DNA damage regulates alternative splicing through inhibition of RNA polymerase II elongation.

DNA damage induces apoptosis and many apoptotic genes are regulated via alternative splicing (AS), but little is known about the control mechanisms. Here we show that ultraviolet irradiation (UV) affects cotranscriptional AS in a p53-independent way, through the hyperphosphorylation of RNA polymerase II carboxy-terminal domain (CTD) and a subsequent inhibition of transcriptional elongation, estimated in vivo and in real time. Phosphomimetic CTD mutants not only display lower elongation but also duplicate the UV effect on AS. Consistently, nonphosphorylatable mutants prevent the UV effect. Apoptosis promoted by UV in cells lacking p53 is prevented when the change in AS of the apoptotic gene bcl-x is reverted, confirming the relevance of this mechanism. Splicing-sensitive microarrays revealed a significant overlap of the subsets of genes that have changed AS with UV and those that have reduced expression, suggesting that transcriptional coupling to AS is a key feature of the DNA-damage response.

Cerebrospinal fluid kappa and lambda free light chains in oligoclonal band-negative patients with suspected multiple sclerosis.

BACKGROUND AND PURPOSE: Cerebrospinal fluid (CSF) kappa free light chains (FLCs) may be a more sensitive marker of intrathecal immunoglobulin (Ig)G synthesis compared with oligoclonal bands (OCBs). Our aim was to retrospectively determine the additional value of the kappa and lambda index (CSF FLC/serum FLC)/(CSF albumin/serum albumin) in predicting a multiple sclerosis (MS) diagnosis in a group of OCB-negative patients with suspected MS. METHODS: The CSF and serum kappa and lambda FLCs were tested using the Freelite kit (serum) and Freelite Mx (CSF) assay (The Binding Site Group, Bimingham, UK) in 391 OCB-negative patients with suspected/possible MS and in 54 OCB-positive patients with MS. RESULTS: The CSF kappa FLC levels were below the detection limit (0.27 mg/L) in 61% of patients. Using quantitative data, we found the best kappa index cut-off value for the prediction of MS to be 5.8. A kappa index ≥5.8 was present in 25% of OCB-negative MS (23/92) and in 98% of OCB-positive patients with MS. Using a qualitative approach and a kappa index cut-off of 5.9, based on literature data, we likewise found that 24% of OCB-negative patients with MS had a kappa index ≥5.9, compared with 5.4% of OCB-negative patients without MS (P < 0.001). No reliable data could be obtained for the lambda index; lambda FLCs were below the detection limit (0.68 mg/L) in 90% of CSF samples. CONCLUSIONS: The kappa index could contribute to the identification of OCB-negative patients with a high probability of an MS diagnosis. Using more sensitive techniques might even improve the diagnostic performance of the kappa index and better define the role of the lambda index.

Characterization of two GH5 endoglucanases from termite microbiome using synthetic metagenomics.

Here, we characterize two novel GH5 endoglucanases (GH5CelA and GH5CelB) from an uncultured bacterium identified in termite gut microbiomes. Both genes were codon-optimized, synthetized, cloned, and expressed as recombinant proteins in Escherichia coli for subsequent purification. Both enzymes showed activity on the pNPC and barley ß-glucan substrates, whereas GH5CelB also showed low activity on carboxymethyl cellulose. The optimum conditions for both enzymes were an acid pH (5) and moderate temperature (35 to 50 °C). The enzymes differed in the kinetic profiles and patterns of the generated hydrolysis products. A structural-based modeling analysis indicated that both enzymes possess a typical (ß/α)8-barrel fold characteristic of GH5 family, with some differential features in the active site cleft. Also, GH5CelB presents a putative secondary binding site. Furthermore, adjacent to the active site of GH5CelA and GH5CelB, a whole subdomain rarely found in GH5 family may participate in substrate binding and thermal stability.Therefore, GH5CelA may be a good candidate for the production of cello-oligosaccharides of different degrees of polymerization applicable for feed and food industries, including prebiotics. On the other hand, GH5CelB could be useful in an enzymatic cocktail for the production of lignocellulosic bioethanol, because of the production of glucose as a hydrolysis product. Key Points • Synthetic metagenomics is a powerful approach for discovering novel enzymes. • Two novel GH5 endoglucanases from nonculturable microorganisms were characterized. • Structural differences between them and other GH5 endoglucanases were observed. • The enzymes may be good candidates for feed, food, and/or bioethanol industries.

