Alendronate combined with Lactobacillus rhamnosus to combat bone loss in ovariectomized mice / 中华创伤骨科杂志

Objective:To study the protective effect of alendronate combined with Lactobacillus rhamnosus on bone loss in ovariectomized mice.Methods:Fifty female C57BL/6 mice were divided into 5 equal groups ( n=10). Ovariotomy was performed in groups A, B, C and D while a sham operation was performed in group E. Group A was subjected to combined administration of alendronate and Lactobacillus rhamnosus, group B to administration of alendronate, group C to administration of Lactobacillus rhamnosus, and groups D and E to administration of physiological saline only. At 3 months after operation, all the mice were sacrificed to harvest their femurs. Micro CT scanning was performed to detect the bone mineral density (BMD), trabecular relative volume, bone surface area/bone volume, and trabecular thickness and number of trabecular bone. Three-point bending test was used to detect the maximum load, stiffness, ultimate load, Young＇s modulus, and fracture energy. Osteocalcin and alkaline phosphatase levels were measured using blood samples from the mice eyeballs. The 2 groups were compared in terms of all the above indexes. Results:The BMD [(669.87±67.87) mg/cm 3], maximum load [(14.35±0.75) N] and fracture energy [(1,497.43±38.29) J/m 2] in group A were significantly higher than those in group B [(520.07±9.01) mg/cm 3, (11.94±0.82) N and(1,277.61±35.12) J/m 2] and group C [(388.15±25.61) mg/cm 3, (11.10±0.93) N and (1,115.27±63.24) J/m 2] (all P<0.05). The osteocalcin level in group A [(22.25±1.78) ng/mL] was significantly higher than that in group B [(19.08±1.45) ng/mL] and group D [(19.33±1.66) ng/mL] (both P<0.05). The alkaline phosphatase level in group A [(83.21±9.69) ng/mL] was significantly lower than that in group C [(113.16±14.44) ng/mL] and group D [(137.96±14.01) g/mL] (both P<0.05). Conclusion:Alendronate combined with Lactobacillus rhamnosus may play a synergistic role in prevention of bone loss in ovariectomized mice, because combined administration of the two is more effective than administration of either of the two.

Ectopic replantation of forearm, wrist and palm in a destructive injury of a young child: A case report / 中华显微外科杂志

Reports a case admitted in the Ward I of Department of Surgery of Zhengzhou Renji Hospital in June 2017. A young child who suffered destructive injury of left forearm, wrist and palm with severed 3rd-5th fingers. Tendon and neurovascular repairs of forearm, wrist and palm were performed with pedicled abdomina flap and the 3rd-5th fingers ectopic replantation in Phase I surgery. In the Phase II surgery, the abdomina flap division was carried out. The replantation of severed fingers after ectopic replantation and the reconstruction of foot defect with free anterolateral thigh flap(ALTF) were carried out in Phase III surgery. In Phase IV surgery, fingers functional reconstruction and foot flap thinning were performed. Four years after surgery, the thumb oppositions to middle, ring and little fingers could be completed, with slightly limitations. The appearance and texture of transferred foot flap were good, and the child could walk and run almost normally.

Vascularised medial femoral condylar osteochondral chimeric tissue flap for repairing a complex tissue defect in metatarsal head: A case report / 中华显微外科杂志

In February, 2019, a patient with a defect of open dorsal cartilage and bone in the first metatarsal head, including the defects of soft tissue, tendon and joint capsule, was treated in our department. After multiple debridement, the vascularised medial femoral condyle osteochondral chimeric tissue flap was transferred to repair the composite tissue defect in the metatarsal head at the second stage. After 18 months of follow-up, the patient felt no pain in the foot and walking, and there was no sign of lameness and discomfort at donor sites. The postoperative functional recovery was satisfactory.

