Elucidating the role of polygalacturonase genes in strawberry fruit softening.

To disentangle the role of polygalacturonase (PG) genes in strawberry softening, the two PG genes most expressed in ripe receptacles, FaPG1 and FaPG2, were down-regulated. Transgenic ripe fruits were firmer than those of the wild type when PG genes were silenced individually. Simultaneous silencing of both PG genes by transgene stacking did not result in an additional increase in firmness. Cell walls from ripe fruits were characterized by a carbohydrate microarray. Higher signals of homogalacturonan and rhamnogalacturonan I pectin epitopes in polysaccharide fractions tightly bound to the cell wall were observed in the transgenic genotypes, suggesting a lower pectin solubilization. At the transcriptomic level, the suppression of FaPG1 or FaPG2 alone induced few transcriptomic changes in the ripe receptacle, but the amount of differentially expressed genes increased notably when both genes were silenced. Many genes encoding cell wall-modifying enzymes were down-regulated. The expression of a putative high affinity potassium transporter was induced in all transgenic genotypes, indicating that cell wall weakening and loss of cell turgor could be linked. These results suggest that, besides the disassembly of pectins tightly linked to the cell wall, PGs could play other roles in strawberry softening, such as the release of oligogalacturonides exerting a positive feedback in softening.

Antisense down-regulation of the strawberry ß-galactosidase gene FaßGal4 increases cell wall galactose levels and reduces fruit softening.

Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by ß-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative ß-galactosidase gene, FaßGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaßGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaßGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaßGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaßGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaßGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness.

The polygalacturonase FaPG1 gene plays a key role in strawberry fruit softening.

The loss of firm texture is one of the most characteristic physiological processes that occur during the ripening of fleshy fruits. It is generally accepted that the disassembly of primary cell wall and middle lamella is the main factor involved in fruit softening. In this process, polygalacturonase (PG) has been implicated in the degradation of the polyuronide network in several fruits. However, the minor effect of PG downregulation on tomato softening, reported during the nineties, minimized the role of this enzyme in softening. Further works in other fruits are challenging this general assumption, as is occurring in strawberry. The strawberry (Fragaria x ananassa) fruit undergoes an extensive and fast softening that limit its shelf life and postharvest. Traditionally, it has also been considered that PG plays a minor role on this process, due to the low PG activity found in ripened strawberry fruits. Transgenic strawberry plants expressing an antisense sequence of the ripening-specific PG gene FaPG1 have been generated to get an insight into the role of this gene in softening. Half of the transgenic lines analyzed yielded fruits significantly firmer than control, without being affected other fruit parameters such as weight, color or soluble solids. The increase on firmness was maintained after several days of posharvest. In these firmer lines, FaPG1 was silenced to 95%, but total PG activity was only minor reduced. At the cell wall level, transgenic fruits contained a higher amount of covalently bound pectins whereas the soluble fraction was diminished. A microarray analysis of genes expressed in ripened receptacle did not show any significant change between control and transgenic fruits. Thus, contrary to the most accepted view, it is concluded that PG plays a key role on pectin metabolism and softening of strawberry fruit.

Antisense down-regulation of the FaPG1 gene reveals an unexpected central role for polygalacturonase in strawberry fruit softening.

The strawberry (Fragaria x ananassa 'Chandler') fruit undergoes a fast softening during ripening. Polygalacturonase (PG) activity is low during this process, but two ripening-related PG genes, FaPG1 and FaPG2, have been cloned. Both genes were up-regulated during fruit ripening and were also negatively regulated by auxin. To further assess the role of FaPG1 on strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the 35S promoter (APG lines) were obtained. Sixteen out of 30 independent transgenic lines showed fruit yields similar to those of the control. Several quality parameters were measured in ripe fruits from these 16 lines. Fruit weight was slightly reduced in four lines, and most of them showed an increase in soluble solid content. Half of these lines yielded fruits significantly firmer than did the control. Four APG lines were selected, their ripened fruits being on average 163% firmer than the control. The postharvest softening of APG fruits was also diminished. Ripened fruits from the four selected lines showed a 90% to 95% decrease in FaPG1 transcript abundance, whereas the level of FaPG2 was not significantly altered. Total PG activity was reduced in three of these lines when compared with control fruits. Cell wall extracts from APG fruits showed a reduction in pectin solubilization and an increase in pectins covalently bound to the cell wall. A comparative transcriptomic analysis of gene expression between the ripened receptacle of the control and those of the APG fruits (comprising 1,250 receptacle expressed sequence tags) did not show any statistically significant change. These results indicate that FaPG1 plays a central role in strawberry softening.

