In Vitro Development and Cell Allocation After Aggregation of Syngeneic Wild Type and Fluorescence-Expressing Bovine Cloned Embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the. 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

KRAS gene mutations in Noonan syndrome familial cases cluster in the vicinity of the switch II region of the G-domain: Report of another family with metopic craniosynostosis

Noonan syndrome (NS) and Noonan-related disorders [cardio-facio-cutaneous (CFC), Costello, Noonan syndrome with multiple lentigines (NS-ML), and neurofibromatosis-Noonan syndromes (NFNS)] are a group of developmental disorders caused by mutations in genes of the RAS/MAPK pathway. Mutations in the KRAS gene account for only a small proportion of affected Noonan and CFC syndrome patients that present an intermediate phenotype between these two syndromes, with more frequent and severe intellectual disability in NS and less ectodermal involvement in CFC syndrome, as well as atypical clinical findings such as craniosynostosis. Recently, the first familial case with a novel KRAS mutation was described. We report on a second vertical transmission (a mother and two siblings) with a novel mutation (p.M72L), in which the proband has trigonocephaly and the affected mother and sister, prominent ectodermal involvement. Metopic suture involvement has not been described before, expanding the main different cranial sutures which can be affected in NS and KRAS gene mutations. The gene alteration found in the studied family is in close proximity to the one reported in the other familial case (close to the switch II region of the G-domain), suggesting that this specific region of the gene could have less severe effects on intellectual ability than the other KRAS gene mutations found in NS patients and be less likely to hamper reproductive fitness. (c) 2012 Wiley Periodicals, Inc.

SLCO1B1 haplotypes are not associated with atorvastatin-induced myalgia in Brazilian patients with familial hypercholesterolemia

Purpose Recent studies reported the association of SLCO1B1 haplotypes with the development of musculoskeletal side effects during simvastatin use. The aim was to evaluate the pharmacogenetic association of SLCO1B1 haplotypes with atorvastatin-induced myalgia in a sample of individuals on high-dose atorvastatin regimens. Methods One hundred and forty-three patients with familial hypercholesterolemia were followed for at least 12 months while receiving atorvastatin. Genotypes for the rs2306283 (c.A388G) and rs4149056 (c.T521C) polymorphisms were detected by high-resolution melting analysis. These markers form four distinct haplotypes (*1A, *1B, *5 and *15). Results During the follow-up period, 14 (9.8%) patients developed myalgia and 16 (11.2%) presented CK levels more than 3 times the upper limit of the normal range. No association of the SLCO1B1 rs2306283 and rs4149056 genotypes or haplotypes with the presence of myalgia or creatine kinase (CK) values was found. Presence of rs2306283 AG+GG genotypes was not associated with increased risks of myalgia or abnormal CK values (OR 2.08, 95% CI 0.62-7.00, p=0.24 and OR 0.51, 95% CI 0.21-1.26, p=0.15 respectively). The presence of rs4149056 TC+CC genotypes was also not associated with increased risk of myalgia or abnormal CK values (OR 2.24, 95% CI 0.47-10.72, p=0.31 and OR 1.51, 95% CI 0.57-3.96, p=0.41 respectively). Conclusions Our findings reaffirm that the SLCO1B1 genetic risk appears to be greater in those patients receiving simvastatin compared with those receiving atorvastatin. This suggests that the importance of SLCO1B1 haplotypes depends on the specific statin that has been used.

Signaling Events During Cyclic Guanosine Monophosphate-Regulated Pigment Aggregation in Freshwater Shrimp Chromatophores

