either one of the two Xpert test results was positive (including both tests were positive, the same below) in 1 639 patients, and 1 502 (91.6%) were positive in the first Xpert tests. The additional 137 (8.4%) test results were positive in the second tests. According to the smear test results, all specimens were divided into the smear negative group and the smear positive group. The second Xpert test was significantly higher than the smear-positive group (14.86%, 3.2%, P<0.001), and the extrapulmonary tuberculosis group was higher than the tuberculosis group (11.2%, 8.0%, P=0.12).Of the susceptibility test results, a total of 371 were rifampicin-resistant specimens. The first Xpert detected 91.4% (339/371), and the second Xpert detected the additional 8.1% (30/371).The cost increase of the second test was very significant. Tests were calculated at 650 yuan per time, the tuberculosis group was 1 184 yuan and 13 696 yuan(P<0.001); the extrapulmonary tuberculosis group was 1 755 yuan and 13 961 yuan(P<0.001). In the test of specimens of tuberculosis and extrapulmonary tuberculosis, the smear-negative specimen cost increase of the second Xpert test was lower than that of the smear-positive specimen. Conclusion: The second xpert test showed significant value-added cost-effectiveness in the diagnosis of tuberculosis.

[The expressions and diagnostic values of miR-18a and miR-21 in esophageal cancer].

Objective: To investigate the expressional levels and diagnostic values of miR-18a and miR-21 in esophageal carcinoma. Methods: The expressions of miR-18a and miR-21 in esophageal cancer tissues and adjacent tissues from 45 esophageal cancer patients, peripheral blood from 45 esophageal cancer patients and 50 healthy donors respectively were detected by RT-PCR. The expressions of miR-18a and miR-21 in normal esophageal epithelial cell HET-1A, esophageal cancer cell lines including ECA109, KYSE150 and TE1 were also detected. Chemiluminescence immunoassay was used to quantitatively detect the concentrations of carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), CYRFA21-1 and TPA (tissue polypeptide antigen) in peripheral blood serum from esophageal cancer patients and healthy controls. Meanwhile, the diagnostic effects of miR-18a and miR-21 on esophageal cancer were compared with those of tumor markers in serum. Results: The expression levels of miR-18a and miR-21 in esophageal cancer cells ECA109, KYSE150 and TE1 were 1.64±0.17, 1.62±0.19, 1.46±0.12 and 20.52±1.48, 6.73±0.73, 1.43±0.19, respectively, higher than those in normal esophageal epithelial cells (both P<0.01). The expressions of miR-18a and miR-21 in esophageal cancer tissues were 32.48±28.62 and 8.67±11.98, respectively, significantly higher than those in adjacent tissues (all P<0.001). The expression levels of miR-18a and miR-21 in peripheral blood of patients with esophageal cancer were 12.66±11.92 and 9.15±8.14, respectively, significantly higher than those in the normal control group (both P<0.001). The receiver operating characteristic (ROC) curve analysis showed that the area under the curve of miR-18a and miR-21 for diagnosis of esophageal cancer were 0.948 and 0.913 5, respectively. Compared with traditional esophageal tumor markers, the expressions of miR-18a and miR-21 were more sensitive in the diagnosis of esophageal cancer. The sensitivity and accuracy of the expressions of miR-18a and miR-21 combined with traditional esophageal tumor markers in diagnosis of esophageal cancer can be further improved to 97.8% and 68.4%, respectively. Conclusion: Our study reveals that the expressions of miR-18a and miR-21 play important roles in the diagnosis of esophageal cancer and may be potentially novel biomarkers.

[Antimicrobial susceptibility and genotyping of Mycobacterium intracellulare].

