The two-component system ActJK is involved in acid stress tolerance and symbiosis in Sinorhizobium meliloti.

The nitrogen-fixing α-proteobacterium Sinorhizobium meliloti genome codifies at least 50 response regulator (RR) proteins mediating different and, in many cases, unknown processes. RR-mutant library screening allowed us to identify genes potentially implicated in survival to acid conditions. actJ mutation resulted in a strain with reduced growth rate under mildly acidic conditions as well as a lower capacity to tolerate a sudden shift to lethal acidic conditions compared with the parental strain. Mutation of the downstream gene actK, which encodes for a histidine kinase, showed a similar phenotype in acidic environments suggesting a functional two-component system. Interestingly, even though nodulation kinetics, quantity, and macroscopic morphology of Medicago sativa nodules were not affected in actJ and actK mutants, ActK was required to express the wild-type nitrogen fixation phenotype and ActJK was necessary for full bacteroid development and nodule occupancy. The actJK regulatory system presented here provides insights into an evolutionary process in rhizobium adaptation to acidic environments and suggests that actJK-controlled functions are crucial for optimal symbiosis development.

Codon Usage Heterogeneity in the Multipartite Prokaryote Genome: Selection-Based Coding Bias Associated with Gene Location, Expression Level, and Ancestry.

Prokaryotes represent an ancestral lineage in the tree of life and constitute optimal resources for investigating the evolution of genomes in unicellular organisms. Many bacterial species possess multipartite genomes offering opportunities to study functional variations among replicons, how and where new genes integrate into a genome, and how genetic information within a lineage becomes encoded and evolves. To analyze these issues, we focused on the model soil bacterium Sinorhizobium meliloti, which harbors a chromosome, a chromid (pSymB), a megaplasmid (pSymA), and, in many strains, one or more accessory plasmids. The analysis of several genomes, together with 1.4 Mb of accessory plasmid DNA that we purified and sequenced, revealed clearly different functional profiles associated with each genomic entity. pSymA, in particular, exhibited remarkable interstrain variation and a high density of singletons (unique, exclusive genes) featuring functionalities and modal codon usages that were very similar to those of the plasmidome. All this evidence reinforces the idea of a close relationship between pSymA and the plasmidome. Correspondence analyses revealed that adaptation of codon usages to the translational machinery increased from plasmidome to pSymA to pSymB to chromosome, corresponding as such to the ancestry of each replicon in the lineage. We demonstrated that chromosomal core genes gradually adapted to the translational machinery, reminiscent of observations in several bacterial taxa for genes with high expression levels. Such findings indicate a previously undiscovered codon usage adaptation associated with the chromosomal core information that likely operates to improve bacterial fitness. We present a comprehensive model illustrating the central findings described here, discussed in the context of the changes occurring during the evolution of a multipartite prokaryote genome.IMPORTANCE Bacterial genomes usually include many thousands of genes which are expressed with diverse spatial-temporal patterns and intensities. A well-known evidence is that highly expressed genes, such as the ribosomal and other translation-related proteins (RTRPs), have accommodated their codon usage to optimize translation efficiency and accuracy. Using a bioinformatic approach, we identify core-genes sets with different ancestries, and demonstrate that selection processes that optimize codon usage are not restricted to RTRPs but extended at a genome-wide scale. Such findings highlight, for the first time, a previously undiscovered adaptation strategy associated with the chromosomal-core information. Contrasted with the translationally more adapted genes, singletons (i.e., exclusive genes, including those of the plasmidome) appear as the gene pool with the less-ameliorated codon usage in the lineage. A comprehensive summary describing the inter- and intra-replicon heterogeneity of codon usages in a complex prokaryote genome is presented.

Open inguinal hernia repair with self-gripping Parietex ProGrip mesh: a retrospective study of 204 cases.

