Biomechanical study on the treatment of Pauwels type III femoral neck fracture by open reduction and internal fixation with hollow nail internal fixation.

OBJECTIVE: We aimed to explore the therapeutic effect of open reduction and internal fixation with hollow nail internal fixation for Pauwels type Ⅲ femoral neck fracture. PATIENTS AND METHODS: From January 2016 to February 2021, a total of 100 eligible patients with Pauwels type III femoral neck fracture were involved in this study and divided into two groups randomly: the combined remedy group and the closed therapy group, with 50 patients in each group. After that, 50 subjects in the combined remedy group were treated with open reduction and support plate combined with hollow screw internal fixation, and the treatment conditions were observed and recorded. The closed therapy group received routine treatment. RESULTS: Among the 100 patients selected, the operation time of the combined remedy group was significantly lower than that of the closed therapy group, and the intraoperative bleeding was also significantly less. In the closed therapy group, the time of getting out of bed after the operation and the excellent and good rate were better; moreover, the functional score and pain score of three months after the operation were significantly better than that of one month after the operation. The functional score and pain score of one month after the operation were not statistically significant for the combined remedy group or the closed therapy group. CONCLUSIONS: In the treatment of Pauwels type Ⅲ femoral neck fracture with open reduction and internal fixation combined with hollow nail internal fixation, the operation time and intraoperative bleeding volume were significantly decreased, but the postoperative recovery time was enhanced compared to that of total joint replacement. After the operation, the functional score and pain score had a significant relationship with the recovery time, and there was no significant relationship with the type of treatment. Therefore, in clinical treatment, doctors should take appropriate treatment methods for their patients.

[Comparison of epidermal growth factor receptor (EGFR) gene T790M mutation by droplet digital PCR and Super-ARMS PCR in plasma ctDNA samples of non-small cell lung cancer patients with the resistance to EGFR-tyrosine kinase inhibitor].

Objective: To compare droplet digital PCR (ddPCR) and Super-amplification refractory mutation system (ARMS) in the detection of T790M mutation of epidermal growth factor receptor (EGFR) in the plasma of non-small cell lung cancer patients who had developed resistance to EGFR tyrosine kinase inhibitor (TKI) , and to investigate the clinical application of ddPCR. Methods: Plasma samples were collected from non-small cell lung cancer patients who had acquired EGFR-TKI resistance at Shanghai Pulmonary Hospital, Tongji University, from May 2017 to November 2017. Extracted ctDNA was analyzed by ddPCR and Super-ARMS to evaluate the T790M mutation status of EGFR gene. Results: A total of 37 patients with activating EGFR mutation that acquired resistance to EGFR-TKI were selected in the study, including 17 male and 20 female with a median age of 64 years (range 40-83 years). Before TKI treatment, all the patients harbored EGFR inhibitor sensitive mutations but without T790M mutation. After acquiring resistance to EGFR-TKI treatment, the T790M mutation rate detectable by ddPCR was 45.9% (17/37). In contrast, the mutation rate of T790M detectable by Super-ARMS was 35.1% (13/37, P<0.05). For the 13 positive cases detected by Super-ARMS (ΔCt<8), they were all positive by ddPCR assay; Among the 10 negative cases detected by Super-ARMS (ΔCt≥8), there were 3 cases positive by ddPCR assay. For patients without ΔCt by Super-ARMS assay, there was one weak positive case detectable by ddPCR assay. Among 17 EGFR T790M positive patients, 9 received EGFR inhibitor Osimertinib treatment, and 7 of them had good therapeutic response after the treatment. Conclusions: While a significant correlation between the two methods is shown. ddPCR is more sensitive than Super-ARMS in the detection of EGFR T790M mutation, indicating that it is a better method in guiding target drug therapy of non-small cell lung cancer patients after acquiring the resistance to EGFR-TKI.

[The application of modified cosmetic incision and skin adhesive in parotid benign tumor functional surgery].

