Progress in the study of reproductive toxicity of platinum-based antitumor drugs and their means of prevention.

Platinum-based antitumor drugs are broad-spectrum agents with unique mechanisms of action. Combination chemotherapy regimens based on platinum drugs are commonly used in cancer treatment. However, these drugs can cause various adverse reactions in the human body through different routes of administration, including reproductive toxicity, genetic toxicity, and embryonic developmental toxicity. Preventing adverse effects is crucial to enhance patients' quality of life and reduce healthcare costs. This article discusses the types and developmental history of antitumor active platinum compounds, their mechanisms of action, routes of administration, and their potential reproductive, genetic, and embryonic developmental toxicity. This text explores preventive measures based on animal experimental results. Its aim is to provide references for personalized treatment and occupational protection when using platinum drugs. The continuous progress of science and technology, along with the deepening of medical research, suggests that the application of platinum drugs will broaden. Therefore, the development of new platinum drugs will be an important direction for future research.

Metabonomic study of blood of mice with high-voltage electrical injury / 局解手术学杂志

Objective To explore the changes of metabonomics in blood of mice after high-voltage electric shock,then screen out the significantly changed differential metabolites and metabolic pathways.Methods The head of C57BL/6J mice was subjected to high-voltage electric shock(electric shock group)or exposed to acoustic and optical stimulation of high-voltage electric(control group),then the whole blood from mice were collected to separate serum.The dual platform combined metabonomic analysis based on gas chromatography-mass spectrometer(GC-MS)and liquid chromatography-mass spectrometer(LC-MS)was performed and orthogonal partial least squares discriminant analysis(OPLS-DA)was used to screen the differential metabolites and related metabolic pathways.Results A total of 415 differential metabolites were screened out in metabonomics in blood of mice after high-voltage electric shock,including 187 up-regulated and 228 down-regulated metabolites.These differentially metabolites were significantly enriched in metabolic pathways including central carbon metabolism in cancer,glucagon signaling pathway,etc.Conclusion By establishing the model of high-voltage electrical injury on experimental mice,this study reveals the significant change of metabolite content and metabolic pathway in blood by high-voltage electrical injury.Which provides a basis for the damage of blood metabolic activity by high-voltage electrical injury,and suggests the potential harm of high-voltage electrical injury to blood metabolic activity in the whole body.

Systems pharmacology dissection of action mechanisms for herbs in osteoporosis treatment / 中草药·英文版

Objective: Osteoporosis has become the biggest cause of non-fatal health issue. Currently, the limitations of traditional anti-osteoporosis drugs such as long-term ill-effects and drug resistance, have raised concerns toward complementary and alternative therapies, particularly herbal medicines and their natural active compounds. Thus, this study aimed to provide an integrative analysis of active chemicals, drug targets and interacting pathways of the herbs for osteoporosis treatment. Methods: Here, we introduced a systematic pharmacology model, combining the absorption, distribution, metabolism, and excretion (ADME) screening model, drug targeting and network pharmacology, to probe into the therapeutic mechanisms of herbs in osteoporosis. Results: We obtained 86 natural compounds with favorable pharmacokinetic profiles and their 58 targets from seven osteoporosis-related herbs. Network analysis revealed that they probably synergistically work through multiple mechanisms, such as suppressing inflammatory response, maintaining bone metabolism or improving organism immunity, to benefit patients with osteoporosis. Furthermore, experimental results showed that all the five compounds (calycosin, asperosaponin VI, hederagenin, betulinic acid and luteolin) enhanced osteoblast proliferation and differentiation in vitro, which corroborated the validity of this system pharmacology approach. Notably, gentisin and aureusidin among the identified compounds were first predicted to be associated with osteoporosis. Conclusion: Herbs and their natural compounds, being characterized as the classical combination therapies, might be engaged in multiple mechanisms to coordinately improve the osteoporosis symptoms. This work may contribute to offer novel strategies and clues for the therapy and drug discovery of osteoporosis and other complex diseases.

