Confirmatory validation analysis of the PROFFIT questionnaire to assess financial toxicity in cancer patients.

BACKGROUND: The Patient Reported Outcome for Fighting FInancial Toxicity (PROFFIT) questionnaire was developed to measure financial toxicity (FT) and identify its determinants. The aim of the present study was to confirm its validity in a prospective cohort of patients receiving anticancer treatment. PATIENTS AND METHODS: From March 2021 to July 2022, 221 patients were enrolled at 10 Italian centres. Selected items of the EORTC-QLQ-C30 questionnaire represented the anchors, specifically, question 28 (Q-28) on financial difficulties, and questions 29-30 measuring global health status/quality of life (HR-QOL). The study had 80% power to detect a 0.20 correlation coefficient (r) between anchors and PROFFIT-score (items 1-7, range 0-100, 100 indicating maximum FT) with bilateral alpha 0.05 and 80% power. Confirmatory factor analysis was conducted. FT determinants (items 8-16) were described. RESULTS: Median age of patients was 65 years, 116 (52.5%) were females, 96 (43.4%) had low education level. Confirmatory factor analysis confirmed goodness of fit of the PROFFIT-score. Significant partial correlation of PROFFIT-score was found with Q-28 (r = 0.51) and HR-QOL (r = -0.23). Mean (SD) PROFFIT-score at baseline was 36.5 (24.9); it was statistically significantly higher for patients living in South Italy, those with lower education level, those who were freelancer/unemployed at diagnosis and those who reported significant economic impact from the COVID-19 pandemic. Mean (SD) scores of determinants ranged from 17.6 (27.1) for item 14 (support from medical staff) to 49.0 (36.3) for item 10 (expenses for medicines or supplements). PROFFIT-score significantly increased with worsening response to determinants. CONCLUSIONS: External validation of PROFFIT-score in an independent sample of patients was successful. The instrument is now being used in clinical studies.

Mindfulness-based stress reduction in cancer patients: impact on overall survival, quality of life and risk factor.

Mindfulness-based stress reduction, a complementary and alternative therapy, is able to decrease cancer-related fatigue, and stress and to improve the quality of life in cancer patients. Some studies evaluated if mindfulness-based stress reduction could improve some cardiometabolic and cancer risk factors, including systemic chemokines, growth factors, and pro-inflammatory biomarkers (e.g., C-reactive protein, Interleukin-1). In this narrative review, we highlight the pleiotropic beneficial effects of mindfulness-based stress reduction and its clinical impact on cardiovascular and cancer risk factors among patients with cancer in different stages. Moreover, improvements in the overall quality of life, sleep quality, and immune functions [changes in plasma levels of interleukin-4 (IL-4), interferon-γ (INF-γ), and interleukin-10 (IL-10)] will also be discussed. Albeit few clinical studies available in the literature, evidenced the beneficial effects of mindfulness-based stress reduction on the immune and cardiometabolic profile in cancer patients, providing important insights into the closest collaboration between psycho-oncologists, oncologists, and cardiologists.

The effect of slope on repeated sprint ability in young soccer players.

This study aimed to describe a gradient repeated sprint ability (RSA) test in comparison with a standard level one by investigating performance, metabolic demand and muscular jumping performance as a proxy for running mechanics. Eighteen athletes performed two level RSA tests (40 m × 6) - for reliability evaluation - and one ±5% gradient RSA test, second leg downhill (RSAgrad). Rating of perceived exertion (RPE), blood lactate concentration (BLa) concentration, vertical jump heights were assessed as well. Level test measures resulted highly reliable (Intra-class correlation coefficient (ICC) ≥0.96). RSAgrad worsened only first sprints' performance (-2%) but not overall test performance (~45 s). RSAgrad resulted to be less deteriorating in terms of fatigue index (FI) (-36%), BLa (-23%), RPE (-11%), jumping performance (RSAgrad post-/pre-squat jump, countermovement jump heights (CMJh): -3%, -6%, respectively). RSAgrad could be used to diversify common training protocol without stressing excessively athletes' current metabolic-anaerobic capacity. Such physical conditioning procedures could improve acceleration/braking capability.

The effects of one-week training camp on motor skills in Karate kids.