Objective:To investigate the clinical application value and related technology of modified cosmetic incision and cyanoacrylate octyl ester skin adhesive (Dermabond) in functional surgery of parotid benign tumor. Method:Forty-two cases of clinical cases were collected and randomly divided into groups of A and B. Group A used the approach of modified cosmetic incision,intradermal suture with 6-0 absorbable Vicryl stitches and gluing the skin with Dermabond. Group B used the approach of traditional S shaped incision and continuous suture of skin with 4-0 Prolene stitches.The operative time, postoperative drainage volume, facial nerve function, salivary leakage, Frey syndrome, postoperative numbness, scar and aesthetic satisfaction were compared between the two groups. Result:The operation time, facial nerve function,Frey syndrome, salivary fistula, facial deformity and tumor recurrence in the two groups had no significant difference (P>0.05); but for postoperative drainage, postoperative numbness, facial appearance satisfaction, scar assessment, Group A was lower than Group B and there was statistical significance difference (P<0.05). Conclusion:The application of modified cosmetic incision and Dermond is safe and feasible. Its complication is lower than the traditional operation. The scar is smaller and more hidden, and the patient's satisfaction is higher. It doesn't affect the daily social interaction of patients, and achieves the effect of minimally invasive treatment.

[The influence of chemotherapy-induced leucopenia on the disease-free survival of gastric cancer patients after radical gastrectomy].

Objective: To investigate the association between chemotherapy-induced leucopenia and patients' disease-free survival in gastric cancer patients who received radical gastrectomy. Methods: The clinical data of 273 gastric cancer patients who received radical gastrectomy and postoperative adjuvant chemotherapy between January, 2010 and December, 2015 in PLA 309(th) hospital was reviewed retrospectively. Results: A total of 195 (71.4%) patients experienced at least one time of leucopenia, while it was absent in the other 78 (28.6%) patients. The median disease-free survival of patients with or without leucopenia was 49.7 and 44.0 months respectively (P=0.009), leucopenia was an independent factor influencing patients' disease-free survival (HR=2.758, P=0.022), but there was no statistical difference between the disease-free survival of patients with different degrees and frequency of leucopenia (P=0.446, 0.123). Conclusion: Chemotherapy-induced leucopenia is a predictor of good prognosis for gastric cancer patients who receive radical gastrectomy.

Impact of human activities on subaqueous topographic change in Lingding Bay of the Pearl River estuary, China, during 1955-2013.

Estuaries have been sites of intensive human activities during the past century. Tracing the evolution of subaqueous topography in estuaries on a decadal timescale enables us to understand the effects of human activities on estuaries. Bathymetric data from 1955 to 2010 show that land reclamation decreased the subaqueous area of Lingding Bay, in the Pearl River estuary, by ~170 km2 and decreased its water volume by 615 × 106 m3, representing a net decrease of 11.2 × 106 m3 per year and indicating the deposition of approximately 14.5 Mt/yr of sediment in Lingding Bay during that period. Whereas Lingding Bay was mainly governed by natural processes with slight net deposition before 1980, subsequent dredging and large port engineering projects changed the subaqueous topography of the bay by shallowing its shoals and deepening its troughs. Between 2012 and 2013, continuous dredging and a surge of sand excavation resulted in local changes in water depth of ± 5 m/yr, far exceeding the magnitude of natural topographic evolution in Lingding Bay. Reclamation, dredging, and navigation-channel projects removed 8.4 Mt/yr of sediment from Lingding Bay, representing 29% of the sediment input to the bay, and these activities have increased recently.

Security assessment of magnesium alloys used as biodegradable implant material.

The security risk of magnesium alloys used as biodegradable implant material was evaluated in this study. Dose-response assessment was conducted by using toxicological data from authoritative public health agencies (World Health Organization) and assuming 1~3 years of uniform corrosion. Through modification calculation, the tolerable corrosion rate of biodegradable magnesium alloys in vivo was proposed, which theoretically ensured the bio-safety of the degradation products. The tolerable limits corresponding to various component elements in magnesium alloys were considered separately, although there are deficits in the toxicological data of some component elements. The influence of corrosion on the strength of magnesium alloys was evaluated, which would contribute to the rationally utilization of magnesium alloys as degradable implant materials. This study illustrates that not only toxicological calculations but also mechanical performance should be taken into consideration when developing novel degradable metallic implant.

Alteration of coenzyme specificity of malate dehydrogenase from Streptomyces coelicolor A3(2) by site-directed mutagenesis.

