Objective To establish a reversed -phase HPLC m ethod for the determination of psora len and isopsoralen in Fukangbao capsule.Methods The contents of Psoralen and Isopsoralen were assayed on a ODS -C 18 column with a mobile phase of methanol -water(40∶60)at a column temperature of 35℃.The fl ow rate was 1.0mL /min.The detection wave-length was at 247nm.Results The linear ranges of Psoralen and Iso psoralen were 1.05～10.52?g /mL(r =0.9999)and1.02～10.20?g /mL(r =0.9999)respectively.Their recoveries were within 97.5%(Psoralen)and 100.8%(Isopsoralen),and both of their RSD were 0.6%.Conclusion This method is simple,rapid and accu rate and suitable for quantity -limiting control of Fukan gbao capsule.

Objective To comparatively evaluate the effects of a recombinant Mtb protein ESAT 6-CFP10 ( rESAT6-CFP10 ) and a purified protein derivative ( PPD ) as skin test reagents in guinea pigs . Methods Guinea pigs were sensitized with different Mycobacteria species .After sensitization , all guinea pigs were intradermally injected with rESAT6-CFP10 and PPD.At 48 h after the injection, the size of ery-thema at injection sites was measured by using a double-blind method .For guinea pigs sensitized with viable Mtb, the size of erythema at injection sites were measured at 24 h after the injection .The positive conversion rates of skin test with rESAT 6-CFP10 and PPD were calculated .Results The results of PPD skin test were positive in all guinea pigs sensitized with viable Mtb , killed Mtb and BCG with erythema diameters of (11.4 ±0.9) mm, (11.8±1.1) mm and (13.2±0.8) mm, respectively.Positive skin test with rESAT6-CFP10 was only observed in guinea pigs infected by viable Mtb-showing erythema diameters of (13.7±5.7) mm. The skin test with rESAT6-CFP10 was negative in guinea pigs sensitized by killed Mtb-and vaccinated by BCG.The skin tests by using rESAT6-CFP10 and PPD were performed on randomly selected guinea pigs at ninth day after infection by Mycobacterium tuberculosis H37Rv.At the 2nd week, totally 24 selected guinea pigs showed positive skin test results with rESAT6-CFP10 (24/24) with erythema diameters of (19.9± 3.0) mm, while only 15 out of 24 had positive PPD skin test with erythema diameters of (6.1±5.5) mm. At the 4th week, all guinea pigs showed positive PPD skin test (3/3) with erythema diameters of (12.7± 2.5) mm.Conclusion The skin test by using recombinant ESAT 6-CFP10 protein can effectively distin-guish viable Mtb infection from BCG vaccination and killed Mtb sensitization , which is a more suitable anti-gen than PPD for the early diagnosis of Mtb infection .

Objective To establish a suitable guinea pig model for evaluating the protective effects of new therapeutic TB vaccines in preclinical study .Methods The guinea pigs were subcutaneously injected with single-cell suspension of multi-drug resistant M.tuberculosis at a dose of 1000 CFU, 0.2 ml per animal.The study was divided into experimentⅠand experimentⅡ.In experimentⅠ, the guinea pigs were given immuno-therapy and/or chemical treatment on day 3 after infection .Four guinea pigs in each group were dissected at weeks 5, 7 and 9 after infection for evaluating lesion scores and histopathology changes of liver , spleen and lung, as well as bacterial load in spleen .In experimentⅡ, the guinea pigs were given immunotherapy and/or chemical treatment with different doses on day 14 after infection .All guinea pigs were dissected at week 5 after infection for evaluating lesion scores of liver , spleen and lung , as well as bacterial load in spleen .Results In experimentⅠ, all of the three treatments including immunotherapy combined with chemotherapy , immunotherapy alone and chemotherapy alone could effectively prevent organ lesion and reduce bacterial load in spleen , which were significantly different from negative control group .The immunotherapy combined with chemotherapy showed better treatment effects than other two treatments .Along with a prolonged period after drug withdrawal , the de-gree of organ lesion in immunotherapy group and chemotherapy group rebounded sharply , but only slightly in im-munotherapy combined with chemotherapy group .In experimentⅡ, animals in all treatment groups showed alle -viated organ lesion and reduced bacterial load in spleen .A relatively better treatment effect was observed in im-munotherapy combined with chemotherapy group .Conclusion The established guinea pig model of M.tubercu-losis infection showed an advantage of good repeatability .It might be used to evaluate the protective effects of new therapeutic vaccines against tuberculosis in preclinical study .