Characteristics of immune function of full-term infants with different feeding patterns at the age of 3 months: a prospective cohort study / 中国当代儿科杂志

OBJECTIVES@#To explore the characteristics of immune function of healthy full-term infants at the age of 3 months, and to analyze the relationship of immune function with feeding pattern and sex.@*METHODS@#A total of 84 healthy full-term infants born in four hospitals in Beijing and Hohhot, China were prospectively recruited. Their feeding patterns remained unchanged within 4 months after birth. They were divided into a breast-feeding group and a milk powder feeding group according to their feeding patterns. At the age of 3 months after birth, peripheral venous blood samples of the two groups were collected to evaluate cellular immunity and humoral immunity and perform routine blood test. The laboratory indices were compared between infants with different feeding patterns and sexes.@*RESULTS@#Compared with the milk powder feeding group, the breast-feeding group had significantly lower proportion of T cell second signal receptor CD28, immunoglobulin M, and proportion and absolute count of neutrophils (@*CONCLUSIONS@#Sex has no significant effect on the proportion of lymphocyte subsets in 3-month-old full-term infants, but feeding patterns are associated with the proportion of CD28

Forensic Analysis of 52 Corpses Found in Wells / 法医学杂志

OBJECTIVES@#To explore the forensic pathological characteristics of corpses in wells, and to summarize the differences in corpses between homicide and suicide, so as to provide references for forensic analysis of such cases.@*METHODS@#Data of 52 corpses found in wells （51 cases） in Xuchang, Henan Province from 2004 to 2016 were retrospectively collected, and descriptive statistics were performed on the dead individuals, time of death, wells, autopsies, and diatom testings.@*RESULTS@#The proportion of males and females in the 52 corpses was 1∶2.5, and 42 people were at the age of >20-50 years （80.8%）. The accuracy of the death time inference were 75.0% and 54.2% within 8 d and 8 d or more after the actual death time, respectively. Most of the wells （84.3%） were small ones with big wellhead diameters of 60-100 cm. The death causes in homicide cases were mainly mechanical injury and suffocation （90.3%） with heads downwards （58.1%）, but that in suicide cases was mostly drow-ning （85.0%） with heads upwards （65.0%） and body surface abrasions （95.0%）.@*CONCLUSIONS@#Cases of corpses in wells should be comprehensively analyzed according to scene inspections, autopsies, and auxi-liary tests combined with inspection results.

Analysis of malaria cases in Haidian District of Beijing from 2005 to 2017 / 中华疾病控制杂志

Objective To understand the epidemiological character of malaria in Haidian District of Beijing from 2005 to 2017. Methods The epidemiological data of malaria was collected from the infectious disease reporting system of medical institutions at various levels in Haidian District of Beijing from 2005 to 2017, and the epidemiological methods was used to analyze the distribution of malaria in population, time and region. Results From 2005 to 2017, 111 malaria cases were reported in Haidian District of Beijing, the annual average incidence rate was 0.26/100 000 and one death case was reported in 2014. Among the four reported types of falciparum malaria, vivax malaria, three-day malaria and untyped malaria, the most common falciparum malaria (54.5%, 60/111), no mixed infection; The peak incidence was concentrated in the summer and autumn of June-September (52.0%, 58/111); the cases were mainly occurred in young adults aged from 20 to 59(93.7%,104/111), and the incidence of males was higher than that of females ( 2=52.9, P<0.001); Cadres were the main ward population (33.3%, 33/111). Malaria cases were reported in 26 streets and towns in Haidian District. 81 cases were imported from abroad, accounting for 71.4% of the total cases, of which 74 (91.36%) were originated from Africa. Conclusions In the past 13 years, the incidence of malaria was sporadic, mainly in imported cases. The monitoring of malaria should be strengthened by entry and exit to prevent the second-generation cases of malaria.

