Establishment of patient-derived tumor organoids to functionally inform treatment decisions in metastatic colorectal cancer.

BACKGROUND: Metastatic colorectal cancer (mCRC) patients tend to have modest benefits from molecularly driven therapeutics. Patient-derived tumor organoids (PDTOs) represent an unmatched model to elucidate tumor resistance to therapy, due to their high capacity to resemble tumor characteristics. MATERIALS AND METHODS: We used viable tumor tissue from two cohorts of patients with mCRC, naïve or refractory to treatment, respectively, for generating PDTOs. The derived models were subjected to a 6-day drug screening assay (DSA) with a comprehensive pipeline of chemotherapy and targeted drugs against almost all the actionable mCRC molecular drivers. For the second cohort DSA data were matched with those from PDTO genotyping. RESULTS: A total of 40 PDTOs included in the two cohorts were derived from mCRC primary tumors or metastases. The first cohort included 31 PDTOs derived from patients treated in front line. For this cohort, DSA results were matched with patient responses. Moreover, RAS/BRAF mutational status was matched with DSA cetuximab response. Ten out of 12 (83.3%) RAS wild-type PDTOs responded to cetuximab, while all the mutant PDTOs, 8 out of 8 (100%), were resistant. For the second cohort (chemorefractory patients), we used part of tumor tissue for genotyping. Four out of nine DSA/genotyping data resulted applicable in the clinic. Two RAS-mutant mCRC patients have been treated with FOLFOX-bevacizumab and mitomycin-capecitabine in third line, respectively, based on DSA results, obtaining disease control. One patient was treated with nivolumab-second mitochondrial-derived activator of caspases mimetic (phase I trial) due to high tumor mutational burden at genotyping, experiencing stable disease. In one case, the presence of BRCA2 mutation correlated with DSA sensitivity to olaparib; however, the patient could not receive the therapy. CONCLUSIONS: Using CRC as a model, we have designed and validated a clinically applicable methodology to potentially inform clinical decisions with functional data. Undoubtedly, further larger analyses are needed to improve methodology success rates and propose suitable treatment strategies for mCRC patients.

Bi- and three-dimensional fractal analysis of the brown seaweed Gongolaria montagnei and their relationship with gastropod molluscs assemblage.

Habitat complexity is one of the main influences on biodiversity in marine environments, particularly in coastal areas where foundation seaweeds provide substrate for highly diverse communities. We studied the 2D and 3D fractal dimensions of Gongolaria montagnei (Fucales) over the vegetative season and examine their relationship with the abundance, species richness and morpho-functional groups of the gastropod associated. Overall, the 3D fractal analysis method used here better describes seaweeds structural complexity compared to the traditional 2D fractal analysis, as highlighted by the higher relationship with gastropod assemblage associated to the alga in terms of abundance, number of species and morpho-functional groups. We propose this new method as a valuable tool for understanding the relationship between seaweeds and associated fauna, which is critical for gaining a better understanding of the role that algal species play in a specific habitat and the consequences of their loss.

The invasive seaweed Asparagopsis taxiformis erodes the habitat structure and biodiversity of native algal forests in the Mediterranean Sea.