Objective To establish a guinea pig model of latent Mycobacterium tuberculosis infec-tion for evaluating the effects of therapeutic vaccines .Methods Guinea pigs were subcutaneously inocula-ted with 5.0×103 CFU Mtb.The skin test was performed with 0.5μg recombinant ESAT6-CFP10 protein to detect positive conversion rates at different time points .Two weeks after Mtb inoculation , guinea pigs in model group received 5 mg isoniazid treatment ( three times a week for four weeks ) by oral gavage , while those in control group received normal saline .At the sixth week after Mtb infection , guinea pigs with and without isoniazid treatment were dissected for pathology examination .The pathological scores of liver , spleen and lung, as well as bacteria loads in spleen were compared between two groups .The established guinea pig model of latent infection was then validated by testing two reference vaccines ( AEC/BC02 and AEC/BC03 ) . Results Two weeks after Mtb inoculation , all guinea pigs showed positive EC skin test with induration area of (19.9±3.0) mm.Upon four weeks of isoniazid treatment , the guinea pigs in model group showed no pathological changes with zero scores in the examined organs .No bacterium was detected in spleen of ani-mals from model group.However, the total pathological score was 38.8±16.5 and bacteria load in spleen was (5.1±0.3) Log10 CFU with the guinea pigs from control group .Natural recurrence of tuberculosis in model group was observed after drug withdrawal .The total pathological scores were 48.5±23.9 and 51.3± 23.41.The bacterial loads in spleen were (4.5±1.3) and (4.2±1.1) Log10 CFU and bacterial loads in lung were (4.1±1.2) and (3.4±1.3) Log10 CFU respectively as verified with reference vaccines of AEC /BC02 and AEC/BC03.Conclusion Isoniazid treatment inhibited the proliferation of inoculated Mtb in guinea pigs.A guinea pig model of latent Mycobacterium tuberculosis infection is successfully established with an advantage of good repeatability .Therefore, it can be used to evaluate the effects of therapeutic vaccines on latent Mycobacterium tuberculosis infection.

Objective To establish a suitable guinea pig model for evaluating the protective effects of new TB vaccines in BCG prime-boost regimen .Methods Two different immunization strategies by using the recombinant TB vaccine were employed to boost BCG primed guinea pigs in this study .One was for short-term evaluation with 14 weeks interval between prime and boost immunization and another was for long -term evaluation with 54 weeks interval .In the short-term evaluation group , guinea pigs were boosted twice with the recombinant TB vaccine ( AEC/BC02 ) in every two weeks , while guinea pigs in the long-term evaluation group were boosted for three times with two weeks interval between each injection .A negative con-trol group ( NS→NS) and a BCG control group ( BCG→NS) were both set up in two evaluation groups .One week after the last immunization , all guinea pigs were challenged with M.tuberculosis.Six to seven weeks after bacteria challenge , all animals were euthanized and dissected to evaluate lesion scores of liver , spleen and lung, as well as the viable bacterial load in spleen .Results In the short-term evaluation group , the le-sion scores in those boosted with vaccine (3.33±5.00) was lower than that of BCG control group (5.56± 7.27) (P>0.05) and negative control group (47.00±28.11) (P=0.0001).The difference between BCG control group and negative control group in lesion score was also significant .The animals in vaccine boosted group had lower bacterial loads (0.78±1.55 log10 ) in spleen than that in BCG control group (1.06±1.87) (P>0.05) and negative control group (5.47±0.61) (P=0.0003).In the long-term evaluation group, the lesion score in those boosted with vaccine was lower (5.0±7.6) than that in BCG control group (14.4± 13.5) (P=0.0394) and negative control group (56.9±14.1) (P<0.0001).The animals in vaccine boos-ted group (1.00±1.86 log10) had lower bacterial loads in spleen than that in BCG control group (1.46± 1.94) (P>0.05) and negative control group (5.43±0.56) (P=0.01).There was a significant difference in bacterial load between BCG control group and negative group (P=0.0089).Conclusion The results suggest that the interval time between BCG-prime and boost immunization should be properly prolonged in the guinea pig model used for evaluating the protective effects of new TB vaccines in BCG prime -boost regimen .

Frontal fibrosing alopecia is a primary lymphocytic cicatricial alopecia, and is generally considered to be a subtype of lichen planopilaris due to similar histopathological changes. Its etiology is still unclear. With the deepening of research on this disease, more and more cases of frontal fibrosing alopecia have been reported in China and other countries. This review summarizes research progress in pathogenesis, clinical and pathological characteristics, and treatment of frontal fibrosing alopecia.

Alopecia is a common disease in dermatologic clinics.The diagnosis of some hair diseases does not rely on pathological examinations,and with the development of science and technology,the role of many new non-invasive detection instruments,such as dermoscopy,hair scanners and skin CT,is increasingly valued in the diagnosis and treatment of hair diseases.However,the role of pathological examination is still irreplaceable.Because of the particularity of hair diseases,hair pathology differs from common skin pathology in the aspects of scalp biopsy sites,biopsy methods and techniques,tissue-slicing methods and pathological report content.This review systematically discusses the role of scalp pathological biopsy in the diagnosis and treatment of alopecia.