Chronic pain and recurrence rates are the main challenge in modern inguinal hernia surgery. Several trials have investigated the role of self-adhesive mesh repair for inguinal hernia, with special attention to the incidence of chronic postoperative inguinal pain and recurrence. The purpose of our study was to retrospectively evaluate the early and long-term results using a self-gripping mesh (Parietex Progrip® , Covidien) in our institution. A total of 204 patients, mean age 50.3 standard deviation (SD) 15.3, was included in the study. The repair was performed under local anaesthesia in 159 (78%) cases and locoregional anaesthesia in remaining 45 (22%). Mean operative time was 39 ± 20 minutes. The time for self-gripping mesh placement ranged from 5 to 9 minutes (mean 7 ± 2 minutes). There were no intraoperative complications. Clinical follow-up was performed at 1 month, 1 year and 2 years and consisted in the evaluation of complications, discomfort/pain and recurrence. One case of cutaneous infection and three cases of seroma were observed at one-month follow-up and were all treated conservatively. 8 patients were lost at one year follow-up, and another 4 were lost at 2 years. 3 patients died for other causes during follow-up. At 1 year and 2 years follow-up no cases of seroma, testicular complications or mesh infection were observed. Two cases of recurrence were recorded at 2 years follow up. No patient reported VAS score > 2 at one month, 1 year and 2 years follow-up. There were no readmissions, systemic complications or death during 2 years follow-up. Lichtenstein open repair using Parietex Progrip® mesh is a simple, rapid, effective and safe method for inguinal hernia repair. The main advantage of self-fixing mesh is the reduced operative time. A suturless fixation seems to prevent the development of postoperative chronic pain, without increasing recurrence rate in the majority of the trials.

Thermal ablation combined with high ligation of sapheno-femoral junction for lower limb primary varicosity.

AIM: For long time the traditional surgical treatment for lower limb varicose veins has been high ligation of sapheno-femoral junction and stripping of great saphenous vein. Surgery, however, has been frustrated by postoperative pains, discomfort and recurrences so that it has been challenged by minimally invasive endovenous techniques such as laser treatment and radiofrequency ablation. The aim of the article is to assess the feasibility of a combined approach to greater saphenous vein reflux: high ligation of sapheno-femoral junction and thermal treatment of the great saphenous vein. METHODS: A retrospective analysis on 95 patients treated with high ligation and thermal ablation at our institution was performed, assessing duration of surgery, post-operative pain and analgesics requirements, early complications and resumption of activities. RESULTS: Two patients (5,4%), in the laser group experienced skin burns in the course of the GSV. Moderate ecchymosis, by laser fibre-Romainduced perforation of the vein wall, were observed in another two patients (5.4%). Four limbs (10.8%) in the EVLT group developed transient paraesthesias. Analgesic requirement on POD 3 was nil for RFA group; conversely half of the EVLT patients did take analgesics, either 2 or 3 tabs were required. On POD 7, the patients of RFA group continued to not ask for any analgesics, but the same half of the patients in EVLT group still needed 1-2 tabs to carry out their normal activities smoothly. On POD 15, no patient did require analgesics. Resumption of routine activities was earlier for RFA group patients than for those in the EVLT group. The RFA group resumed their activities within 3 days, whereas EVLT group in 8-9 days. High ligation of the SFJ didn't add too much time or morbidities. CONCLUSION: High ligation of saphenous femoral arc combined with catheter delivered thermal energies for saphenous ablation, even when combined with high ligation of saphenous femoral arc, demonstrated to be minimally invasive, easy to learn and easy to perform, with early resumption and return to normal activity. EVLT achieved similar results to RFA and both techniques were considered equally effective and safe; the results we obtained were not statistically significant but RFA showed less pain, ecchymosis and haematomas, as well as provided better short-term quality of life.

Utility of continuous intraoperative neural monitoring in thyroid surgery in a low volume centre.