AIM: The purpose of this study was to evaluate the effects of short-term increased hours of specific high-intensity karate training on motor skills in young karate athletes aged between 8 and 12 years. METHODS: Seventy-three children, who regularly trained three karate sessions per-week were divided in two groups: high-intensity karate group (HG=53) and low-intensity karate group (LG=20). HG trained for 7 days: with two sessions per-day (one hour per-session) including specific karate techniques as well as coordination, balance, and flexibility exercises. LG, however, followed the same number of karate training sessions as before the beginning of the study (i.e., three sessions per-week with one hour per-session). Participants performed a battery of tests, 24 hours pre- and one week post-training: a medicine ball throw (MBT), standing long jump (SLJ), active joint flexibility (JM) and lateral/frontal jumps (JLT). RESULTS: Significant differences between results of pre and post karate training (ANOVA with repeated measures) included: MBT (P<0.05), SLJ (P<0.0001), JM (P<0.0001), JLT (P<0.0001); whilst the interaction training × time was: MBT (P=0.145), SLJ (P<0.0001), JM (P<0.0001), JLT (P< 0.0001). The HG significantly improved their performance on MBT by 3.23% (P<0.05), SLJ by 5.09% (P<0.001), JM by 1.51% (P<0.001), and JLT by 21.36% (P<0.001). For LG group, there were no significant differences between pre and post-testing in all fitness tests. CONCLUSION: Muscular power, flexibility and coordination represent the basics of karate fitness component. In this regard, short term high-intensity karate training represents an effective method for enhancing muscular power and range of motion (i.e. flexibility) in young karate athletes aged between 8 and 12 years. Further studies are needed to support these findings with deeper data.

The acute effect of whole body vibration on repeated shuttle-running in young soccer players.

The aim of this study was to investigate the acute effects of whole-body vibration (WBV) on Repeated Sprint Ability (RSA). Seventeen male soccer players (16.71±0.47 y) performed three RSA tests (Randomized crossover study design). The second RSA test was done with WBV (RSA2) to assess the effect of WBV. The studied variables were: best time (BT), worst time (WT), total time (TT), the fatigue index (FI) of RSA, and post-test blood lactate (BLa). ANOVA with repeated measures showed no differences between RSA1 and RSA3, while there were significant differences in all variables studied. TT= [RSA2 0.93% and 1.68% lower than RSA1 and RSA3 respectively; p<0.05], BLa= [RSA2 16.97% and 14.73% greater than RSA1 and RSA3 respectively; p<0.001], WT= [RSA2 1.90% and 2.93% lower than RSA1 and RSA3 respectively; p<0.01], and FI = [RSA2 30.64% and 40.15% lower than RSA1 and RSA3 respectively; p<0.0001]. When comparing individual sprints, WBV showed a significant effect at the 5th sprint: RSA2 2.29% and 2.95% lower than RSA1 and RSA3 respectively (p<0.005), while at the 6th sprint: RSA2 2.75% and 4.09% lower than RSA1 and RSA3 respectively; p<0.005. In conclusion, when applying WBV during the recovery periods of Repeated Sprint Ability efforts, most of the performance variables improved.

Measurement of pulmonary gas exchange variables and lactic anaerobic capacity during field testing in elite indoor football players.

AIM: The aims of this study were: 1) to examine the gas exchange responses of elite indoor football players to a repeated sprint ability (RSA) test; and 2) to verify whether or not the excess of carbon dioxide production (CO2excess) correlates with blood lactate accumulation during RSA field testing. METHODS: Eleven elite male indoor football players were recruited. A preliminary incremental exercise test on a treadmill was performed to elicit V'O2max. Then, participants underwent an RSA test consisting in a shuttle running through a course with various changes of direction while wearing a portable gas analyzer able to provide values of oxygen uptake, carbon dioxide production, and CO2excess. BLa concentrations during recovery were also measured. RESULTS: The main results were that: 1) during the RSA test subjects did not reached the V'O2max level achieved in the preliminary test; 2) during the RSA test BLa levels were higher compared with the preliminary test; 3) the peak BLa concentration during recovery was significantly correlated with the average CO2excess CONCLUSION: It was concluded that the RSA test did not appear to be useful to elicit V'O2max. Rather, it seemed suitable to recruit subjects' lactic anaerobic capacity. Moreover, CO2excess appeared suitable for qualitatively estimate BLa accumulation during field testing.