Objective To evaluate the therapeutic effect of antibiotics combined with recombinant tuberculosis vaccine AEC/BC02 on Mycobacterium tuberculosis infection in guinea pigs. Methods Two weeks after guinea pigs were challenged subcutaneously with a high dose of Mycobacterium tuberculosis,the guinea pigs with the positive skin test responses to the recombinant ESAT6-CFP10 allergen were randomly di-vided into four groups:normal saline (NS),AEC/BC02,antibiotics and antibiotics+AEC/BC02. In antibiotics+AEC/BC02 group,guinea pigs firstly received isoniazid ( INH) and rifapentine ( RFT) treatment once a week for a total of three times,and then were immunized with a single dose of AEC/BC02 vaccine six times at 10-day intervals. Guinea pigs in AEC/BC02 and antibiotics groups were respectively vaccinated with AEC/BC02 vaccine and given INH and RFT treatment at the same dose and frequency as given to antibiotics+ AEC/BC02 group. Thirteen weeks after challenge,all guinea pigs were sacrificed for necropsy. Results The gross pathological scores of NS,AEC/BC02,antibiotics and antibiotics+AEC/BC02 groups were 83±8,77± 22,45±28 and 19±14,respectively. Antibiotics+AEC/BC02 group had a significantly lower gross pathological score than antibiotics,AEC/BC02 and NS groups (P<0. 05,P<0. 01,P<0. 01,respectively). Moreover,the gross pathological scores of antibiotics and AEC/BC02 groups were significantly decreased as compared with that of NS group (both P<0. 01). However,there was no significant difference between AEC/BC02 and NS groups. The spleen bacterial load of antibiotics+AEC/BC02 group was (2. 50±1. 26) lg CFU,which was sig-nificantly lower than those of NS [(4. 92+0. 52) lg CFU],AEC/BC02 [(4. 78+0. 84) lg CFU] and antibi-otics [(4. 39+0. 50) lg CFU] groups (P<0. 01). Compared with NS group,antibiotic and AEC/BC02 groups showed no significant difference in spleen bacterial load. Histopathological changes indicated different levels of granulomatous lesions appeared in lung tissues of all groups and the most severe change was ob-served in AEC/BC02 group,followed by that in NS,antibiotics and antibiotics+AEC/BC02 groups. Conclu-sion INH and RFT treatment in combination with AEC/BC02 vaccine in the treatment of guinea pigs with Mycobacterium tuberculosis infection was superior to either treatment alone as it significantly alleviated organ lesions and lowered the bacterial loads in spleen and lung.

Self-assembling amphipathic peptides (SAPs) have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes. In this study, a novel multifunctional tag, S1vw (HNANARARHNANARARHNANARARHNARARAR) was developed to modify fused enzymes. After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker, the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1- and 1.89-fold, respectively, compared to the wild-type proteins. The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold. All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates. Moreover, S1vw also induced the thermostabilities enhancement of the fusions, with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16- and 3.2-fold increase compared with the corresponding wild-type, respectively. In addition, S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris. These results indicated that S1vw could be used as a multifunctional tag to benefit the production, thermal stability and purification of the fusion protein in prokaryotic expression system.
Escherichia coli ; Green Fluorescent Proteins ; Hydrophobic and Hydrophilic Interactions ; Peptides ; Pichia ; Recombinant Fusion Proteins ; metabolism

ObjectiveTo compare the effects of different doses and withdrawal time of 4-vinylcyclohexene diepoxide （VCD） on the reproductive endocrine levels of female rats， and to explore the effective， stable， and safe dosage of VCD for constructing a premature ovarian insufficiency （POI） rat model. MethodSD rats with regular estrous cycles were randomly divided into three groups： blank group， low-dose VCD group （80 mg·kg^-1·d^-1）， and high-dose VCD group （160 mg·kg^-1·d^-1）， with 24 rats in each group. After drug intervention， samples were collected on the 15th day （D15） and the 45th day （D45） after intervention. The general condition， rate of estrous cycle disturbance， serum hormone levels， ovarian histomorphology， follicle count， pregnancy outcome， and the protein and mRNA expression of transforming growth factor （TGF）-β and Smad2/3 were assessed. ResultCompared with the blank group， the low-dose VCD group showed no significant differences in the rate of estrous cycle disturbance or serum follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and estradiol （E₂） levels. Ovarian tissue was damaged. Specifically， the number of primordial and primary follicles decreased on D15 （P<0.01）， and the number of secondary follicles （P<0.01） and antral follicles （P<0.05） further decreased on D45. The litter number decreased on D15 （P<0.05）， but there was no significant difference on D45. Furthermore， TGF-β protein levels increased on D15 （P<0.05） and D45 （P<0.01）. The Smad2/3 levels increased on D45 （P<0.01）， and TGF-β and Smad2/3 mRNA levels increased on D45 （P<0.05）. Compared with the results in the blank group， the disturbance rate of the estrous cycle increased on D45 in the high-dose VCD group （P<0.01）. The serum of FSH and LH increased （P<0.01）， while E₂ decreased （P<0.05）. Ovarian tissue was damaged， and the downward trend of follicles at all levels was similar to that in the low-dose VCD group. The litter number significantly decreased on D15 and D45. TGF-β and Smad2/3 protein levels increased （P<0.05，P<0.01）， and TGF-β mRNA increased on D45 （P<0.05）. ConclusionHigh-dose VCD is an ideal method for constructing a POI rat model， being effective， stable， and safe.