Invasive seaweeds are listed among the most relevant threats to marine ecosystems worldwide. Biodiversity hotspots, such as the Mediterranean Sea, are facing multiple invasions and are expected to be severely affected by the introduction of new non-native seaweeds in the near future. In this study, we evaluated the consequences of the shift from the native Ericaria brachycarpa to the invasive Asparagopsis taxiformis habitat on the shallow rocky shores of Favignana Island (Egadi Islands, MPA, Sicily, Italy). We compared algal biomass and species composition and structure of the associated epifaunal assemblages in homogenous and mixed stands of E. brachycarpa and A. taxiformis. The results showed that the biomass of primary producers is reduced by 90% in the A. taxiformis invaded habitat compared to the E. brachycarpa native habitat. The structure of the epifaunal assemblages displayed significant variations among homogenous and mixed stands. The abundance, species richness and Shannon-Wiener diversity index of the epifaunal assemblages decreased by 89%, 78% and 40%, respectively, from homogenous stands of the native E. brachycarpa to the invasive A. taxiformis. Seaweed biomass was the structural attribute better explaining the variation in epifaunal abundance, species richness and diversity. Overall, our results suggest that the shift from E. brachycarpa to A. taxiformis habitat would drastically erode the biomass of primary producers and the associated biodiversity. We hypothesize that a complete shift from native to invasive seaweeds could ultimately lead to bottom-up effects on rocky shore habitats, with negative consequences for the ecosystem structure, functioning, and the services provided.

The invasive Asparagopsis taxiformis hosts a low diverse and less trophic structured molluscan assemblage compared with the native Ericaria brachycarpa.

Invasive seaweeds threaten biodiversity and socio-economics values of worldwide marine ecosystems. Understanding to what extent invasive seaweeds can modify local biodiversity is one of the main priorities in conservation ecology. We compared the molluscan assemblage of the invasive Asparagopsis taxiformis with that of the native Ericaria brachycarpa and explore if variation in the molluscan assemblage diversity was related to the substrate attributes (biomass, and thallus, canopy, and interstitial volumes) of the algae. Results showed that A. taxiformis harboured lower diversity and trophic structure of the molluscan assemblage compared to E. brachycarpa. Biomass was the variable that better explained the variation of abundance and number of species as well as the multivariate structure of the molluscan assemblage. Overall, our results suggest that a complete habitat shift from native to invasive species can potentially trigger bottom-up effects in rocky shores habitats, reducing the biodiversity and the services provided by the invaded habitat.

Early evolutionary divergence between papillary and anaplastic thyroid cancers.

Background: Papillary thyroid cancer (PTC) is the most common thyroid carcinoma and exhibits an almost uniformly good prognosis, while anaplastic thyroid cancer (ATC) is less frequent and is one of the most aggressive cancers usually resistant to conventional treatment. Current hypothesis posits that ATC derives from PTC through the progressive acquisition of a discrete number of genomic alterations and implies that the mutational landscape of ATC resembles that of PTC. However, the clinical behaviour of ATC and PTC is radically different. We decided to address the disconnection between the clinical behaviour of ATC and PTC and the proposed model of the progressive development of ATC from PTC. Patients and methods: We carried out exome sequencing of DNA from 14 ATC specimens including three cases of concomitant ATC and PTC as well as their corresponding normal DNA from 14 patients. The sequencing results were validated using droplet digital PCR. We carried out immunohistochemistry and immunofluorescence studies of the concomitant ATC and PTC cases. In addition, we integrated our sequencing results with the existing TCGA data. Results: Most of the somatic mutations identified in the ATC component differed from the ones in PTC in the cases of concomitant ATC and PTC. The trunks of the phylogenetic trees representing the somatic mutations were short with long branches. In one case of concomitant PTC and ATC specimens, we observed an infiltration of PTC cells within the ATC component. Moreover, we integrated our results with data obtained from TCGA and observed that the most frequent mutations found in ATC presented high cancer cell fraction values and were significantly different from the PTC ones. Conclusion: ATC diverge from PTC early in tumour development and both tumour types evolve independently. Our work allows the understanding of the relationship between ATC and PTC facilitating the clinical management of these malignancies.

RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer.

Background: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. Patients and methods: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. Results: RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor. Conclusion: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.

Status of vulnerable Cystoseira populations along the Italian infralittoral fringe, and relationships with environmental and anthropogenic variables.

