Semen impairment and occurrence of SARS-CoV-2 virus in semen after recovery from COVID-19.

STUDY QUESTION: How is the semen quality of sexually active men following recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection? SUMMARY ANSWER: Twenty-five percent of the men with recent SARS-Cov-2 infections and proven healing were oligo-crypto-azoospermic, despite the absence of virus RNA in semen. WHAT IS KNOWN ALREADY: The presence of SARS-CoV-2 in human semen and its role in virus contagion and semen quality after recovery from coronavirus disease 2019 (COVID-19) is still unclear. So far, studies evaluating semen quality and the occurrence of SARS-CoV-2 in semen of infected or proven recovered men are scarce and included a limited number of participants. STUDY DESIGN, SIZE, DURATION: A prospective cross-sectional study on 43 sexually active men who were known to have recovered from SARS-CoV2 was performed. Four biological fluid samples, namely saliva, pre-ejaculation urine, semen, and post-ejaculation urine, were tested for the SARS-CoV-2 genome. Female partners were retested if any specimen was found to be SARS-CoV-2 positive. Routine semen analysis and quantification of semen leukocytes and interleukin-8 (IL-8) levels were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Questionnaires including International Index of Erectile Function and Male Sexual Health Questionnaire Short Form were administered to all subjects. The occurrence of virus RNA was evaluated in all the biological fluids collected by RT-PCR. Semen parameters were evaluated according to the World Health Organization manual edition V. Semen IL-8 levels were evaluated by a two-step ELISA method. MAIN RESULTS AND THE ROLE OF CHANCE: After recovery from COVID-19, 25% of the men studied were oligo-crypto-azoospermic. Of the 11 men with semen impairment, 8 were azoospermic and 3 were oligospermic. A total of 33 patients (76.7%) showed pathological levels of IL-8 in semen. Oligo-crypto-azoospermia was significantly related to COVID-19 severity (P < 0.001). Three patients (7%) tested positive for at least one sample (one saliva; one pre-ejaculation urine; one semen and one post-ejaculation urine), so the next day new nasopharyngeal swabs were collected. The results from these three patients and their partners were all negative for SARS-CoV-2. LIMITATIONS, REASONS FOR CAUTION: Although crypto-azoospermia was found in a high percentage of men who had recovered from COVID-19, clearly exceeding the percentage found in the general population, the previous semen quality of these men was unknown nor is it known whether a recovery of testicular function was occurring. The low number of enrolled patients may limit the statistical power of study. WIDER IMPLICATIONS OF THE FINDINGS: SARS-CoV-2 can be detected in saliva, urine, and semen in a small percentage of men who recovered from COVID-19. One-quarter of men who recovered from COVID-19 demonstrated oligo-crypto-azoospermia indicating that an assessment of semen quality should be recommended for men of reproductive age who are affected by COVID-19. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Selecting patients for resection after primary chemotherapy for non-metastatic pancreatic adenocarcinoma.

BACKGROUND: Patients with borderline (BL) or locally advanced (LA) pancreatic adenocarcinoma are usually treated with primary chemotherapy (CT), followed by resection when feasible. Scanty data are available about the criteria to candidate patients to resection after CT. PATIENTS AND METHODS: Between 2002 and 2016 overall 223 patients diagnosed with BL or LA pancreatic adenocarcinoma were primarily treated with Gemcitabine combination (4-drugs or nab-paclitaxel-gemcitabine) for 3-6 months followed by surgery and/or chemoradiation. Resection was carried out when radical resection could be predicted by imaging studies and intraoperative findings. The prognostic value of both pre-treatment factors and treatment response was retrospectively evaluated, searching for criteria that could improve the selection of patients for surgery. RESULTS: Median survival (MS) for the whole population was 18.3 months. Surgical resection was carried out in 61 patients; MS in resected patients was significantly longer (30.0 months) as compared with 162 non-resected patients (16.5 months) (P < 0.00001). According to response criteria, 48% had a radiological partial response, 47% a stable disease and 5% a disease progression); CA19.9 response (reduction >50%) was obtained in 77.8% of patients. Among resected patients, neither pre-treatment factors, including BL/LA distinction, nor radiological response, were able to prognosticate survival differences. Survival of resected patients having no CA19.9 response was significantly lower as compared with responders (MS 15.0 versus 31.5 months, P = 0.04), and was similar to non-responders patients that did not undergo resection (MS 10.9 months, P= 0.25). Multivariate analysis carried out on the overall population, showed that Karnofsky performance status, T3-T4 status, resection and CA19.9 response were independent prognostic factors, while radiological response, BL/LA distinction and baseline CA19.9 had not significant influence on survival. CONCLUSIONS: CA19.9 response may allow a better selection of patients who will benefit from resection after primary CT for BL or LA pancreatic adenocarcinoma.