Crustacean color change results partly from granule aggregation induced by red pigment concentrating hormone (RPCH). In shrimp chromatophores, both the cyclic GMP (3', 5'-guanosine monophosphate) and Ca2+ cascades mediate pigment aggregation. However, the signaling elements upstream and downstream from cGMP synthesis by GC-S (cytosolic guanylyl cyclase) remain obscure. We investigate post-RPCH binding events in perfused red ovarian chromatophores to disclose the steps modulating cGMP concentration, which regulates granule translocation. The inhibition of calcium/calmodulin complex (Ca2+/CaM) by N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) induces spontaneous aggregation but inhibits RPCH-triggered aggregation, suggesting a role in pigment aggregation and dispersion. Nitric oxide synthase inhibition by N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) strongly diminishes RPCH-induced aggregation; protein kinase G inhibition (by rp-cGMPs-triethylamine) reduces RPCH-triggered aggregation and provokes spontaneous dispersion, disclosing NO/PKG participation in aggregation signaling. Myosin light chain phosphatase inhibition (by cantharidin) accelerates RPCH-triggered aggregation, whereas Rho-associated protein kinase inhibition (by Y-27632, H-11522) reduces RPCH-induced aggregation and accelerates dispersion. MLCP (myosin light chain kinase) and ROCK (Rho-associated protein kinase) may antagonistically regulate myosin light chain (MLC) dephosphorylation/phosphorylation during pigment dispersion/aggregation. We propose the following general hypothesis for the cGMP/Ca2+ cascades that regulate pigment aggregation in crustacean chromatophores: RPCH binding increases Ca2+ (int), activating the Ca2+/CaM complex, releasing NOS-produced nitric oxide, and causing GC-S to synthesize cGMP that activates PKG, which phosphorylates an MLC activation site. Myosin motor activity is initiated by phosphorylation of an MLC regulatory site by ROCK activity and terminated by MLCP-mediated dephosphorylation. Qualitative comparison reveals that this signaling pathway is conserved in vertebrate and invertebrate chromatophores alike.

Ecological implications on the aggregation of Amblyomma fuscum (Acari: Ixodidae) on Thrichomys laurentius (Rodentia: Echimyidae), in northeastern Brazil

We investigated the Amblyomma fuscum load on a pullulating wild rodent population and the environmental and biological factors influencing the tick load on the hosts. One hundred and three individuals of Thrichomys laurentius were caught in an Atlantic forest fragment in northeastern Brazil, as part of a longitudinal survey on ticks infesting non-volant small mammals. Ticks (n = 342) were found on 45 individuals and the overall mean intensity of infestation was 7.6 ticks per infested rodent. Ticks were highly aggregated in the host population and the negative binomial distribution model provides a statistically satisfactory fit. The aggregated distribution was influenced by sex and age of the host. The microhabitat preference by T. laurentius probably increases contact opportunities between hosts and aggregated infesting stages of the ticks and represents important clues about the habitat suitability for A. fuscum.

A familial case with interstitial 2q36 deletion: Variable phenotypic expression in full and mosaic state

Submicroscopic chromosomal anomalies play an important role in the etiology of craniofacial malformations, including midline facial defects with hypertelorism (MFDH). MFDH is a common feature combination in several conditions, of which Frontonasal Dysplasia is the most frequently encountered manifestation; in most cases the etiology remains unknown. We identified a parent to child transmission of a 6.2 Mb interstitial deletion of chromosome region 2q36.1q36.3 by array-CGH and confirmed by FISH and microsatellite analysis. The patient and her mother both presented an MFDH phenotype although the phenotype in the mother was much milder than her daughter. Inspection of haplotype segregation within the family of 2q36.1 region suggests that the deletion arose on a chromosome derived from the maternal grandfather. Evidences based on FISH, microsatellite and array-CGH analysis point to a high frequency mosaicism for presence of a deleted region 2q36 occurring in blood of the mother. The frequency of mosaicism in other tissues could not be determined. We here suggest that the milder phenotype observed in the proband's mother can be explained by the mosaic state of the deletion. This most likely arose by an early embryonic deletion in the maternal embryo resulting in both gonadal and somatic mosaicism of two cell lines, with and without the deleted chromosome. The occurrence of gonadal mosaicism increases the recurrence risk significantly and is often either underestimated or not even taken into account in genetic counseling where new mutation is suspected. (C) 2012 Elsevier Masson SAS. All rights reserved.

Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Early Amyloid-like Oligomers and Their Implication in alpha-Synuclein Aggregation

Lewy bodies and Lewy neurites, neuropathological hallmarks of several neurological diseases, are mainly made of filamentous assemblies of alpha-synuclein. However, other macromolecules including Tau, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, and glycosaminoglycans are routinely found associated with these amyloid deposits. Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that can form fibrillar aggregates in the presence of acidic membranes, but its role in Parkinson disease is still unknown. In this work, the ability of heparin to trigger the amyloid aggregation of this protein at physiological conditions of pH and temperature is demonstrated by infrared and fluorescence spectroscopy, dynamic light scattering, small angle x-ray scattering, circular dichroism, and fluorescence microscopy. Aggregation proceeds through the formation of short rod-like oligomers, which elongates in one dimension. Heparan sulfate was also capable of inducing glyceraldehyde-3-phosphate dehydrogenase aggregation, but chondroitin sulfates A, B, and C together with dextran sulfate had a negligible effect. Aided with molecular docking simulations, a putative binding site on the protein is proposed providing a rational explanation for the structural specificity of heparin and heparan sulfate. Finally, it is demonstrated that in vitro the early oligomers present in the glyceraldehyde-3-phosphate dehydrogenase fibrillation pathway promote alpha-synuclein aggregation. Taking into account the toxicity of alpha-synuclein prefibrillar species, the heparin-induced glyceraldehyde-3-phosphate dehydrogenase early oligomers might come in useful as a novel therapeutic strategy in Parkinson disease and other synucleinopathies.