We analyzed the occurrence and status of infralittoral fringe populations of Cystoseira spp. (Fucales) at thirteen rocky sites around the Italian coastline, and explored the relationships with relevant environmental and anthropogenic variables. We found Cystoseira populations at 11 sites: most were scattered and comprised monospecific stands of C. compressa, and only 6 sites also supported sparse specimens of either C. amentacea var. stricta or C. brachycarpa. Coastal human population density, Chlorophyll a seawater concentrations, sea surface temperature, annual range of sea surface temperature and wave fetch explained most of the variation of the status of C. compressa. We hypothesize a generally unhealthy state of the Italian Cystoseira infralittoral fringe populations and identify multiple co-occurring anthropogenic stressors as the likely drivers of these poor conditions. Extensive baseline monitoring is needed to describe how Cystoseira populations are changing, and implement a management framework for the conservation of these valuable but vulnerable habitats.

Lateral unicompartmental knee replacement for the treatment of arthritis progression after medial unicompartmental replacement.

PURPOSE: Lateral progression of arthritis following medial unicompartmental knee arthroplasty (UKA), although infrequent, is still the most common reason for revision surgery. Treatment options normally include conversion to total knee arthroplasty. An alternative strategy for some patients may be addition of a lateral UKA. We report the first results of staged bi-compartmental UKA (Bi-UKA) strategy. METHODS: We retrospectively selected from our UKA database patients who underwent a lateral UKA to treat a symptomatic lateral osteoarthritis progression after a medial UKA. The analysis included a clinical and radiological assessment of each patient. RESULTS: Twenty-five patients for a total of 27 knees of staged Bi-UKA were carried out in a single centre. The mean time interval between primary medial UKA and the subsequent lateral UKA was 8.1 years (SD ± 4.6 years). The mean age at the time of the Bi-UKA was 77.1 years (SD ± 6.5 years). The median hospital stay was 3 (range 2-9 days) days, and the mean follow-up after Bi-UKA was 4 years (SD ± 1.9 years). The functional scores showed a significant improvement as compared to the pre-operative status (paired t test, p = 0.003). There were no radiological evidences of failure. None of the patients needed blood transfusion, and there was no significant complications related to the surgical procedure without further surgeries or revisions at final follow-up. CONCLUSIONS: These results suggest that addition of a lateral UKA for arthritis progression following medial UKA is a good option in appropriately selected patients. LEVEL OF EVIDENCE: Observational study without controls, Level IV.

Arthroscopic treatment of isolated subscapolaris lesions: our experience.

BACKGROUND AND AIM OF THE WORK: Subscapularis tendon lesions, in particular the isolated ones, are often not recognized and undervalued, so in the literature they are described with a variable incidence. Aim of the work is presenting our experience with the short to medium term follow up results of the arthroscopic repair of isolated subscapularis lesions. METHODS: We retrospectively analyzed 311 shoulder arthroscopies performed by a single senior surgeon, from which we have found 10 isolated subscapularis lesions. After the arthroscopic repair of subscapularis tendon the patients have been evaluated with a median follow up of 17.7 months with specific tests for the subscapularis (Napoleon's and lift off tests) and clinical scores (Constant and UCLA scores). RESULTS: We have obtained the tests negativization with an internal rotation level up to D8. The Constant score reached 86.7 with a median improvement of 49.4 points. The UCLA score at the last follow up was 30.8 with a median improvement of 20.1 points. CONCLUSIONS: Isolated subscapularis lesions are uncommon and often they are not correctly diagnosed. Arthroscopy has a decisive role in both the diagnostic and therapeutic side, with good short to medium term results.

Toxicity of cadmium on Sertoli cell functional competence: an in vitro study.