We describe here for the first time the alteration of coenzyme specificity of malate dehydrogenase (MDH) from Streptomyces coelicolor A3(2) (ScMDH). In the present study, we replaced four amino acid residues in the Rossmann fold (ßB-αC) region of NADH-dependent ScMDH by site-directed mutagenesis with those of NADPH-dependent MDH (Glu42Gly, Ile43Ser, Pro45Arg, and Ala46Ser). The coenzyme specificity of the mutant enzyme (ScMDH-T4) was examined. Coenzyme specificity of ScMDH-T4 was shifted 2231.3-fold toward NADPH using kcat/Km(coenzyme) as the measurement of coenzyme specificity. Accordingly, the effect of the replacements on coenzyme specificity is discussed. Our work provides further insight into the coenzyme specificity of ScMDH.

First Report of Elm Yellows Subgroup 16SrV-B Phytoplasma as the Cause of Rose Balsam Phyllody in China.

Rose balsam (Impatiens balsamina L.) is an ornamental species frequently cultivated in China and the red flower is often used as nail polish in rural regions. The phytoplasmas previously reported with rose balsam phyllody in China have been classified as aster yellows group (16SrI) (1). In August 2012, some rose balsams were observed with typical phytoplasma symptoms in Handan City, Hebei Province, China, with an incidence of about 70% in the fields. The flowers turned green and petals fascicled. The new leaves wrinkled and deformed and internodes shortened. Infected plants were stunted, matured prematurely, and failed to produce seeds. To confirm phytoplasma infection, 100 mg of plant tissue (leaves, petals) was collected from five symptomatic and four asymptomatic plants and total DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method (2). The 16S rDNA gene was amplified by nested PCR using primer pair P1/P7 followed by R16F2n/R16R2 (3). No amplicons were generated with DNA from asymptomatic samples, but amplicons of approximately 1.2 kb were obtained with DNA from five symptomatic samples. The amplified products were purified with aTIANgel midi purification kit (Tiangen, Beijing) and sequenced at the Sangon Biotech facility (Shanghai, China). The sequences of the amplicons were 100% identical and deposited in NCBI GenBank (Accession No. KC993832). The 16S rDNA gene sequence from this phytoplasma was 99% similar to Jujube witches broom phytoplasma (JQ675716), Puna chicory flat stem phytoplasma (JN582266), Plum yellows phytoplasma (FJ459914), and other elm yellows group phytoplasmas by BLAST search of the NCBI database. Restriction fragment length polymorphism (RFLP) analyses were carried out by digesting the 1.2-kb R16F2n/R16R2 nested PCR product with restriction enzymes AluI, RsaI, HhaI, HpaI, Eco RI, TaqI, HaeIII, HinfI, and KpnI (Takara, Dalian). The 16S rDNA RFLP patterns matched that of Jujube witches broom phytoplasma (JWB, subgroup 16SrV-B) (4). Nucleotide sequences of rose balsam phyllody were analyzed by iPhyClassifier software, which revealed that it had maximum similarity to the reference pattern of 16Sr group V, subgroup B (AB052876). All samples were detected with transmission electron microscopy. The results showed phytoplasma-like cells in phloem sieve element of symptomatic plants, while no phytoplasma-like cells were observed in healthy phloem tissues. The phytoplasma cells ranged from 230 to 470 nm in diameter and were ellipsoidal or orbicular with visible membranes. Combining the RFLP pattern and sequence analysis by iPhyClassifier, we classified the phytoplasma causing rose balsam phyllody into subgroup 16SrV-B. To our knowledge, this is the first report of 16SrV-B group phytoplasmas infecting rose balsam in China. References: (1) Z. N. Li et al. J. Phytopathol. 159:799, 2011. (2) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. 81:8014, 1984. (3) I. M. Lee et al. Phytopathology 83:834, 1993. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

First Report of Fusarium equiseti Causing a Sheath Rot of Corn in China.

