Expression of complement components and regulators by different subtypes of bone marrow-derived macrophages

Under inflammatory conditions, macrophages can differentiate into different functional subtypes. We show that bone marrow-derived macrophages constitutively express different levels of various complement-related genes. The relative expression levels are C1qb > Crry > CFH > C3 > C1r > CFB > DAF1 > CD59a > C2 > C1INH > C1s > C4. Upon activation, the expression of C1r, C1s, C3, C2, CFB, and C1INH was up-regulated, and CFH, CD59a, and DAF1, down-regulated in M1 (induced by interferon-? + lipopolysaccharides (LPS)) and M2b (induced by immune complex + LPS) macrophages. The expression of C4 and CFH was slightly up-regulated in interleukin (IL)-10-induced M2c macrophages. Complement gene expression in IL-4-induced M2a macrophages was weakly down-regulated as compared to resting M0 macrophages. Higher levels of C3, C1INH, and CFB but lower levels of CFH expression in M1 and M2b macrophage suggests that they may be involved in the alternative pathway of complement activation during inflammation.

Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2-RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC(50) values for inhibition of cAMP, 5.83 ± 0.11; Ca(2+) mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.-Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs.

The molecular dynamics of Trypanosoma brucei UDP-galactose 4'-epimerase:a drug target for African sleeping sickness

During the past century, several epidemics of human African trypanosomiasis, a deadly disease caused by the protist Trypanosoma brucei, have afflicted sub-Saharan Africa. Over 10 000 new victims are reported each year, with hundreds of thousands more at risk. As current drug treatments are either highly toxic or ineffective, novel trypanocides are urgently needed. The T. brucei galactose synthesis pathway is one potential therapeutic target. Although galactose is essential for T. brucei survival, the parasite lacks the transporters required to intake galactose from the environment. UDP-galactose 4'-epimerase (TbGalE) is responsible for the epimerization of UDP-glucose to UDP-galactose and is therefore of great interest to medicinal chemists. Using molecular dynamics simulations, we investigate the atomistic motions of TbGalE in both the apo and holo states. The sampled conformations and protein dynamics depend not only on the presence of a UDP-sugar ligand, but also on the chirality of the UDP-sugar C4 atom. This dependence provides important insights into TbGalE function and may help guide future computer-aided drug discovery efforts targeting this protein.

Complement expression in retinal pigment epithelial cells is modulated by activated macrophages

Complement activation is involved in a variety of retinal diseases. We have shown previously that a number of complement components and regulators can be produced locally in the eye, and that retinal pigment epithelial (RPE) cells are the major source of complement expression at the retina-choroidal interface. The expression of complement components by RPE cells is regulated by inflammatory cytokines. Under aging or inflammatory conditions, microglia and macrophages accumulate in the subretinal space, where they are in close contact with RPE cells. In this study, we investigated the effect of activated macrophages on complement expression by RPE cells. Mouse RPE cells were treated with the supernatants from un-activated bone marrow-derived macrophages (BM-DMs), the classically activated BM-DMs (M1) and different types of the alternatively activated BM-DMs (M2a by IL-4, M2b by immune complex and lipopolysaccharide (LPS), M2c by IL-10). The expression of inflammatory cytokines and complement genes by RPE cells were determined by real-time RT-PCR. The protein expression of CFB, C3, C1INH, and C1r was examined by Western blot. Our results show that un-stimulated RPE cells express a variety of complement-related genes, and that the expression levels of complement regulators, including C1r, factor H (CFH), DAF1, CD59, C1INH, Crry, and C4BP genes are significantly higher than those of complement component genes (C2, C4, CFB, C3, and C5). Macrophage supernatants increased inflammatory cytokine (IL-1ß, IL-6, iNOS), chemokine (CCL2) and complement expression in RPE cells. The supernatants from M0, M2a and M2c macrophages mildly up-regulated (2~3.5-fold) CFB, CFH and C3 gene expression in RPE cells, whereas the supernatants from M1 and M2b macrophages massively increased (10~30-fold) CFB and C3 gene expression in RPE cells. The expression of other genes, including C1r, C2, C4, CFH, Masp1, C1INH, and C4BP in RPE cells was also increased by the supernatants of M1 and M2b macrophages; however, the increment levels were significantly lower than CFB and C3 genes. M1 and M2b macrophage supernatants enhanced CFB (Bb fragment) protein expression and C3 secretion by RPE cells. M1 macrophages may affect complement expression in RPE cells through the STAT1 pathway. Our results suggest that under inflammatory conditions, activated macrophages could promote the alternative pathway of complement activation in the retina via induction of RPE cell CFB and C3 expression.