Preparation of long-circulating dihydromyricetinliposomes and its pharmacokinetics in rats / 中草药

Objective To prepare dihydromyricetin (DMY) long-circulating liposomes and evaluate in vitro release dynamics and in vivo pharmacokinetics in rats. Methods Film-ultrasonic method was used to prepare DMY liposomes by single factor experiment and orthogonal test to optimize the formulation and preparation of DMY liposomes. The particle size and zeta potential of liposomes were determined by laser particle size analyzer. The morphological examination of liposomes was performed by using transmission electron microscopy. The liposome release in vitro was studied using dialysis method. DMY concentration in rat plasma was determined by the established LC-MS/MS method. Results The optimal prescription was 75∶20∶5 for soybean phospholipid-cholesterol-mPEG 2000-DSPE, and 1∶12 for DMY-lipid (wt/wt) with the ultrasonic time of 20 min and loading temperature of 60 ℃ in pH 5.0 PBS buffer. Under the optimized conditions, DMY liposomes was sphere with mean particle size of (117.9 ± 5.5) nm and mean zeta potential of (−2.6 ± 1.7) mV, the encapsulation efficiency and drug-loading content was (54.7 ± 3.3) % and (4.3 ± 0.2) %, respectively. The in vitro accumulative release rate of 48 h was 86% in pH 1.2 and pH 6.8 dissolve medium. Compared with free DMY, the t1/2z and AUC0-∞ of DMY liposome were increased by 2.7-fold and 1.8-fold, respectively. Conclusion Compared with free DMY, DMY liposomes released gently and slowly in vitro, eliminated slowly in vivo, and had higher bioavailability of oral administration.

A case report of the old patient with congenital aortic coarctation and severe coronary heart disease / 天津医药

Congenital coarctation of the aorta(CoA)is a congenital macrovascular disease.Most of the untreated CoA patients died before the age of 30 years. This study presented a 67-year-old patient who was first diagnosed as aortic coarctation with severe coronary heart disease and was long-term survival.This paper reviewed the diagnosis and treatment of the patient and reviewed the relevant literature of the diagnosis and treatment.

Identification of Fel Serpentis and its adulterants with specific PCR method / 中国中药杂志

The study aims at developing a convenient and specific method for the identification of Fel Serpentis DNA. The methods of Fel Serpentis genomic DNA purification were tested and optimized, four pairs of specific primers for the amplification of COⅠ, Cyt b and 16S were designed. Then the best pair of primers were selected according to the specificity and efficiency. The DNA fragment about 400 bp was amplified from 20 kinds of Fel Serpentis, whereas no DNA fragment was amplified from other animal samples under the same condition. This method is specific,accurate and reproducible, which provides a useful tool for the quality control of Fel Serpentis.

Psoralen inhibits RAW264.7 differentiation into osteoclasts and bone resorption by regulating CD4+T cell differentiation / 中国中药杂志

This paper aimed to investigate whether psoralen inhibits the differentiation and bone resorption by regulating CD4+T cell differentiation in RANKL-induced osteoclastogenesis in RAW264.7 cells, and elucidate its mechanism for osteoporosis. CD4+T cells were isolated from spleen cells of Balb/c mice by immunomagnetic separation method. The cells were divided into blank control group and psoralen group. The cells were cultured in 24-well plates and cultured for 3 days, and then they were collected for co-culture experiments after 4 days. Co-culture experiments were divided into RAW264.7 cell group, psoralen+RAW264.7 cell group, without psoralen treatment of CD4+T cells+RAW264.7 cell group, psoralen treatment of CD4+T cells+RAW264.7 cell group. After 5 days of co-culture, TRAP staining was used to detect the number of osteoclasts, and after 8 days of co-culture, bone resorption was evaluated by toluidine blue staining. The expressions of RORγt, Foxp3, IL-17, TNF-α, TGF-β and IL-10 in CD4+T cells and osteoclast differentiation-related genes MMP-9, TRAP and Cat-K were detected by Real-time polymerase chain reaction (RT-PCR); ELISA kit was used to detect IL-17, TNF-α, TGF-β and IL-10 and other cytokines levels. Our data confirmed that the psoralen significantly promoted the expression of Foxp3, TGF-β and IL-10 in CD4+T, and inhibited the expression of RORγt, IL-17 and TNF-α in CD4+T, the CD4+T cells without treatment by psoralen can significantly promote RANKL-induced differentiation of RAW264.7 to osteoclasts, and psoralen treatment of CD4+T can significantly inhibit RANKL-induced RAW264.7 osteoclast differentiation and bone resorption. Taken together, psoralen inhibits the differentiation and bone resorption of RAW264.7 into osteoclasts by promoting the development of CD4+ CD25+ Treg/Th17 balance in CD4+T cells to CD4+CD25+T.