Genetic diversity of Mycobacterium avium complex strains isolated in Argentina by MIRU-VNTR.

Mycobacterium avium sp. avium (MAA), M. avium sp. hominissuis (MAH), and M. avium sp. paratuberculosis (MAP) are the main members of the M. avium complex (MAC) causing diseases in several hosts. The aim of this study was to describe the genetic diversity of MAC isolated from different hosts. Twenty-six MAH and 61 MAP isolates were recovered from humans and cattle, respectively. GenoType CM® and IS1311-PCR were used to identify Mycobacterium species. The IS901-PCR was used to differentiate between MAH and MAA, while IS900-PCR was used to identify MAP. Genotyping was performed using a mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) scheme (loci: 292, X3, 25, 47, 3, 7, 10, 32) and patterns (INMV) were assigned according to the MAC-INMV database (http://mac-inmv.tours.inra.fr/). Twenty-two (22/26, 84·6%) MAH isolates were genotyped and 16 were grouped into the following, INMV 92, INMV 121, INMV 97, INMV 103, INMV 50, and INMV 40. The loci X3 and 25 showed the largest diversity (D: 0·5844), and the global discriminatory index (Hunter and Gaston discriminatory index, HGDI) was 0·9300. MAP (100%) isolates were grouped into INMV 1, INMV 2, INMV 11, INMV 8, and INMV 5. The HGDI was 0·6984 and loci 292 and 7 had the largest D (0·6980 and 0·5050). MAH presented a higher D when compared with MAP. The MIRU-VNTR was a useful tool to describe the genetic diversity of both MAH and MAP as well as to identify six new MAH patterns that were conveniently reported to the MAC-INMV database. It was also demonstrated that, in the geographical region studied, human MAC cases were produced by MAH as there was no MAA found among the human clinical samples.

Genomic comparison of two strains of Mycobacterium avium subsp. paratuberculosis with contrasting pathogenic phenotype.

In a previous study, we evaluated the degree of virulence of Mycobacterium avium subsp. paratuberculosis (Map) strains isolated from cattle in Argentina in a murine model. This assay allowed us to differentiate between high-virulent MapARG1347 and low-virulent MapARG1543 strains. To corroborate whether the differences in virulence could be attributed to genetic differences between the strains, we performed Whole Genome Sequencing and compared the genomes and gene content between them and determined the differences related to the reference strain MapK10. We found 233 SNPs/INDELS in one or both strains relative to Map K10. The two strains share most of the variations, but we found 15 mutations present in only one of the strains. Considering NS-SNP/INDELS that produced a severe effect in the coding sequence, we focus the analysis on four predicted proteins, putatively related to virulence. Survival of MapARG1347 strain in bMDM was higher than MapARG1543 and was more resistant to acidic pH and H2O2 stresses than MapK10. The genomic differences between the two strains found in genes MAP1203 (a putative peptidoglycan hydrolase), MAP0403 (a putative serine protease) MAP1003c (a member of the PE-PPE family) and MAP4152 (a putative mycofactocin binding protein) could contribute to explain the contrasting phenotype previously observed in mice models.

Inventory of landslides triggered by an extreme rainfall event in Marche-Umbria, Italy, on 15 September 2022.