Longitudinal familial analysis of blood pressure involving parametric (co)variance functions

Abstract Background For analyzing longitudinal familial data we adopted a log-linear form to incorporate heterogeneity in genetic variance components over the time, and additionally a serial correlation term in the genetic effects at different levels of ages. Due to the availability of multiple measures on the same individual, we permitted environmental correlations that may change across time. Results Systolic blood pressure from family members from the first and second cohort was used in the current analysis. Measures of subjects receiving hypertension treatment were set as censored values and they were corrected. An initial check of the variance and covariance functions proposed for analyzing longitudinal familial data, using empirical semi-variogram plots, indicated that the observed trait dispersion pattern follows the assumptions adopted. Conclusion The corrections for censored phenotypes based on ordinary linear models may be an appropriate simple model to correct the data, ensuring that the original variability in the data was retained. In addition, empirical semi-variogram plots are useful for diagnosis of the (co)variance model adopted.

1031-1034delTAAC (Leu125Stop): a novel familial UBE3A mutation causing Angelman syndrome in two siblings showing distinct phenotypes

Background More than 50 mutations in the UBE3A gene (E6-AP ubiquitin protein ligase gene) have been found in Angelman syndrome patients with no deletion, no uniparental disomy, and no imprinting defect. Case Presentation We here describe a novel UBE3A frameshift mutation in two siblings who have inherited it from their asymptomatic mother. Despite carrying the same UBE3A mutation, the proband shows a more severe phenotype whereas his sister shows a milder phenotype presenting the typical AS features. Conclusions We hypothesized that the mutation Leu125Stop causes both severe and milder phenotypes. Potential mechanisms include: i) maybe the proband has an additional problem (genetic or environmental) besides the UBE3A mutation; ii) since the two siblings have different fathers, the UBE3A mutation is interacting with a different genetic variant in the proband that, by itself, does not cause problems but in combination with the UBE3A mutation causes the severe phenotype; iii) this UBE3A mutation alone can cause either typical AS or the severe clinical picture seen in the proband.

Gagueira desenvolvimental persistente familial: perspectivas genéticas

A gagueira é uma desordem da comunicação oral que tem uma característica multidimensional. A predisposição biológica no desenvolvimento da gagueira ainda não é bem compreendida, mas contribuições genéticas para esta predisposição são reforçadas tanto por referências à agregação familial da gagueira, quanto à gagueira familial, que têm aparecido na literatura há mais de 70 anos. Assim, procuramos estabelecer uma revisão quanto aos prováveis fatores genéticos envolvidos com a manifestação da gagueira desenvolvimental persistente familial. A identificação de genes relacionados à gagueira, bem como de alterações em suas estruturas (por exemplo, mutações), contribuem significativamente para sua compreensão. O modelo exato de transmissão da herança genética para a gagueira ainda não está claramente definida e, provavelmente pode ser diferente entre diferentes famílias e populações. As análises genômicas demonstram, concomitantemente, a relevância dos componentes genéticos envolvidos e sua complexidade, sugerindo assim tratar-se de uma doença poligênica, na qual diversos genes de efeitos variados podem estar envolvidos com o aumento da susceptibilidade de ocorrência da gagueira. O clínico deverá estar alerta ao fato de que uma criança com histórico familial positivo para gagueira poderá ter uma forte tendência a desenvolver o distúrbio de forma crônica. É importante que o clínico esteja atento, de modo a fornecer às famílias orientações precisas sobre o distúrbio. As avaliações objetivas e os tratamentos controlados têm um papel muito importante para o domínio da evolução do distúrbio.