Corn is the most important cereal crop in China. Over 34.94 million ha of corn is cultivated in the country annually. However, fungal diseases are a major limiting factor in corn production. In August 2008, 50 ha in several corn fields in Liaoning, Jilin, and Heilongjiang provinces were observed to be severely affected by a disease causing a yield loss of 30%. Results from field surveys suggested an epidemic during late corn growth stages that affected corn sheaths, causing irregularly circular spots with grayish brown to dark brown lesions. Lesions ranged from 2.5 to 3 × 3 to 5 cm. To isolate the causal agent, tissue was removed from the border of lesions and surface sterilized in 75% ethanol for 30 sec and 0.1% HgCl2 for 1 min. The sample was then triple rinsed in sterile distilled water. The isolate was purified and subcultured on potato dextrose agar (PDA) at 25 ± 2°C. The initial color of the mycelium was white, turning brown after being cultured for 7 days. A pale brown to dark brown pigment developed in the agar beneath the colony. Chlamydospores, solitary but also in short chains, measuring 7.2 to 15.3 µm, were produced on carnation leaf agar (CLA) after 10 days and became verrucose 20 days later. Macroconidia were produced on CLA in orange sporodochia from monophialides on branched conidiophores, usually 5- to 7-septate, and apical cells were tapered and elongate. Basal cells were prominent, foot-shaped, and elongated in appearance. Microconidia were not observed (1). These morphological characteristics matched the description of Fusarium equiseti reported by Leslie and Summerell (1). A pathogenicity test was conducted with an isolate from each of the 36 corn plants by spraying 2 ml of spore suspension (106 conidia/ml) on 45-day-old corn sheaths (cv. Huang Zao). For the control treatment, 36 corn plants were sprayed with an equal volume of sterilized water. Inoculated plants were placed in a greenhouse at 32 to 34°C and 95% relative humidity. Typical irregularly circular lesions were observed 7 days after inoculation, except in the control samples. Each treatment was replicated three times. The suspected pathogen was consistently re-isolated from diseased tissue according to Koch's postulates, and was found to be morphologically similar to F. equiseti. Preliminary morphological identification of the fungus was confirmed by a PCR assay using genomic DNA extracted from the mycelia of a 7-day-old culture on PDA at 25 ± 2°C. A 750-bp amplified region of the transcription elongation factor (TEF) of rDNA was generated using TEF1 (5'-ATGGGTAAGGAGGACAAGAC-3') and TEF2 (5'-GGAAGTACCAGTGATCATGTT-3') primers. The TEF region (GenBank Accession No. KF754798) was sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) and displayed 99% nucleotide similarity with the rDNA-TEF of F. equiseti (JN127347.1) separately after a BLASTn search in GenBank. Based on the symptoms, fungal morphology, TEF sequence, and pathogenicity testing, this fungus was identified as F. equiseti. To our knowledge, this is the first report of F. equiseti on corn sheaths in China. This report will establish a foundation for further study of F. equiseti to address the disease effectively and to determine the severity of damage caused by F. equiseti. Reference: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006.

First Report of Bipolaris papendorfii Causing Corn Leaf Spot in China.

Corn is the most important cereal crop in China, with over 34.94 million ha being cultivated in the country annually. However, fungal diseases are a major limiting factor in corn production. In August 2012, 20 ha of corn fields in Anhui Province were found to be heavily infected by fungi. The margin of the lesion was achlorotic, and the middle was yellowish white or off-white, which was similar to the corn Curvalaria leaf spot. The oval lesions were approximately 5 to 7 mm. Lesion tissue was removed from the border between symptomatic and healthy tissue. The surface was sterilized in 75% ethanol for 30 s and 0.1% HgCl2 for 1 min, after which the sample was washed three times in sterile distilled water. The isolate was purified and subcultured on potato dextrose agar (PDA) at 25 ± 2°C. The initial color of the colony was light brown, turning dark brown after being cultured for 7 days. The conidia were boat-shaped or inverted pear-shaped and were clearly bent to one side. The cells of both ends were slightly lighter and respectively ranged from 34.5 to 44.0 µm and 12.0 to 21.0 µm away from the base, with the second cell as the widest. The majority conidia had three or four false septates; isolates produced light brown to medium brown conidiophore, scattered or clustered, often branching, and exhibited bending. These morphological characteristics matched with the description of Bipolaris papendorfii reported by Zhang (3). A pathogenicity test was conducted with the two isolates on each of the 36 corns by spraying 2 ml spore suspension (106 conidia/ml). For the control treatment, 36 corns were inoculated with an equal volume of sterilized water. Inoculated plants were placed in a greenhouse from 29 to 33°C and 95% relative humidity. The typical 5 to 7 mm oval lesions were observed 7 days after inoculation, except on the control samples. Three replications of 36 corns were used for each treatment. The isolate was consistently 100% reisolated from the diseased tissue according to Koch's postulate. The isolate was found to be morphologically similar to B. papendorfii. Preliminary morphological identification of the fungus was confirmed by PCR assay using genomic DNA extracted from the mycelium of a 7-day-old culture on PDA at 25 ± 2°C. A 550-bp amplified region of the internal transcribed spacer (ITS) of rDNA was generated using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') universal primers (1). The ITS region (GenBank Accession No. KC592365) was then sequenced by Sangon Biotech (Shanghai, China), and displayed 99% nucleotide similarity with the rDNA-ITS of B. papendorfii (JQ753972.1) separately after BLASTn research in GenBank. Based on the symptoms, fungal morphology, ITS sequence, and pathogenicity testing, this fungus was identified as B. papendorfii. The pathogen could reportedly infect tobacco and cotton (2). To our knowledge, this is the first study to report that B. papendorfii can infect corn in China. This report will establish a foundation for the further study of B. papendorfii to address the disease effectively. Further studies will be conducted to determine the incidence of the disease and the severity of damage caused by B. papendorfii as well as determine a possible mode for controlling the spread of the disease. References: (1) Y. J. Cao et al. Chin. J. Trop. Crops 31:1098, 2010. (2) H. Deng et al. Mycosystema 21:327, 2002. (3) T. Y. Zhang. Chin. Fungi Chi. 30:21, 2010.

