Exploring the short linear motif-mediated protein-protein interactions of CrkL through ProP-PD.

Adaptor proteins play a pivotal role in cellular signaling mediating a multitude of protein-protein interaction critical for cellular homeostasis. Dysregulation of these interactions has been linked to the onset of various cancer pathologies and exploited by viral pathogens during host cell takeover. CrkL is an adaptor protein composed of an N-terminal SH2 domain followed by two SH3 domains that mediate interactions with diverse partners through the recognition of specific binding motifs. In this study, we employed proteomic peptide-phage display (ProP-PD) to comprehensively explore the short linear motif (SLiM)-based interactions of CrkL. Furthermore, we scrutinized how the binding affinity for selected peptides was influenced in the context of the full-length CrkL versus the isolated N-SH3 domain. Importantly, our results provided insights into SLiM-binding sites within previously reported interactors, as well as revealing novel human and viral ligands, expanding our understanding of the interactions mediated by CrkL and highlighting the significance of SLiM-based interactions in mediating adaptor protein function, with implications for cancer and viral pathologies.

Construction of a nomogram for predicting COVID-19 in-hospital mortality: A machine learning analysis.

Background and objectives: We aim to verify the use of ML algorithms to predict patient outcome using a relatively small dataset and to create a nomogram to assess in-hospital mortality of patients with COVID-19. Methods: A database of 200 COVID-19 patients admitted to the Clinical Hospital of State University of Campinas (UNICAMP) was used in this analysis. Patient features were divided into three categories: clinical, chest abnormalities, and body composition characteristics acquired by computerized tomography. These features were evaluated independently and combined to predict patient outcomes. To minimize performance fluctuations due to low sample number, reduce possible bias related to outliers, and evaluate the uncertainties generated by the small dataset, we developed a shuffling technique, a modified version of the Monte Carlo Cross Validation, creating several subgroups for training the algorithm and complementary testing subgroups. The following ML algorithms were tested: random forest, boosted decision trees, logistic regression, support vector machines, and neural networks. Performance was evaluated by analyzing Receiver operating characteristic (ROC) curves. The importance of each feature in the determination of the outcome predictability was also studied and a nomogram was created based on the most important features selected by the exclusion test. Results: Among the different sets of features, clinical variables age, lymphocyte number and weight were the most valuable features for prognosis prediction. However, we observed that skeletal muscle radiodensity and presence of pleural effusion were also important for outcome determination. Integrating these independent predictors was successfully developed to accurately predict mortality in COVID-19 in hospital patients. A nomogram based on these five features was created to predict COVID-19 mortality in hospitalized patients. The area under the ROC curve was 0.86 ± 0.04. Conclusion: ML algorithms can be reliable for the prediction of COVID-19-related in-hospital mortality, even when using a relatively small dataset. The success of ML techniques in smaller datasets broadens the applicability of these methods in several problems in the medical area. In addition, feature importance analysis allowed us to determine the most important variables for the prediction tasks resulting in a nomogram with good accuracy and clinical utility in predicting COVID-19 in-hospital mortality.

ABCB1 c.-6-180T>G polymorphism and clinical risk factors in a multi-breed cohort of dogs with refractory idiopathic epilepsy.

Epilepsy is the most common chronic neurological disorder in dogs. Approximately 20-30% of dogs do not achieve satisfactory seizure control with two or more anti-epileptic drugs at appropriate dosages. This condition, defined as refractory epilepsy, is a multifactorial condition involving both acquired and genetic factors. The P glycoprotein might play and important role in the pathophysiological mechanism and it is encoded by the ABCB1 gene. An association between a single nucleotide variation of the ABCB1 gene (c.-6-180T>G) and phenobarbital resistance has previously been reported in a Border collie population with idiopathic epilepsy. To date, the presence and relevance of this polymorphism has not been assessed in other breeds. A multicentre retrospective, case-control study was conducted to investigate associations between ABCB1 c.-6-180T>G, clinical variables, and refractoriness in a multi-breed population of dogs with refractory idiopathic epilepsy. A secondary aim was to evaluate the possible involvement of the ABCB1 c.-6-180T>G single nucleotide variation this population. Fifty-two refractory and 50 responsive dogs with idiopathic epilepsy were enrolled. Of these, 45 refractory and 50 responsive (control) dogs were genotyped. The G allele was found in several breeds, but there was no evidence of association with refractoriness (P=0.69). The uncertain role of the c.-6-180T>G variation was further suggested by an association between the T/T genotype with both refractoriness and responsiveness in different breeds. Furthermore, high seizure density (cluster seizure) was the main clinical risk factor for refractory idiopathic epilepsy (P=0.003).

