A Method for Using Cell-Penetrating Peptides for Loading Plasmid DNA into Secreted Extracellular Vesicles.

The low bioavailability and high toxicity of plasmid DNA (pDNA)-based therapeutics pose challenges for their in vivo application. Extracellular vesicles (EVs) have great potential to overcome these limitations, as they are biocompatible native cargo carriers. Various methods for loading pDNA into EVs, including electroporation, sonication, and co-incubation, have been previously investigated, but their success has been questionable. In this study, we report a unique method for loading EVs with pDNA through transient transfection using cell-penetrating peptides (CPPs). With this method, we found a 104-fold increase in the expression levels of the luciferase reporter protein in recipient cells compared to the untreated cells. These data point to the high transfection efficacy and bioavailability of the delivered encapsulated nucleic acid. Furthermore, the in vivo experimental data indicate that the use of pDNA-loaded EVs as native delivery vehicles reduces the toxic effects associated with traditional nucleic acid (NA) delivery and treatment.

TM9SF4 expression in tumor tissues: a novel diagnostic biomarker for gastrointestinal tumors.

BACKGROUND: The identification of novel biomarkers for the early detection and monitoring of gastric (GC) and colorectal cancer (CRC) is of paramount importance. TM9SF4 is a newly described V-ATPase interacting protein involved in the malignant progression of cancer cells. While TM9SF4 expression pattern and cellular localization have been described in in vitro in tumor cell lines of different histotypes, its expression in gastrointestinal tumor tissues has never been investigated. METHODS: In this study, we detected by immunohistochemistry (IHC) in tumor and surrounding healthy tissues TM9SF4, in comparison with clinically adopted biomarkers CEA and CA 19-9 to evaluate TM9SF4 potential as a novel tissue marker for early detection and monitoring of GC and CRC cancers. RESULTS: The expression of TM9SF4, CEA and CA 19-9 was evaluated in samples from 108 cancer patients (68 with GC and 40 CRC) and in healthy tissues from 20 non-cancer patients. Our results clearly suggest that TM9SF4 expression was significantly increased in GC and CRC samples and significantly correlated to disease stage in both cancer types. CONCLUSIONS: We propose TM9SF4 as highly specific cancer biomarker, exploitable for disease detection and staging of gastrointestinal cancers patients, with tumor tissue levels of expression outperforming those of clinically adopted markers such as CEA and CA 19-9.

Implant site preparation using a single bur versus multiple drilling steps: 4-month post-loading results of a multicenter randomised controlled trial.

PURPOSE: To compare the clinical outcome of implants inserted in sites prepared with a simplified protocol consisting of one single drill versus multiple conventional drilling steps. MATERIALS AND METHODS: In two private clinics, 40 patients, requiring one single implant and having a residual bone height of at least 10 mm and a thickness of at least 5 mm measured on computerised tomography (CT) scans, were randomised after flap elevation to have the implant site prepared using a single drilling step with a newly designed tapered-cylinder drill (1-drill group) or a conventional procedure with multiple drills (multiple-drill group). Implants were left to heal non-submerged for 3 months and then they were loaded with a final metal-ceramic crown. Outcome measures were: implant failure; any complications; peri-implant marginal bone level changes assessed by a blinded outcome assessor; operation time; operator preference and post-surgical pain, swelling and analgesic consumption. All patients were followed up to 4 months after implant loading. RESULTS: Twenty patients were randomised to the 1-drill group and 20 patients to the multiple-drill group. No implant failed and no complications occurred. Four months after loading, implants in the 1-drill group lost 0.54 mm of peri-implant bone versus 0.41 mm for the implants in the multiple-drill group. There were no statistically significant differences for marginal bone level changes between the two groups (difference 0.13 mm, 95% CI -0.21; 0.47, P = 0.108). Less time which was statistically significant (3.66 mins, 95% CI 2.69; 4.63, P < 0.0001) was required to place the implant with the single bur. Both operators always preferred the single bur technique. Postoperatively, patients in the 1-drill group vs patients in the multiple-drill group reported statistically significant differences for pain level (difference 27.5, 95% CI 3.3; 51.7, P < 0.0001), number of days in which the swelling persisted (difference 3.4, 95% CI 2.4; 4.4, P < 0.0001) and the number of analgesic drugs taken (difference 2.8, 95% CI 1.4; 4.2, P < 0.0001) CONCLUSIONS: Within the limits of this trial, both drilling techniques produced successful results over a 4-month post-loading follow-up period, but the single bur procedure required less surgical time and lead to less postoperative morbidity.

Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches.

Clinical implementation of exosome based diagnostic and therapeutic applications is still limited by the lack of standardized technologies that integrate efficient isolation of exosomes with comprehensive detection of relevant biomarkers. Conventional methods for exosome isolation based on their physical properties such as size and density (filtration, ultracentrifugation or density gradient), or relying on their differential solubility (chemical precipitation) are established primarily for processing of cell supernatants and later adjusted to complex biological samples such as plasma. Though still representing gold standard in the field, these methods are clearly suboptimal for processing of routine clinical samples and have intrinsic limits that impair their use in biomarker discovery and development of novel diagnostics. Immunoisolation (IA) offers unique advantages for the recovery of exosomes from complex and viscous fluids, in terms of increased efficiency and specificity of exosome capture, integrity and selective origin of isolated vesicles. We have evaluated several commercially available solutions for immunoplate- and immunobead-based affinity isolation and have further optimized protocols to decrease non-specific binding due to exosomes complexity and matrix contaminants. In order to identify best molecular targets for total exosome capture from diverse biological sources, as well as for selective enrichment in populations of interest (e.g. tumor derived exosomes) several exosome displayed proteins and respective antibodies have been evaluated for plate and bead functionalisation. Moreover, we have optimized and directly implemented downstream steps allowing on-line quantification and characterization of bound exosome markers, namely proteins and RNAs. Thus assembled assays enabled rapid overall quantification and validation of specific exosome associated targets in/on plasma exosomes, with multifold increased yield and enrichment ratio over benchmarking technologies. Assays directly coupling selective immobilization of exosomes to a solid phase and their immune- and or molecular profiling through conventional ELISA and PCR analysis, resulted in easy-to-elaborate, quantitative readouts, with high low-end sensitivity and dynamic range, low costs and hands-on time, minimal sample handling and downscaling of a working plasma volumes to as few as 100 µl.

Immediate, early (3 weeks) and conventional loading (4 months) of single implants: Preliminary data at 1 year after loading from a pragmatic multicenter randomised controlled trial.

PURPOSE: To compare the clinical outcome of single implants which underwent immediate nonocclusal loading with implants subjected to early non-occlusal loading at 3 weeks, and implants conventionally loaded at 4 months. MATERIALS AND METHODS: One hundred and five patients in five private practices requiring a single implant-supported crown were randomised to immediate loading (35 patients), early loading (35 patients) and conventional loading (35 patients) groups. To be immediately or early loaded, implants had to be inserted with a torque superior to 45 Ncm. Immediately and early loaded implants received non-occluding temporary crows, whereas conventionally loaded implants were directly restored with definitive crowns. Temporary crowns were replaced by definitive ones after 4 months. Outcome measures were crown and implant failures, complications and peri-implant marginal bone level changes recorded by a blinded assessor. RESULTS: Two patients dropped out from the immediate loading group up to 1-year post-loading. Two implants failed, one in the immediately loaded and one in the early loaded group (P=0.601). One immediately loaded implant and two early loaded implants were affected by one complication each (P=0.162). Mean peri-implant marginal bone loss after 1 year was -0.120±0.230 mm (95% CI -0.35, 0.10) for immediate, -0.390±0.840 mm (95% CI -1.23, 0.45) for early and -0.201±0.306 mm (95% CI -0.51; 0.11) for conventionally loaded implants. There were no statistically significant differences for any of the outcome measures between the three loading strategies up to 1-year post-loading. CONCLUSIONS: No major clinical differences were observed with regard to implant survival, complications and marginal bone level changes when loading single implants immediately, early or conventionally.

One abutment-one time versus a provisional abutment in immediately loaded post-extractive single implants: a 1-year follow-up of a multicentre randomised controlled trial.