Isolation and identification of cholesterol-degrading intestinal bacteria by culturomics and evaluation of their functions / 生物工程学报

High cholesterol is one of the important factors inducing cardiovascular and cerebrovascular diseases. Drug therapy is the main method for reducing cholesterol, but has the disadvantages such as high cost and side effects. Studies have shown that intestinal bacteria play important roles in cholesterol metabolism. However, there are few reports on the screening and functional evaluation of cholesterol-lowering intestinal bacteria. In this study, 36 bile-tolerant bacteria were screened from healthy people stool through culturomics using bovine bile acid or artificial mixed bile acids as substrates. Taking Lactobacillus rhamnosus GG (LGG) as a positive control, three bile acid concentration groups (0 g/L, 0.3 g/L, 3 g/L) were set up to evaluate the cholesterol-lowering ability of bile-tolerant bacteria in vitro. Ten bacteria (including Proteus mirabilis, Providencia stuartii, Proteus vulgaris et al) were identified as the dominant cholesterol-lowering bacteria. Six of the above bacteria, Proteus mirabilis, Providencia stuartii, Proteus vulgaris, Proteus penneri, Wohlfahrtiimonas chitiniclastica, Providencia rettger, were evaluated for their ability to reduce triglycerides in vitro and tolerance to artificial gastric juice. Comparing with strain LGG, the six bacteria showed better triglyceride-lowering ability in vitro. With the decrease of pH value of artificial gastric juice and the increase of treatment time, the survival rate of six bacteria decreased. The above screening experiments and functional evaluation provide a basis for further development of potential cholesterol-lowering bacterial products.

Expert consensus on microbiome sequencing and analysis / 生物工程学报

In the past ten years, the research and application of microbiome has continued to increase. The microbiome has gradually become the research focus in the fields of life science, environmental science, and medicine. Meanwhile, many countries and organizations around the world are launching their own microbiome projects and conducting a multi-faceted layout, striving to gain a strategic position in this promising field. In addition, whether it is scientific research or industrial applications, there has been a climax of research and a wave of investment and financing, accordingly, products and services related to the microbiome are constantly emerging. However, due to the rapid development of microbiome sequencing and analysis related technologies and methods, the research and application from various countries have not yet unified on the standards of technology, programs, and data. Domestic industry participants also have insufficient understanding of the microbiome. New methods, technologies, and theories have not yet been fully accepted and used. In addition, some of the existing standards and guidelines are too general with poor practicality. This not only causes obstacles in the integration of scientific research data and waste of resources, but also gives related companies unfair competition opportunity. More importantly, China still lacks national standards related to the microbiome, and the national microbiome project is still in the process of preparation. In this context, the experts and practitioners of the microbiome worked together and developed the consensus of experts. It can not only guide domestic scientific research and industrial institutions to regulate the production, learning and research of the microbiome, the application can also provide reference technical basis for the relevant national functional departments, protect the scale and standardized corporate company's interests, strengthen industry self-discipline, avoid unregulated enterprises from disrupting the market, and ultimately promote the benign development of microbiome-related industries.

Application of free medial sural artery perforator flap to repair soft tissue defects of forearm and foot / 中华显微外科杂志

Objective:To reconstruct and repair forearm and foot injuries with soft tissue defect using medial sural artery perforator flap (MSAP), and to evaluate the curative effects.Method:From May, 2015 to September, 2017, 13 patients (9 males and 4 females) with soft tissue defect on forearm and foot underwent MSAP reconstruction operations. The age was ranged from 19 to 57 (mean 41) years. Six wounds located in forearms and 7 in foot. Ipsilater- al shank was used as donor for the repair of foot. The donor sites were directly sutured. The area of flaps was ranged from 3.0 cm×4.0 cm-7.0 cm×15.0 cm. All cases were followed-up for flap appearance, sensation and donor healing by visit of clinic and WeChat reviews.Results:All 13 flaps survived well without any vascular crisis nor necrosis. Postoperative superficial infections were found in 3 cases, and the wound healed gradually after daily dress changing and anti-infection treatment. Eleven patients were followed-up for 4 to 18 months (average 12 months). Two provincial patients lost to follow-up. No obvious disfunction was found from the donor shanks. The appearance and texture of flaps were in excellent condition and satisfactory. The sensation of 7 flaps was recovered to S 2-S 3. TPD was 6-9 mm. Conclusion:The free MSAP is rational therapeutic strategy for repairing the soft tissue defect of forearm and foot. It has advantages of long vascular pedicle, constant perforation and relatively thin subcutaneous fat.