A double-blind randomized placebo-controlled trial with short-term beta-glucuronidase therapy in children with chronic rhinoconjunctivitis and/or asthma due to dust mite allergy.

BACKGROUND: Enzyme potentiated desensitization, in which beta-glucuronidase (BG) is administered with low doses of mixed allergens, was proposed in the 1970s for specific immunotherapy. The BG currently commercially available in a purified and standardized preparation devoid of any allergen has been suggested as a regulator in the allergic immune response, acting on the cytokine-network of type 2 helper T cells. A double-blind trial with a single-dose of BG proved effective in preventing symptoms in adult patients with rhinoconjunctivitis due to grass pollens. OBJECTIVE: The aim of this randomized double-blind placebo-controlled trial was to confirm the safety and effectiveness of double-dose intradermal BG immunotherapy in preventing symptoms in children suffering from chronic rhinoconjunctivitis and/or asthma due to dust mite. METHOD: We randomized 125 children with dust-mite related chronic rhinoconjunctivitis and/or asthma to the BG treated group (67) or the placebo group (58). All patients were screened before treatment (TO), at BG or placebo administration (T1 and T3), and at 3 and 9 months after T1 (T2 and T4). Drug intake and bronchial, nasal and ocular symptoms were recorded in a diary. RESULTS: Patients in both groups completed the study and BG treatment was well tolerated without side effects. Significant differences in symptoms were observed, in particular for conjunctivitis (P= .008). The total drug intake for allergic symptoms was significantly lower in the treated group than in the placebo group (P<. 01). CONCLUSIONS: BG immunotherapy is efficacious, safe, and well tolerated in allergic children. Moreover, good compliance with the administration of 2 doses per year and the lack of significant side effects makes the benefit/risk ratio of this treatment particularly favorable.

Tumour staging, morphology and p53 overexpression concur in predicting survival in hepatocellular carcinoma.

BACKGROUND/AIMS: The prognosis of hepatocellular carcinoma (HCC) on cirrhosis is hard to predict as it depends on tumour stage, underlying liver disease, type of treatment and, possibly, biological factors of the tumour itself. METHODS: We prospectively evaluated the survival of 91 consecutive patients with HCC on cirrhosis, diagnosed between January 1998 and December 1999. Clinical features and histological/biological aspects, including histotype, grade, p53 overexpression, cytoproliferation and apoptotic markers were analysed. RESULTS: Child-Pugh (P = 0.01), Okuda (P < 0.0001), Cancer of the Liver Italian Program (CLIP) staging (P < 0.0001) and type of treatment (P = 0.0001) were significantly related to survival. In the Cox model, CLIP staging was included as independent predictor of survival at step 1 (P < 0.0001) with Okuda at step 2 (P = 0.013). Amongst the biological factors, p53 overexpression and histotype were significantly related with survival (P = 0.0044 and 0.017 respectively). When clinical and biological variables were examined together in the Cox model, CLIP and Okuda were confirmed as being statistically related with survival (P < 0.0001 and =0.012) followed by histotype and p53 overexpression (P = 0.019 and 0.02). CONCLUSIONS: CLIP, Okuda, histotype and p53 overexpression are the strongest predictors of survival in this series of patients. These data confirm that staging of the tumour and underlying liver disease are strictly related to prognosis but support the concurrent role of clinical and biological factors in upgrading our capacity of predicting the fate of HCC patients.

[Comparison of methodologies to reach 'dry weight']. / Metodiche a confronto per il raggiungimento del peso secco.

BACKGROUND: In clinical practice it is very difficult to determine a final weight that is the expression of normovolemia. In hemodialysis (HD), 'dry weight' is conventionally defined as the weight reached by the patient at the end of that hemodialytic session when the maximum quantity of fluids is removed without inducing any symptomatology. The determination of dry weight has been based on the application of clinical criteria. The use of artificial kidneys with blood volume (BV) sensors has allowed the determination of dry weight through the interpretation of changes in the intradialytic BV curve. Conventional bioimpedance analysis (BIA), or better, the vectorial BIA (BIVA) is a new method for determining dry weight. This study evaluated the use of the above-mentioned method for the proper governing of dry weight. PATIENTS AND METHODS: Twenty HD patients were observed for 4 weeks. In the 1st week, the clinical symptomatology of every patient was monitored during both HD sessions and interdialytic periods. During the 2nd week, intradialytic changes in the BV of each patient were observed on artificial kidneys. In the 3rd week, a cardiologist monitored patients before and after hemodialytic treatments. In the 4th week, the body composition of each patient was analyzed through bioelectrical bioimpedance. RESULTS: Patients, who had clinically shown symptoms of hyperhydration, to the contrary at BIA were dehydrated. Conversely, patients who had dehydration symptoms presented signs of hyperhydration at BIA. CONCLUSIONS: BIVA is the diagnostic instrument that more accurately demonstrates the hydration state of hemodialytic patients. It contributes in defining dry weight more efficiently.