Effect of puerarin in Longmaining formula on pharmacokinetics-pharmacodynamics correlation in rats with myocardial ischemia / 中国中药杂志

To study the pharmacokinetics of puerarin in compound Longmaining(FFLMN) in normal rats and myocardial ischemia rats, and investigate its correlation with anti-myocardial ischemia effect of FFLMN. Models of myocardial ischemia rats were produced by subcutaneous injection of isoproterenol(ISO), then FFLMN extract solution was administered by gavage. Orbital sinus blood sampling was collected at different time points after gavage. HPLC-UV method was applied to determine the concentration of puerarin in plasma, and compare the difference in pharmacokinetics between normal rats and model rats after application of same dose of FFLMN. Meanwhile, microplate reader was used to determine IL-6 and SOD activities in plasma of different time points, and draw dose-effect curve. The results indicated that the pharmacokinetics of puerarin conformed to the two-compartment model in both normal group and myocardial ischemia model group. In the comparison of main pharmacokinetic parameters between two groups: AUC0-∞=(11.451±3.228) mg•h•L⁻¹,AUC0-t=(14.047±3.765) mg•h•L⁻¹, Cmax=(5.623±1.40) mg•L⁻¹ in normal group; AUC0-∞=(68.849±50.396 9) mg•h•L⁻¹, AUC0-t= (58.312±45.802) mg•h•L⁻¹,Cmax=(18.456±7.517) mg•L⁻¹ in treatment group. The SOD level was significantly increased and IL-6 concentration was significantly decreased in plasma, indicating that as compared with the normal group, puerarin in FFLMN had a higher plasma concentration, slower elimination rate and higher bioavailability. Therefore, puerarin concentration in plasma has correlation with the anti-myocardial ischemia effect of FFLMN, which could increase SOD level and inhibit the release of IL-6.

Antiapoptotic Effect of the Leukemia Associated Gene MLAA-34 in HeLa Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the antiapoptotic effect of leukemia-associated gene MLAA-34 in HeLa cells.</p><p><b>METHODS</b>The MLAA-34 recombinant lentiviral expression vector was constructed, and the stably transfected HeLa cell line with high expression of MLAA-34 was set up; As(2)O(3) was used to induce apoptosis; the MTT assay, colony formation test and flow cytometry were used to detect the ability of cell proliferation, colong formation, apoptosis and cell cycle changes respectively.</p><p><b>RESULTS</b>After treatment with As(2)O(3), the survival rate of HeLa cells with MLAA-34 overexpression was significantly higher than that of the control cells, and the colony formation ability of MLAA-34 significantly increased, and the high expression of MLAA-34 gene significantly decreased the apoptosis rate of HeLa cells, and decreased the proportion of G(2)/M phase cells.</p><p><b>CONCLUSION</b>The leukemia-associated gene MLAA-34 has been comfirmed to show antiapoptotic effect in HeLa cells which are induced by As(2)O(3).</p>

Modified titration intratympanic gentamicin injection for unilateral intractable Ménière's disease / 华中科技大学学报（医学）（英德文版）

This study looked into the efficacy of a modified titration protocol of intratympanic gentamicin injection (ITG) in the patients with unilateral intractable Ménière's disease (MD). Modified titration protocol of ITG at a low dose (20 mg/mL) was administered to 10 patients with definite unilateral intractable MD. After initial first two fixed ITGs on weekly basis, the patients might or might not be given any more injections, depending on the appearance of unilateral vestibular loss (UVL). ITG was terminated if the patients satisfied the criteria of UVL. All patients were followed-up for at least two years. The effects of ITG on the vertigo attack, functional level scores and postural balance were evaluated. Of the 10 cases, 8 showed the sign of UVL after receiving initial two ITGs and were not given any more intratympanic injections, and the other 2 patients were administered three ITGs. A two-year follow-up revealed that complete and substantial vertigo control was achieved in 9 cases, and limited vertigo control in 1 patient. Hearing level was lowered in 2 patients. The posture stability and functional level scores were improved. Our study showed that the modified titration protocol of ITG at a low dose could effectively control vertigo in patients with unilateral intractable MD.