This retrospective study, of a single surgeon's experience, evaluates the role of intraoperative neuromonitoring (IONM) for total thyroidectomy, in a low-volume district general hospital. 128 patients with normal preoperative vocal fold function underwent thyroid surgery with routine use of nerve monitoring. Patients were followed for 6 months after surgery, and postoperative Romanerve function was determined by fiberoptic laryngoscopy. One (0,8%) patient was found to have a unilateral vocal fold paralysis, but after 6 months this patient had regained vocal fold motion. The technique of intraoperative neuromonitoring in thyroid surgery is safe and reliable in excluding postoperative recurrent laryngeal nerve palsy; it has high accuracy, specificity, sensitivity and negative predictive value. Neuromonitoring is useful to identify the recurrent laryngeal nerve and it can be a useful adjunctive technique for reassuring surgeons of the functional integrity of the nerve. Its application can be particularly recommended for low-volume thyroid operation centres.

Can Longo stapled haemorrhoidopexy for haemorroids be a day-case procedure?

We retrospectively collected data of 100 consecutives elective Longo's procedures for third- and fourth-degree simptomatic haemorroids, classified ASA grade I and II. All patients stayed overnight, because discharge was scheduled the day after the operation. 98 were discharged the day after the operation. Two patients were not discharged the day after because mild and severe bleeding respectively occurred during the first night after the operation which settled conservatively. All the patients were discharged on oral NSAID and stool softeners. None required rehospitalisation. Our retrospective study, pointing out that, in general patients did not require active intervention on the first postoperative night, represents an encouragement to introduce day-case stapled procedure for haemorroids.

Amyand's hernia with acute phlegmonous appendicitis: case report.

Any inguinal hernia containing the vermiform appendix is called Amyand's hernia. Amyand hernias are very rare and even rarer is the association of Amyand hernia with acute appendicitis. Due to the rarity of this entity, it constitutes a challenging case in terms of diagnosis and treatment. The surgical management is not yet standardized and there are no clear guidelines. There are some controversies regarding whether to perform an appendectomy if appendix appears normal or whether mesh can be used for the hernia repair if appendectomy is performed. We describe a case of Amyand hernia in a 90-year old man with acute appendicitis and we review current literature regarding surgical strategy.

A consolidated analysis of the physiologic and molecular responses induced under acid stress in the legume-symbiont model-soil bacterium Sinorhizobium meliloti.

Abiotic stresses in general and extracellular acidity in particular disturb and limit nitrogen-fixing symbioses between rhizobia and their host legumes. Except for valuable molecular-biological studies on different rhizobia, no consolidated models have been formulated to describe the central physiologic changes that occur in acid-stressed bacteria. We present here an integrated analysis entailing the main cultural, metabolic, and molecular responses of the model bacterium Sinorhizobium meliloti growing under controlled acid stress in a chemostat. A stepwise extracellular acidification of the culture medium had indicated that S. meliloti stopped growing at ca. pH 6.0-6.1. Under such stress the rhizobia increased the O2 consumption per cell by more than 5-fold. This phenotype, together with an increase in the transcripts for several membrane cytochromes, entails a higher aerobic-respiration rate in the acid-stressed rhizobia. Multivariate analysis of global metabolome data served to unequivocally correlate specific-metabolite profiles with the extracellular pH, showing that at low pH the pentose-phosphate pathway exhibited increases in several transcripts, enzymes, and metabolites. Further analyses should be focused on the time course of the observed changes, its associated intracellular signaling, and on the comparison with the changes that operate during the sub lethal acid-adaptive response (ATR) in rhizobia.

Development of new positive-selection RIVET tools: detection of induced promoters by the excision-based transcriptional activation of an aacCI (GmR)-gfp fusion.