Systematic and timely documentation of triggered (i.e. event) landslides is fundamental to build extensive datasets worldwide that may help define and/or validate trends in response to climate change. More in general, preparation of landslide inventories is a crucial activity since it provides the basic data for any subsequent analysis. In this work we present an event landslide inventory map (E-LIM) that was prepared through a systematic reconnaissance field survey in about 1 month after an extreme rainfall event hit an area of about 5000 km2 in the Marche-Umbria regions (central Italy). The inventory reports evidence of 1687 triggered landslides in an area of ~550 km2. All slope failures were classified according to type of movement and involved material, and documented with field pictures, wherever possible. The database of the inventory described in this paper as well as the collection of selected field pictures associated with each feature is publicly available at figshare.

Effect of the disclosure of MS diagnosis on anxiety, mood and quality of life of patients: a prospective study.

BACKGROUND: In the light of the new diagnostic criteria for multiple sclerosis (MS) and currently available early treatment, this study aimed to explore whether, and to what extent, disclosure of the diagnosis of MS or clinically isolated syndrome (CIS) affects patients' anxiety, mood and quality of life (QoL). METHODS: Eligible participants were all patients referred for the first time to the Neurological Unit who had manifested symptoms suggestive of MS for no more than 6 months. All patients were evaluated for (i) QoL (SEIQoL and MS-QoL54), (ii) Anxiety (STAI) and Depression (CMDI) on study inclusion (T0), 30 days after diagnosis disclosure (T30), and after 1 (T1y) and 2 (T2y) years' follow-up. RESULTS: Two hundred and twenty-nine patients were enrolled; 93 of these were unaware of their diagnosis. Patients who already knew their diagnosis (100 with CIS and 22 with MS) were excluded from the main analyses and used to perform control analyses. At the end of the screening, an MS diagnosis was disclosed to 18 of the 93 patients, whereas a CIS diagnosis was disclosed to 62 patients (12 patients received a diagnosis other than MS or CIS). Thirty days after diagnosis disclosure, irrespective of the diagnosis disclosed, both QoL and Anxiety and Depression were significantly rated as better compared to the start of screening, (p(s) < 0.03), and this improvement remained stable over the two annual follow-ups. However, as suggested by a significant 'Time' × 'Diagnosis' interaction with regard to both QoL and Anxiety and Depression (p(s) < 0.02), the effect of the disclosure in the short term differed depending on CIS or MS diagnosis. Specifically, on MSQoL, which is a health-related QoL scale, we found a statically significant improvement, immediately after the diagnosis disclosure, in both the MS and CIS groups (p(s) < 0.01). Differently, on SEIQoL, which is a non health-related QoL measure, and on the anxiety scale, we observed a statistically significant improvement only in the group which received a MS diagnosis (p(s) < 0.03). CONCLUSIONS: This first prospective study provides objective data showing that early disclosure of MS diagnosis improves both the patient's QoL and psychological well-being. In addition, the results seem to suggest that CIS disclosure does not lead to the same favourable effects.

Hemoperitoneum as severe and unusual complication in the stapler recto-anopexy for hemorrhoidal prolapse. Case report.

We report unusual but severe complication after Longo recto-anopexy for hemorrhoidal prolapse, i.e. large intramural hematoma of the rectum and subsequent hemoperitoneum. We make some assessment about the technique.

Preoperative weekly cisplatin, epirubicin, and paclitaxel (PET) improves prognosis in locally advanced breast cancer patients: an update of the Southern Italy Cooperative Oncology Group (SICOG) randomised trial 9908.

BACKGROUND: The present article reports the updated survival outcome of the 200 patients enrolled in the Southern Italy Cooperative Oncology Group 9908 trial, which compared 12 weekly cycles of cisplatin-epirubicin-paclitaxel (PET) with 4 triweekly (once every 3 weeks) cycles of epirubicin-paclitaxel (ET) in patients with locally advanced breast cancer (LABC). METHODS: The effects of treatment, pathologically documented response (pathological response), pre- and post-treatment biomarkers on relapse-free survival (RFS), distant metastasis-free survival (DMFS), and overall survival (OS) are analysed. RESULTS: At a median follow-up of 74 (range 48-105 months) months, the 5-year RFS, DMFS, and OS were 64 % versus 53% (P = 0.11), 73% versus 55% (P = 0.04), and 82% versus 69% (P = 0.07) in PET and ET, respectively. At multivariate analysis, after adjusting treatment effect for pretreatment biomarkers, PET independently predicted better DMFS (P = 0.018) and OS (P = 0.03), whereas the impact on RFS was of borderline significance (0.057). PET treatment was significantly better than ET treatment only in high-grade or highly proliferating tumours. The better outcome in PET arm was the results of both the higher rate of patients with optimal pathological response and the lower rate of patients with biologically aggressive residual tumour. CONCLUSIONS: The PET weekly regimen significantly improves both DMFS and OS in LABC patients, compared with the triweekly ET combination. The therapeutic advantage is limited to patients with highly aggressive tumours.