Cadmium (Cd), an ubiquitous environmental metal, mainly used for industrial purposes, may be toxic at level of the reproductive system. Testis tubular-based Sertoli cells (SC), play a major role in constituting the blood-testis barrier and provide a unique microenvironment for the genesis and differentiation of germ cells. Hence SC strictly control sperm qualitative and quantitative parameters. We aimed to assess whether exposure to Cd would adversely affect superior mammal SC viability and function. We isolated and purified SC from pre-pubertal pig testes according to our method and incubated the retrieved cells with three different Cadmium chloride concentrations (5-10-15 microM). Parameters of SC function such as inhibin B and anti-Mullerian hormone (AMH) were depressed by Cd exposure, contrary to what observed in untreated controls. No impairment of the FSH receptor integrity on the SC, as assessed by 17-beta-estradiol production, upon stimulation with FSH, was observed in either 5 microM Cd-treated or untreated controls. Differences, on the contrary, were observed for higher Cd concentrations (10 and 15 mM), in terms of FSH receptor integrity, that was altered, as compared to untreated controls, in terms of lower production of 17-beta-estradiol. In addition, the apoptotic test showed a significant increase of early (ANNEXIN V-/Propidium Iodide+) (AV-/PI+) and late apoptotic cells (AV+/ PI+) in all Cd -treated SC conditions as compared to controls. In conclusion, the Cd -related toxicity on SC, clearly demonstrated by our study, even at low concentrations, is expected to damage spermatogenesis that directly is dependent upon retention of SC viability and function.

Short-term stimulation studies on neonatal pig pancreatic duct-derived cell monolayers.

Short-term stimulation with insulinotropic factors may induce morphologic and functional changes in primary ductal cell cultures as a potential source of stem cells. We sought to assess the capacity of hepatocyte growth factor (HGF) to induce expression and maturation of proteins--PDX-1 and GLUT-2--and the subsequent beta-cell secretory profiles. HGF, which is involved in pancreatic development, may induce islet beta-cell neogenesis. Primary ductal cell monolayers were cultured in Click's + FBS 10% at 37 degrees C until tissue confluence. The medium was enriched with HGF (10 ng/mL for different periods); controls were treated for similar times with normal culture medium. At the end of the study, three-dimensional islet-like cell aggregates were observed in both conditions. In all conditions immunostaining studies showed positivity for the major endocrine-phenotype cell markers: insulin, PDX-1, glucokinase, and GLUT-2. Furthermore, treatment with HGF for short periods induced the expression of a functionally active, phosphorylated isoform of PDX-1. Finally, we observed that under basal conditions the cells initially and progressively released proinsulin throughout 5 days in all settings. Thereafter proinsulin was gradually replaced by insulin in the culture medium, reflecting a maturation progress. This pattern of insulin maturation and release was more evident when the cells were continuously stimulated with HGF for 12 days. The employed stimuli seemed to differentiate the original ductal cell layers toward endocrine cell phenotypes that synthesize and release proinsulin and subsequently insulin. HGF seems to provide a more efficient differentiation.

Optimized parameters for microencapsulation of pancreatic islet cells: an in vitro study clueing on islet graft immunoprotection in type 1 diabetes mellitus.

Alginate (AG)-based microcapsules may provide a selective permeable and biocompatible physical barrier to prevent islet graft (TX)-directed immune destruction. However, extent of the achieved immunoprotection will continue to be variable and unpredictable until the role of the individual mechanisms involved with TX-related inflammatory cell and immune reactivity are clarified. Macrophages (M) are believed to play a pivotal role in controlling the host/TX interaction and its consequences. We then have studied the effects of isolated rat M and their secretory products on allogeneic islets enveloped in variably sized and configured microcapsules, within in vitro mixed islet-M cocultures. In particular, we aimed to determine the sequence of immune or not immune specific cascade of early events that derive from such on interaction. One of the specific aims was to assess whether the membrane's physical intactness and conversely its even minimal rupture, along with the microcapsules' size (i.e., large vs. small) would significantly impact M reactivity and, thereby, the encapsulated islet viability and function. Special care was taken to evaluate extent of the elicited reactivity by meticulously monitoring cytokine, N2 derivative, and other proinflammatory protein curve profiles during the early M activation process. The study has preliminarily shown that, for equally formulated microcapsules, the capsular size and membrane's morphologic thoroughness are key to prevent M reactivity and possibly avoid the intracapsular islet cell damage. While elucidation of pathways involved with the encapsulated islet TX-directed host's responsiveness actually is in progress, it has clearly emerged that microcapsules should comply with well-defined physical properties and formulation specifications in order to obviate the primum movens of the inflammatory reaction process.