Proctocolectomy and ileal J-pouch anal anastomosis on the surgical treatment of familial adenomatous polyposis and ulcerative colitis: analysis of 49 cases

OBJECTIVE: To evaluate the results of ileal J-pouch anal anastomosis in ulcerative colitis and familial adenomatous polyposis. METHOD: Retrospective analysis of medical records of 49 patients submitted to ileal J-pouch anal anastomosis. RESULTS: Ulcerative colitis was diagnosed in 65% and familial adenomatous polyposis in 34%. Mean age was 39.5 years. 43% were male. Among familial adenomatous polyposis, 61% were diagnosed with colorectal cancer. Thirty-one percent of patients with ulcerative colitis was submitted to a previous surgical approach and 21% of these had toxic megacolon. Average hospital stay was 10 days. Post-operative complications occurred in 50% of patients with ulcerative colitis and 29.4% with familial adenomatous polyposis. Intestinal diversion was performed in 100% of ulcerative colitis and 88% of familial adenomatous polyposis. Pouchitis occurred in eight cases (seven ulcerative colitis and one FAP), requiring excision of the pouch in three ulcerative colitis. Mortality rate was 7.6%: two cases of carcinoma on the pouch and two post-operative complications. Late post-operative complications occurred in 22.4%: six familial adenomatous polyposis and five ulcerative colitis). Two patients had erectile dysfunction, and one retrograde ejaculation. One patient with severe perineal dermatitis was submitted to excision of the pouch. Incontinence occurred in four patients and two reported soil. Mean bowel movement was five times a day. CONCLUSION: Ileal J-pouch anal anastomosis is a safe surgery with acceptable morbidity and good functional results, if well indicated and performed in referral centers.

Inhibition of human platelet aggregation by eosinophils

AIMS: The relationship between the activity of eosinophils and platelets has been observed in recent decades by many scientists. These observations include increased numbers of eosinophils associated with platelet disorders, including changes in the coagulation cascade and platelet aggregation. Based on these observations, the interaction between eosinophils and platelets in platelet aggregation was analyze. MAIN METHODS: Human platelets were incubated with eosinophil cytosolic fraction, promyelocytic human HL-60 clone 15 cell lineage, and eosinophil cationic protein (ECP). Platelet rich plasma (PRP) aggregation was induced by adenosine diphosphate, platelet activating factor, arachidonic acid, and collagen, and washed platelets (WP) were activated by thrombin. KEY FINDINGS: Aggregation induced by all agonists was dose dependently inhibited by eosinophil cytosolic fraction. This inhibition was only partially reversed by previous incubation of the eosinophils with l-Nitro-Arginine-Methyl-Ester (l-NAME). Previous incubation with indomethacin did not prevent the cytosolic fraction induced inhibition. The separation of eosinophil cytosolic fraction by gel filtration on Sephadex G-75 showed that the inhibitory activity was concentrated in the lower molecular weight fraction. HL-60 clone 15 cells differentiated into eosinophils for 5 and 7 day were able to inhibit platelet aggregation. The ECP protein inhibited the platelet aggregation on PRP and WP. This inhibition was more evident in WP, and the citotoxicity MTT assay proved the viability of tested platelets, showing that the observed inhibition by the ECP protein does not occur simply by cell death. SIGNIFICANCE: Our results indicate that eosinophils play a fundamental role in platelet aggregation inhibition

Untersuchungen zur Beteiligung der Preseniline an den molekularen Mechanismen der Amyloid-ß Entstehung