Geochemical records of palaeoenvironmental controls on peat forming processes in the Mfabeni peatland, Kwazulu Natal, South Africa since the Late Pleistocene

The Mfabeni peatland is the only known sub-tropical coastal fen that transcends the Last Glacial Maximum (LGM). This ca. 10m thick peat sequence provides a continuous sedimentation record spanning from the late Pleistocene to present (basal age c. 47kcalyr BP). We investigated the paleaeoenvironmental controls on peat formation and organic matter source input at the Mfabeni fen by: 1) exploring geochemical records (mass accumulation rate, total organic carbon, carbon accumulation rate, δC, δN and C/N ratio) to delineate primary production, organic matter source input, preservation and diagenetic processes, and 2) employ these geochemical signatures to reconstruct the palaeoenvironmental conditions and prevailing climate that drove carbon accumulation in the peatland. We established that the Mfabeni peat sediments have undergone minimal diagenetic alteration. The peat sequence was divided into 5 linear sedimentation rate (LSR) stages indicating distinct changes in climate and hydrological conditions: LSR stage 1 (c. 47 to c. 32.2kcalyr BP): predominantly cool and wet climate with C4 plant assemblages, interrupted by two short warming events. LSR stage 2 (c. 32.2 to c. 27.6kcalyr BP): dry and windy climate followed by a brief warm and wet period with increased C4 sedge swamp vegetation. LSR stage 3 (c. 27.6 to c. 20.3kcalyr BP): initial cool and wet period with prevailing C4 sedge plant assemblage until c. 23kcalyr BP; then an abrupt change to dry and cool glacial conditions and steady increases in C3 grasses. LSR stage 4 (c. 20.3 to c. 10.4kcalyr BP): continuation of cool and dry conditions and strong C3 grassland signature until c. 15kcalyr BP, after which precipitation increases. LSR stage 5 (c. 10.4kcalyr BP to present): characterised by extreme fluctuations between pervasive wet and warm to cool interglacial conditions with intermittent abrupt millennial-scale cooling/drying events and oscillations between C3 and C4 plant assemblages. In this study we reconstructed a high-resolution record of local hydrology, bulk plant assemblage and inferred climate since the Late Pleistocene, which suggest an anti-phase link between Southern African and the Northern Hemisphere, most notably during Heinrich (5 to 2) and Younger Dryas events. © 2013 Elsevier B.V.

Stable Isotope Dietary Analysis of Prehistoric Populations from the Minusinsk Basin, Southern Siberia, Russia: A new chronological framework for the introduction of millet to the eastern Eurasian steppe

We report the results of stable carbon and nitrogen isotope analysis of 354 human and faunal samples from five archaeological cultures of the Minusinsk Basin, Southern Siberia – Afanasyevo, Okunevo, Andronovo, Karasuk and Tagar (ca. 2700–1 BC) – a key location in Eurasia due to its position on a northern corridor linking China and central Eurasia. The results indicate that the diet of Eneolithic to Middle Bronze Age (Afanasyevo to Andronovo) populations was primarily C3-based, with C4 plants only becoming an important component of the diet in the Late Bronze Age Karasuk and Early Iron Age Tagar cultures. Freshwater fish seems to have been an important constituent of the diets in all groups. The findings constitute the earliest concrete evidence for the substantial use of millet in the eastern Eurasian steppe. We propose that it was probably introduced from Northwestern China during the Karasuk culture at the start of the Late Bronze Age, ca. 1500 BC. We conclude with a discussion of the implications for the nature of pastoralist economies on the steppes.

Freshwater Reservoir Effect on Re-Dating of Eurasian Steppe Cultures: First Results for Eneolithic and Early Bronze Age North-East Kazakhstan

Freshwater reservoir effects (FRE) can cause a major problem with radiocarbon dating human skeletal material in the Eurasian steppe. We present the first results of research into the extent of the FRE in the sites of Borly 4 (Eneolithic), and Shauke 1 and 8b (Early Bronze Age), North-Eastern Kazakhstan. AMS 14C dating and stable isotope (δ13C, δ15N) analysis of associated groups of samples (32 samples, 11 groups in total) demonstrate that: a) the diet of the humans and fauna analysed was based on the C3 foodchain with no evidence of a C4 plant (such as millet) contribution; aquatic resources apparently were a continuous dietary feature for the humans; b) the first 14C dates obtained for the Upper and Middle Irtysh River region attribute the Eneolithic period of the area to the 34th-30th c. BC, and the Early Bronze Age – to the 25th-20th c. BC; there is a ca. 450 years hiatus between the two periods; c) the maximum fish-herbivore freshwater reservoir offset observed equals 301±47 14C yrs. As such, 14C dates from aquatic and human samples from the area need to be interpreted with caution as they are likely to be affected by the offset (i.e. appear older).
The paper also discusses the effect of a sodium hydroxide (NaOH) wash on δ13C, δ15N, C:Natomic levels and collagen yields of the bone samples. Our results indicate a minor but significant effect of NaOH treatment only on C:Natomic ratios of the samples.