The big toe wrap-around flap combination of the second phalange with the metatarsal to reconstruct the thumb / 中华显微外科杂志

Objective To explore the surgical technique and treatment outcomes of the big toe wrap-around flap combination of the second phalange with the metatarsal to reconstruct the thumb. Methods From June,2014 to December, 2016, 6 patients of the thumb defects onⅤdegree, we took the big toe wrap-around flap with the second toe and the metatarsal to reconstruct the thumb. The metatarsal head was truncated nearby the metatarsophalangeal joint,and the metatarsal head was turned 70°-80° from the dorsal side to the plantar side, then recombinated the metatarsal after dealed with the fracture, so it can rebuild the metacarpophalangeal joints and the metacarpal. 6 cases were followed up. Results All cases survived,and they were followed up duing 4 to 24 months after operation. The shape was similar with uninjured sides and the two-point discrimination was 1.0-2.0 cm.The function recovered satis-factorily and the maximum flexion of the metacarpophalangeal joints can reach 50 degrees,at the same time,it has the function of dorsiflexion. They were got bone healing and there was no bone absorption and joint degeneration. The donor foot has no ulceration,and walking without the pain and lameness. According to the Upper Extremity Functional functional Evaluation Standard set up by Hand Surgery Branch of Chinese Medical Association,there were excellent in 3 cases and good in 3 cases. Conclusion Combined the big toe wrap-around flap with the second toe and the metatarsal to reconstruct the thumb, it can rebuild the metacarpophalangeal joints and metacarpal, we can get the thumb which have the physiological curvature and the suitable length,the configuration and the function were satisfac-tory.It is an effective method for reconstruction of the thumb defect onⅤdegree.

Repair of large area of soft tissue defect of calf by the retrograde anterolateral thigh flap with single high cu-taneous perforator / 中华显微外科杂志

Objective To explore the clinical effect of repairing the large area of soft tissue defect of the calf by the retrograde anterolateral thigh flap with single high cutaneous perforator. Methods From January, 2014 to July, 2017, 9 cases of large area of soft tissue defects were repaired by the retrograde anterolateral thigh flap with sin-gle high cutaneous perforator.There were 7 males and 2 females, aged 24-48 years.Soft tissue defects area of the calf was 10.0 cm×7.0 cm to 35.0 cm×15.0 cm, including skin grafting and skin stretch to repair the area. The perforating point of the high cutaneous artery branches was designed at the proximal end of the flap, which was used as the single nutrient vessel of the flap. The rotation point of the flap was moved upward to the proximal thigh, which not only in-creased the blood supply of the flap, but also made the flap repair range to the distal calf. The flap range was 15.0 cm×10.0 cm to 22.0 cm×12.0 cm. Results All flaps were cut smoothly, and no vascular crisis occurred. All flaps survived smoothly.All patients were followed-up for 6-12 months. The appearance of flaps was plump, slightly bloat-ed, and their color was similar to the recipient area. The texture was soft, and no active disorder in the donor site. Conclusion The retrograde anterolateral thigh flap with single high cutaneous perforator can be designed at a high rotation point.By increasing the number and caliber of the anastomotic branch between the pedicle and lateral superi-or genicular artery, the blood supply and reflux of flap can be improved, and the survival rate is not affected. Com-pared with the traditional anterolateral thigh flap, it has great advantages.

Two-plasmid scarless genetic modification in Escherichia coli hfq and rne-710 / 生物工程学报

Gene modification is an important technique to understand gene function. We firstly constructed Δhfq::Spe and Δrne-710::Spe mutant strains of Escherichia coli MG1655. The fragment lacking of hfq and rne-710 was ligated to the auxiliary plasmid and separately replace the spectinomycin box by homologous recombinase system to obtain the Δhfq and Δrne-710 mutant strains. The combination of two-plasmid scarless genetic modification and fusion PCR led to a new way for the long DNA fragment gene deletions.