RIVET (Recombination Based in vivo Expression Technology) is a powerful genetic tool originally conceived for the identification of genes induced in complex biological niches where conventional transcriptomics is difficult to use. With a broader application, genetic recombination-based technologies have also been used, in combination with regulatory proteins and specific transcriptional regulators, for the development of highly sensitive biosensor systems. RIVET systems generally comprise two modules: a promoter-trap cassette generating genomic transcriptional fusions to the tnpR gene encoding the Tn-γδ TnpR resolvase, and a reporter cassette carrying res-flanked selection markers that are excised upon expression of tnpR to produce an irreversible, inheritable phenotypic change. We report here the construction and validation of a new set of positive-selection RIVET systems that, upon induction of the promoter-trap module, generate the transcriptional activation of an antibiotic-resistant and a green-fluorescent phenotype. Two classes of promoter-trap tools were constructed to generate transcriptional fusions to tnpR: one based on the use of a narrow-host-range plasmid (pRIVET-I), integrative in several Gram-negative bacteria, and the other based on the use of a broad-host-range plasmid (pRIVET-R). The system was evaluated in the model soil bacterium Sinorhizobium meliloti, where a clear-cut phenotypic transition from Nm(R)-Gm(S)-GFP(-) to Nm(S)-Gm(R)-GFP(+) occurred upon expression of tnpR. A S. meliloti integrative RIVET library was constructed in pRIVET-I and, as expected, changes in the extracellular conditions (e.g., salt stress) triggered a significant increase in the appearance of Gm(R)-GFP(+) (excised) clones. The sacB-independent positive-selection RIVET systems here described provide suitable basic tools both for the construction of new recombination-based biosensors and for the search of bacterial markers induced when microorganisms colonize and invade complex environments and eukaryotic hosts.

Identification and characterization of a nodH ortholog from the alfalfa-nodulating Or191-like rhizobia.

Nodulation of Medicago sativa (alfalfa) is known to be restricted to Sinorhizobium meliloti and a few other rhizobia that include the poorly characterized isolates related to Rhizobium sp. strain Or191. Distinctive features of the symbiosis between alfalfa and S. meliloti are the marked specificity from the plant to the bacteria and the strict requirement for the presence of sulfated lipochitooligosaccharides (Nod factors [NFs]) at its reducing end. Here, we present evidence of the presence of a functional nodH-encoded NF sulfotransferase in the Or191-like rhizobia. The nodH gene, present in single copy, maps to a high molecular weight megaplasmid. As in S. meliloti, a nodF homolog was identified immediately upstream of nodH that was transcribed in the opposite direction (local synteny). This novel nodH ortholog was cloned and shown to restore both NF sulfation and the Nif+Fix+ phenotypes when introduced into an S. meliloti nodH mutant. Unexpectedly, however, nodH disruption in the Or191-like bacteria did not abolish their ability to nodulate alfalfa, resulting instead in a severely delayed nodulation. In agreement with evidence from other authors, the nodH sequence analysis strongly supports the idea that the Or191-like rhizobia most likely represent a genetic mosaic resulting from the horizontal transfer of symbiotic genes from a sinorhizobial megaplasmid to a not yet clearly identified ancestor.

[Autologous peripheral stem cell transplantation in a patient with diffuse systemic sclerosis: our experience]. / Trapianto autologo di cellule staminali periferiche in un paziente con sclerosi sistemica diffusa: nostra esperienza.

The diffuse form of systemic sclerosis (SSc) can often lead to a rapidly progressive course with the involvement of the visceral organs which causes a severe prognosis. The 5-years cumulative mortality is between 30 and 60%, depending on the clinic form at the onset. Until now, no drug treatment has been proved to be efficacious against the progression of the disease or the regression of the fibrosis. Recently autologous peripheral blood stem cell (PBSC) transplantation has been found to be promising. We introduce the case of a patient, male, 56 years old, who came under our observation on February 2001, suffering from a SSc with a severe multisystem involvement of lungs, skin, heart and gastrointestinal tract, and a positive antibodies anti-Scl-70. The 8 months therapy, at first with iloprost and cyclophosphamide, then with bolus of cyclophosphamide, was ineffective, with a rapid worsening of the cutaneous and pulmonary involvement. Under the patient agreement we decided to carry out an autologous PBSC transplantation. On December 2001, we obtained the PBSC mobilization after the administration of cyclophosphamide and lenograstim and the PBSC recovery with two leucoaferesis procedures. On February 2002, we gave the conditioning therapy with: thiotepa, cyclophosphamide, fludarabine, rabbit antilymphocytic globulin; then we made the infusion of PBSC. The bone marrow recovery (GN >500 and PLT >20.000) arrived at the day + 10. For three months after the transplantation we made an antibacterial, antiviral and antifungin prophylaxis with valacocyclovir, co-trimoxazole and fluconazole. The one-year follow-up has shown an essentially good response with the improving of the skin involvement and of the subjective indicators of the disease, while the pulmonary involvement don't seen modified from the high dose therapy.