Objective: To investigate the antimicrobial susceptibility and genotyping of Mycobacterium intracellulare. Methods: A total of 150 M. intracellulare isolates were collected. The susceptibility against 15 antimicrobial agents widely used for treatment of non-tuberculosis mycobacteria (NTM) infections, was tested by broth microdilution assay. Variable number of tandem repeats (VNTR) assay was also performed using the 16-loci genotyping method. Results: The drug susceptibility test revealed that clarithromycin (97.3%, 146/150), moxifloxacin (94.0%, 141/150) and amikacin (90.0%, 135/150) had the best antimicrobial activities in vitro against the M. intracellulare isolates. Secondly, 75.3%(113/150), 64.0%(96/150), 52.7%(79/150) and 8.7%(13/150) of the strains were susceptible to rifampicin, linezolid, capreomycin, and ethambutol, respectively. The MIC(50) and MIC(90) values of the 3 injectable anti-tuberculosis drugs were as follows: amikacin 4 mg/L and 16 mg/L, streptomycin 4 mg/L and 16 mg/L, capreomycin 8 mg/L and 16 mg/L. The MIC(50) and MIC(90) values of the 5 different fluoroquinolones were 0.5 mg/L and 2 mg/L for moxifloxacin , 1 mg/L and 8 mg/L for ciprofloxacin, 1 mg/L and 8ug/ml for levofloxacin, 2 mg/L and 16 mg/L for antoflolxacin, 2 mg/L and 16 mg/L for ofloxacin. The Hunter-Gaston Discriminatory Index (HGDI) value for the 16-loci VNTR typing of M. intracellulare isolates was 0.994. VNTR differentiated the 150 isolates into 21 clusters and acquired a total of 121 unique patterns. Drug resistance profile was not independently associated with cluster strains. Conclusions: Clarithromycin, moxifloxacin and amikacin had the best antimicrobial activities in vitro against M. intracellulare isolates. The 16-loci VNTR typing revealed a highly discriminatory power and drug resistance profile was not independently associated with cluster strains.

Engineering the porosity and acidity of H-Beta zeolite by dealumination for the production of 2-ethylanthraquinone via 2-(4'-ethylbenzoyl)benzoic acid dehydration.

Environmentally-friendly zeolites have been used commercially to replace concentrated sulfuric acid and oleum in the alkylation reactions and dehydration of alcohols. However, moderate activity, associated with access and diffusion limitations, low intramolecular dehydration selectivity, associated with unsatisfactory acidity, and unknown reusability have hampered their industrial implementation in the dehydration of bulky 2-(4'-ethylbenzoyl)benzoic acid (E-BBA) to 2-ethylanthraquinone (2-EAQ). Herein, we have discovered that after being treated with mild HNO3, nano-sized H-Beta zeolite showed outstanding catalytic activity, selectivity and reusability, compared with a commercial oleum catalyst. A number of techniques, such as XRD, XPS, XRF, 29Si MAS NMR, 27Al MQ MAS NMR, FTIR, NH3-TPD, argon physisorption and HR-TEM, have been employed to decouple the interdependence between acidity, porosity and catalytic performance. It was found that mild HNO3 treatment could clean out the extra-framework aluminium deposits and selectively extract the aluminium species on the outer surface of Beta zeolites, which strengthened the acidity of the Brønsted acid sites (Si(OH)Al) inside the H-Beta micropores, thus increasing the possibility of intramolecular dehydration of E-BBA. Moreover, this mild HNO3 treatment also dredged the network of intercrystalline mesopores, alleviating the diffusion constraints. Therefore, through the dual adjustment of acidity and porosity, dealuminated H-Beta zeolite has a promising future in the green synthesis of 2-EAQ.

Sensititre® MYCOTB MIC plate for drug susceptibility testing of Mycobacterium tuberculosis complex isolates.