Choline kinase-alpha by regulating cell aggressiveness and drug sensitivity is a potential druggable target for ovarian cancer.

BACKGROUND: Aberrant choline metabolism has been proposed as a novel cancer hallmark. We recently showed that epithelial ovarian cancer (EOC) possesses an altered MRS-choline profile, characterised by increased phosphocholine (PCho) content to which mainly contribute over-expression and activation of choline kinase-alpha (ChoK-alpha). METHODS: To assess its biological relevance, ChoK-alpha expression was downmodulated by transient RNA interference in EOC in vitro models. Gene expression profiling by microarray analysis and functional analysis was performed to identify the pathway/functions perturbed in ChoK-alpha-silenced cells, then validated by in vitro experiments. RESULTS: In silenced cells, compared with control, we observed: (I) a significant reduction of both CHKA transcript and ChoK-alpha protein expression; (II) a dramatic, proportional drop in PCho content ranging from 60 to 71%, as revealed by (1)H-magnetic spectroscopy analysis; (III) a 35-36% of cell growth inhibition, with no evidences of apoptosis or modification of the main cellular survival signalling pathways; (IV) 476 differentially expressed genes, including genes related to lipid metabolism. Ingenuity pathway analysis identified cellular functions related to cell death and cellular proliferation and movement as the most perturbed. Accordingly, CHKA-silenced cells displayed a significant delay in wound repair, a reduced migration and invasion capability were also observed. Furthermore, although CHKA silencing did not directly induce cell death, a significant increase of sensitivity to platinum, paclitaxel and doxorubicin was observed even in a drug-resistant context. CONCLUSION: We showed for the first time in EOC that CHKA downregulation significantly decreased the aggressive EOC cell behaviour also affecting cells' sensitivity to drug treatment. These observations open the way to further analysis for ChoK-alpha validation as a new EOC therapeutic target to be used alone or in combination with conventional drugs.

Afatinib demonstrates remarkable activity against HER2-amplified uterine serous endometrial cancer in vitro and in vivo.

BACKGROUND: Uterine serous carcinomas (USCs) are an aggressive form of uterine cancer that may rely on HER2/neu amplification as a driver of proliferation. The objective of this paper is to assess the sensitivity of USC cell lines with and without HER2/neu gene amplification to afatinib, an irreversible ErbB tyrosine kinase inhibitor, and to test the efficacy of afatinib in the treatment of HER2-amplified USC xenografts. METHODS: Eight of fifteen primary USC cell lines (four with HER2 amplification and four without) demonstrating similar in vitro growth rates were treated with scalar concentrations of afatinib. Effects on cell growth, signalling and cell cycle distribution were determined by flow cytometry assays. Mice harbouring xenografts of HER2/neu-amplified USC were treated with afatinib by gavage to determine the effect on tumour growth and overall survival. RESULTS: Primary chemotherapy-resistant USC cell lines harbouring HER2/neu gene amplification were exquisitely sensitive to afatinib exposure (mean ± s.e.m. IC50=0.0056 ± 0.0006 µM) and significantly more sensitive than HER2/neu-non-amplified USC cell lines (mean ± s.e.m. IC50=0.563 ± 0.092 µM, P<0.0001). Afatinib exposure resulted in abrogation of cell survival, inhibition of HER2/neu autophosphorylation and S6 transcription factor phosphorylation in HER2/neu overexpressing USC and inhibited the growth of HER2-amplified tumour xenografts improving overall survival (P=0.0017). CONCLUSIONS: Afatinib may be highly effective against HER2/neu-amplified chemotherapy-resistant USC. The investigation of afatinib in patients harbouring HER2/neu-amplified USC is warranted.

Vascular involvement in periampullary tumors: MDCT, EUS, and CDU.