Tratamiento del cáncer de cérvix localmente avanzado (estadíos IB BULKY-IIIB) con dosis bajas bisemanales de gemcitabina más cisplatino semanal en forma concurrente con radioterapia / Treatment cervix uteri carcinoma (IB-BULKY IIIB) with low close biweekly gemcitabine and weekly cisplatin combined with radiation therapy

El tratamiento estándar para el CCLA consiste en radioterapia más qiomioterapia en forma concurrente. El agregado de gemcitabina al cisplatino es factible sin agregarle toxicidad (Abs.2150 ASCO 2001). Basándonos en esos resultados, diseñamos un estudio evaluando la eficacia y toxicidad de la radioterapia con dosis bajas bisemanales de gemcitabina más cisplatino en el CCLA. Se incluyeron 60 pacientes; la edad media fue de 49 años (r:25-76). Los estadíos en el momento del diagnóstico se distribuyeron de la siguiente manera: 17 (28 por ciento): estadíos IIIB; 40 (66,6 por ciento): IIAB; y 3: IB Bulky (la paciente tenía contraindicada la cirugía). Se administró radioterapia externa a la pelvis total en 23 fracciones, alcanzando 46 Gys. en 5 semanas, con 2 inserciones de braquiterapia al final de la tercera y quinta semana. El total de la dosis administrada al punto A fue de 85 to 90 Gys. La quimioterapia consistió en gemcitabina 20 mg/m²/d 2 veces por semana (comenzando 3 días previo a la radioterapia) y cisplatino 30 mg/m² semanalmente. Toxicidad: 60 pacientes son evaluables para toxicidad y respuesta. Tres pacientes tuvieron que demorar 1 semana la primera braquiterapia debido a toxicidad gastrointestinal. Seis pacientes debieron omitir una aplicación de QT por toxicidad hematológica y gastrointestinal. Hubo 1 muerte durante el tratamiento, no relacionada con el tratamiento. No hubo alopecia ni mucositis. Cinco pacientes tuvieron trombocitopenia G2; 1 paciente: G3. Ocho pacientes experimentaron neutropenia G2, mientras que una sola paciente tuvo G3, y una G4...

Tratamiento del cáncer de cérvix localmente avanzado (estadíos IB BULKY-IIIB) con dosis bajas bisemanales de gemcitabina más cisplatino semanal en forma concurrente con radioterapia / Treatment cervix uteri carcinoma (IB-BULKY IIIB) with low close biweekly gemcitabine and weekly cisplatin combined with radiation therapy

El tratamiento estándar para el CCLA consiste en radioterapia más qiomioterapia en forma concurrente. El agregado de gemcitabina al cisplatino es factible sin agregarle toxicidad (Abs.2150 ASCO 2001). Basándonos en esos resultados, diseñamos un estudio evaluando la eficacia y toxicidad de la radioterapia con dosis bajas bisemanales de gemcitabina más cisplatino en el CCLA. Se incluyeron 60 pacientes; la edad media fue de 49 años (r:25-76). Los estadíos en el momento del diagnóstico se distribuyeron de la siguiente manera: 17 (28 por ciento): estadíos IIIB; 40 (66,6 por ciento): IIAB; y 3: IB Bulky (la paciente tenía contraindicada la cirugía). Se administró radioterapia externa a la pelvis total en 23 fracciones, alcanzando 46 Gys. en 5 semanas, con 2 inserciones de braquiterapia al final de la tercera y quinta semana. El total de la dosis administrada al punto A fue de 85 to 90 Gys. La quimioterapia consistió en gemcitabina 20 mg/m²/d 2 veces por semana (comenzando 3 días previo a la radioterapia) y cisplatino 30 mg/m² semanalmente. Toxicidad: 60 pacientes son evaluables para toxicidad y respuesta. Tres pacientes tuvieron que demorar 1 semana la primera braquiterapia debido a toxicidad gastrointestinal. Seis pacientes debieron omitir una aplicación de QT por toxicidad hematológica y gastrointestinal. Hubo 1 muerte durante el tratamiento, no relacionada con el tratamiento. No hubo alopecia ni mucositis. Cinco pacientes tuvieron trombocitopenia G2; 1 paciente: G3. Ocho pacientes experimentaron neutropenia G2, mientras que una sola paciente tuvo G3, y una G4...(AU)

Liver transplantation: the Italian experience.