Construction of a Sinorhizobium meliloti strain carrying a stable and non-transmissible chromosomal single copy of the green fluorescent protein GFP-P64L/S65T.

A single copy of the green fluorescent protein (GFP)-encoding gene gfp-P64L/S65T under the control of the constitutive nptII promoter was introduced in a neutral region of the Sinorhizobium meliloti chromosome, between the genes recA and alaS. Within the same chromosomal region downstream of gfp-P64L/S65T a tetracycline (Tc) resistant cassette was also inserted. Both markers were very stable during at least 40 bacterial generations without any selective pressure. Similarly, the gfp-Tc cassette was stable and functional in all rhizobia that were recovered from alfalfa nodules. The GFP-associated fluorescence derived from the (single copy) chromosomal gfp-P64L/S65T allowed detection of rhizobia during the colonisation of the root, infection thread formation, and nodule development. The gfp-Tc rhizobia showed indistinguishable phenotypes for nodulation, competitiveness, and nitrogen-fixation from the parental strain. The labelling system described here can be used for the stable fluorescent tagging of S. meliloti strains allowing their detection in biologically complex soil environments.

Neck hibernoma: case report and literature review.

The case history of a 23 year old man with a large hibernoma of the cervical region is reported. Th tumour showed all the clinical and pathologic characteristic of these rare benign neoplasms. A review of the most recent literature supports the Authors' conclusion that local excision is curative.

Successfully managed genital necrotizing fasciitis with multiple debilitating diseases. A case report.

Genital necrotizing fasciitis is a rapidly progressive bacterial infection of soft tissues with a reported average mortality of about 36%; associated debilitating diseases increase the mortality rate. The Authors report a case of successful management, due to an aggressive medical and surgical therapy, despite the presence of multiple debilitating diseases and an advanced necrosis.

[The chance finding of gallbladder carcinoma during cholecystectomy for long-term symptomatic lithiasis. A clinical case]. / Riscontro occasionale di carcinoma colecistico durante colecistectomia per litiasi sintomatica di lunga durata. Caso clinico.

The case of a patient who underwent conventional cholecystectomy for gallbladder adenocarcinoma is reported. Gallbladder carcinoma is rare, but since laparoscopic cholecystectomy has quickly emerged as a popular alternative to open cholecystectomy an increasing number of gallbladder carcinomas are discovered. Most of the time it is detected as an incidental tumor, the patient being with no or few symptoms and often the lesion too far advanced for any type of curative surgery. The most important questions related to the therapy are focused and a partial review of the literature is made.

[Myocardial revascularization surgery via a left minithoracotomy]. / Cirurgia de revascularização do miocárdio através de minitoracotomia esquerda.