Footstep manipulation during uphill running.

The present study investigated the effects of step frequency manipulation during training on slopes (2%) on biomechanical parameters at Iso-Efficiency Speed (without increasing the metabolic demand). 24 male marathon runners were randomly allocated to one of 2 training groups for 3 weeks: step frequency manipulation group (SFM, n=12) and free step frequency group (SFF, n=12). Lower limb kinematic parameters were measured before and after the 3 weeks training. The SFM group increased step length 4.30% (p<0.001), flight time 29.48% (p<0.001) and decreased contact time 14% (p<0.01). These findings coincide with characteristics of better running performances. The SFF group did not elicit such results. The results from the study could help coaches to devise training methods which could improve an athlete's performance through increasing step length. The method provided may aid faster race times for athletes.

Assessment of circulatory adjustments during underwater apnoea in elite divers by means of a portable device.

AIM: Considering that sympathetic activation is induced by exercise, it is reasonable to assume that hemodynamic adjustments to exercise act in opposition to those elicited by the diving response. However, cardiovascular measurements have never been performed during underwater dynamic apnoea (DA), and this hypothesis remains speculative. METHODS: Data concerning heart rate (HR), stroke volume (SV) and cardiac output (CO) during static apnoea (SA) and DA were collected from 12 elite divers by means of an impedance cardiograph adapted to the underwater environment. Mean arterial pressure (MBP), systemic vascular resistance (SVR) and arterial oxygen saturation (SaO(2)) were also assessed. Five trials were performed by the divers: head-out immersion during normal breathing (test A); 3 min of SA immersed at the surface (B) and at 3 m depth (C); DA till exhaustion immersed at the surface (D) and at 3 m depth (E). RESULTS: Both B and C conditions led to bradycardia (-17%) compared to A and also induced a decrement in SV (-8%) and in CO (-25%), while MBP was maintained because of an increase in SVR. A significant MBP increment (+11%) was detected only during tests D and E, when a SaO(2) drop was also present, whereas HR, SV and CO remained unchanged. CONCLUSION: We concluded that typical diving response was present only during SA, while sympathetic activation was induced by exercise during DA, which partially obscured the effects of the diving response.

Uphill running at iso-efficiency speed.

The purpose of this study was to investigate the effects of slopes (0%, 2% and 7%) on temporal gait kinematics during running at iso-efficiency speed (IES). 65 male marathon runners were selected for this study. A single digital camera (210 Hz) was used to record motion; Dartfish5.5Pro was used to perform 2-dimensional (2D) video analysis and heart rate was recorded during the test. The parameters considered in this study were: step length (SL), flight time (FT), step frequency (SF), contact time (CT) and heart rate (HR). The results showed SL, FT and SF decreased as a result of the increasing treadmill gradient; SL=[(0-2%=8.38%, p<0.0001), (0-7%=23.61%, p<0.0001)]; FT=[(0-2%=8.92%, p<0.02), (0-7%=23.40%, p<0.0001)]; SF=[(0-2%=1.18%), (0-7%=4.02%, p<0.001)]. The CT and HR however increased with the increasing gradient CT=[(0-2%=9.06% p<0.0001), (0-7%=25.64%, p<0.0001)]; HR=[(0-2%=1.65%), (0-7%=3.58%)]. These results show a different trend of the footstep's kinematic parameters when running on a slope at IES. Moreover, we can calculate the optimal run speed on a slope without increasing the metabolic demand.

Cell-based treatments: advanced therapies and transplants.

The introduction of Regulation 1394/2007/EC managing the use of tissue engineering products for the purpose of repairing, regenerating, or replacing missing tissue and cells has created a gray area where transplant and medicinal products overlap. The classification of such borderline products depends on the European Medicines Agency at the moment of marketing application. However, when these products first enter into the clinical development, the burden of definition falls on National competent authorities. The concept of "minimal manipulation" and of "heterologous use" are introduced to describe currently used criteria.

Development of humanized mice for the study of hepatitis C virus infection.