Autoregulation of PhoP/PhoQ in Yersinia pestis under different conditions / 军事医学

Objective To investigate the transcriptional autoregulation of PhoP/PhoQ under different growth conditions in Yersinia pestis.Methods The entire promoter region of YPO1635 was amplified and cloned into the pRW50 vector containing a promoterless lacZ reporter gene.The recombinant LacZ reporter plasmid was transformed into the wild-type strain (WT) and the phoP mutant strain (ΔphoP),respectively,to measure the promoter activity (the β-galactosidase activity) of the target gene in WT and ΔphoP by using the β-galactosidase enzyme assay system.Total RNAs were extracted from WT and ΔphoP strains,and primer extension assay was employed to detect the promoter activity by examining the amount of primer extension products of YPO1635 in WT and ΔphoP.Results The LacZ fusion results showed that the transcription of YPO1635 was positively regulated by PhoP under L-TMH and brain-heart infusion(BHI) conditions,but it was not regulated in H-TMH medium.The primer extension assay detected two transcriptional start sites located at 90 and 118 bp upstream of the translation initiation site of phoP,named P1 and P2,respectively.Under low Mg2+ TMH conditions,the promoter activity of P1 rather than P2 was positively regulated by PhoP.Under high Mg2+ TMH conditions,the promoter activities of both P1 and P2 showed no obvious difference in the WT and ΔphoP strains.Under rich BHI conditions,both promoters were under negative control of PhoP.Conclusion Different autoregualtion patterns of PhoP/PhoQ under different growth conditions would help Y.pestis to quickly adapt to the changing living environment.

Development of web-based literature database for Yersinia pestis / 军事医学

Objective To build a literature database that systematically sorts out and integrates related knowledge of Yersinia pestis researches, which will improve the efficiency of future researches on Y.pestis.Methods EndNote X5 software was used as a literature collection tool in this research.Computer languages, including Perl and PHP, were employed to standardize the text data, establish the entity relationship model, code the webpage and build the basic literature database.The tool combination of Apache+PHP+MySQL that is usually used in development of small or medium-scale websites was selected to build a dynamic website.Results and Conclusion The Y.pestis literature database(YPKD) was constructed (accessed by http://101.201.51.148/ypkd/.), from which users can quickly search or browse the required literature, download the required full text, and obtain the latest online information on Y.pestis.The database can serve as a model for establishing literature databases of other crucial pathogens.

Advance in research on regulation of sRNAs in bacterial biofilm formation / 军事医学

Small RNAs(sRNAs) play a significant role in the regulation of bacterial growth.When sensing certain environmental cues such as fluctuation of nutrient concentration, temperature, pH, and osmolarity, sRNAs can influence the expression of target genes.The formation of biofilms is initiated by bacteria transitioning from the planktonic to the surface-associated mode of growth, which is a self-produced extracellular matrix composed of proteins, polysaccharides, and DNA.Recent evidences have shown that small RNA plays an important role in the regulation of bacterial biofilm formation.sRNAs have key roles in biofilm formation process by base pairing with target mRNAs or interaction with modulating proteins.This review discussed the regulation mechanism and pathway of sRNAs in bacterial biofilms formation, and summarized three classical regulatory models of sRNAs in bacterial biofilms formation, this review also gives the research status and development direction of sRNAs in bacterial biofilms formation.

Preparation of monoclonal antibodies against ricin toxin and development of up-converting phosphor technology-based lateral flow assay for its quantitative detection / 军事医学

Objective To develop an up-converting phosphor technology based lateral flow assay ( UPT-LF) to detect ricin toxin ( RT) quickly, accurately and quantitatively.Methods Ricin-monoclonal antibodies were prepared and their affinity was evaluated before four types of monoclonal antibodies with the highest titer were applied to couple with the up-converting phosphor nano-particles ( UCP-NPs) as the bio-conjugate and disperse on the analysis membrane as the test line, respectively.Following systematic optimization to establish the RT-UPT-LF strip, the sensitivity, precision, quantita-tive ability and specificity of RT-UPT-LF were evaluated.Results The detection could be accomplished within 15 min and the detection limit of the RT-UPT-LF assay could reach 0.5 ng/ml within the quantitative detection range of 0.5-1000 ng/ml.Other non-specific toxins at a concentration of 1000 ng/ml did not cause any non-specific reactions.Conclusion The developed RT-UPT-LF strip provides a new means for on-site quantitative detection of ricin toxin.