Process for the preparation of water-soluble cellulose hydrolysis products.

The title process comprises admixing cellulose with an ionic liq. capable of solvating or dissolving at least some of the cellulose, the ionic liq. being a compd. comprised solely of cations and anions (e.g., 1-ethyl-3-methylimidazolium sulfate) and which exists in a liq. state at a temp. at or below 150°, the cations in the ionic liq. having the general formula R1Z(R2)(R3)R4: in which Z represents a nitrogen or phosphorus atom, R1 represents a Me or Et group, each of R2 and R3, which may be the same or different, is selected from C4-8alkyl, optionally-substituted benzyl, optionally-substituted Ph, and C5-7cycloalkyl, and R4 represents C1-8 alkyl, optionally-substituted benzyl, optionally-substituted Ph or C5-7cyclohexyl; in which the optional substituents on a benzyl or Ph ring are one, two or three substituents selected from C1-4alkyl or alkoxy groups, halogen atoms and nitro groups; and treating the resulting solvate or soln. with an acid in the presence of water, the acid having a pKa in water of less than 2 at 25°. [on SciFinder(R)]

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Schizophrenia risk from complex variation of complement component 4

Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia's strongest genetic association at a population level involves variation in the major histocompatibility complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.

Effect of elevated CO2 concentrations and irradiance on the photosynthetic performance of the seagrass Cymodocea nodosa

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Exigências funcionais em pavimentos de madeira

Durante séculos a madeira foi dos materiais mais privilegiados e usados na construção. Ainda hoje, existem construções antigas em madeira em bom estado de conservação e que, desempenham as suas funções adequadamente. Com o aparecimento do betão e do aço, os projetistas deixaram de usar este tipo de estruturas. Isto provocou um desinteresse na indústria da madeira, adiando a criação de regulamentos e normas relativamente às exigências funcionais deste tipo de estruturas. Com a necessidade da reabilitação do património edificado, verifica-se uma inversão desta tendência, devido às edificações no centro histórico serem constituídas por estruturas de madeira, essencialmente os pavimentos e coberturas. Na maioria das vezes estas estruturas apresentam um elevado nível de degradação e a solução mais rentável é demolir. Os pavimentos de madeira são então substituídos por lajes aligeiradas ou de betão armado provocando alterações estruturais inadequadas nas fachadas a manter. Pretende-se com esta dissertação mostrar as potencialidades dos pavimentos de madeira e assim incentivar ao seu uso, principalmente nas áreas a reabilitar. São analisados todos os regulamentos e normas aplicáveis às exigências estruturais, térmicas, acústicas e contra incêndio dos pavimentos de madeira. A análise da normalização aplicável vai ser sintetizada, ou seja, só vão ser referidos os aspetos a ter em conta para a verificação das exigências funcionais em pavimentos de madeira. A aplicação dos conceitos e das verificações necessárias são aplicadas a um pavimento de madeira a ser construído na parcela C4 do Quarteirão das Cardosas. Os resultados obtidos neste caso de estudo são encorajadores, e abrem uma perspetiva das potencialidades que este sistema construtivo apresenta.

Relaciones tróficas, espectro alimentario y ración de alimentación de las principales especies pelágicas en el verano 1996

Se analiza la dieta de Engraulis ringens, Sardinops sagax sagax, Trachurus picturatus murphyi, Scomber japonicus peruanus durante el verano de 1996. La ración diaria se determinó mediante el modelo de SAINSBURY (1986), utilizando el Software MAXIMS y el espectro alimentario siguiendo lo descrito por HYSLOP (1980). En la anchoveta y sardina los items presa estuvieron compuestos por copépodos de los géneros Centropages, Oncaea, Eucalanus y el crustáceo planctónico Euphausia mucronata entre los más importantes y por los grupos diatomeas, dinoflagelados y silicoflagelados. La fracción zooplanctónica observada durante la época, ha sido superior a la observada en los estudios de serie de tiempo. En las especies jurel y caballa predominaron los crustáceos planctónicos e incluyeron en su dieta peces de las familias Myctophidae y Sciaenidae. No se observaron anchoveta ni sardina, a pesar que, tradicionalmente, estas especies has sido sus presas favoritas. La ración diaria de anchoveta ha sido determinada en 0,97 g.día-1, sardina 14,52 g.día-1, jurel 37,75 g.día-1 y caballa 49,17 g.día-1.

