RESUMO
Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is widely used as a starter for yogurt and cheese worldwide. Despite the economic importance of this bacterium in the dairy industry, there have been few genetic studies involving knockout or overexpression mutants to identify the functions of L. bulgaricus genes. One of the main reasons for this gap is the low transformation efficiency of available L. bulgaricus chromosome-integrating vectors upon performing conventional electroporation. We previously proposed the conjugal plasmid pAMß1 as an integration vector for L. bulgaricus, as conjugation could avert the need for a restriction modification system; pAMß1 does not replicate and integrate into the chromosome of L. bulgaricus. Here, we describe an effective chromosomal manipulation system involving a novel shuttle vector pGMß1, which could improve the operability of the broad host-range conjugal plasmid pAMß1. We further developed an enhanced filter-mating method for conjugation. To validate this system, the effectiveness of conversion of the lactate dehydrogenase gene D-ldh of L. bulgaricus to the L-ldh form of Streptococcus thermophilus was examined. As pGMß1 and pAMß1 are unable to replicate in L. delbrueckii subsp. delbrueckii, they were chromosomally integrated. However, these plasmids could replicate in L. delbrueckii subsp. indicus and sunkii. This integration system could unearth important gene functions in L. bulgaricus and thus improve its applications in the dairy industry. Moreover, this conjugation system could be used as a stable vector for the transformation of long cluster genes in several species of lactic acid bacteria.
RESUMO
OBJECTIVE: To examine chronological changes in urethral and bladder functions before, immediately after, and 1 year after robot-assisted radical prostatectomy (RARP), urodynamic studies were prospectively performed. METHODS: Sixty-three consecutive patients underwent pressure-flow studies, urethral pressure profiles, and abdominal leak point pressure (ALPP) tests 1-2 days before, immediately after, and 1 year after RARP. RESULTS: The mean bladder compliance was 28.3 mL/cm H2O before RARP; it worsened to 16.3 mL/cm H2O immediately after RARP and recovered to 27.1 mL/cm H2O at 1 year. The mean detrusor pressure at maximum flow rate was 61.9 cm H2O before RARP; it decreased to 34.3 cm H2O immediately after RARP and remained at 35.6 cm H2O at 1 year. The mean maximum urethral closure pressure was 84.2 cm H2O before RARP; it decreased to 33.4 cm H2O immediately after RARP and recovered to 63.0 cm H2O at 1 year. Intrinsic sphincter deficiency (ISD) evaluated by the ALPP test was observed in 53 patients immediately after RARP, although no patient showed ISD before RARP. ISD remained in 7 patients at 1 year. Both ALPP and maximum urethral closure pressure at 1 year were significant factors for continence in multivariate analysis. CONCLUSION: Urethral sphincter and bladder function worsen immediately after RARP and recover over time. The bladder storage function after RARP returns to almost the same level before RARP, and the voiding function improves compared with the condition before RARP; however, the urethral sphincter function does not return to its preoperative level. Urethral sphincter dysfunction is considered the main factor for urinary incontinence after RARP.
