Small Vessel Disease in the Heart and Brain: Current Knowledge, Unmet Therapeutic Need and Future Directions

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Fat area and lipid droplet morphology of porcine oocytes during in vitro maturation with trans-10, cis-12 conjugated linoleic acid and forskolin

Lipid droplets (LD) in porcine oocytes form a dark mass reaching almost all cytoplasm. Herein we investigated changes in fat areas, cytoplasmic tone and LD morphology during in vitro maturation (IVM) of porcine oocytes cultured with 100mM trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA) or 10mM forskolin at different time periods. Four groups were constituted: control, excipient, t10,c12 CLA and forskolin, with drugs being supplemented during 44 to 48h and the initial 22 to 24h in Experiments 1 and 2, respectively. In Experiment 3, forskolin was supplemented for the first 2 h. Matured oocytes were inseminated with frozen-thawed boar semen and cleavage rate recorded. Before and during IVM, samples of oocytes were evaluated for LD, total and fat areas and fat gray value or for meiotic progression. Results showed that forskolin supplementation during 44 to 48 h or 22 to 24 h inhibits oocyte maturation (exp. 1: forskolin = 5.1±8.0%, control = 72.6±5.0%; exp. 2: forskolin =24.3±7.4%, control =71.6±5.6%) and cleavage (exp. 1: forskolin=0.0±0.0%, control=55.4±4.1%; exp. 2: forskolin=8.3±3.3%, control=54.5±3.0%). Forskolin also reduced oocyte and fat areas. In Experiment 3, forskolin negative effect on oocyte maturation and cleavage disappeared, although minor (P<0.03) LD and oocyte fat areas were identified at 22 to 24 h of IVM. Oocytes supplemented with t10,c12 CLA during 44 to 48h presented a lighter (P<0.04) colour tone cytoplasm than those of control and forskolin. In conclusion, t10,c12 CLA and forskolin were capable of modifying the distribution and morphology of cytoplasmic LD during porcine oocyte maturation, thus reducing its lipid content in a time-dependent manner

The use of oxalate to reduce dentin permeability under adhesive restorations

Abstract no. 1705published_or_final_versio

The inflammatory response in the regression of lumbar disc herniation

Lumbar disc herniation (LDH) is highly associated with inflammation in the context of low back pain. Currently, inflammation is associated with adverse symptoms related to the stimulation of nerve fibers that may lead to pain. However, inflammation has also been indicated as the main factor responsible for LDH regression. This apparent controversy places inflammation as a good prognostic indicator of spontaneous regression of LDH. This review addresses the molecular and cellular mechanisms involved in LDH regression, including matrix remodeling and neovascularization, in the scope of the clinical decision on conservative versus surgical intervention. Based on the evidence, a special focus on the inflammatory response in the LDH context is given, particularly in the monocyte/macrophage role. The phenomenon of spontaneous regression of LDH, extensively reported in the literature, is therefore analyzed here under the perspective of the modulatory role of inflammation.This work was financed by project “Bioengineered Therapies for infectious diseases and tissue regeneration” (NORTE-01-0145-FEDER-000012), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), by FEDER/COMPETE 2020 (POCI), Portugal 2020, and by Portuguese funds through FCT/MCTES in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01- 0145-FEDER-007274). Cunha C and Gonçalves RM acknowledge FCT by their postdoc fellowship (SFRH/BDP/87071/2012) and FCT Investigator Grant (IF/ 00638/2014), respectively. Silva AJ acknowledges her fellowship under the framework of the project Norte-01-0145-FEDER-000012

Therapeutic strategies for IVD regeneration through hyaluronan/SDF-1-based hydrogel and intravenous administration of MSCs

Intervertebral disc (IVD) degeneration involves a complex cascade of events, including degradation of the native extracellular matrix, loss of water content, and decreased cell numbers. Cell recruitment strategies for the IVD have been increasingly explored, aiming to recruit either endogenous or transplanted cells. This study evaluates the IVD therapeutic potential of a chemoattractant delivery system (HAPSDF5) that combines a hyaluronan-based thermoreversible hydrogel (HAP) and the chemokine stromal cell derived factor-1 (SDF-1). HAPSDF5 was injected into the IVD and was combined with an intravenous injection of mesenchymal stem/stromal cells (MSCs) in a pre-clinical in vivo IVD lesion model. The local and systemic effects were evaluated two weeks after treatment. The hydrogel by itself (HAP) did not elicit any adverse effect, showing potential to be administrated by intradiscal injection. HAPSDF5 induced higher cell numbers, but no evidence of IVD regeneration was observed. MSCs systemic injection seemed to exert a role in IVD regeneration to some extent through a paracrine effect, but no synergies were observed when HAPSDF5 was combined with MSCs. Overall, this study shows that although the injection of chemoattractant hydrogels and MSC recruitment are feasible approaches for IVD, IVD regeneration using this strategy needs to be further explored before successful clinical translation.Funding: This research was funded by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia (IUD/BIM/04293/2019) and by EUROSPINE TRF (2017_05)