Changes of ADAMTS13 activity and vWF antigen level in patients with acute myelogenous leukemia and their significance / 中国实验血液学杂志

This study was purposed to investigate the changes of von Willebrand factor cleaving protease (ADAMTS13) activity and vWF antigen level in patients with acute myelogenous leukemia (AML) before and after treatment and evaluate their clinical significance. Seventy-three AML patients were enrolled in this study, the sodium citrate anticoagulated plasma was collected before and after their induction chemotherapy. Fluorescence resonance energy transfer substrate vWF73 (FRETS-vWF73) assay was established to detect the plasma ADAMTS13 activity while vWF antigen level was measured by ELISA. The results showed that the ADAMTS13 activity in newly diagnosed patients with AML before induction therapy was obviously lower than that in normal controls (63.3 ± 25.5)% vs (105.1 ± 37.7)(P < 0.01), while the vWF antigen level was higher than that in normal controls (226.6 ± 127.0)% vs (111.4 ± 39.7)% (P < 0.01). After standard induction chemotherapy, the ADAMTS13 activity of AML patients in complete remission period was higher than that in AML patients before therapy (P < 0.01), and was not significant difference with that in normal controls; the vWF antigen was significantly lower than that in AML patients before therapy (P < 0.01), but it still was higher than that in controls (P < 0.05). The ADAMTS13 activity in newly diagnosed AML patients complicated with infection before therapy was obviously lower than that in AML patients without infection (52.2 ± 20.6)% vs (73.9 ± 24.7)% (P < 0.01), while the vWF antigen level was significantly higher than that in AML patients without infection (262.2 ± 135.7)% vs (193.8 ± 110.2)% (P < 0.05). The ADAMTS13 activity in AML patients with disseminated intravascular coagulation (DIC) was significantly lower than that in AML patients without DIC (42.0 ± 14.5)% vs (73.4 ± 22.7)% (P < 0.01), while the vWF antigen level was obviously higher that in AML patients without DIC (274.2 ± 140.0)% vs (204.7 ± 115.5)% (P < 0.01). It is concluded that the ADAMTS13 activity in newly diagnosed AML patients befor induction therapy has been confiremed to be lower and the vWF antigen level to be higher, especially in AML patients with infection or DIC. The ADAMTS13 and vWF antigen may play a role in the pathogenesis of AML and the formation of infection and DIC.

Three-dimensional finite element analysis of different reactive force direction of maxillary protraction on temporomandibular joint / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To analyze the influence of different reactive force direction of protractions on temporomandibular joint (TMJ) by establishing a three-dimensional finite element model (FEM) of craniomaxillofacial complex.</p><p><b>METHODS</b>The CT image of the head of a healthy young male volunteer was obtained.With the help of Mimics software, we established a three-dimensional finite element model of craniomaxillofacial complex which included TMJ. The force pattern of maxillary protraction appliance was imitated. The force (5 N) was applied on the chin and the direction of force was from 22° to 49° relative to the occlusal plane. The displacement and stress distribution of TMJ were analyzed.</p><p><b>RESULTS</b>The contact stress on the maxilla decreased with the angle of the force direction increased from 22° to 40°, and increased with the angle increased from 40° to 49°. The stress on the condyle decreased with the angle of the force direction increased. The stress on the condylar neck decreased initially and then increased with the angle of the force direction increased. Comprehensively, the stress was the smallest when the angle of the force direction was 40°. The clockwise rotation of the mandible was found when the angle of the force direction was smaller than 40°. The displacement value was relatively small when the angle was 40°.</p><p><b>CONCLUSIONS</b>Stress and displacement were relatively small when the angle of the force direction was 40° relative to the occlusal plane.</p>