In patients affected by periampullary tumors, surgical resection represents the only treatment with curative intent. Preoperative evaluation of vascular involvement is necessary to avoid surgical treatments unable of curative intent resection. The aim of our update article is to assess the performance of multidetector computed tomography (MDCT), endoscopic ultrasonography (EUS), and color Doppler ultrasonography (CDU) in the evaluation of vascular involvement of major peripancreatic vessels, in periampullary tumors, analyzing the current and past literature.

First Report of Crown and Root Rot Caused by Rhizoctonia solani AG-4 on Orange Jessamine in Italy.

Murraya paniculata (L.) Jack, commonly called orange jessamine or orange jasmine (Rutaceae), is a small tropical tree that is native to Asia. This species, closely related to Citrus, is grown as an ornamental tree or hedge. During October of 2007, crown and root rot was observed on approximately 12,000 pot-grown, 4-month-old plants in a nursery in eastern Sicily, Italy. Basal leaves turned yellow and gradually became necrotic, and infected plants often died. Disease symptoms were observed on 1,800 (15%) plants. Isolations from affected tissues on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 mg/liter recovered a fungus with mycelial and morphological characteristics consistent with Rhizoctonia solani Kühn. Fungal colonies were initially white, turned brown with age, and produced irregularly shaped, brown sclerotia. Microscopic examination revealed that hyphae had a right-angle branching pattern, were constricted at the base of the branch near the union with main hyphae, and were septate near the constriction. The nuclear condition of hyphal cells was determined on cultures grown at 25°C on 2% water agar (WA) when stained with 3% safranin O solution and examined at ×400. Anastomosis groups were determined by pairing isolates on 2% WA in petri plates (4). Pairings were made with tester strains AG-1 IA, AG-2-2-1, AG-2-2IIIB, AG-2-2IV, AG-3, AG-4, AG-5, AG-6, and AG-11. Anastomosis was observed only with tester isolates of AG-4 producing both C2 and C3 reactions. The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and cell death of adjacent cells was observed. These results were consistent with other reports on anastomosis reactions (1). The identification of group AG-4 within R. solani has been confirmed by electrophoretic patterns of pectic enzymes (polygalacturonases) in vertical pectin-acrylamide gel stained with ruthenium red (2). Pathogenicity tests were conducted on potted, healthy, 6-month-old seedlings of orange jessamine. Twenty-five plants were inoculated by placing 1-cm2 PDA plugs from 5-day-old mycelial cultures near the base of the stem. The same number of plants inoculated with PDA plugs served as controls. Plants were maintained at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Wilt symptoms, identical to ones observed in the nursery, developed 3 months after inoculation because of crown and root rot. Control plants remained disease free. The pathogen was reisolated from symptomatic tissues, completing Koch's postulates. Collar rot due to R. solani was previously detected on M. koenigii (3). To our knowledge, this is the first report of R. solani causing disease on M. paniculata. References: (1) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reactions. Kluwer Academic Publishers, the Netherlands, 1996. (2) R. H. Cruickshank and G. C. Wade. Anal. Biochem. 107:177, 1980. (3) A. C. Jain and K. A. Mahmud. Rev. Appl. Mycol. 32:460, 1953. (4) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

First Report of Crown Rot, Stem Rot, and Root Rot Caused by Binucleate Rhizoctonia AG-G on Viburnum tinus in Italy.