Activity and isoenzyme profile of N-acetyl-beta-D-glucosaminidase in urine from workers exposed to cadmium.

The urinary excretion of N-acetyl-beta-D-glucosaminidase (U-NAG) and urinary Cadmium (U-Cd) concentration, a measure of the metal load in the body, were evaluated in 28 workers exposed to Cd, to determine the relation between the two parameters. In urine from 22 exposed workers with U-Cd<2 microg/g creatinine (Cr) there was no significant difference in U-NAG value (0.98+/-0.59 U/gCr) compared to non-exposed (0.73+/-0.48 U/gCr). In the six workers with 2 microg/gCr < or =U-Cd<10 microg/gCr the U-NAG (2.32+/-0.61 U/gCr) was statistically (P<0.05) higher than in other workers. In both the U-Cd intervals examined there were no altered values of beta2-microglobulin from urine of exposed workers compared to non-exposed (<0.30 mg/l). The U-NAG isoenzymes were separated by DEAE-cellulose chromatography from urine of non-exposed subjects and exposed workers. The U-NAG isoenzyme profile in urine of non-exposed subjects showed a high percentage (about 95%) of the A (acid) form, a much lower percentage (about 4.5%) of B (basic) form and a negligible percentage (about 0.5%) of I (intermediate) form. In the urine of 22 exposed workers with U-Cd<2 microg/gCr, the percentages of U-NAG isoenzymes were not different from non-exposed. In the urine of six workers with 2 microg/gCr< or =U-Cd<10 microg/gCr the percentage (8.34+/-0.91) of isoenzyme B (U-NAG-B), a marker of lesional enzymuria, was statistically increased (P<0.05) compared to non-exposed (4.42+/-0.56). These results suggest that adopting a biological limit for U-Cd equal to 10 microg/gCr might not be sufficiently protective. The increase in U-NAG-B content at 2 microg/gCr

Recorrência da infecçäo por H pylori em portadores de úlcera péptica submetidos à erradicaçäo da bactéria / Recurrence of H pilori infection in patients peptic ulcer disease submited to eradication treatment

A maioria dos autores executa controle de erradicaçäo após um mês do término do tratamento, todavia, avolumam relatos de taxas crescentes de recorrência da infecçäo pelo Hp na mewdida em que aumenta o período de avaliaçäo pós-tratamento. Foi avaliada a evoluçäo quanto à infecçäo pelo Hp em adultos portadores de úlcera peptica (UP) que presentaram pesquisa negativa após 3 meses do trataemtno de erradicaçäo. O diagnóstico de UP baseou-se na endoscopia digestiva alta e histologia, no caso de lesäo gástrica. A pesquisa do Hp foi realizada em duas biópsias endoscópicas de antro gástrico, através do teste da urease ou da análise histológica. No periódo de março/93 a setembro/97, 57 dos 92 pacientes que receberam integralmente o tratamento de erradicaçäo retornaram para controle após 3 meses do término do tratamento, sendo que 21 permaneceram infectados pelo Hp. Os 36 pacientes Hp negativo foram avaliados trimestralmente durante 15 meses, para determinaçäo de infecçäo pelo Hp. Dos 36 pacientes avaliados, 11 mantiveram-se Hp negativo, 11 sofreram positivaçäo e 14 pacientes näo completaram o acompanhamento. O indice de recorrência da infecçäo aos 6, 9, 12 e 15 meses do término do tratamento foi de 3,3 por cento, 4,5 por cento, 15,8 por cento e 50 por cento respectivamente. A baixa taxa de erradicaçäo provavalmente implicou a elevada recorrência, sendo imperativo que os tratamentos em nosso meio atinjam melhores níveis de eficácia

Increase in the basal tone of guinea pig thoracic aorta induced by ouabain is inhibited by spironolactone canrenone and potassium canrenoate.