The development of a small animal model for hepatitis C virus (HCV) infection is a critical issue for the development of novel anti-HCV drugs. To this aim, we have tried many different approaches for generating mice carrying humanized liver. Main efforts were focused on the transplantation of human hepatocytes into immunocompromised mice (SCID-/-, Bg-/-) carrying a genetic lethal liver disease (Alb-uPA). Survival of homozygotic animals should largely depend on early transplantation with healthy hepatocytes. In parallel to establishing a colony of Alb-uPA/SCID/Bg mice, we developed a microsurgical procedure for intrasplenic xenotransplantation of healthy hepatocytes in 1-week-old mice. So far, we generated several chimeras by xenotransplanting human hepatocytes in Alb-uPA+/+/SCID-/-/Bg-/- mice at 1 week after birth. In a first step, identification of successfully engrafted animals is possible by quantification of human serum albumin and human alpha 1 antitrypsin in mouse sera. Additional preliminary histomorphological analysis of liver sections from chimeric animals was also carried out. One of the mice was transiently infected with HCV, reaching viremia levels of approximately 10(5) genomes/mL. However, the efficiency of this system to generate chimeric mice is still very limited. We are currently exploring the use of more robust models of hepatic disease. Moreover, we have been also exploring novel strategies for the generation of chimeric mice by xenotransplanting human adult stem cells, instead of human hepatocytes, at preimmune stages of development.

Preclinical pharmacokinetics and metabolism of a potent non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

The disposition of compound A, a potent inhibitor of the hepatitis C virus (HCV) NS5B polymerase, was characterized in animals in support of its selection for further development. Compound A exhibited marked species differences in pharmacokinetics. Plasma clearance was 44 ml min-1 kg-1 in rats, 9 ml min-1 kg-1 in dogs and 16 ml min-1 kg-1 in rhesus monkeys. Oral bioavailability was low in rats (10%) but significantly higher in dogs (52%) and monkeys (26%). Compound A was eliminated primarily by metabolism in rats, with biliary excretion accounting for 30% of its clearance. Metabolism was mainly mediated by cyclohexyl hydroxylation, with N-deethylation and acyl glucuronide formation constituting minor metabolic pathways. Qualitatively, the same metabolites were identified using in vitro systems from all species studied, including humans. The low oral bioavailability of compound A in rats was mostly due to poor intestinal absorption. This conclusion was borne out by the findings that hepatic extraction in the rat was only 30%, intraperitoneal bioavailability was good, and compound A was poorly absorbed from the rat isolated intestinal loop, with no detectable intestinal metabolism. Compound A was not an inhibitor of major human cytochrome P450 enzymes, indicating minimal potential for clinical drug-drug interactions. The metabolic clearance of compound A in rat, dog and monkey hepatocytes correlated with the systemic clearance observed in these species. Since compound A was very stable in human hepatocytes, the results suggest that it will be a low clearance drug in humans.

[Experimental study on efficacy of thermal muds of Ischia Island combined with balneotherapy in the treatment of psoriasis vulgaris with plaques]. / Studio sperimentale sull'efficacia dei fanghi termali dell'isola di Ischia associati a balneoterapia nella cura della psoriasi volgare a placche.

A lot of studies show the efficacy of thermal therapy in psoriasis. The main indications concern all forms of psoriasis (except for pustolosa and eritrodermic) especially diffuse psoriasis, intensive itchy, artropatic psoriasis and gutted psoriasis child. Benefit effects of thermal water and muds, which are important parameters in psoriasis' treatment, they are excellent to reduce inflammation, scaling, and erythema. In this study we evaluated the clinical efficacy of thermal bath plus muds in patients affected by psoriasis. 18 patients has been examined and randomly divided in 3 group (A-B-C). All the patients received thermal therapy for 4 weeks. To evaluate the behaviour of the disease we performed (corneometry, sebometry, phmetry), as well as evaluation of P.A.S.I. At the end of the study, values of P.A.S.I. showed a reduction from 10 to 6 unites P.A.S.I. for patients belong to group A, more 12 to less 10 for patients belong to group C e no significant variations for group B (because represent control group). Results obtained can be considered useful, considering that thermal treatment was used alone in the treatment of all patients.

Amplification of T cells from human cord blood in serum-deprived culture stimulated with stem cell factor, interleukin-7 and interleukin-2.