A healthy dietary pattern associates with a lower risk of a first clinical diagnosis of central nervous system demyelination

Background: The evidence associating diet and risk of multiple sclerosis is inconclusive. Objective: We investigated associations between dietary patterns and risk of a first clinical diagnosis of central nervous system demyelination, a common precursor to multiple sclerosis. Methods: We used data from the 2003–2006 Ausimmune Study, a case–control study examining environmental risk factors for a first clinical diagnosis of central nervous system demyelination, with participants matched on age, sex and study region. Using data from a food frequency questionnaire, dietary patterns were identified using principal component analysis. Conditional logistic regression models (n = 698, 252 cases, 446 controls) were adjusted for history of infectious mononucleosis, serum 25-hydroxyvitamin D concentrations, smoking, race, education, body mass index and dietary misreporting. Results: We identified two major dietary patterns – healthy (high in poultry, fish, eggs, vegetables, legumes) and Western (high in meat, full-fat dairy; low in wholegrains, nuts, fresh fruit, low-fat dairy), explaining 9.3% and 7.5% of variability in diet, respectively. A one-standard deviation increase in the healthy pattern score was associated with a 25% reduced risk of a first clinical diagnosis of central nervous system demyelination (adjusted odds ratio 0.75; 95% confidence interval 0.60, 0.94; p = 0.011). There was no statistically significant association between the Western dietary pattern and risk of a first clinical diagnosis of central nervous system demyelination. Conclusion: Following healthy eating guidelines may be beneficial for those at high risk of multiple sclerosis

Gastric microbial community profiling reveals a dysbiotic cancer-associated microbiota

Objective Gastric carcinoma development is triggered by Helicobacter pylori. Chronic H. pylori infection leads to reduced acid secretion, which may allow the growth of a different gastric bacterial community. This change in the microbiome may increase aggression to the gastric mucosa and contribute to malignancy. Our aim was to evaluate the composition of the gastric microbiota in chronic gastritis and in gastric carcinoma. Design The gastric microbiota was retrospectively investigated in 54 patients with gastric carcinoma and 81 patients with chronic gastritis by 16S rRNA gene profiling, using next-generation sequencing. Differences in microbial composition of the two patient groups were assessed using linear discriminant analysis effect size. Associations between the most relevant taxa and clinical diagnosis were validated by real-time quantitative PCR. Predictive functional profiling of microbial communities was obtained with PICRUSt. Results The gastric carcinoma microbiota was characterised by reduced microbial diversity, by decreased abundance of Helicobacter and by the enrichment of other bacterial genera, mostly represented by intestinal commensals. The combination of these taxa into a microbial dysbiosis index revealed that dysbiosis has excellent capacity to discriminate between gastritis and gastric carcinoma. Analysis of the functional features of the microbiota was compatible with the presence of a nitrosating microbial community in carcinoma. The major observations were confirmed in validation cohorts from different geographic origins. Conclusions Detailed analysis of the gastric microbiota revealed for the first time that patients with gastric carcinoma exhibit a dysbiotic microbial community with genotoxic potential, which is distinct from that of patients with chronic gastritis.This research was supported by a Worldwide Cancer Research grant to CF and JCM (Reference 16-1352). RMF, JPM and IPR have fellowships from Fundacao para a Ciencia e a Tecnologia (FCT; SFRH/BPD/84084/2012, PD/BD/114014/2015 and SFRH/BD/110803/2015, respectively) through Programa Operacional Capital Humano (POCH) and the European Union. JPM's fellowship is in the framework of FCT's PhD Programme BiotechHealth (Ref PD/0016/2012). i3S-Instituto de Investigacao e Inovacao em Saude is funded by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through Fundacao para a Ciencia e a Tecnologia (FCT)/Ministerio da Ciencia, Tecnologia e Inovacao (POCI-01-0145-FEDER-007274)

A utilização de sémen fresco na fertilização in vitro de embriões ovinos melhora a qualidade dos blastocistos na raça portuguesa Merino