[Comparative analysis of various techniques for the repair of stress urinary incontinence in women. Review of our experience]. / Análisis comparativo de diferentes técnicas para la corrección de la incontinencia de stress en la mujer. Revisión de nuestra experiencia.

OBJECTIVE: We aim to expose our experience in the surgical correction of stress urinary incontinence (SUI) and to analyze the possible factors that could modify the outputs of this type of surgery. METHODS: We have studied 114 women who underwent surgery (60 Raz, 36 Burch, and 18 vaginal wall sling), with a mean follow-up time of 10.5 months. RESULTS: We have not succeeded in demonstrating that preoperative factors such as age, irritative voiding symptoms; history of prior hysterectomy or urethropexia, neurological disease, diabetes or recurrent urinary tract infections; the finding of cistocele, a positive Bonney-Marchetti test or bladder unstability, play any role in the outputs. The failure rate was 16.7% for vaginal wall sling procedure, 35% Raz, and 33.3% Burch. "Survival" analysis did not demonstrate differences related to the procedure or the surgeon. We discovered and important decrease of continence rate with time from the intervention. Higher incidence of postoperative pain, urinary retention and greater residual urine were detected with transvaginal procedures. There was no difference in the incidence of wound infection. CONCLUSIONS: We don't believe that the selection of candidates for this type of surgery should be carried out in base to the above mentioned preoperative factors. Also, we observed a consistent decrease of postoperative continence with time. Finally, we have detected a greater incidence of complications after transvaginal procedures.

[Limitations of ultrasonography in the clinical course monitoring of bladder tumors]. / Limitaciones de la ecografía en el control evolutivo de los tumores vesicales.

Ultrasonography is a fairly innocuous test in the follow-up of bladder tumours. Its results, however, can not be superposed to those of cystoscopy. This study aims to identify the risk factors for failure of transabdominal ultrasonography in the FU of bladder tumors. The influence of the primary tumour, sex and age of patients on the ability of ultrasonography to detect relapses was analyzed. Chi square and Student's t tests were used to associate the characteristics of primary tumours and patients to the results of ultrasonography. Student's t test was used to estimate the effect of diagnostic oversight in terms of annual recurrence rate and progression. The characteristics of primary tumours where relapse was detected or overlooked had no influence on the results of ultrasound follow-up as neither did age and sex. No differences were detected in recurrence rate of patients with anticipated (0.57) or overlooked (0.58) tumours. Although differences in progression rates (4.76% and 9% for overlooked and identified tumours, respectively) were substantial, they did not reach statistical significance. There are no features in the original tumour or the patient to anticipate the failure of ultrasound monitoring. Multiple and/or small relapses are overlooked more frequently that single and/or large ones, and tumours located in lateral walls, base and dome may be unnoticed. In spite of oversights, alternate ultrasound/cystoscopic monitoring does not compromise the outcome of the disease.

[A paratesticular myxoid liposarcoma as a simulator of a hernial process. A case report]. / Liposarcoma mixoide paratesticular como simulador de proceso herniario. Descripción de un caso.

OBJECTIVE: To describe a case of paratesticular liposarcoma in a 69-year-old male. The literature is briefly reviewed and the prognostic and therapeutic aspects are discussed. METHODS/RESULTS: The patient was submitted to surgery. Pathological analysis of the surgical specimen disclosed a myxoid liposarcoma with undifferentiated pleomorphic areas. Patient follow up has shown no recurrence or metastasis. CONCLUSIONS: This tumor type develops in scrotal soft tissue, slowly increasing scrotal size, and should be distinguished from inguinoscrotal hernia. Together with physical examination, ultrasonography is generally useful. Treatment is by radical orchiectomy through an inguinal approach; the role of adjuvant therapy remains unclear. It has a good prognosis, with frequent local relapses; metastatic spread is uncommon.