Western area surge for controlling Ebola hemorrhagic fever outbreak in Sierra Leone and evaluation of its effect / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the Western Area Surge (WAS) program in the Ebola outbreak of Sierra Leone, and to analyze its implementing effect.</p><p><b>METHODS</b>The subject of this study was 3,813 laboratory confirmed Ebola hemorrhagic fever (EHF) cases reported in Sierra Leone from November 19, 2014 through January 27, 2015, a period before and after the implementation of the WAS program. To analyze and make conclusions according to the working experience of China Mobile Laboratory Reponses Team in the fight of Ebola outbreak, using WHO published EHF case definition to make diagnosis and compare the number of bed numbers, confirmed EHF cases, samples tested, and positive rates before and after implementation of WAS program.</p><p><b>RESULTS</b>From the implementation of WAS program on 17th December 2014 to half a month later, the total numbers of Ebola holding and treatment centers increased from 640 to 960, six additional laboratories were established. On January, 2015, another two laboratories from America and The Netherlands were established. The numbers of samples tested one month before and after WAS program were 7,891 and 9,783, respectively, with an increase of 24.0 percent, while the positive rate of Ebola virus decreased from 22.2% (1,752/7,891) to 11.0% (1,077/9,783). The positive rate of blood samples decreased from 39.6% (248/626) in the month before WAS program to 27.4% (131/478) (χ2=17.93, P<0.001) in the mother after WAS program, the positive rate of blood samples 22.7% (103/454) to 10% (62/609) (χ2=31.03, P<0.001), accordingly. After 3 weeks of WAS program, in addition to Western Area, another four hotspots in Sierra Leone had also reported a significant decrease of the numbers of confirmed EVD cases. Forty-two days after implementation of WAS program, the daily number of laboratory confirmed EHF cases decreased from 63 to 10.</p><p><b>CONCLUSION</b>WAS program played a vital role in controlling the EHF outbreak rapidly in Sierra Leone. It could also provide guidance for the control similar large infectious diseases outbreak in the future.</p>

Development and systematical evaluation of an up-converting phosphor technology based lateral flow assay for quantitative detection of Burkholderia pseudomallei / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop an up-converting phosphor technology-based lateral-flow (UPT-LF) assay for rapid quantitative detection of Burkholderia pseudomallei on site.</p><p><b>METHODS</b>The strip Bps-UPT-LF strip was prepared with up-converting phosphor (UCP) particles as the bio-label using double-antibody sandwich method. Detection performance, including sensitivity, quantitative accuracy, precision, and specificity, were first evaluated using bacterial suspensions of Burkholderia pseudomallei, the related species and the strains which had similar routes of transmission with serial standard concentrations diluted by phosphate buffer, then biological and chemical reagents and simulated samples with series concentrations were employed for sample tolerance evaluation, while the operation error during on site detection was also evaluated through adjusting liquid measure.</p><p><b>RESULTS</b>The whole detection was accomplished within 20 minutes, and the sensitivity was 10(4) CFU/ml with linear quantitative range from 10(4) CFU/ml to 10(7) CFU/ml, which covered four orders of magnitude. Bps-UPT-LF strip demonstrated high specificity with the absence of any false-positive result even at 10(7) and 10(8) CFU/ml of non-specific bacterial contamination. Not only Bps-UPT-LF strip could tolerate to high concentration of the extreme acid and basic matter (pH 1-12), saline matter (≤ 2 mol/L mixture of NaCl and KCl), viscous materials (≤ 50 g/L of PEG 20000 and ≤ 20% of glycerol) and bio-macromolecule (≥ 400 g/L of bovine serum albumin or ≥ 80 g/L of casein), but also it can directly detect animal, environmental and powder specimen, such as ≥ 400 g/L of milk powder, flour powder, fruit juice, fresh and decomposed viscera, and ≤ 200 g/L of putty powder, sucrose, gourmet powder, and soil. Operation errors of liquid measure had few effects on sensitivity and specificity, including -50%-200% of sample, -22%-44% of sample-treating buffer and -30%-30% of loading mixture.</p><p><b>CONCLUSION</b>The good detection performance and tolerance performance bring the bright future for Bps-UPT-LF strip to detect Burkholderia pseudomallei on site rapidly and quantitatively for nature foci surveillance and anti-bioterrorism.</p>