SETTING: National Tuberculosis Clinical Laboratory, China. OBJECTIVE: To evaluate the accuracy and feasibility of the MYCOTB MIC plate in anti-tuberculosis drug susceptibility testing. DESIGN: MYCOTB testing of Mycobacterium tuberculosis isolates, the Löwenstein-Jensen (LJ) proportion method and the resurazine microtitre assay (REMA), which is extensively used for in-house assay for minimal inhibition concentration (MIC) testing, were performed simultaneously for comparison. A total of 126 clinical isolates were tested using both MYCOTB and the LJ proportion method against 12 anti-tuberculosis drugs; 80 were also tested using REMA. RESULTS: Categorical agreement between MYCOTB and the LJ proportion method was 99.2% for rifampicin, ofloxacin, amikacin, kanamycin and cycloserine, and 98.4% for isoniazid and para-aminosalicylic acid; ethambutol (EMB) had the lowest agreement (86.5%). The overall categorical agreement between MYCOTB and REMA ranged between 98.8% and 100%. MYCOTB outcomes, interpreted on day 10 and 21, were stable for all drugs except EMB. CONCLUSION: MYCOTB is a rapid, convenient, quantitative and accurate method for testing both first- and second-line anti-tuberculosis drugs.

A TRPA1 channel that senses thermal stimulus and irritating chemicals in Helicoverpa armigera.

Sensing and responding to changes in the external environment is important for insect survival. Transient receptor potential (TRP) channels are crucial for various sensory modalities including olfaction, vision, hearing, thermosensation and mechanosensation. Here, we identified and characterized a transient receptor potential gene named as HarmTRPA1 in Helicoverpa armigera antennae. HarmTRPA1 was abundantly expressed in the antennae and labial palps. Transcripts of HarmTRPA1 could also be detected in the head and proboscis. Furthermore, functional analyses of HarmTRPA1 were conducted in the Xenopus Oocyte system. The results showed that the HarmTRPA1 channel could be activated by increasing the temperature from 20 to 45 °C. No significant adaptation was observed when the stimulus was repeated. In addition to thermal stimuli, pungent natural compounds including allyl isothiocyanate, cinnamaldehyde and citronellal also activated HarmTRPA1. Taken together, we infer that HarmTRPA1 may function as both a thermal sensor involved in peripheral temperature detection and as a chemical sensor detecting irritating chemicals in vivo. Our data provide valuable insight into the TRPA1 channel in this moth and lay the foundation for developing novel strategies for pest control.

Functional characterization of sex pheromone receptors in the purple stem borer, Sesamia inferens (Walker).

The sex pheromone communication system in moths is highly species-specific and extremely sensitive, and pheromone receptors (PRs) are thought to be the most important factors in males. In the present study, three full-length cDNAs encoding PRs were characterized from Sesamia inferens antennae. These three PRs were all male-specific in expression, but their relative expression levels were very different; SinfOR29 was 17- to 23-fold higher than the other two PRs. Phylogenetic and motif pattern analyses showed that these three PRs were allocated to different PR subfamilies with different motif patterns. Functional analysis using the heterologous expression system of Xenopus oocytes demonstrated that SinfOR29 specifically and sensitively responded to the major pheromone component, Z11-16:OAc [concentration for 50% of maximal effect (EC50 ) = 3.431 × 10(-7) M], while SinfOR21 responded robustly to a minor pheromone component Z11-16:OH (EC50 = 1.087 × 10(-6) M). SinfOR27, however, displayed no response to any of the three pheromone components, but, interestingly, it was sensitive to a non-sex pheromone component Z9,E12-14:OAc (EC50 = 1.522 × 10(-6) M). Our results provide insight into the molecular mechanisms of specificity and sensitivity of the sex pheromone communication system in moths.

There can be turbulence in microfluidics at low Reynolds number.

Turbulence is commonly viewed as a type of macroflow, where the Reynolds number (Re) has to be sufficiently high. In microfluidics, when Re is below or on the order of 1 and fast mixing is required, so far only chaotic flow has been reported to enhance mixing based on previous publications since turbulence is believed not to be possible to generate in such a low Re microflow. There is even a lack of velocimeter that can measure turbulence in microchannels. In this work, we report a direct observation of the existence of turbulence in microfluidics with Re on the order of 1 in a pressure driven flow under electrokinetic forcing using a novel velocimeter having ultrahigh spatiotemporal resolution. The work could provide a new method to control flow and transport phenomena in lab-on-a-chip and a new perspective on turbulence.