We report the effects exerted by cytokine combinations, including stem cell factor (SCF), interleukin-7, interleukin-4 and interleukin-2, on the amplification of T cells from cord blood (CB) mononuclear cells cultured for 10-11 days under serum-deprived conditions. Of all the combinations investigated, SCF+interleukin-7 sustained the best fold increase (FI) of total nucleated cells (FI=6.4+/-1.17), amplifying preferentially CD4(+) over CD8(+) T-cell subsets (FI=4.72+/-0.79 vs 2.73+/-1.2, respectively, P<0.05). The addition of interleukin-2 to this combination did not significantly increase the total number of cells generated (FI=7.4+/-2.27), but allowed preferential amplification of CD8(+) over CD4(+) T cells (FI=6.04+/-0.14 vs 1.67+/-0.6, respectively, P<0.05). Single-strand conformation polymorphism analysis of the T-cell receptor V(beta)-chain rearrangements expressed by the expanded T cells indicated that the complexity of the T-cell repertoire had increased after 10 days of culture in the presence of SCF and IL-7. Interestingly, a modest expansion (FI=8.67+/-1.5) of myeloid progenitor cells was also observed in these cultures. These results indicate that it is possible to expand specific T-cell subsets for adoptive immunotherapy without losing myeloid progenitor cells necessary for neutrophil recovery after CB transplantation, by modulating the cytokines added to the cultures.

Circulating hematopoietic progenitor cells in a fetus with alpha thalassemia: comparison with the cells circulating in normal and non-thalassemic anemia fetuses and implications for in utero transplantations.

Our aim was to evaluate the number of progenitor cells circulating in an alpha-thalassemic fetus during its infusion in utero with paternal CD34(+) and adult red cells and to compare those values with those circulating in normal and non-thalassemic anemic fetuses of matched gestational age. The treatment of the alpha-thalassemic fetus has been described elsewhere. Fetal blood was obtained from normal and anemic fetuses by fetal blood sampling for diagnostic or therapeutic purposes according to a protocol approved by the human subject committee. The number of progenitor cells in fetal blood was estimated on the basis of the number of colonies they gave rise to in semisolid cultures. The alpha-thalassemic fetus, as did the other fetuses analyzed, contained high numbers (10(6)-10(7) depending on the age) of progenitor cells, values which were higher than the number (10(4)-10(5)) of paternal progenitor cells being transplanted. Progenitor cells with adult characteristics (adult kinetics of differentiation) were detected rapidly (10 min) after the CD34(+) cell infusion, but were not detectable 2-3 weeks after the transplant. These results indicate that adult progenitor cells do not have a numerical advantage when transplanted into alpha-thalassemic fetuses.

Accentuated response to phenylhydrazine and erythropoietin in mice genetically impaired for their GATA-1 expression (GATA-1(low) mice).

The response of mice genetically unable to up-regulate GATA-1 expression (GATA-1(low) mice) to acute (phenylhydrazine [PHZ]-induced anemia) and chronic (in vivo treatment for 5 days with 10 U erythropoietin [EPO] per mouse) erythroid stimuli was investigated. Adult GATA-1(low) mice are profoundly thrombocytopenic (platelet counts [x 10(9)/L] 82.0 +/- 28.0 vs 840 +/- 170.0 of their control littermates, P <.001) but have a normal hematocrit (Hct) (approximately.47 proportion of 1.0 [47%]). The spleens of these mutants are 2.5-fold larger than normal and contain 5-fold more megakaryocytic (4A5(+)), erythroid (TER-119(+)), and bipotent (erythroid/megakaryocytic, TER-119(+)/4A5(+)) precursor cells. Both the marrow and the spleen of these animals contain higher frequencies of burst-forming units-erythroid (BFU-E)- and colony-forming units-erythroid (CFU-E)-derived colonies (2-fold and 6-fold, respectively) than their normal littermates. The GATA-1(low) mice recover 2 days faster from the PHZ-induced anemia than their normal littermates (P <.01). In response to EPO, the Hct of the GATA-1(low) mice raised to.68 proportion of 1.0 (68%) vs the.55 proportion of 1.0 (55%) reached by the controls (P <.01). Both the GATA-1(low) and the normal mice respond to PHZ and EPO with similar (2- to 3-fold) increases in size and cellularity of the spleen (increases are limited mostly to cells, both progenitor and precursor, of the erythroid lineage). However, in spite of the similar relative cellular increases, the increases of all these cell populations are significantly higher, in absolute cell numbers, in the mutant than in the wild-type mice. In conclusion, the GATA-1(low) mutation increases the magnitude of the response to erythroid stimuli as a consequence of the expansion of the erythroid progenitor cells in their spleen.