PURPOSE: There is, today, a global tendency towards a surgical approach privileging very small incisions, the so-called minimally invasive intervention, which results in a less aggressive action. The introduction of this new technique makes it possible to dissect the left internal thoracic artery (LITA) and to perform in the anastomosis with the anterior interventricular artery (AIA) through a left minithoracotomy. METHODS: From May of 1996 to october of 1997, 11 patients with ischemic heart disease and a single proximal lesion of the AIA were submitted to a myocardial revascularization (MR). The surgical approach consisted of a left anterolateral thoracotomy through the 4th left intercostal space, of approximately 10 cm, and in the last 6 cases, resection of part of the cartilage of the 4th and 5th ribs, dissection of the LITA, as well as opening and repair of the pericardium adjacent to the AIA was done. All patients received a single bypass to the AIA with a graft of the LITA, without extracorporeal circulation. RESULTS: The patients' age varied between 46 and 76 years (mean = 58.55). Ten patients (90.90%) were males and 1 (9.09%) was a female. Hospital stay ranged from 4 to 8 days, with the average of 5.2 days. None of the patients presented any electrocardiographic change in the immediate post-operatory period. During the control period one patient developed a clot in the distal LITA, with important compromise of the flow. In another patient the stenosis was at the level of the anastomosis. Both were successfully submitted to angioplasty. There were no deaths in the groups studied. CONCLUSION: The absence of deaths suggests to us that MR surgery carried out with this minimally invasive technique, in selected groups and is an excellent alternative to the revascularization of the AIA.

[A 3-year experience of the repair of voluminous abdominal laparoceles with patches of prosthetic material]. / Un'esperienza di tre anni nella riparazione dei voluminosi laparoceli addominali con pezze di materiale protesico.

The authors deal with huge ventral hernias having a defect of more than 10 cm in diameter. They report 16 cases in three years of experience: 10 treated with Marlex patches and 6 treated with Goretex prosthetic material.

A phase II randomised trial of 5-fluorouracil with or without interferon alpha-2a in advanced colorectal cancer.

With the association of 5-fluorouracil (5-FU) and alpha-interferon (IFN), objective responses as high as 26 63% have been reported in untreated patients with advanced colorectal cancer. However, grade 3-4 toxicity has also been reported. We have conducted a prospective phase II randomised study comparing 5-FU to 5-FU + IFN, to investigate whether the addition of IFN to a weekly 5-FU regimen devoid of significant toxicity used at our institutions could improve the effectiveness of 5-FU while maintaining acceptable toxicity. Patients with histologically proven advanced colorectal carcinoma were randomised to receive 5-FU 500 mg m-2 intravenous (i.v.) bolus on days 1-5 followed by 5-FU 500 mg m-2 i.v. bolus weekly from day 15, with or without IFN alpha-2a intramuscularly (i.m.) 1.5 mU daily on days 6-12 and 3 mU i.m. daily thereafter. The treatment was administered on an outpatient basis. Response was evaluated every 3 months, and treatment continued until progression or after two consecutive judgements of stable disease. Response rate was the main end point of the study. Of 141 patients eligible, 72 were randomised to 5-FU alone (arm A) and 69 to 5-FU + IFN (arm B). Responses were 9/72 (12.5%) in arm A and 6/69 (8.7%) in arm B; complete responses were three in arm A and two in arm B. Progression-free survival (median 4 months) and survival (median 12 months) were identical in the two arms. Toxicity was almost absent in arm A and moderate in arm B, represented mainly by haematological toxicity (usually leucopenia). In conclusion, overall survival was good in both arms of treatment and toxicity was moderate. While the response rate with 5-FU alone was in accord with the literature data, response to 5-FU + IFN was lower than expected. At least at this dosage and schedule, the association of 5-FU and IFN is no better than 5-FU alone and is of no clinical interest.

Enfermedad de Dowling Degos / Dowling Degos 'disease

Se comunica un caso clínico de la enfermedad de Dowling Degos en una mujer de 68 años,la cual presentó hallazgos clínicos e histopatológicos típicos de la enfermedad. Realizamos una revisión bibliográfica, abarcando la literatura desde el año 1974 a 1994, dentro de los cuales incluimos revistas nacionales y extranjeras. Los casos argentinos publicados son escasos; debiodo a esto, creemos de interés incorporar a la casuística nuestro paciente. Después de haber revisado la literatura, concluímos al igual que otros autores, que la enfermedad de Dowling Degos es una variante de un mismo complejo genodermatológico