Preliminary analysis of aberrant expression of plasma miR-223 in pediatric acute lymphoblastic leukemia with a direct RT-PCR assay / 中国实验血液学杂志

This study was aimed to investigate the expression of plasma miR-223 in pediatric acute lymphoblastic leukemia (ALL) in different treatment time point. A total of 64 pediatric ALL samples were selected from patients treated in Beijing Children's Hospital from May 2005 to January 2012, including 30 samples at new diagnosis (ND), 30 samples at complete remission (CR) and 4 samples at relapse. Without RNA extraction, the miR-223 levels in plasma were directly detected by a reverse-transcription quantitative real-time PCR assay. The results indicated that the expression of plasma miR-223 in pediatric ALL was lower at ND but elevated after CR. The miR-223 expression in plasma of relapse patients didn't show significant difference probably due to a few cases of relapse. The miR-223 levels in plasma had not displayed significant difference between TEL-AML1 positive patients and no fusion gene B lineage ALL patients either at ND or at CR. It is concluded that the plasma miR-223 decreases at ND and increases in CR of children with ALL. miR-223 may act as an anti-oncogene and may be taken as a potential predictive biomarker for evaluating the therapeutic effect of leukemia.

Influence of transplantation and some clinical factors on prognosis of patients with diffuse large B-cell lymphoma / 中国实验血液学杂志

This study was aimed to analyze the survival status of patients with diffuse large B-cell lymphoma (DLBCL) and to investigate the influence of autologous hematopoietic stem cell transplantation (auto-HSCT), different pathological types, International Prognosis Idex (IPI) on prognosis. One hundred and sixteen cases of DLBCL were analyzed retrospectively. The treatment efficacy of R-CHOP alone and R-CHOP combined with auto-HSCT as well as the influence of different immunopathologic types, IPI, hypersensitive C-reactive protein (HSCRP), α-hydroxybutyric acid deaminase (HBDH) on the prognosis of DLBCL patients including overall survival (OS) rate, progression-free survival (PFS) rate were analyzed. The results indicated that the 5-year OS for all patients was 72.4%. in which 30 patients with Ann Arbor staging III-IV received auto-HSCT plus R-CHOP. The prognosis of the 30 patients was better than that of 86 cases received R-CHOP chemotherapy alone (5-year OS was 82.5% vs 69.0%, 5-year PFS was 77.1% vs 68.3%) (P < 0.05). The prognosis of patients in germinal center B-cell-like group (GCB group) was better than that of patients in activated B-cell-like group (ABC group). Some clinical features were associated with poor prognosis including OS and PFS, such as age, B symptoms, IPI scores, the level of LDH, HSCRP and HBDH (P < 0.05) in which the level of LDH, age ≥ 60 years and B symptoms were independent prognostic factors in DLBCL patients (P < 0.05). It is concluded that auto-HSCT combined with R-CHOP can improve the long-term survival of DLBCL patients. The prognosis of patients in GCB group is better than that of patients in the ABC group. The clinical features such as age, B symptoms, IPI scores and LDH are associated with prognosis.

Effect of hyperventilation on cerebral blood flow, brain metabolism and cerebral perfusion in patients with traumatic brain injury by PETCO2 monitoring / 中华神经医学杂志