Establishing an employee baseline purified protein derivative status in the division of immigration health Service Processing Centers.

While the incidence of reportable tuberculosis in United States born persons declined, the number of cases among foreign-born persons increased by 6 percent in 1998. The Immigration and Naturalization Service (INS) processes about 95,000 undocumented aliens annually from countries with a high prevalence of tuberculosis. An effort was made to establish a baseline Purified Protein Derivative (PPD) status of employees of the Division of Immigration Health (DIH) and INS, Services Processing Centers (SPC). This was achieved through a special operation (project) of a one time, two-step, mass PPD testing of all SPC employees on all eleven sites in the United States and Puerto Rico. A reading of > 10mm was considered positive. The operation was optional and open to all SPC employees. Exclusion criteria for the study included a history of PPD skin testing within six months of the operation, past history of positive PPD test and past history of tuberculosis. Preliminary results from El Paso SPC, Texas, which is the largest SPC, showed that of the 148 employees which were tested (67 percent of all employees), 17 (11 percent) were PPD positive. Security officers constituted 100 percent of all positive cases. Eighty-one percent of the employees at El Paso are security officers, eighty-seven percent of whom participated in the study. Only 20 (23 percent) of administrative staff participated in the study. Results from El Paso are suggestive of differences in the pattern PPD positivity among SPC employees. The complete results of the study should provide sound evidence for formulating appropriate policies for establishing an effective employee tuberculosis prevention and surveillance program in the Service Processing Centers.

Standardization of progenitor cell assay for cord blood banking.

Cord blood has proved itself, if correctly stored with rational criteria, an excellent source of stem cells for related and unrelated transplants. It has been recently proven that the factor which predicts the best the speed of engraftment in cord blood transplants in the dose of progenitor cells injected per kg of body weight of the recipient. This result has been obtained thanks to a careful standardization of the neonatal progenitor cell assay. This manuscript describes such a standardization realized as a joined effort by the Istituto Superiore di Sanità, Rome, and the pivotal cord blood bank founded as a feasibility study by the National Institutes of Health, Bethesda at the New York Blood Center.

Erythropoietin-dependent suppression of the expression of the beta subunits of the interleukin-3 receptor during erythroid differentiation.

To clarify how erythroid cells lose their response to interleukin-3 (IL-3), we analyzed the expression of the alpha (alpha(IL-3)) and beta (beta(IL-3)/beta(com)) subunits of its receptor in a panel of murine cell lines immortalized at different stages of hemopoietic differentiation. The panel was composed by the mast cell line 32D and by its granulo-monocytic (32D GM), granulocytic (32D G), and erythroid (32D Epo1.1 and Epo) subclones. The 32D Epo cells grow only in erythropoietin (EPO) while the Epo1.1 subclone grows in either EPO or IL-3. The phenotype of these cells is that of early (expression of globins and erythroid-specific carbonic anhydrase II) and late (also expression of the erythroid-specific band 4.1 mRNA) erythroblasts when they grow in IL-3 or EPO, respectively. All the cell lines expressed comparable levels of alpha(IL-3). In contrast, the expression of beta(IL-3)/beta(com) was restricted to cells growing in IL-3 and was barely detectable in 32D Epo and 32D Epo1.1 cells growing in EPO. When switched from EPO to IL-3, 32D Epo1.1 cells expressed 10 times more beta(IL-3)/beta(com) by rapidly activating (within 1 h) their transcription rate. When reexposed to EPO, 32D Epo1.1 cells first expressed (1-6 h) more beta(IL-3)/beta(com) (2 times) but suppressed such an expression at later time points (by 48 h). The beta(IL-3)/beta(com) mRNA half-life was also different when 32D Epo1.1 cells grew in EPO or IL-3 (2-3 h vs >5 h, respectively). These results indicate that EPO specifically induces transcriptional and posttranscriptional downmodulation of beta(IL-3)/beta(com) expression in late erythroid cells.

In vivo selection of protease cleavage sites by using chimeric Sindbis virus libraries.

Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.