A produção de embriões em ovinos é uma tarefa difícil, exigindo experiência e condições onerosas, principalmente na produção de embriões in vivo. A recolha sistemática de oócitos em animais de matadouro ou em animais vivos por ovum pick-up, permite a produção in vitro de embriões (IVP), em larga escala e menos dispendiosa, nos pequenos ruminantes. Esta possibilidade é importante não só como fonte de embriões mas também de oócitos e zigotos para fins comerciais ou de investigação, facilitando a sua disponibilidade em tecnologias emergentes tais como a clonagem ou a transgénese. Para IVP foram desenvolvidos vários protocolos de maturação, utilizando fertilização in vitro (IVF) com sémen fresco ou congelado. Em Portugal, a produção de embriões in vitro foi somente realizada com sémen congelado dada a sua disponibilidade em condições de rotina. Contudo, o sémen fresco poderá melhorar a produção de embriões frescos ou criopreservados. Este trabalho teve como objectivo comparar a eficiência da IVP em ovinos usando diferentes protocolos de maturação de oócitos e IVF com sémen fresco ou congelado. Oócitos (n=1768) recolhidos em matadouro foram maturados em meio TCM199 com 100 μM cisteamine, 10 ng mL-1 EGF, 10 μg mL-1 E2 e gentamicina (mat A, n=692) ou suplementada com 10 μg mL-1 FSH e 0,3 mM piruvato de sódio (mat B, n=707) a 39 ºC e 5% CO2 durante 22h. O sémen fresco (FS) e congelado/descongelado (TS) de carneiros de raça Merino Branco (n=3) foi lavado ou submetido a swim-up, respectivamente. Após a fertilização (18h p.i.), os presumíveis zigotos foram cultivados em meio de fluido sintético do oviducto (SOF) enriquecido com aminoácidos e BSA a 38,5 ºC, em atmosfera humidificada com 5% O2, 5% CO2 e 90% N2 até ao estadio de 2-4-8 células. Após clivagem, o desenvolvimento embrionário prosseguiu até ao estadio de blastocisto em meio SOF, BSA e 10% FCS. A qualidade foi avaliada no dia 6-7, classificando-se como bons, médios e maus, baseado nos parâmetros IETS. Os dados das taxas de produção embrionária foram analisados utilizando ANOVA. Foi utilizado o teste de Mann-Whitney U para avaliação da qualidade dos embriões. Os diferentes protocolos de maturação não interferiram (p>0,05) quer com as taxas de maturação quer com as taxas de produção de embriões. A qualidade embrionária foi superior (p=0,004) na fertilização com sémen fresco (bom: FS=40,1±8,0% vs TS=32,9±5,6%; média: FS=20,1±4,7% vs TS=35,7±5,8%; má: FS=39,8±9,8% vs TS=31,4±7,6%). Em conclusão, estes resultados preliminares mostram que o sémen fresco de carneiro pode ser facilmente utilizado para fertilização in vitro e melhora a qualidade dos embriões produzidos.#Embryo production in sheep is a difficult task demanding experience and expensive facilities, particularly when dealing with in vivo embryo production. Easy ways to obtain ovine embryos consist of collecting oocytes at slaughterhouses or systematically pick them up from live animals, allowing a large scale and cheaper in vitro embryo production (IVP) for small ruminants. Those are important sources of embryos, oocytes and zygotes for commercial, laboratorial and research proposes, making easier the availability of resources for emerging techniques like cloning or transgenesis. For IVP, several oocyte maturation protocols have been developed using fertilization (IVF) either with fresh or frozen-thawed semen. In Portugal, IVP has been done through IVF using cryopreserved semen because it is easily available for routine use. However, the use of fresh semen could improve embryo production and cryopreservation results. The aim of this work was to compare the efficiency of in vitro embryo production in ovine using different oocyte maturation protocols and fresh or frozen semen for IVF. Abattoir-derived oocytes (n=1768) were matured in TCM199, 10 μM cysteamine, 10 ng mL-1 EGF, 10 μg mL-1 E2 and gentamicin (mat A, n=692) or plus 10 μg mL-1 FSH and 0.3 mM sodium piruvate (mat B, n=707) at 39 ºC and 5% CO2 for 22h. Prior to fertilization, either fresh (FS) or frozen/thawed (TS) semen from Merino rams (n=3) was washed or submitted to swim-up respectively. Presumptive zygotes (18h p.i.) were cultured in synthetic oviductal fluid (SOF) enriched with aminoacids and 6 mg mL-1 BSA at 38.5 ºC, under 5% O2, 5% CO2 and 90% N2 in an humidified atmosphere until the stage of 2-4-8 cell embryos. After assessing cleavage, embryo development proceeded until the blastocyst stage in SOF+BSA and 10% FCS. Quality was evaluated on D6-7 by scoring embryos as good, fair and bad based on IETS guidelines. Data from embryo production rates were analysed using ANOVA. Mann-Whitney U test was used for embryo quality evaluation. Different maturation protocols did not interfere (P>0.05) either on maturation or on embryo quality or production rates. Embryo quality was higher (P=0.004) when fertilization was accomplished with fresh semen (good: FS=40.1±8.0% vs TS=32.9±5.6%; fair: FS=20.1±4.7% vs TS=35.7±5.8%; bad: FS=39.8±9.8% vs TS=31.4±7.6%). Preliminar results show that ram fresh semen can be easily used for in vitro fertilization and improves the quality of produced embryos