Laurustinus (Viburnum tinus L.), native to the Mediterranean Region, is an evergreen shrub belonging to the Caprifoliaceae that is commonly cultured as an ornamental shrub or small tree. During the summer and autumn of 2007 and 2008, a widespread yellowing, partial foliar necrosis, or death of the whole plant was observed on 3- to 4-year-old potted plants of V. tinus in a commercial nursery in eastern Sicily (Italy). More than 20% of the plants showed disease symptoms. Infected roots, crowns, and stems turned dark brown, leaves gradually became necrotic, and infected plants were often killed. Diseased tissues were disinfested for 1 min in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 mg/liter, and then incubated at 25°C. A binucleate Rhizoctonia (BNR) species was consistently isolated from affected tissue of Laurustinus. Fungal colonies were initially white, then turned light brown or brown with age, and formed irregularly shaped, light brown sclerotia after 10 days. Hyphal cells were determined to be binucleate when stained with 1% safranin O and 3% KOH solution (1) and examined with a microscope at ×400. Anastomosis groups were determined by pairing isolates on 2% water agar in petri plates (4). Pairings were made with tester strains of binucleate Rhizoctonia AG-A through AG-S, except AG-J and AG-M. Anastomosis was observed only with tester isolates of AG-G. The rDNA-ITS of two isolates of BNR (DISTEF-Vt 31 and DISTEF-Vt 32) was sequenced (GenBank Accession Nos. AB478783 and AB478784, respectively) (3). The sequence from these two isolates exhibited 100% homology with BNR AG-G (GenBank Accession No. AY927334). Pathogenicity tests were conducted on potted, healthy, 6-month-old laurustinus. Twenty plants were inoculated by placing 1-cm2 plugs of PDA from 5-day-old mycelial cultures near the base of the stem. The same number of plants was treated with 1-cm2 PDA plugs as control. Plants were kept at 25°C and 95% relative humidity with a 12-h fluorescent light/dark regimen. Stem, crown, and root rot symptoms, identical to ones observed in nursery, appeared 20 days after inoculation, and all the inoculated plants showed symptoms within 1 month. Control plants remained healthy. Binucleate Rhizoctonia was reisolated from symptomatic tissues, completing Koch's postulates. R. solani was previously reported on Viburnum sp. in the United States (2). To our knowledge, this is the first report of binucleate Rhizoctonia causing disease on V. tinus. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) D. F. Farr et al. Page 1252 in: Fungi on Plants and Plant Products in the United States. The American Phytopathological Society. St. Paul, MN, 1989. (3) M. Hyakumachi et al. Phytopathology 95:784, 2005. (4) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

First Report of Damping-Off on African Daisy Caused by Rhizoctonia solani AG-4 in Italy.

Osteospermum (African Daisy or Cape Daisy) is a genus belonging to the Calendulae with a large number of perennial plant species. In February of 2007, a severe damping-off occurred on 3- to 4-month-old potted cuttings of Osteospermum 'Impassion Rose Purple', 'Impassion White', 'Impassion Purple', and 'Impassion White Rose' cultivated in a nursery in eastern Sicily. More than 30% of the plants were infected. Disease symptoms consisted of extensive water-soaked lesions at the base of the stem followed by wilt and collapse of the plant. Isolations from diseased tissues on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 mg/l consistently recovered a fungus with morphological characteristics of Rhizoctonia solani. Fungal colonies were initially white, turned brown after 2 to 3 days, and produced irregularly shaped, brown sclerotia after 1 week. Microscopic examination revealed that hyphae had right angle branching patterns, were constricted at the base of the branch near the union with main hyphae, and septate near the constriction. The number of nuclei per hyphal cell was determined on cultures grown at 25°C on 2% water agar in petri plates. Mycelium was stained with 0.5% aniline blue solution (4) and examined with a microscope at ×400. The hyphal cells were all multinucleate. Anastomosis groups were determined by pairing isolates (3) with tester isolates of AG-1 IA, AG-2-2-1, AG-2-2IIIB, AG-2-2IV, AG-3, AG-4, AG-5, AG-6, and AG-11. Anastomosis was observed only with tester isolates of AG-4, giving C2 reactions (1) at a high frequency. The identification of group AG-4 within R. solani had been obtained by electrophoretic patterns of pectic enzymes (polygalacturonases) in vertical pectin-acrylamide gel stained with ruthenium red (2). All isolates of R. solani collected from infected plants were paired in all combinations on PDA plus 1% activated charcoal and examined for somatic interaction. All paired colonies merged without producing visible tufts of aerial mycelium. Absence of tufts and the lack of formation of heterokaryon at the hyphal interaction zone indicated that all isolates belonged to the same mating type with the same mating alleles (3). Pathogenicity tests were performed by placing plugs of PDA from 5-day-old mycelial cultures in the soil near the base of the stem on 20 potted, healthy, 2-month-old cuttings of Osteospermum cv. Impassion Rose Purple. The same number of plants treated with 1/cm2 PDA plugs served as controls. Following inoculation, all plants were maintained in a growth chamber at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Wilt symptoms and lesions at the base of stem identical to those observed in the nursery developed 7 days after inoculation, and all inoculated plants died within 20 days. Control plants remained symptomless. R. solani AG-4 was consistently reisolated from symptomatic tissues, completing Koch's postulates. To our knowledge, this is the first report of damping-off on the genus Osteospermum caused by R. solani. References: (1) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reactions. Kluwer Academic Publishers, the Netherlands, 1996. (2) R. H. Cruickshank and G. C. Wade. Anal. Biochem. 107:177, 1980. (3) M. C. Juliàn et al. Phytopathology 86:566, 1996. (4) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

First Report of Damping-Off Caused by Rhizoctonia solani AG-4 on Lagunaria patersonii in Italy.