PURPOSE: To compare immediately loaded post-extractive single implants using a definitive abutment versus provisional abutment later replaced by custom-made abutment. MATERIALS AND METHODS: In two private clinics, 28 patients in need of one single post-extractive implant in the maxilla or mandible from the left second premolar to the right second premolar area were randomised shortly before tooth extraction to provisional abutment (PA) and definitive abutment (DA) groups. Three patients had to be excluded for buccal wall fracture after tooth extraction. In the PA group, implants were immediately restored using a platform-switched provisional titanium abutment and definitive platform-switched titanium abutments were used in the DA group. In both groups, a non-occluding provisional single crown was provided. Implants were definitively restored after 4 months. In the PA group, the abutment was removed and the impression was made directly on the implant platform. In the DA group an impression of the abutment was made using a retraction cord. Outcome measures were: implant failures; complications; and marginal peri-implant bone level changes. Patients were followed up to 1 year after loading. RESULTS: Twelve patients were randomised to the DA group and 13 patients to the PA group. At the 12-month follow-up, no implant failed. One biological complication occurred in the DA group and one mechanical complication occurred in the PA group. All complications were successfully treated. One year after loading, implants in the DA group lost an average of 0.11 mm (SD: 0.06) of periimplant bone and implants in PA group about 0.58 mm (SD: 0.11). At the 12-month follow-up, there was a statistically significant difference in bone level change between groups (mean difference: 0.48 mm, CI 95% 0.40; 0.55, P < 0.0001). CONCLUSIONS: Within the limits of this study, the non-removal of abutments placed at the time of surgery resulted in the maintenance of 0.5 mm more bone levels around immediately restored postextractive single implants than repeated abutment removal, although this amount of bone maintenance may not have a clinical impact. Conflicts of interest notification: Dr Tommaso Grandi and Dr Paolo Guazzi serve as consultants for JDentalCare. This study was completely self-financed and no funding was sought or obtained, not even in the form of free materials.

A 3-year report from a multicentre randomised controlled trial: immediately versus early loaded implants in partially edentulous patients.

PURPOSE: To compare implant failure, prosthesis failure and radiographic bone level changes of immediate non-occlusal loading versus early loading in partially edentulous patients 3 years after implant placement. MATERIALS AND METHODS: A total of 80 patients with partial edentulism were selected for a two implant-supported immediate restoration and randomised to immediate loading (test group) or early loading (control group) after 2 months, 40 patients for each group. To be included in the study, implants had to be inserted with a torque ≥ 30 Ncm. In the test group, implants were provided with non-occluding temporary restorations. In the control group, healing abutments were attached and implants were left to heal nonsubmerged. Definitive prostheses were delivered 2 months after surgery with full occlusal contacts. The outcome variables were implant failure, prosthesis failure, complications and radiographic bone level changes at implants 3 years after loading. The assessor was blinded to group assignment. RESULTS: Eighty-one implants were immediately loaded and 80 were early loaded. Two immediately loaded patients and 1 early loaded patient dropped out at 3 years. No implant failed. Two complications occurred in the immediate loading group and 1 in the early loading group. There were no significant differences in bone levels changes at 36 months between the two groups (P = 0.67; difference 0.2 mm; 95% CI -0.23, 0.63). Immediately loaded implants lost 0.90 mm (95% CI 0.63, 1.17) and early loaded implants 1.10 mm (95% CI 0.81, 1.39). CONCLUSION: If adequate primary stability was achieved, no statistically significant difference in failure rates, complications or bone level changes between implants loaded immediately or early were observed 3 years after placement in partially edentulous patients.

Immediate provisionalisation of single post-extractive implants versus implants placed in healed sites in the anterior maxilla: 1-year results from a multicentre controlled cohort study.