1H-NMR signal assignments and secondary structure analysis of martentoxin.

Martentoxin is a peptide of 37 amino acid residues purified from the venom of the Chinese scorpion Buthus martensi Karch, which has been demonstrated to be an inhibitor of voltage-dependent sodium channel and voltage-dependent delayed rectifier potassium channel. To elucidate the molecular mechanism of this interaction, the structure of martentoxin was studied by 2D-NMR. The secondary structure of martentoxin consists of a triple-stranded beta-sheet connected to a alpha-helical structure. This helix encompasses 10 residues from Ser11 to Lys20. The three strands of beta-sheet probably comprise residues Gly2-Asp5, Q27-N30 and Glu33-Cys36, Cys30-Asn33 with a type I'beta turn centered on Asn31-Asn32. The results indicate that martentoxin possesses the conserved beta alpha beta beta structure of all the potassium channel toxins.

Purification and characterization of a new peptide with analgesic effect from the scorpion Buthus martensi Karch.

Anew peptide, designated as Buthus martensi Karch (BmK) AngM1, with an isoelectric point (pI) of 5.8 was purified and characterized from the venom of Buthus martensi Karch. The molecular mass was calculated as 7040.5 Da from multiple-charged ions by elelctrospray ionization mass spectroscopy (ESI/MS). The complete amino acid sequence of BmK AngM1 of 64 amino acid residues was determined by automatic sequencing of N-terminal part of the native peptide and the fragments of reduced and S-carboxymethylated (RCM)-peptide degraded by Staphylococcus aureaus V(8) protease and TPCK(N-p-Tosyl-L-phenylalanine chloromethyl ketone)-treated trypsin. Bioactivity tested using mouse-twisting model showed an evident analgesic effect with 63.0% (P < 0.001) inhibition efficiency at the dose of 0.8 mg/kg, but the LD(50) was larger than 50 mg/kg. Electrophysiological studies showed that BmK AngM1 at the concentration of 1 microm obviously inhibit voltage-dependent Na(+) current (I(Na)) and voltage-dependent delayed rectifier K(+) current (I(K)) but had no effects on transient K(+) current.

Gene transfer of pro-IL-18 and IL-1beta converting enzyme cDNA induces potent antitumor effects in L1210 cells.

We report in a murine model of acute lymphoid leukemia L1210 the potent antitumor efficiency of a combinatorial delivery of pro-IL-18 gene modified L1210 (Lp18) and IL-1beta converting enzyme (ICE) gene modified L1210 (LpICE). Live leukemia cells Lp18 or Lp18 plus LpICE showed apparently reduced leukemogenicity with a survival rate of 40 or 50% at 50 days after intraperitoneal (i.p.) inoculation of a lethal dose of cells, respectively. Combination of Lp18 and LpICE was capable of inhibiting accumulation of bloody ascites, synergistically superior to Lp18 or LpICE alone. All surviving mice were rechallenged with parental L1210 cells at day 50, and all survived up to day 80, suggesting that gene-modified cells induced immune protection. Moreover, NK cytotoxicity and CTL activity were both enhanced in mice injected with Lp18, especially Lp18 plus LpICE. Levels of IFN-gamma were not altered significantly by inoculation of Lp18 or Lp18 plus LpICE. Our results demonstrate that IL-18 is a useful candidate gene in gene therapy of lymphoma or lymphoid leukemia, and ex vivo combinatorial delivery of Lp18 plus LpICE either as a single approach or as an adjunct to concomitant radiotherapy or chemotherapy, may be more efficient in a situation of minimal residual disease.