Lagunaria patersonii (Adr.) G. Don (cow itch tree) is native to Australia and tolerates salted winds. During July 2007, damping-off of cow itch tree was observed on 4-month-old seedlings growing in a commercial nursery in eastern Sicily, Italy. More than 20% of the seedlings showed disease symptoms. First symptoms consisting of water-soaked lesions at the seedling base that expand rapidly girdle the stem and collapse the seedling in a few days. Diseased tissues were disinfested for 1 min in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with streptomycin sulphate at 100 mg/l, and then incubated at 25°C. A fungus with mycelial and morphological characteristics of Rhizoctonia solani Kühn was consistently yielded. Fungal colonies were initially white, turned brown with age, and produced irregularly shaped, brown sclerotia. Microscopic examination revealed that hyphae had a right-angle branching pattern, were constricted at the base of the branch near the union with main hyphae, and septate near the constriction. Basidia were not observed in the greenhouses or on the plates. Hyphal cells were determined to be multinucleate when stained with 0.5% aniline blue solution and examined at ×400 magnification with a microscope. Anastomosis groups were determined by pairing isolates on 2% water agar in petri plates (3). Pairings were made with tester strains of AG-1 IA, AG-2-2-1, AG-2-2IIIB, AG-2-2IV, AG-3, AG-4, AG-5, AG-6, AG-11. Anastomosis was observed only with tester isolates of AG-4 producing both C2 and C3 reactions. The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and cell death of adjacent cells was observed. These results were consistent with other reports on anastomosis reactions (1). The identification of group AG-4 within R. solani has been confirmed by electrophoretic patterns of pectic enzymes (polygalacturonases) in vertical pectin-acrylamide gel stained with ruthenium red (2). Pathogenicity tests were conducted on potted, healthy, 3-month-old seedlings of cow itch tree. Twenty plants were inoculated by placing plugs of PDA from 5-day-old mycelial cultures near the base of the stem. The same number of plants was treated with 1 cm2 PDA plugs as control. Plants were kept at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Wilt symptoms due to basal stem rot, identical to ones observed in the nursery, appeared 10 days after inoculation and all inoculated plants showed symptoms within 1 month. Control plants remained healthy. The pathogen was reisolated from symptomatic tissues, completing Koch's postulates. To our knowledge, this is the first report in the world of R. solani causing disease on L. patersonii. References: (1) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reactions. Kluwer Academic Publishers, the Netherlands, 1996. (2) R. H. Cruickshank and G. C. Wade. Anal. Biochem. 107:177, 1980. (3) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

Dose-intense PEFG (cisplatin, epirubicin, 5-fluorouracil, gemcitabine) in advanced pancreatic adenocarcinoma: a dose-finding study.

The aim of this study was to assess the maximum tolerated dose (MTD) of an intensified PEFG regimen administered every 14 days to patients with Stage III or metastatic pancreatic adenocarcinoma. Twenty-nine patients received fixed doses of both epirubicin (30 mg/m2) and 5-fluorouracil (200 mg/m2/day on Days 1-14) and of escalating doses of cisplatin and gemcitabine. The MTD was cisplatin 30 mg/m2 and gemcitabine 800 mg/m2. With respect to classical PEFG, intensified regimen potentially improved the dose-intensity of both cisplatin and epirubicin by 50 percent and of gemcitabine by 33 percent, reduced Grade 3-4 haematological toxicity and the number of outpatient accesses.

Dose-intense PEFG (cisplatin, epirubicin, 5-fluorouracil, gemcitabine) in advanced pancreatic adenocarcinoma.