Regulation of motility of Salmonella enterica serovar Typhi by QseBC / 军事医学

Objective To study the impact of QseBC on the motility of Salmonella enterica serovar Typhi ( S.Typhi ) . Methods The motility of wild-type ( WT) and null mutants (ΔqseB and ΔqseC) at mid-log phase was investigated by swimming assay.Quantitative RT-PCR was carried out to calculate the transcriptional variation of flhD and qseB among WT,ΔqseB andΔqseC.QseB overexpressing strain was constructed to compare its motility and flhD expression with the wild-type control.Results The result of motility assay showed that the motility of ΔqseB was similar to that of the WT strain , while the motility of ΔqseC was much lower than that of WT .qRT-PCR revealed that compared with WT , the expression of flhD was significantly decreased in ΔqseC while the expression of qseB was increased considerably .The motility of QseB overex-pressing strain was lower .Conclusion The expression of flhD may be regulated by QseBC which has an effect on the motil-ity of S.typhi, and the overexpression of QseB may inhibit the motility .

Experimental study of ectopic activation about allograft segment tibia of rabbits / 中华显微外科杂志

Objective To compare of the difference about ectopic activation between autogenous bone graft and allograft from large segment tibia of rabbits.Methods Eighty healthy adult Chinese rabbits (6 months of age),weighing (2.5 ±-0.5)kg,were randomly divided into experimental group (allogeneic bone group) and the control group (autograft group),40 rabbits in each group.Another 10 rabbits were allogeneic bone donor.In experimental group,when 1.5 cm long rabbit tibial allograft were finishied,they were implanted into spatium intermusculare between the musculus rectus femoris and medial vastus muscle of the rabbit around the saphenous artery and were fastened to the femur by 1.0 mm Kirschner-wire.In control group,autologous tibias were done,the same as experimental group including length and position and method.Four weeks and 8 weeks and 12 weeks postoperative,respectively,the postmortem specimens were examined gross and immunohistochemistry and the expression of BMP-2 and collagen type Ⅰ of transplanted bone tissue were detected.Results BMP-2 mainly exist in cytoplasm of osteoblasts and chondrocytes undifferentiated mesenchymal cells.Collagen type Ⅰ primarily exist in the bone matrix around the pit of bone.The expression level of BMP-2 of experimental group in postoperative 4,8,12 and 16 weeks were 85.25 ± 4.47,109.44 ± 14.69,141.85 ± 9.45,116.25 ± 14.18,respectively,and the expression level of BMP-2 of control group were 103.78 ±-6.59,124.95 ± 14.94,145.46 ± 8.10,112.48 ± 13.27,respectively.The expression level of collagen type Ⅰ of experimental group in postoperative 4,8,12 and 16 weeks were 78.74 ± 7.99,95.95 ± 6.99,139.91 ± 4.32,137.76 ± 3.48,respectively,and the expression level of BMP-2 of control group were 88.87 ± 11.26,102.45 ± 2.82,140.76 ± 4.62,139.05 ± 4.55.Compared with control group,there was a significant difference in the expression level of the BMP-2 and collagen type Ⅰ of experimental group in postoperative 4,8 weeks (P ＜ 0.05),but,there was no significant difference in the expression level of the BMP-2 and collagen type Ⅰ of experimental group in postoperative 12,16 weeks (P ＞ 0.05).The amount increased gradually during 4 weeks,8 weeks,12weeks,peaked at 12 weeks,BMP-2 displayed a downward trend at 16 weeks,and collagen type Ⅰ basiclly maintain the level of 12 weeks.Conclusion Allograft segment could complete activation while they are implanted into spatium intermusculare containing famous blood supply within 3 months,there is no significant difference between autologous bone and allograft,it shows the feasibility of ectopic activation about allograft segment.