ZusammenfassungMorbus Alzheimer ist eine progressive, neurodegenerative Erkrankung, die weltweit die häufigste Form der Demenz darstellt und im mittleren bis späten Lebensabschnitt auftritt. Die neuropathologischen Merkmale beinhalten das Auftreten von extrazellulären Ablagerungen aus fibrillogenem Aß42 Peptiden in senilen Plaques und intraneuronalen Akkumulationen von hyperphosphoryliertem Tau in sogenannten neurofibrillären Bündeln. Obwohl die meisten Alzheimer Fälle sporadisch und Alters-assoziiert auftreten, gibt es eine autosomal dominant vererbte Form (FAD; Familial Alzheimer Disease), die schon in einem frühen Lebensabschnitt (ab 28 Jahren) ausbrechen kann. Diese aggressive Alzheimer Form wird durch Mutationen im Amyloid-Precursor-Protein-Gen (APP) oder den Presenilin-Genen (PS-1 und PS-2) ausgelöst. Die Presenilin (PS) Proteine sind entscheidend an der Entstehung von Aß beteiligt. So erhöhen FAD-assoziierte Mutationen in PS-1 und PS-2 die Bildung von Aß42. Außerdem verhindern sowohl homozygote PS-1 Null-Mutationen (PS-1-/-) in transgenen Mäusen, als auch dominant negative PS-1 Mutationen in Kulturzellen die Ab Bildung. Diese Belege sprechen für die zur Zeit favorisierte Amyloid Hypothese, in der die toxische Wirkung des Aß-Peptides in der Entstehung der Alzheimer Erkrankung eine zentrale Rolle einnimmt. Die y-Sekretase ist eine Protease, deren Aktivität für die Entstehung von Ab aus dem Vorläuferprotein APP essentiell ist. Damit bildet sie einen möglichen Ansatzpunkt, um grundlegend in den Prozeß der Ab Bildung einzugreifen. Die y-Sekretase ist allerdings noch nicht identifiziert oder kloniert. Es gibt Hinweise, daß die Preseniline y-Sekretase Aktivität besitzen könnten. Diese Theorie ist bis heute jedoch nicht eindeutig belegt. In dieser Arbeit sollten die molekularen Mechanismen der Ab Entstehung und insbesondere die Beteiligung der Preseniline an diesem Prozeß untersucht werden. Dazu wurde zunächst die subzelluläre Verteilung der endogenen Preseniline analysiert. Es konnte erstmalig ein Unterschied in der subzellulären Verteilung zwischen PS-1 und PS-2 festgestellt werden. PS-1 war vorwiegend im ER lokalisiert, wogegen PS-2 stark im Golgi-Apparat angereichert war. Im zweiten Teil der Arbeit wurde nach möglichen Interaktionen der Preseniline mit C-terminalen APP Fragmenten gesucht, die die Substrate der y-Sekretase darstellen. Es konnte gezeigt werden, daß die Preseniline mit einem 21 kDa großen C-terminalen APP Fragment interagieren. Dabei band die Mutante-Form der Preseniline mehr C-terminales APP Fragment als die Wildtyp-Form. Weiterhin wurde ein zellfreies System zur indirekten Bestimmung der y-Sekretase Aktivität etabliert. Mit Hilfe dieses Systems wird es möglich, Inhibitoren der y-Sekretase zu identifizieren. Die Spezifität des zellfreien Testsystems konnte dadurch deutlich gemacht werden, daß das PS-1, das schon in Zellkultur als essentielle Proteinkomponente zur Entstehung von Aß beschrieben wurde, auch in diesem zellfreien y-Sekretase System notwendig war. Allgemeine Proteaseinhibitoren, die alle bekannten Proteasemechanismen abdeckten, zeigten keinen Einfluß auf die de novo Bildung von Aß. Es konnte festgestellt werden, daß neben der y-Sekretase als Aß produzierende Protease auch Aß abbauende Proteasen vorlagen. Das pH-Optimum der y-Sekretase wurde im neutralen Bereich festgestellt. Weiterhin konnte gezeigt werden, daß die y-Sekretase eine transmembrane oder zumindest membranassoziierte Protease ist, die keine cytosolischen Komponenten benötigt.

Impact of ocean acidification on phytoplankton growth and aggregation

Since the industrial revolution, the ocean has absorbed around one third of the anthropogenic CO2, which induced a profound alteration of the carbonate system commonly known as ocean acidification. Since the preindustrial times, the average ocean surface water pH has fallen by 0.1 units, from approximately 8.2 to 8.1 and a further decrease of 0.4 pH units is expected for the end of the century. Despite their microscopic size, marine diatoms are bio-geo-chemically a very important group, responsible for the export of massive amount of carbon to deep waters and sediments. The knowledge of the potential effects of ocean acidification on the phytoplankton growth and on biological pump is still at its infancy. This study wants to investigate the effect of ocean acidification on the growth of the diatom Skeletonema marinoi and on its aggregation, using a mechanistic approach. The experiment consisted of two treatments (Present and Future) representing different pCO2 conditions and two sequential experimental phases. During the cell growth phase a culture of S. marinoi was inoculated into transparent bags and the effect of ocean acidification was studied on various growth parameters, including DOC and TEP production. The aggregation phase consisted in the incubation of the cultures into rolling tanks where the sinking of particles through the water column was simulated and aggregation promoted. Since few studies investigated the effect of pH on the growth of S. marinoi and none used pH ranges that are compatible with the OA scenarios, there were no baselines. I have shown here, that OA does not affect the cell growth of S. marinoi, suggesting that the physiology of this species is robust in respect to the changes in the carbonate chemistry expected for the end of the century. Furthermore, according to my results, OA does not affect the aggregation of S. marinoi in a consistent manner, suggesting that this process has a high natural variability but is not influenced by OA in a predictable way. The effect of OA was tested over a variety of factors including the number of aggregates produced, their size and sinking velocity, the algal, bacterial and TEP content. Many of these variables showed significant treatment effects but none of these were consistent between the two experiments.