An odorant receptor from the common cutworm (Spodoptera litura) exclusively tuned to the important plant volatile cis-3-hexenyl acetate.

Olfaction plays an important role in insect behaviours. The odorant receptor (OR) repertoire, housed within the dendritic membrane of sensory neurons, is one of the primary determinants of odour recognition. ORs in moths could be classified into pheromone receptors (PRs) and non-pheromone receptors (non-PR ORs). Much research in the field of insect olfaction recently has been focused on PRs of the male moth, but few Lepidoptera studies have been done on the functional study of non-PR ORs. In the present study, we identified and characterized four non-PR ORs from Spodoptera litura (Lepidoptera: Noctuidae) antennae. The tissue expression pattern showed that the four ORs were mainly expressed in adult antennae and further in situ hybridization revealed SlituOR12 was expressed in both long and short sensilla trichodea and sensilla basiconica. A functional analysis of the four SlituORs was conducted in the heterologous expression system Xenopus oocytes. SlituOR12 was exclusively and sensitively tuned to cis-3-Hexenyl acetate and SlituOR19 slightly responded to 4'-Ethylacetophenone; however, SlituOR44 and SlituOR51 did not respond to any chemicals tested in this study. It is proposed that SlituOR12 might partially account for some key behaviours of the female, such as detection of host location and oviposition site.

Molecular interaction of α-synuclein with tubulin influences on the polymerization of microtubule in vitro and structure of microtubule in cells.

Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Evidences demonstrate that α-synuclein may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between α-synuclein and tubulin was confirmed by GST pull-down assay and co-immunoprecipitation. The interacting regions within α-synuclein with tubulin were mapped at the residues 60-100 of α-synuclein that is critical for the binding activity with tubulin. Microtubule assembly assays and sedimentation tests demonstrated that α-synuclein influenced the polymerization of tubulin in vitro, revealing an interacting region-dependent feature. Confocal microscopy detected that exposures of α-synuclein proteins inhibited microtubule formation in the cultured cells, with a length-dependent phenomenon. Our data highlight a potential role of α-synuclein in regulating the microtubule dynamics in neurons. The association of α-synuclein with tubulin may further provide insight into the biological and pathophysiological function of synuclein.

Transient expressions of doppel and its structural analog prionDelta32-121 in SH-SY5Y cells caused cytotoxicity possibly by triggering similar apoptosis pathway.

Doppel (Dpl) is a recently identified prion (PrP)-like protein due to the structural and biochemical similarities, however, its natural function and pathogenic role in neurodegenerative diseases remains unclear. To investigate the possible pathogenic pathway of Dpl and its structural analog for cell apoptosis, mammalian expressing recombinant plasmids containing human PRND gene encoding the full-length Dpl and a truncated human PRNP gene deleting the sequences encoding the peptide from aa 32 to 121 (PrPDelta32-121) were generated. MTT assays showed the cell viabilities of the human neuroblastoma cell line SH-SY5Y receiving Dpl and PrPDelta32-121 expressing plasmids were remarkably lower. Obvious apoptosis phenomena were observed to be associated with the cells transient expressing Dpl and PrPDelta32-121, including reduced mitochondrial transmembrane potential (psim), decreased pro-caspase-3 quantity, more numbers of annexin V- and annexin V/PI-double-stained cells and depressed Bcl-2 level. Moreover, we also found that the Dpl- and PrPDelta32-121-induced cytotoxicities and relevant apoptotic events in SH-SY5Y cells could be fully antagonized by co-expression of the human full-length PrP. These data highly indicate that cytotoxicity induced by the expression of Dpl and truncated PrP in neural derived cells are closely related with the apoptosis process, probably triggering the mitochondrial pathway. It also implies that the cell-benefit activity of the full-length PrP may result from its anti-apoptosis capacity.

Laser induced fluorescence photobleaching anemometer for microfluidic devices.