BACKGROUND: Liver transplantation is the standard treatment for patients with end-stage liver disease no longer responsive to conventional medical treatment AIMS: To report the long-term experience of liver transplantation in Italy. PATIENTS AND METHODS: Data were obtained retrospectively by means of a multiple-item form collected from 15 Italian liver transplant centres. The filing centre was centralized. RESULTS: A total of 3323 liver transplants were performed on 3026 patients, with a cumulative proportional survival of 72.4%. Three, 5 and 10 years' patient survival rates were 72.3%, 68.8% and 61.3%, respectively. The most common indication for liver transplantation were hepatitis B virus (+/- hepatitis D virus)- and hepatitis C virus-related cirrhosis (59.4%). Excellent survival rates were observed particularly in controversial indications, such as alcoholic cirrhosis, hepatitis B virus-related cirrhosis and hepatocellular carcinoma. Retransplantation was required in 8.9% of the cases. The overall prevalence of acute cellular rejection episodes was 43.5%. In our study population, primary non-function and disease recurrence were the most common causes of graft failure (28.7% and 25.4%, respectively). Infections and/or sepsis were the most common causes of death after transplantation (42%). CONCLUSION: This study confirms that patients with controversial indications to liver transplantation such as alcoholic cirrhosis, HBV-related cirrhosis and hepatocellular carcinoma can achieve excellent survival when properly selected.

Does the choice of treatment influence survival of patients with small hepatocellular carcinoma in compensated cirrhosis?

OBJECTIVE: Untreated patients with small, single hepatocellular carcinoma (HCC) in compensated cirrhosis are characterized by a relatively good prognosis. METHODS: We report the findings generated in a retrospective study on a cohort of 186 consecutive patients with small (< 5 cm) HCC in Child A or B cirrhosis, who were transplanted (four), underwent surgery (15), or were treated with percutaneous ethanol injection (117), lipiodol chemoembolization (44) or best supportive care (six), depending on their clinical features. RESULTS: Overall survival was 26% at 5 years (31% Child A, 20% Child B), with a mean and median survival of 44 and 38 months, respectively. The longest survival was obtained with transplantation and surgery, and the worst with best supportive care. When untreated patients were not considered, no significant differences were observed between the different types of treatment, however, even when patients in the Child A group were considered alone. Almost all the patients who underwent surgery relapsed. No significant difference was observed in relation to the stage of the disease, while alpha-fetoprotein levels were singled out as the only relevant prognostic factor in a multivariate Cox's regression model. Costs per year of life saved were extremely high for transplantation and lowest for ethanol injection, with surgery being less expensive than chemoembolization. CONCLUSIONS: This study confirms that patients with single, small HCC nodules in well compensated cirrhosis should be treated. The choice of type of treatment should be based on the availability of local resources and expertise, and on the patients' preference, after they have been properly informed on the survival, morbidity and mortality related to each treatment option. The relative cost of the procedures should also be considered.

Fast coordination changes in cytochrome c do not necessarily imply folding.

Folding of globular proteins occurs with rates that range from microseconds to minutes; consequently, it has been necessary to develop new strategies to follow the faster processes that exceed stopped-flow capabilities. Rapid photochemical methods have been employed to study the rate of folding of reduced cytochrome c. In this protein, the iron of the covalently bound heme binds a His and a Met, proximal and distal. Unfolding by guanidine or urea weakens the Fe-Met bond, and the reduced unfolded cytochrome c easily binds CO and other heme ligands, which would react slowly or not at all with the native protein. Therefore in the presence of CO, reduced cytochrome c unfolds at lower denaturant concentrations than in the absence of this ligand, and rapid photochemical removal of CO from unfolded cytochrome c, is expected to trigger at least an incomplete refolding. This approach is complicated by the breakage of the proximal His-Fe bond that may occur as a consequence of CO photodissociation in the unfolded cytochrome c because of the so-called base elimination mechanism. Rebinding of CO to the four-coordinate heme yields kinetic intermediates unrelated to folding. Our hypothesis is supported by parallel observations carried out with protoheme and microperoxidase.