BACKGROUND: PEFG regimen (cisplatin and epirubicin 40 mg/m2 day 1, gemcitabine 600 mg/m2 days 1 and 8, 5-fluorouracil (FU) 200 mg/m2/day continuous infusion) significantly improved the outcome of patients with advanced pancreatic adenocarcinoma (PA) with respect to standard gemcitabine in a previous phase III trial. This regimen was subsequently modified in a dose-finding study by increasing dose intensity of cisplatin and epirubicin (both at 30 mg/m2 every 14 days) and of gemcitabine (at 800 mg/m2 every 14 days). Results of a consecutive series treated by dose-intense PEFG regimen are herewith reported. MATERIAL AND METHODS: Dose-intense PEFG was administered to chemotherapy-naive patients with stages III-IV PA, < 75 years, performance status (PS) > 50, till progressive disease or for a maximum of 6 months. RESULTS: Between January 2004 and June 2005, 49 (31 or 63% metastatic) patients, median age 62 years, median PS 80, were treated with dose-intense PEFG. Partial response and stable disease was observed in 24 (49%) and 16 (33%) patients, respectively; 31 patients were progression-free at 6 months (PFS-6 = 63%). Median survival was 10.5 months and 1-year overall survival (OS) was 48% (95% confidence interval: 33-61%). Main grade 3-4 toxicity was: neutropenia in 26% of patients, stomatitis and fatigue in 8%, anaemia, diarrhoea, nausea/vomit in 6%, febrile neutropenia and thrombocytopaenia in 4%, hand-foot syndrome in 2%. CONCLUSION: When compared with 84 patients treated by classical PEFG at the same institution, dose-intense PEFG was not inferior in terms of PFS-6 (63 versus 57%), 1-year OS (48 versus 42%) and response rate (49 versus 49%); it allowed to increase dose intensity for gemcitabine by 32%, for cisplatin and epirubicin by 36% (FU reduced by 3%), to significantly reduce grade 3-4 hematological toxicity (neutropenia: 26 versus 86%; P < 0.00001; thrombocytopaenia: 4 versus 58%; P < 0.00001) and to reduce by one-third the number of outpatient accesses. The new PEFG schedule appears more suitable for clinical use and should be preferred as a basis for further development of therapeutic strategies against pancreatic cancer.

Co-Culture of Plant Beneficial Microbes as Source of Bioactive Metabolites.

In microbial cultures the production of secondary metabolites is affected by experimental conditions, and the discovery of novel compounds is often prevented by the re-isolation of known metabolites. To limit this, it is possible to cultivate microorganisms by simulating naturally occurring interactions, where microbes co-exist in complex communities. In this work, co-culturing experiments of the biocontrol agent Trichoderma harzianum M10 and the endophyte Talaromyces pinophilus F36CF have been performed to elicit the expression of genes which are not transcribed in standard laboratory assays. Metabolomic analysis revealed that the co-culture induced the accumulation of siderophores for both fungi, while production of M10 harzianic and iso-harzianic acids was not affected by F36CF. Conversely, metabolites of the latter strain, 3-O-methylfunicone and herquline B, were less abundant when M10 was present. A novel compound, hereby named harziaphilic acid, was isolated from fungal co-cultures, and fully characterized. Moreover, harzianic and harziaphilic acids did not affect viability of colorectal cancer and healthy colonic epithelial cells, but selectively reduced cancer cell proliferation. Our results demonstrated that the co-cultivation of plant beneficial fungi may represent an effective strategy to modulate the production of bioactive metabolites and possibly identify novel compounds.

Secondary metabolites from the endophytic fungus Talaromyces pinophilus.

Endophytic fungi have a great influence on plant health and growth, and are an important source of bioactive natural compounds. Organic extracts obtained from the culture filtrate of an endophytic strain of Talaromyces pinophilus isolated from strawberry tree (Arbutus unedo) were studied. Metabolomic analysis revealed the presence of three bioactive metabolites, the siderophore ferrirubin, the platelet-aggregation inhibitor herquline B and the antibiotic 3-O-methylfunicone. The latter was the major metabolite produced by this strain and displayed toxic effects against the pea aphid Acyrthosiphon pisum (Homoptera Aphidiidae). This toxicity represents an additional indication that the widespread endophytic occurrence of T. pinophilus may be related to a possible role in defensive mutualism. Moreover, the toxic activity on aphids could promote further study on 3-O-methylfunicone, or its derivatives, as an alternative to synthetic chemicals in agriculture.

Definitive results of a phase II trial of cisplatin, epirubicin, continuous-infusion fluorouracil, and gemcitabine in stage IV pancreatic adenocarcinoma.