Anastomotic pseudoaneurysm complicating renal transplantation: treatment options.

INTRODUCTION: Anastomotic pseudoaneurysm following renal transplantation is uncommon. Indications for repair, treatment options and outcomes remain controversial. REPORT: We present 6 renal transplant recipients with large anastomotic pseudoaneurysms. Five of the patients underwent open repair while one had a stent-grafting and delayed transplant nephrectomy for a ruptured pseudoaneurysm. A transplant nephrectomy was needed in all cases but one. Arterial reconstruction enabled limb salvage in all cases. One patient died of sepsis postoperatively. No patient presented late infection, failure of vascular reconstruction, nor pseudoaneurysm recurrence. CONCLUSIONS: Surgical excision of anastomotic pseudoaneurysms results in high rates of allograft loss. Less invasive techniques have a place in selected cases.

Effects of voriconazole on tacrolimus metabolism in a kidney transplant recipient.

Infection occurs frequently in the organ transplant recipients during the post-transplant period because of immunosuppression. Therefore, prophylactic antimicrobial agents are often used. The azole antifungals, widely prescribed prophylactically, are known to have many drug-drug interactions. This report presents a case of drug-drug interaction between voriconazole and tacrolimus in a kidney transplant recipient. Voriconazole treatment led to a dramatic increase in tacrolimus concentration that required its discontinuation in spite of the manufacturer's guidelines that recommend a reduction of tacrolimus dosage by one-third. The present drug-drug interaction can be attributed to a strong inhibitory effect on cytochrome P450-3A4 activity by voriconazole. When voriconazole and tacrolimus are coadministered, close monitoring of tacrolimus blood levels is recommended as the rule-of-thumb reduction of tacrolimus dose by one-third may not be satisfactory.

Development and diagnostic validation of an ELISA based on an antigenic mixture for the detection of bovine tuberculosis.

Programs for the eradication of bovine tuberculosis (bTB) focus on the tuberculin skin test (TST) and slaughter of reactor cattle. However, the disease remains an animal health concern in several countries and improving the efficiency of the TST has become a critical issue. The detection of Mycobacterium bovis antibodies in serum, within weeks after the TST, may be a rapid and inexpensive way to improve bTB control. This study reports the validation of an enzyme-linked immunosorbent assay (ELISA) to detect bovine tuberculosis as an ancillary test to TST in dairy farms in Argentina. The estimated validation parameters were within the established requirements of the World Organization for Animal Health (OIE). The test demonstrated high repeatability, with coefficients of variation <25%. High test reproducibility through interlaboratory testing was also found, with an estimated Pearson coefficient of 0.9648 (95% confidence intervals 0.9315-0.9820). The ELISA detected tuberculous cattle unidentified by the TST. Of 43 animals sent to slaughterhouses that were ELISA positive 15-17 days after a negative TST, 36 were confirmed as infected with M. bovis by histopathology and IS6110 PCR. According to ROC curve analysis of results of 145 cattle from M. bovis-free herds and the 36 M. bovis-infected cattle, at a corrected optical density cut-off point of 0.3853, specificity was 95.95% and the positive predictive value at this cut-off was 83.72%. The ELISA detection test validated in this study could be readily applied in dairy farms, to complement a prior TST and improve livestock health.

Spontaneous cervical extradural hematoma in a cutaneo-meningospinal angiomatosis (Cobb syndrome): case report.