Refolding kinetics of cytochrome c(551) reveals a mechanistic difference between urea and guanidine.

The energetic parameters for the folding of small globular proteins can be very different if derived from guanidine hydrochloride (GdnHCl) or urea denaturation experiments. A study of the equilibrium and kinetics of the refolding of wild-type (wt) cytochrome c(551) (cyt c(551)) from Pseudomonas aeruginosa and of two site-directed mutants (E70Q and E70V) shows that the nonionic nature of urea reveals the role of a salt bridge between residues E70 and K10 on the transition state, which is otherwise completely masked in GdnHCl experiments. Mixed denaturant refolding experiments allow us to conclude that the masking effect of GdnHCl is complete at fairly low GdnHCl concentrations ( congruent with 0.1 M). The fact that potassium chloride is unable to reproduce this quenching effect, together with the results obtained on the mutants, suggests a specific binding of the Gdn(+) cation, which involves the E70-K10 ion pair in wt cyt c(551). We propose, therefore, a simple kinetic test to obtain a mechanistic interpretation of nonlinear dependences of DeltaG(w) on GdnHCl concentration on the basis of kinetic refolding experiments in the presence of both denaturants.

Snapshots of protein folding. A study on the multiple transition state pathway of cytochrome c(551) from Pseudomonas aeruginosa.

Cytochrome c(551) (cyt c(551)) from Pseudomonas aeruginosa is a small protein (82 residues) that folds via a three-state pathway with the accumulation in the microsecond time-range of a compact collapsed intermediate. The presence of a single His residue, at position 16, permits the study of the refolding at pH 7.0 in the absence of miscoordination events. Here, we report on folding kinetics in the millisecond time-range as a function of urea under different pH conditions. Analysis of this process (over-and-above proline cis-trans isomerization) at pH 7.0, suggests the existence of a multiple transition state pathway in which we postulate three transition states. Taking advantage of site-directed mutagenesis we propose that the first "unfolded-like" transition state (t(1)) originates from the electrostatic properties of the collapsed state, while the second transition state (t(2)) involves the interaction between the N and C-terminal helices and is stabilized by the salt bridge between Lys10 and Glu70 ( approximately 1 kcal mol(-1)). Our results suggest that, contrary to other cytochromes c, the roll-over effect observed for cyt c(551) at low denaturant concentration can be interpreted in terms of a broad energy barrier without population of any intermediates. The third and more "native-like" transition state (M) can be associated with the breaking/formation of the Fe(3+)-Met61 bond. This strong interaction is stabilized by the hydrogen bond between Trp56 and heme propionate 17 (HP-17) as suggested by the increase in the unfolding rate at high denaturant concentration of the Trp56Phe site-directed mutant.

How should patients with hepatocellular carcinoma be staged? Validation of a new prognostic system.

BACKGROUND: The life expectancy of a patient with hepatocellular carcinoma (HCC) in cirrhosis is hard to predict, making it difficult to decide whether a certain treatment is indicated and what to say to the patient regarding prognosis. A new score recently has been proposed, which includes the parameters involved in the Child-Pugh stage, plus macroscopic tumor morphology, alpha-fetoprotein levels, and the presence or absence of portal thrombosis. The score has been validated in internal control series, but its general applicability has yet to be confirmed. The authors compared the discriminatory ability of the Cancer of the Liver Italian Program (CLIP) score with those of the Okuda and TNM staging systems and the Child-Pugh classification in a group of cirrhotic patients with HCC, diagnosed and followed up by their unit. METHODS: One hundred fifty-four patients with histologically ascertained HCC in cirrhosis were recruited (median age, 62.5 years; male/female ratio, 122/32) and prospectively followed up. Staging was performed at the baseline using the Child-Pugh, Okuda, TNM, and CLIP systems. RESULTS: The CLIP score was able to predict survival better than the Okuda or TNM staging system, as confirmed by the Kaplan-Meier comparison of survival curves and by the Cox regression analysis, with a median survival rate of 31, 27, 13, 8, 2, and 2 months in patients with CLIP Stages 0, I, II, III, IV, and V-VI, respectively. The Child-Pugh classification performed as well as the Okuda. The predictive capacity of CLIP score was confirmed in the subgroup of patients undergoing chemoembolization. Overall, the survival rate in the authors' series was higher than predicted on the basis of previous reports. CONCLUSIONS: The CLIP score, which is based on simple features of the patient and of the tumor, can accurately identify patients with different prognoses, particularly in the early phases of HCC, thus representing a useful tool in the management of the disease and of the affected patient.