Impacto de la variación oceanográfica estacional e interanual sobre los ensambles de microfitoplancton, mesozooplancton, ictioplancton y macrozoobentos de dos áreas costeras del norte del Perú entre 1994 y 2002

Se ha analizado la variación oceanográfica y las respuestas de los ensambles de microfitoplancton, mesozooplancton, ictioplancton y macrobentos en las áreas costeras (<20 mn) frente a Paita (05°S) y a San José (06°45’S) durante el período 1994 a 2002. La variación oceanográfica presentó componentes a varias escalas temporales, moduladas por el ciclo ENOS, la propagación de ondas atrapadas hacia la costa y la intensificación estacional del afloramiento costero. La sucesión de los eventos El Niño (EN) 1997-98 y La Niña (LN) 1998-99 presentó características bien diferenciadas en las condiciones físicas superficiales y en la estructura vertical de la columna de agua. El evento EN 1997-98 fue antecedido por el impacto de una onda Kelvin en febrero de 1997, provocando anomalías positivas de temperatura, profundización de la estructura vertical y presencia de algunos indicadores de masas de agua cálida en el plancton, entre febrero y abril de 1997. Estas condiciones se mantuvieron, o se acentuaron, hasta el final del evento. El evento LN 1998-99 se caracterizó por la ausencia de masas de agua cálidas cerca de la costa y la dominancia de aguas costeras frías, la no propagación de ondas Kelvin, la posición somera de aguas frías y pobres en oxígeno, así como la hegemonía de indicadores planctónicos de aguas costeras frías. Estacionalmente, durante otoño-invierno tendieron a desarrollarse condiciones subsuperficiales más oxigenadas (una oxiclina más profunda), mientras que durante el verano las condiciones tendieron a ser menos oxigenadas (hipóxicas, con una oxiclina más somera). Tal patrón no responde a la estacionalidad del afloramiento costero y más bien coincide con la dinámica esperada de la Extensión Sur de la Corriente de Cromwell (ESCC). En general, se determinó un muy buen ajuste de los rangos de tolerancia de algunos organismos planctónicos a las características de las masas de agua dominantes en la capa superficial: Aguas Costeras Frías (ACF), Aguas Ecuatoriales Superficiales (AES) y Aguas Subtropicales Superficiales (ASS), validando la utilidad de estas especies como eficaces indicadores biológicos de masas de agua. Se determinaron los rangos de tolerancia en temperatura y salinidad de los dinoflagelados Protoperidinium obtusum (ACF), Ceratium breve (AES) y Ceratium praelongum (ASS), así como de los copépodos Centropages brachiatus (ACF), Eucalanus inermis (ACF), Centropages furcatus (AES) y Mecynocera clausi (ASS), entre otras. Los indicadores presentaron variaciones en su distribución a lo largo del período estudiado. Los indicadores de ACF fueron detectados durante la mayor parte del estudio, pero ocurrieron hechos sobresalientes durante EN 1997-98: (a) entre los dinoflagelados, Goniodoma polyedricum alcanzó su mayor frecuencia en San José y Pyrocystis lunula frente a Paita; (b) el copépodo Centropages furcatus (AES) incrementó su abundancia frente a Paita y fue hallado frente a San José; (c) se evidenciaron cambios en la composición específica del plancton, detectándose el ingreso de especies no residentes y aumento de la riqueza de especies; (d) las biomasas fitoplanctónica y zooplanctónica tendieron a mostrar una relación directa bajo condiciones neutras del ENOS; sin embargo, al ocurrir variaciones ambientales (EN y LN) presentaron una tendencia contraria; (e) las comunidades del macrobentos en estas dos áreas, mostraron disminuciones significativas en los parámetros comunitarios, contrastando con la respuesta de la macrofauna bentónica frente a la costa central. Este comportamiento frente a Paita pudo obedecer a la alteración del ambiente sedimentario por las muy altas des- cargas del río Chira; y frente a San José, pudo resultar de la disminución local de la producción primaria y del flujo de alimento particulado al bentos. Se sugiere que la disponibilidad de alimento, influenciada por los procesos erosivos sobre el fondo, marca una diferencia clave en la dinámica de estas comunidades en relación a las registradas frente a la costa central, ya que estas últimas habitan en áreas donde pre- dominan los procesos deposicionales y la acumulación de materia orgánica en los sedimentos. Los anfípodos gamáridos, especialmente de la familia Ampeliscidae, mostraron ser más sensibles a los cambios ambientales en el fondo (interfase sedimento-agua) al disminuir significativamente sus poblaciones, en ambas áreas costeras.