PURPOSE: To evaluate the efficacy and toxicity of a cisplatin, epirubicin, gemcitabine, and fluorouracil (PEF-G) schedule on stage IV pancreatic adenocarcinoma. PATIENTS AND METHODS: Patients < or = 70 years, with no prior chemotherapy and with bidimensionally measurable stage IV pancreatic adenocarcinoma, Eastern Cooperative Oncology Group performance status < or = 2, and adequate bone marrow, kidney, and liver function were eligible for this trial. Eligibility criteria for clinical benefit assessment were pain with at least a daily analgesic consumption of two nonsteroidal anti-inflammatory drugs or Karnofsky performance status between 50 and 70. Treatment consisted of 40 mg/m2 each of cisplatin and epirubicin day 1, gemcitabine 600 mg/m2 on days 1 and 8 every 4 weeks, and fluorouracil 200 mg/m2/d as a protracted venous infusion. RESULTS: Between April 1997 and April 1999, 49 patients from a single institution were eligible for the study. Altogether, 203 cycles (median, four cycles) of PEF-G were delivered. The objective response rate was 58% in 43 assessable patients and 51% in the intent-to-treat population. Fourteen patients had stable disease. Grade 3 or 4 World Health Organization neutropenia occurred in 51% of cycles, thrombocytopenia in 28%, anemia in 7%, stomatitis in 5%, and diarrhea, and nausea, and vomiting in 2%. The median duration of response was 8.5 months. The median time to tumor progression was 7.5 months. The median survival was 11 months in the assessable population and 10 months in the intent-to-treat population. Clinical benefit was achieved in 22 (78%) of 28 assessable patients. CONCLUSION: PEF-G is a well-tolerated and safe regimen; it obtained a very high rate of durable responses and deserves further evaluation in a phase III trial.

3-O-methylfunicone, a secondary metabolite produced by Penicillium pinophilum, induces growth arrest and apoptosis in HeLa cells.

3-O-Methylfunicone (OMF) is a secondary metabolite produced by the soil fungus Penicillium pinophilum which has cytostatic properties. The aim of this study was to investigate the mechanisms by which such properties are exerted, with special reference to any anti-proliferative and apoptotic potential, on HeLa cells. OMF treatment caused about 44% inhibition of cell growth after 24 h, and modifications in the tubulin fibre organization. In addition, a significant increase in p21 mRNA expression and a decrease in cyclin D1 and Cdk4 mRNA expression resulted at the same time. Apoptosis induction was demonstrated by the annexin V assay, cytofluorimetric analysis of the DNA content of the sub-G1 fraction and DNA laddering. Taken together, our data showed that the compound inhibits proliferation of HeLa cells by several mechanisms, such as disruption of tubulin fibres, cell cycle arrest and apoptosis induction. The capacity of the compound to affect the cell cycle and to modulate apoptosis is indicative of a potential for the development of a new agent for cancer chemotherapy.

Is interhemispheric transfer of visuomotor information asymmetric? Evidence from a meta-analysis.

Using a meta-analytic procedure we have analysed 16 studies employing a simple unimanual reaction time (RT) paradigm and lateralized visual stimuli to provide an estimate of interhemispheric transfer time in normal right-handed subjects. We found a significant overall RT advantage of the left visual field over the right and of the right hand over the left. These asymmetries can be explained by a superiority of the right hemisphere for the detection of simple visual stimuli and by a corresponding superiority of the left hemisphere for the execution of the manual response, respectively. Alternatively, they may be interpreted as related to an asymmetry of interhemispheric transmission of visuomotor information, with transfer from the right hemisphere (side of stimulus entry) to the left (side of response generation) faster than in the reverse direction. Although a direct test of these hypotheses is still lacking, we think that the evidence available is more in keeping with the latter possibility.

Right hemisphere interference during negative affect: a reaction time study.

Two experiments are described in which simple reaction times (RTs) to lateralized visual stimuli were measured in normal subjects during negative affect and during recollection of everyday life scenes. In both experiments the mental activity produced a remarkable increase of RTs with respect to a control situation without interfering activity. During production of the negative affect there was a selective lengthening of RTs mediated by the right hemisphere. The role of the right hemisphere in producing emotions is discussed.

Kinematic analysis of the reach to grasp movement in Parkinson's and Huntington's disease subjects.