PURPOSE: The purpose of the study was to compare the clinical and aesthetic outcome of single post-extractive implants versus implants placed in a preserved socket after 4 months of healing in the anterior maxilla. All of the implants were immediately non-occlusally loaded. MATERIALS AND METHODS: A total of 50 patients were treated in the two groups of study. The Delayed Group had a maxillary tooth (premolar, canine, lateral or central incisor) removed, with immediate socket grafting, followed by implant placement and provisionalisation 4 months later. The Immediate Group had immediate implant placement and provisionalisation. Outcome measures were implant failures, biological and biomechanical complications, peri-implant radiographic bone level changes, and gingival aesthetics. RESULTS: At the 12-month follow-up, two implants failed in the Immediate Group (8%) versus one in the Delayed Group (4%), with a comparable rate of implant failure (P = 0.55). No complications occurred for either group. The 12-month peri-implant bone resorption was similar in both groups (P = 0.23): 0.71 mm (95% CI 0.45, 0.97) in the Immediate Group versus 0.60 mm (95% CI 0.38, 0.82) in the Delayed Group. The mean difference in bone resorption was 0.13 mm (95% CI -0.21, 0.47). An ideal gingival marginal level was reached most frequently in the Delayed Group (83.3% versus 52.1%, P = 0.04). Rates of full closure of the papilla were similar between the two groups (82.6% for the Immediate Group versus 62.5% for the Delayed Group, P = 0.12). CONCLUSIONS: Given the limitation that this was not a randomised controlled trial, there were no differences in complications or crestal bone response at immediate post-extractive implants when compared to delayed implants. A delayed protocol might be considered in the aesthetic zone due to the gingival recession that occurs after post-extractive implant placement.

Identification of the Kelch family protein Nd1-L as a novel molecular interactor of KRIT1.

Loss-of-function mutations of the KRIT1 gene (CCM1) have been associated with the Cerebral Cavernous Malformation (CCM) disease, which is characterized by serious alterations of brain capillary architecture. The KRIT1 protein contains multiple interaction domains and motifs, suggesting that it might act as a scaffold for the assembly of functional protein complexes involved in signaling networks. In previous work, we defined structure-function relationships underlying KRIT1 intramolecular and intermolecular interactions and nucleocytoplasmic shuttling, and found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. Here we report the identification of the Kelch family protein Nd1-L as a novel molecular interactor of KRIT1. This interaction was discovered through yeast two-hybrid screening of a mouse embryo cDNA library, and confirmed by pull-down and co-immunoprecipitation assays of recombinant proteins, as well as by co-immunoprecipitation of endogenous proteins in human endothelial cells. Furthermore, using distinct KRIT1 isoforms and mutants, we defined the role of KRIT1 domains in the Nd1-L/KRIT1 interaction. Finally, functional assays showed that Nd1-L may contribute to the regulation of KRIT1 nucleocytoplasmic shuttling and cooperate with KRIT1 in modulating the expression levels of the antioxidant protein SOD2, opening a novel avenue for future mechanistic studies. The identification of Nd1-L as a novel KRIT1 interacting protein provides a novel piece of the molecular puzzle involving KRIT1 and suggests a potential functional cooperation in cellular responses to oxidative stress, thus expanding the framework of molecular complexes and mechanisms that may underlie the pathogenesis of CCM disease.

Immediate loading of four (all-on-4) post-extractive implants supporting mandibular cross-arch fixed prostheses: 18-month follow-up from a multicentre prospective cohort study.

AIM: To evaluate the outcome of immediately loaded mandibular cross-arch prostheses according to the 'all-on-4' concept supported by implants placed in fresh extraction sockets up to 18 months after loading. MATERIALS AND METHODS: In total, 47 patients with a mean age of 62.3 years (range 52 to 78) were rehabilitated with an immediately loaded fixed cross-arch prosthesis supported by four post-extractive implants. A total of 188 implants were inserted. Patients received a provisional fixed dental prosthesis with a metal framework within 48 hours after surgery and a permanent one 6 months later. The patients were evaluated clinically and radiographically at implant placement and at 6-, 12- and 18-month follow-up examinations. RESULTS: At the 18-month follow-up, no implant failed and all restorations were stable. Peri-implant bone levels amounted to 0.31 ± 0.12 mm after 6 months, 0.58 ± 0.112 mm after 12 months and 0.7 ± 0.107 mm after 18 months. No significant differences in bone loss were found between axially placed and tilted implants at the 6-month (0.06 mm; P = 0.115), the 12-month (0.12 mm; P = 0.062) and the 18-month follow-up (0.08 mm; P = 0.146). Three patients had a fracture of the provisional restoration, but all of the definitive prostheses remained stable throughout the study period without any complications. CONCLUSIONS: Within the limits of this study, it can be suggested that immediately loaded mandibular cross-arch fixed dental prostheses can be supported by four post-extractive implants, however larger and longer follow-ups are needed.