We have developed a novel, non-intrusive fluid velocity measurement method based on photobleaching of a fluorescent dye for microfluidic devices. The residence time of the fluorescent dye in a laser beam depends on the flow velocity and approximately corresponds to the decaying time of the photobleaching of the dye in the laser beam. The residence time is inversely proportional to the flow velocity. The fluorescence intensity increases with the flow velocity due to the decrease of the residence time. A calibration curve between fluorescence intensity and known flow velocity should be obtained first. The calibration relationship is then used to calculate the flow velocity directly from the measured fluorescence intensity signal. The new method can measure the velocity very quickly and is easy to use. It is demonstrated for both pressure driven flow and electroosmotic flow.

Regularity of distribution of immunoreactive pulmonary surfactant protein A in rat tissues.

Existing data has shown that SP-A-like protein or mRNA is widely distributed in lamellar bodies such as tissues and mucosal surfaces. Using immunohistochemistry method with a polyclonal antibody against human SP-A, in this study we investigated distribution of immunoreactive pulmonary surfactant protein A (IR-SP-A) in a number of rat tissues. The SP-A-like immunoreactivity was found in alveolar, parenchyma, pleura of lung; myelin sheath of brain; epithelia of Bowman's capsule, glomerulus and renal tubules of kidney; epithelia of colon, stomach, duct of salivary gland, pharynx; and blood vessel wall and connective tissue of extracellular matrix. The positive signal was blocked by pre-absorbed SP-A antigen from recombinant or bronchoalveolar lavage (BAL). SP-A has long been considered as an important frontier host defense molecule which participates in immune and inflammatory regulation of lung. With every inhalation, small particles, viruses, bacteria, and antigens from environment are continuously deposited onto the vast pulmonary epithelial surface. While a proper host defense is required to protect the lung, an over-exuberant response can disrupt the appropriate balance between pro- and anti-inflammatory. Traditional Chinese medicine believes that body is an open system relevant to the external environment. The physical, chemical and biological environmental factors constantly affect the open system, and the body properly reacts to maintain homeostasis of body machinery. The Chinese traditional medicine scholars have thus hypothesized that 'Qi' (meaning air) is the communication way between the body and external environment. What is 'Qi'? The results from our study suggest that IR-SP-A is a candidate of 'Qi'. It is compatible with the sites, theoretically containing collagenous and lectin domain molecules, also compatible with the primary injury sites of some autoimmune diseases. SP-A may be as one of 'Qi' molecules mentioned in traditional Chinese medicine that trigger some of autoimmune diseases.

Cloning, expression and immunocytochemical localization of a general odorant-binding protein gene from Helicoverpa armigera (Hübner).

A cDNA clone coding for general odorant-binding protein2 was isolated from the antenna of Helicoverpa armigera by RT-PCR and (5'/3')-RACE technique. Results of sequencing and structural analyses showed that the full-length of GOBP2Harm was 636 bp, possessing 162 amino acid residues and a signal peptide of 21 amino acids. Its predicted molecular weight and isoelectric point were 18.2 kDa and 5.21, respectively. This deduced amino acid sequence shared some common structural features with odorant-binding proteins from several moth species, including the six conserved cysteine motif, typical of insect's OBPs. Northern blot showed that GOBP2Harm is specifically expressed in the antenna of Helicoverpa armigera at similar levels in both sexes. In order to obtain sufficient GOBP2 for further determining its biochemical and physiological properties, a bacterical expression vector of GOBP2 was constructed and successfully expressed. The protein was obtained mainly as insoluble inclusion bodies, that, however, could be solubilized and refolded. The rGOBP2 was purified by affinity chromatography and gel filtration. The rGOBP2 was shown to cross-react with an anti-GOBP antiserum from Antheraea polyphemus. Finally, polyclonal antibodies against GOBP2Harm were used to mark the distribution of the protein in olfactory sensilla and were tested by immuno-electron microscopy. In the male, GOBP2Harm is mainly expressed in sensilla basiconica, while in the female, it is equally expressed in sensilla basiconica and in sensilla trichodea.