This experiment investigates the kinematic characteristics of the reach to grasp movement of Parkinson's and Huntington's disease subjects under two different experimental conditions. In the first condition subjects were required to perform the movement at a normal speed, while in the second condition they were required to perform the movement as fast as possible. Results showed that the kinematic parameterization of movement in Parkinson's disease subjects did not differ from that of age-matched control subjects for both the normal and the fast condition. However, the performances of Huntington's disease subjects appeared to be different when compared to the other two groups. Differences were mainly related to Huntington's disease patients' inability to properly define the temporal features of the movements.

Can expectancy influence hemispheric asymmetries?

We carried out three experiments with the aim of verifying a critical assumption of Kinsbourne's (Acta Psychol., 33 (1970), 193-201; Attention and Performance V, London: Academic press, (1975), pp. 81-96) 'dynamic' attentional hypothesis of hemispheric asymmetries, namely, that asymmetries arise only when subjects know in advance what type of stimulus and/or cognitive mode they are about to be engaged with. We used a paradigm modified from Posner (J. Exp. Psychol., 109 (1980), 160-174) to study the effects of non-spatial 'cognitive' cueing on hemispheric asymmetries using a lexical decision and a visuo-spatial discrimination task (acute vs. obtuse angles). While we did not find significant overall hemispheric asymmetries with the spatial material, we found a consistent advantage of the left hemisphere in the lexical decision task. In Experiment 2 where the cue was presented in central vision and only the stimuli were lateralised and in Experiment 3 where both cue and stimuli were lateralised to the same hemisphere, the left hemisphere advantage did not interact with the effect of cueing. In contrast, in Experiment 4, where only the cue was lateralised and the stimuli were centrally presented, the left hemisphere advantage in the lexical decision task emerged only following invalid cueing. While the results of Experiments 2 and 3 are not in keeping with Kinsbourne's hypothesis, the result of Experiment 4 shows that some pre-exposural mechanisms may indeed affect the emergence of hemispheric asymmetries. A differential susceptibility in 'disengaging' from the processing mode induced by an invalid cue might represent another interesting example of hemispheric difference.

Renography before heart transplantation in patients with cardiomyopathy.

UNLABELLED: In patients with ischemic cardiomyopathy (CM), abnormal renograms may result not only from circulatory failure (which should reverse after transplantation) but also from intrinsic renal disease (which contraindicates heart transplantation). Here, the outcome of heart transplantation was related to preoperative renograms, and the differentiating and prognostic value of renography was analyzed. METHODS: The study population consisted of 50 patients with ischemic CM expecting heart transplantation. Anatomical renal pathology was excluded in all patients. Dynamic renal scintigraphy was performed with 99mTc-mercaptoacetyltriglycine. Background-subtracted renograms were inspected visually and characterized numerically. Mean parenchymal transit time (mPTT), renal tracer content at 15 min (RTC15) and retention index (RI) were determined. The parametric renogram values were related to a normal reference group of 64 patients. The preoperative renograms were matched with the postoperative outcome. RESULTS: Three characteristic types of symmetrical findings in the kidneys were found: no pathological findings, mildly delayed peak and excretion phase and severely delayed peak and excretion phase. Pathological renograms were observed in 36 of 50 (72%) patients. The mean parametric renogram values in ischemic CM were as follows: Group A (normal kidney function), mPTT = 142+/-26.6 sec, RTC15 = 22.3%+/-4.6% and RI = 24.7+/-11.9; Group B (mild dysfunction), mPTT = 210+/-44.0 sec, RTC15 = 42.6%+/-10.3% and RI = 101.4+/-50.5; Group C (severe dysfunction), mPTT = 320+/-94.2 sec, RTC15 = 79.6%+/-15.9% and RI = 347.7+/-194.7; and reference patients (normal kidney function), mPTT = 137+/-31.1 sec, RTC15 = 22.8%+/-3.8% and RI = 24.6+/-7.9. Postoperative serum creatinine levels were <1.5 mg/dl in all Group A patients, between 1.5 and 2.5 mg/dl in 78% of Group B patients and >2.5 mg/dl in 75% of Group C patients. CONCLUSION: Renography revealed abnormal kidney function when structural pathology was excluded. The renographic abnormalities in ischemic CM did not reflect simply the circulatory failure. The numerical grading of renograms allowed patient stratification, suggestive of possible renal insufficiency after cardiac transplantation and immunosuppressive therapy. With further experience, renography may become a useful tool for predicting postoperative outcome in ischemic CM.