Immediate positioning of definitive abutments versus repeated abutment replacements in immediately loaded implants: effects on bone healing at the 1-year follow-up of a multicentre randomised controlled trial.

PURPOSE: To compare bone resorption around implants immediately loaded and restored using definitive abutments versus provisional abutments later replaced by custom-made abutments up to 12 months after implant placement. MATERIALS AND METHODS: 28 patients with partial edentulism were selected for a two-implant supported immediate restoration and randomised to provisional abutment (PA) and definitive abutment (DA) groups (14 patients for each group). In the PA group, implants were immediately restored using a platform-switched provisional titanium abutment. In the DA group, definitive platform-switched titanium abutments were tightened. In both groups, a provisional restoration was adapted, avoiding occlusal contacts. All implants were definitively restored after 3 months. In the PA group, patients underwent the standard prosthetic protocol: the abutments were removed and impressions were made directly on the implant platform. In the DA group, patients underwent the 'one abutment at one time' protocol: impressions were made of the abutments using a retraction cord. Peri-implant marginal bone levels were assessed immediately after surgery, and at 6- and 12-month follow-up examinations. RESULTS: At the 12-month follow-up no implant failed. In the PA group, peri-implant bone resorption was 0.359 mm after 6 months and 0.435 mm after 12 months. In the DA group, peri-implant bone resorption was 0.065 mm after 6 months and 0.094 mm after 12 months. There were statistically significant differences between the two groups for peri-implant bone level changes at the 6-month (P < 0.001) and the 12-month (P < 0.001) follow-up: 0.294 mm (CI 95% 0.276; 0.312) and 0.341 mm (CI 95% 0.322; 0.36), respectively. CONCLUSIONS: Within the limits of this study, it can be suggested that the non-removal of abutments placed at the time of surgery results in a statistically significant reduction of the crestal bone resorption around the immediately restored implants in cases of partial edentulism, however a difference of 0.3 mm may not have a clinical impact.

Immediate loading of two unsplinted implants retaining the existing complete mandibular denture in elderly edentulous patients: 1-year results from a multicentre prospective cohort study.

PURPOSE: To evaluate the outcome of two freestanding implants immediately loaded retaining an existing mandibular complete denture in elderly edentulous patients up to 1 year after loading. MATERIALS AND METHODS: 42 patients with a mean age of 76.5 years (range 71 to 89) were selected for stabilising their mandibular complete denture with two implants placed mesial to the mandibular canine position bilaterally. Individual ball abutments were connected and torqued at 30 Ncm and the existing complete denture was immediately attached. No postoperative limitations to chewing function were given. The patients were evaluated clinically and radiographically at implant placement and at 6- and 12-month follow-up examinations. RESULTS: At the 12-month follow-up no implant failed. Peri-implant bone resorption was 0.203 mm (CI 95% 0.322; 0.086) after 6 months and 0.298 mm (CI 95% 0.425; 0.173) after 12 months. Of the 42 cases, 3 had major prosthetic complications and 5 patients required minor extra maintenance appointments. CONCLUSIONS: Within the limits of this study, it can be suggested that the immediate loading of two unsplinted implants retaining the existing complete mandibular denture in elderly patients can result in favourable implant survival and peri-implant bone healing, however larger and longer follow-ups of 5 years or more are needed.

Immediate loading of single post-extractive implants in the anterior maxilla: 12-month results from a multicenter clinical study.

The purpose of the study was to evaluate survival and peri-implant bone levels of single, immediately loaded post-extractive implants in the anterior maxilla 12 months after implant placement. Thirty-six consecutive patients from 3 study centers were included in the study. The concerned sites were upper premolars, canines, and incisors. For each patient the following data were recorded: reason for tooth extraction, bone quality, implant size, and final insertion torque. Implants were placed using a flapless technique and immediately loaded with a nonoccluding temporary restoration. Final restorations were provided 4 months later. Peri-implant bone resorption was evaluated radiographically after 6 and 12 months. The average final insertion torque was 70.55 Ncm. One implant inserted in D3 quality bone with a 35-Ncm seating torque was lost. All other implants had a final insertion torque ranging between 50 and 80 Ncm. The average peri-implant bone loss was 0.437 and 0.507 mm at 6 and 12 months, respectively. All the sites maintained excellent papillae and peri-implant soft-tissue conditions. The resulting 1-year success rate was 97.2%. Immediate nonfunctional loading of single post-extractive implants in the anterior maxilla is a predictable treatment. And it seems that achieving high insertion torques by placing self-tapping/self-condensing implants in an underprepared osteotomy is favorable.