Purification, characterization of two peptides from Buthus martensi Karch.

A new peptide named Martentoxin I and an analogue Martentoxin were purified and characterized from the venom of Buthus martensi Karch. Martentoxin I consisted of 36 amino acid residues with molecular mass as 3908.0 Da determined by matrix-assisted laser desorption ionization time-of-flight-MS. The amino acid sequence was determined as GLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY by Edman degradation. Martentoxin consisted of 37 amino acid residues with a molecular mass as 4055.3 Da and it showed highly sequence identity to Martentoxin I as FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY. Estimation from circular dichroism spectra indicated Martentoxin I owned 18.0% alpha-helix, 53.0% beta-sheet structure and 3.9% turn while Martentoxin contained 13.3% alpha-helix, 64.3% beta-sheet structure and 1.1% turn. The toxicity assay showed both peptides had no toxic effects on mice up to the dose of 10 mg/kg. Electrophysiological studies showed that Martentoxin I and Martentoxin at the concentration of 1 microm significantly inhibited voltage-dependent Na+ current (INa) and voltage-dependent delayed rectifier K+ current (IK) but had no effects on transient K+ current (IA). Both interactions with Na+ and K+ channels were irreversible.

[The values of urine cysteine protein activity in the diagnosis of ovarian cancer].

The levels of urine cysteine protease (UCP) were detected in the urines of 70 gynecological cancers, 50 gynecological benign tumors and 50 normal women. The values of UCP assay of gynecological cancers were much higher than those of benign and normal samples (P < 0.01). Best cut off point for diagnosis of gynecological cancers was P95 and UCP cut off value was 0.24 pmol.min-1/L by using percentile and ROC curve. The sensitivity of UCP assay was 90%, specificity 80% and accuracy 86%. The sensitivity for ovarian cancers was 95%, but 77% and 85% for corpus and cervical cancers respectively. There were no differences between UCP and CA125 (sensitivity 85%, specificity 87%, accuracy 84%) in the diagnosis of ovarian cancers. In 7 cases of 8 cases of stage I ovarian cancers, UCP were abnormal. In 6 cases of the same group, CA125 were normal (< 35,000 U/L). So UCP may be better than CA125 in the diagnosis of early ovarian cancer. The sensitivity of lactate dehydrogenase (LDH) isoenzyme was 75% in ovarian cancer which was lower than UCP and CA125, but the specificity 85%. LDH isoenzyme still was one of important tumor markers for diagnosis. Combined assays with UCP, CA125 and LDH isoenzyme may reach the sensitivity 96% and specificity 100% evidently. These data implied that UCP may be a good tumor marker in gynecological cancers especially for ovarian cancers in future.

Isolation of a Degeneration-Resistant Mutant of Clostridium acetobutylicum NCIMB 8052.

Unless periodically grown from germinated spores, Clostridium acetobutylicum tends to degenerate (that is, to spontaneously lose the capacity both to produce solvents and to develop into spores). To obtain mutants that are deficient in degeneration, C. acetobutylicum NCIMB 8052 was mated with Enterococcus faecalis BM4110 harboring transposon Tn1545. We developed a degeneration resistance assay based on a secondary effect of degeneration, the production of toxic levels of acetic and butyric acids. Erythromycin-resistant transconjugant clones were tested individually for longevity by repeated and timely subculturing. One long-lived mutant, A10, survived 18 +/- 3 transfers (mean +/- standard deviation; n = 20) before extinction, while the wild type (parental cells) survived 6.6 +/- 1.5 transfers (n = 11). The three-fold difference in longevity is statistically significant. In a batch culture in a rich medium, the wild-type cells degenerated within 24 h after inoculation with 1% of an overnight culture derived from germinated spores. In contrast, A10 cells were able to switch to solventogenesis and to sporulate. In a minimal medium with greater buffering capacity, both cell types produced solvents and spores. Southern blots of EcoRI and HindIII restriction digests of A10 chromosomal DNA (but not parental DNA) showed that only one copy of Tn1545 was inserted into the clostridial chromosome. Our findings are consistent with the hypothesis that there was an alteration at a regulatory locus that was effected by the insertion of the transposon.