Guinea pig aorta rings repeatedly stimulated with phenylephrine (1 microM) in the presence of Krebs solution containing ouabain (0.8 microM) or low K+ (0.5 mM) concentration produced an increase in basal tone. This effect is due to an increase in intracellular Ca2+ as a consequence of Na(+)-K+ATPase pump inhibition induced by receptorial (ouabain) or ion imbalance (low K+) mechanism. We investigated the effect of spironolactone and its metabolites canrenone and potassium canrenoate on the increase in basal tone of guinea pig aorta rings. Spironolactone, canrenone, and potassium canrenoate, in a concentration-dependent manner (3-30 microM), inhibited the increase in basal tone induced by ouabain, most likely acting as antagonist for ouabain binding site on Na(+)-K+ATPase pump. Indeed, this effect appears to be a feature of these drugs since structurally related drugs, such as aldosterone and hydrocortisone, were ineffective. Conversely, all the drugs tested reduced, to a certain degree, the increase in basal tone produced by low K+ Krebs solution, implying that this could be a non-specific effect. Our results may indicate that spironolactone, canrenone, and potassium canrenoate act in hypertension by interfering with mechanisms in which an ouabain-like factor is involved.

Endogenous nitric oxide modulates behavioural effects elicited by substance P in rat.

Several studies have shown that the undecapeptide, substance P, alters behaviour following central or peripheral administration in the rat. Here we report that L-arginine administration increases substance P-induced locomotion and changes in food intake in rats. NG-Nitro-L-arginine methyl ester, a specific inhibitor of nitric oxide synthase, reduces substance P-induced effects. These results suggest that endogenous nitric oxide plays a role in the modulation of the catecholaminergic effect of substance P on motor behaviour. They also clarify the mechanism underlying food intake induced by substance P.

Biological activities of CNBr fragments of a major protein secreted from the rat seminal vesicle epithelium.

Two fragments of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, were produced in vitro by protein cleavage with CNBr at level of the single methionine residue (Met-70) occurring in its polypeptide chain. After their purification by reversed-phase chromatography, SV-IV/A (1-70 fragment) and SV-IV/B (71-90 fragment) were assayed as transglutaminase substrates, and their anti-inflammatory, anti-thrombotic and immunosuppressive properties were evaluated in comparison with native SV-IV. Both fragments retained the SV-IV ability to act as transglutaminase substrates in vitro; fast atom bombardment mass spectrometry analyses of the reaction products pointed to Gln-9 and Gln-86 as acyl donor sites, and to Lys-59, -79 and -80 as acyl acceptor sites. In contrast, only SV-IV/A was shown to possess, like SV-IV, the property of inhibiting both the intensity of the carrageenin-induced rat foot edema and the platelet aggregation induced in vivo by different agents. Finally, the two protein fragments were found to be completely unable to inhibit both the mitogen-induced proliferation of human T cells and the mixed lymphocyte reaction.

Endogenous nitric oxide modulates morphine-induced changes in locomotion and food intake in mice.

Opioids increase the dopaminergic turnover in nucleus striatum and nucleus accumbens of mice, causing behavioural changes such as increased locomotion and food intake. We have now shown that L-arginine administration increases morphine-induced locomotion and changes in food intake in mice. D-Arginine had no effect, suggesting a stereospecific mechanism. Furthermore NG-nitro-L-arginine methyl ester, a specific inhibitor of nitric oxide synthase, reduced the morphine-induced effects. These results suggest that endogenous nitric oxide could play a role in the modulation of dopaminergic effects elicited by morphine.