Survival and success rates of immediately and early loaded implants: 12-month results from a multicentric randomized clinical study.

Our objective was to compare survival and peri-implant bone levels of immediately nonocclusally vs early loaded implants in partially edentulous patients up to 12 months after implant placement. Eighty patients (inclusion criteria: general good health, good oral hygiene, 30-65 years old; exclusion criteria: head and neck irradiation/cancer, pregnancy, uncontrolled diabetes, substance abuse, bruxism, lack of opposing occluding dentition, smokers >10 cigarettes/day, need for bone augmentation procedures) were selected in 5 Italian study centers and randomized into 2 groups: 40 patients in the immediately loaded group (minimal insertion torque 30 Ncm) and 40 patients in the early loaded group. Immediately loaded implants were provided with nonoccluding temporary restorations. Final restorations were provided 2 months later. Early loaded implants were provided with a definitive restoration after 2 months. Peri-implant bone resorption was evaluated radiographically with software (ImageJ 1.42). No dropout occurred. Both groups gradually lost peri-implant bone. After 12 months, patients of both groups lost an average of 0.4 mm of peri-implant bone. There were no statistically significant differences (evaluated with t test) between the 2 loading strategies for peri-implant bone level changes at 2 (P = .6730), 6 (P = .6613) and 12 (P = .5957) months or for survival rates (100% in both groups). If adequate primary stability is achieved, immediate loading of dental implants can provide similar success rates, survival rates, and peri-implant bone resorption as compared with early loading, as evaluated in the present study.

Structural and functional differences between KRIT1A and KRIT1B isoforms: a framework for understanding CCM pathogenesis.

KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal beta-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.

G-protein binding features and regulation of the RalGDS family member, RGL2.

RGL2 [RalGDS (Ral guanine nucleotide dissociation stimulator)-like 2] is a member of the RalGDS family that we have previously isolated and characterized as a potential effector for Ras and the Ras analogue Rap1b. The protein shares 89% sequence identity with its mouse orthologue Rlf (RalGDS-like factor). In the present study we further characterized the G-protein-binding features of RGL2 and also demonstrated that RGL2 has guanine-nucleotide-exchange activity toward the small GTPase RalA. We found that RGL2/Rlf properties are well conserved between human and mouse species. Both RGL2 and Rlf have a putative PKA (protein kinase A) phosphorylation site at the C-terminal of the domain that regulates the interaction with small GTPases. We demonstrated that RGL2 is phosphorylated by PKA and phosphorylation reduces the ability of RGL2 to bind H-Ras. As RGL2 and Rlf are unique in the RalGDS family in having a PKA site in the Ras-binding domain, the results of the present study indicate that Ras may distinguish between the different RalGDS family members by their phosphorylation by PKA.

Bronchogenic adenocarcinoma in a cat: an unusual case of metastasis to the skin.

A 6-year-old, spayed, female, domestic shorthair cat was presented for decreased activity. A nodular lesion was found in the skin extending into the subcutaneous tissue of the right abdominal flank. On lateral and ventrodorsal radiographs of the thorax, an opacity involving the entire right caudal lung lobe and pleural effusion were noted. Cytologic evaluation of cells in the thoracic fluid and in the mass revealed a population of atypical epipthelial cells with marked anisocytosis and high N:C ratios, organized in acinar-like clusters. Multinucleated cells and several mitotic figures were found. The cytologic interpretation was carcinoma. Because of the progressive severity of clinical signs, the cat was euthanized. Histologic evaluation of tissues obtained at necropsy indicated a bronchogenic adenocarcinoma in the lung, with metastasis to the skin of the right flank, but no involvement of the digits. Based on immunohistochemical stains, the neoplastic cells strongly co-expressed cytokeratin and vimentin, and were negative for S-100 and actin-specific antigen. Bronchogenic adenocarcinoma is an uncommon neoplasm in cats, and the digits are the most common sites of metastasis. This case was unusual in that the skin of the abdominal wall was the primary site of metastasis, with no involvement of the digits.