Spontaneous spinal extradural hematoma is a rare clinical entity, commonly associated with coagulopathies, tumours or vascular malformations. They are often a neurosurgical emergency, therefore prompt diagnosis and early treatment are necessary. The Cobb syndrome is a neurocutaneous syndrome in which there are metameric vascular skin nevus and spinal arteriovenous malformation. The authors report the case of a 52-year-old woman with acute cervical myelopathy and a cervical cutaneous hemangioma on clinical examination. It is stressed the importance of clinical suspicion of cutaneo-meningospinal angiomatosis based on a spinal cord syndrome in the presence of a vascular skin nevus of the same metameric level.

Extrapleural solitary fibrous tumor: a clinicopathologic study of 19 cases.

This study reports a series of 19 extrapleural solitary fibrous tumors. The patients included 6 men and 13 women with age ranging from 27 to 86 years. Three patients showed local recurrence. In 2 tumors, a diagnosis of malignancy was made. All of the tumors were strongly positive for CD34, and 3 of them expressed high levels of progesterone receptor. Solitary fibrous tumors are fairly rare, occurring in many parts of the body, and their behavior is unpredictable.

Ethical Issues in the Use of Suboptimal Kidneys for Transplants: An Italian Point of View.

The shortage of organs leads to the need for utilizing suboptimal kidneys for transplantation. The distinction between optimal, marginal, and suboptimal kidneys leads surgeons to face not only technical problems but also ethical and legal issues related to clinical advantages offered by the transplant of a nonstandard kidney and the acquisition of consent. Between 1999 and 2015, we performed 658 transplants, 49 (7.5%) using suboptimal kidneys. All patients were alive and with vital graft throughout follow-up. We did not encounter any major surgical complications. From a technical point of view, our experience and literature review confirm that transplant of suboptimal kidney leads to good clinical results but exposes patients to a increased risks of surgical complications. Therefore, these interventions must take place in hospitals fully prepared for this type of surgery and performed by experienced transplant surgeons with proper matching between organ and recipient. Considering the insufficient resources available, from an ethical and legal point of view, doctors play an essential role in optimizing the use of these kidneys by avoiding wastage of organs, ensuring that transplants are done in suitable patients, and that patients are fully informed and aware of the risks and benefits associated with the specific suboptimal kidney being transplanted. We believe that, in highly specialized centers, the number of suboptimal kidney transplants should be increased, as their use has shown good clinical results and carries fewer ethical issues compared with marginal kidneys. Further, suboptimal kidneys may also be proposed for use in young patients with end-stage renal disease.

Our Timing to Remove Peritoneal Catheter Dialysis After Kidney Transplant.

BACKGROUND: Patients on peritoneal dialysis treatment represent 15% of the global dialysis population. The major complication of peritoneal dialysis is catheter and peritoneal infection. Peritoneal dialysis patients who receive kidney transplants are at increased risk of infection because of immunosuppressive therapy. AIM: The purpose of this study is to show our ideal timing to remove peritoneal catheter after kidney transplant, which gives adequate security on renal function recovery and reduction of septic risk. METHOD OF STUDY: We analyzed the outcomes of 65 patients on peritoneal dialysis who underwent kidney transplant between 2000 and 2016. RESULTS: In 61 cases there was an immediate graft functional recovery. In 4 cases there was a delayed graft function (DGF), and we performed a hemodialysis with temporary placement of a venous catheter. In all patients we removed peritoneal dialysis catheter 30 to 45 days after transplant. There has been 1 case of catheter infection, which was treated with antibiotic therapy. DISCUSSION: Our average time to remove the peritoneal dialysis catheter was shorter than times in previous studies, between the 30th and 45th postoperative day. In the 4 cases in which there has been a DGF, we performed hemodialysis treatment to avoid, in the immediate postoperative period, direct insults to the peritoneum by local dialysis procedures. CONCLUSION: Our experience show that the 30th to 45th postoperative day is a good time frame, better yet a good watershed between the safe removal of peritoneal catheter when patients have a stabilized renal function and the possibility of leaving it in situ, to resume peritoneal dialysis in case of persistent DGF.