Objective To investigate the effect ofhyperventilation on cerebral blood flow,brain metabolism and cerebral perfusion in patients with traumatic brain injury by end tidal pressure of CO2 (PETCO2) monitoring.Methods Seventy patients with traumatic brain injury,admitted to our hospital from January 2009 to June 2012 and performed decompressive craniectomy,were chosen in our study; they were randomly divided into group A and group B (n=35); patients in the group A accepted PETCO2 within 20-25 mm Hg and arterial partial pressure of carbon dioxide (PaCO2) within 22-25 mm Hg,and patients in the group B received PETCO2 within 25-30 mm Hg and PaCO2 within 30-45 mm Hg.The blood gas pressure,mean artery pressure,cerebral oxygen partial pressure,cerebral perfusion pressure,cerebral oxygen supply and demand balance,and changes of neuron specific enolase (NSE),glucose and lactic acid were compared between the two groups at the beginning of craniectomy.Results The PaCO2 level,mean artery pressure,intracranial pressure,and levels of NSE,glucose and lactic acid in group A were significantly lower than those in group B (P＜0.05); while the brain oxygen partial pressure,cerebral perfusion pressure,arterial oxygen content (CaO2),venous oxygen content (CjvO2) and cerebral oxygen uptake rate (CERO2) showed no significant difference between the two groups (P＞0.05).Conclusion PETCO2 controlling within 20-25 mm Hg can effectively maintain the cerebral blood perfusion and cerebral oxygen supply and demand balance of the patients; it can also reduce the pathological metabolite effect on the brain tissue; therefore,its clinical value is worthy for attention.

Function of luxS gene in sulfurmetabolism of Streptococcus mutans / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the function of luxS in sulfurmetabolism of Streptococcus mutans (Sm).</p><p><b>METHODS</b>The growth with absorbency (A) of the standards and mutant strains was measured and analyzed in the sulfur-limited defined medium at different periods. The laser scanning confocal microscopy (LSCM) was used to observe and compare the biofilm thickness of the two kinds of strains at different culture conditions.</p><p><b>RESULTS</b>The significant increases in the thickness of mutant strain biofilm and its growth were observed after the addition of cysteine, but did not reach the standards strain levels (P < 0.05). The growth and the biofilm thickness of the mutant strains were (1.301 ± 0.009) and (45.009 ± 0.429) µm. When methionine and S-adenosylhomocysteine of certain concentrations were respectively added, the biofilm thickness and the growth of mutant strain were raised but did not reach the level of the standards strain at 24 h (P < 0.05), but at 48 h they did. When the methionine was added in the mutant strains for 24 h, the biofilm thickness and the growth of mutant strain were (0.448 ± 0.028) and (37.068 ± 2.392) µm, as for the adding of S-adenosylhomocysteine were (0.460 ± 0.005) and (27.343 ± 1.107) µm. When adding the supernatant fluid of standard strains, the biofilm thickness and the growth levels of mutant strain were much higher than those of the standards strain. The biofilm thickness and growth of both kinds of strains decreased after the addition of S-adenosylmethionine.</p><p><b>CONCLUSIONS</b>luxS gene plays not only a role in quorum sensing but also a role in sulfurmetabolism.</p>

Application of Cre-loxP(*) system in constructing the markerless double-gene-deletion strain in Streptococcus mutans / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To construct double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans (Sm) and to remove the antibiotic resistance markers with the Cre-loxP(*) site-specific recombination system.</p><p><b>METHODS</b>The htrA gene was cloned into the pGEM-T-Easy TA cloning vector and then inactivated via the insertion of a kanamycin resistance cassette (lox71-Km-lox66), yielding pGEM-T-ΔhtrA/Km for deleting the htrA gene. Using the same method, the pGEM-T-ΔclpP/Sp was constructed for deleting the clpP gene. Following the transformation of pGEM-T-ΔhtrA/Km in Sm, the homologous recombination event was selected. One such mutant was transformed with a cre expression plasmid (pCrePA). The kanamycin resistance gene was then excised. The pCrePA was then easily eliminated at nonpermissive temperatures, resulting in a mutant strain (MSΔhtrA) carrying a deletion at the htrA loci without a selectable marker. This mutant was verified by PCR and DNA sequencing. Then, the clpP and spectinomycin resistance gene were deleted from MSΔhtrA, yielding markerless mutant strain lacking clpP and htrA.</p><p><b>RESULTS</b>The deletion of htrA, clpP and antibiotic resistance markers were confirmed by PCR analysis and DNA sequencing.</p><p><b>CONCLUSIONS</b>A mutant of Sm was constructed successfully which contained a deletion of the htrA and clpP gene without selectable marker. The Cre-loxP(*) system can be applied to Sm, which provides experimental evidence for generating markerless multiple gene deletion mutants.</p>