Expression and purification of virulence protein Pla of Yersinia pestis and its activity / 军事医学

Objective To prepare recombinant plasminogen activator(Pla) protein in E.coli BL21 cells that can be used in studying interactions between Yersinia pestis proteins and immunologic diagnosis of plague.Methods The pla gene was amplified by PCR and cloned into the pET28a expression vector.E.coli BL21 competent cells were transformed with the recombinant vectors, and isopropyl-β-D-thiogalactopyranoside ( IPTG) was added to induce expression of Pla protein. The expressed protein was detected by SDS-PAGE electrophoresis.The inclusion bodies of Pla protein were denatured in 8 mol/L urea, and then refolded using gradient urea solutions.The purified protein was identified by SDS-PAGE electrophoresis and Western blot.Results and Conclusion The constructed expression vector was demonstrated to be correct through agarose gel electrophoresis and sequencing.The recombinant Pla protein was accumulated as an inclusion body in E.coli, and the overexpression product was mainly a target protein, the yield of which was very high.SDS-PAGE purity of the bioactive Pla protein was obtained by denaturing and refolding the inclusion bodies.This study provides a simple and quick method for highly efficient preparation of biologically active Pla protein.

Loop-mediated isothermal amplification for quick detection of Yersinia pestis / 军事医学

Objective To establish a simple and quick loop-mediated isothermal amplification (LAMP)method for detection of Yersinia pestis.Methods LAMP Primers were designed based on the specific sequence 3a in Y.pestis chromosome.LAMP reaction results were detected using turbidity meter or visual method.The specificity of the constructed method was evaluated by detecting Y.pestis and its closely-related bacteria.The different dilution DNA template was detected with LAMP and PCR to evaluate the sensitivity of the method.Results Thirty strains of bacteria closely related to Y.pestis were detected by the constructed LAMP,and all the results were negative,indicating that the method had a very high specificity.The detection sensitivity of this LAMP assay was 20 pg of DNA per reaction,which was ten-fold that of the regular PCR.The detection reaction was completed in 25 min.Conclusion This LAMP method is quick,sensitive, specific and simple,which is expecked to become an effective method for rapid detection of Y.pestis on the scene.

Screening, identification and function of pPCP1 plasmid-derived sRNA in Yersinia pestis:a preliminary study / 军事医学

Objective To identify small non-coding RNAs encoded by plasmid pPCP1 and investigate their roles in biofilm formation, stress tolerance and/or virulence in Yersinia pestis.Methods Seven plasmid pPCP1-encoded sRNAs were identified by RNA-seq results in Y.pestis in our previous studies.Northern blot was used to validate the presence of the seven sRNAs.The sRNA-deletion mutants were constructed via λ-Red homologous recombination system.The biofilm formation, high salt tolerance and virulence of the phenotypes were compared between Y.pestis WT strain and sRNA mutants.Results and Conclusion The expression of seven pPCP1-encoded sRNAs was validated and the transcript length detected by Northern blotting corresponded to the length observed by RNA-seq.On this basis, five sRNA-deletion mutants were obtained.The capacity of biofilm formation was weakened upon deletion of sR3446.The tolerance of sR3446, sR3457, sR4338 and sR4340 mutants was found weakened in vitro compared to that of wild-type strain,but the tolerance of sR6143 was found increased.Slight virulence attenuation was found in two sRNA mutants ( sR4338 and sR4340 ) .The results suggest that pPCP1-deriving sRNA might be implicated in stress response, biofilm and virulence in Y.pestis.