In situ elongation patch in right kidney transplantation.

BACKGROUND: A short right renal vein complicates transplantation causing traction and difficulties during anastomosis. When we perform a kidney transplantation from a cadaveric donor, this problem may be resolved by using the vena cava to create a venous duct. This elongation technique is proposed to be performed during bench surgery. We propose a small change in the technique: execution of an "elongation patch" during harvesting and under cold perfusion. MATERIALS AND METHODS: From January 2004 to June 2006, we performed 12 in situ (during the harvesting procedure) vena cava elongation patches. In these cases, the right renal vein was too short. The elongation patch was used in only 8 transplantations. In the other 4 cases we sacrificed the patch to perform a direct venous anastomosis due to favorable recipient anatomical characteristics. RESULTS: The 8 transplantations performed with the elongation patch did not show vascular complications and the venous anastomosis was easy to perform. CONCLUSIONS: The "extension patch" was not associated with a greater incidence of vascular complications. Using the elongation patch during the harvest showed some advantages: performed during cold perfusion with a reduction in bench ischemia; the anatomical relationships are preserved so we can perform a calibrated suture; the perfusion of the organ allows us to observe the integrity of the anastomosis. This technique did not significantly increase the harvesting time.

Integrating printed microfluidics with silicon photomultipliers for miniaturised and highly sensitive ATP bioluminescence detection.

Bioluminescence has been widely used for important biosensing applications such as the measurement of adenosine triphosphate (ATP), the energy unit in biological systems and an indicator of vital processes. The current technology for detection is mainly based on large equipment such as readers and imaging systems, which require intensive and time-consuming procedures. A miniaturised bioluminescence sensing system, which would allow sensitive and continuous monitoring of ATP, with an integrated and low-cost disposable microfluidic chamber for handling of biological samples, is highly desirable. Here, we report the design, fabrication and testing of 3D printed microfluidics chips coupled with silicon photomultipliers (SiPMs) for high sensitive real-time ATP detection. The 3D microfluidic chip reduces reactant consumption and facilitates solution delivery close to the SiPM to increase the detection efficiency. Our system detects ATP with a limit of detection (LoD) of 8nM and an analytical dynamic range between 15nM and 1µM, showing a stability error of 3%, and a reproducibility error below of 20%. We demonstrate the dynamic monitoring of ATP in a continuous-flow system exhibiting a fast response time, ~4s, and a full recovery to the baseline level within 17s. Moreover, the SiPM-based bioluminescence sensing system shows a similar analytical dynamic range for ATP detection to that of a full-size PerkinElmer laboratory luminescence reader.

The finding of vascular and urinary anomalies in the harvested kidney for transplantation.

INTRODUCTION: In kidney transplantation, anatomical vascular and excretory anomalies may represent causes of failure. Today's surgical techniques have made the most of the organs with anatomic anomalies and iatrogenic injury successfully used for transplantation. MATERIALS AND METHODS: From January 2000 to June 2006, we harvested 230 kidneys, of including 88 kidneys (20%) with vascular, urinary, or vascular-urinary anomalies; 64 kidneys were implanted and 15 were sent to other transplantation centers. Only 9 kidneys were not appropriate for transplantation. RESULTS: All patients who received kidneys with the above-mentioned anomalies were carefully examined after the transplantation and short-term and long-term complications were evaluated with respect to controls without anomalies. DISCUSSION: Renal anatomic anomalies are frequently observed during kidney transplantation and may produce postsurgical complications. However, the presence of these anomalies does not necessarily imply the impossibility of using the kidney for a transplant, especially because of improved surgical techniques. Our experience in transplantation procedures showed that even if kidneys present the above-mentioned anomalies they can still be considered appropriate for transplantation when we perform a correct harvesting/back-table transplant surgery. So vascular and urinary anomalies have to be considered always an incentive to research new surgical solutions and to perform a careful surgical technique.