Importancia diagnóstica del C4d en la nefropatía membranosa / Diagnostic significance of C4d in membranous ne phropathy

Resumen Introducción : La nefropatía membranosa (NM) es la causa más frecuente de síndrome nefrótico primario en adultos (20-30%). En la microscopia óptica se obser va engrosamiento de membrana basal glomerular con aparición de espigas. Estos hallazgos histológicos no son evidentes en formas tempranas, en cuyo caso el patrón de depósito granular de IgG y/o C3 en la membrana basal por inmunofluorescencia (IF) permite diferenciarla de enfermedad por cambios mínimos (ECM). El sistema del complemento juega un papel central en la fisiopatología de la NM. C4d es producto de degradación y un marcador de la activación del complemento. La marcación con C4d en muestras de biopsias re nales, por técnica de inmunohistoquímica (IH) puede colaborar en el diagnóstico diferencial entre ambas glomerulopatías. Nuestro objetivo fue explorar el poder de discriminación del C4d para diferenciar NM de ECM en material de biopsias renales. Métodos : Se recuperaron muestras en parafina de biopsias renales con diagnóstico de NM y ECM realizados entre 1/1/2008 y 1/4/2019. Se realizaron tinciones de IH por técnica de inmunoperoxidasa con C4d usando un anticuerpo policlonal antihumano de conejo. Resultados : En todos los casos con NM (n = 27, 15 hombres) con mediana de edad de 63 (rango: 18-86) años se detectaron depósitos de C4d. En los 21 casos con ECM (12 hombres) con mediana de edad de 51 (rango: 18-87) años la marcación de C4d fue negativa. Conclusión : Los resultados indican que la marcación de la biopsia renal con C4d es una herramienta útil para el diagnóstico diferencial entre NM y ECM.

Abstract Introduction : Membranous nephropathy (MN) is the most common cause of primary nephrotic syndrome in adults (20-30%). Light microscopy shows thickening of glomerular basement membrane with appearance of spikes. These histological findings are not evident in early forms, in which case the granular deposition pattern of IgG and/or C3 in the basement membrane by immunofluorescence (IF) constitutes the diagnostic tool that allows to differentiate it from minimal change disease (MCD). Complement system plays a key role in the pathophysiology of MN. C4d is a degradation prod uct and a marker of the complement system activation. C4d labelling by immunohistochemical (HI) technique can help in the differential diagnosis between both glomerulopathies NM and MCD when the material for IF is insufficient and light microscopy is normal. Our objective was to explore the discrimination power of C4d to differentiate between MN and MCD in renal biopsy material. Methods : Paraffin-embedded samples were recovered from renal biopsies with a diagnosis of MN and MCD performed between 1/1/2008 and 4/1/2019. IH staining was performed by immunoperoxidase technique using a rabbit anti-human C4d polyclonal antibody. Results : In all cases with MN (n = 27, 15 males) with a median age of 63 (range: 18-87) years, C4d deposits were detected. In 21 cases with MCD (12 males) with a median age of 51 (range: 18-87) years, the C4d marking was negative in every samples. Conclusion : The results indicate that the marking of the renal biopsy with C4d is a useful tool for the dif ferential diagnosis between NM and MCD.

Different Proteomic Profiles Regarding Antihypertensive Therapy in Preeclampsia Pregnant.

Preeclampsia (PE) is a hypertensive pregnancy syndrome associated with target organ damage and increased cardiovascular risks, necessitating antihypertensive therapy. However, approximately 40% of patients are nonresponsive to treatment, which results in worse clinical outcomes. This study aimed to compare circulating proteomic profiles and identify differentially expressed proteins among 10 responsive (R-PE), 10 nonresponsive (NR-PE) patients, and 10 healthy pregnant controls (HP). We also explored correlations between these proteins and clinical data. Plasma protein relative quantification was performed using mass spectrometry, followed by bioinformatics analyses with the UniProt database, PatternLab for Proteomics 4.0, and MetaboAnalyst software (version 6.0). Considering a fold change of 1.5, four proteins were differentially expressed between NR-PE and R-PE: one upregulated (fibronectin) and three downregulated (pregnancy-specific beta-1-glycoprotein 1, complement C4B, and complement C4A). Between NR-PE and HP, six proteins were differentially expressed: two upregulated (clusterin and plasmin heavy chain A) and four downregulated (apolipoprotein L1, heparin cofactor II, complement C4B, and haptoglobin-related protein). Three proteins were differentially expressed between R-PE and HP: one downregulated (transthyretin) and two upregulated (apolipoprotein C1 and hemoglobin subunit beta). These findings suggest a complex interplay of these proteins involved in inflammatory, immune, and metabolic processes with antihypertensive therapy responsiveness and PE pathophysiology.

Time-to-growth: photoperiod and photosynthesis make the call.

The intricate regulation of flowering time in response to day length has been extensively shown. A recent study has now revealed a similar mechanism for regulating vegetative growth. Wang et al. observed that plants measure daylength as the duration of photosynthesis and metabolite production to modulate vegetative growth.

C4d expression in focal segmental glomerulosclerosis.

BACKGROUND: There is a little information about of expression of C4d (complement fragment) in Focal segmental glomerulosclerosis (FSGS) subtypes. Our aim was to determine the expression of C4d in FSGS subtypes in percutaneous native renal biopsies in a second-level hospital and its correlation with clinical, biochemical and histological variables. MATERIAL AND METHODS: A retrospective study in paraffin blocks of patients with biopsy with FSGS aged 16-65 years, indistinct sex, not diabetic or obese. Immunohistochemistry was performed for C4d and their expression was analyzing in non-sclerosed glomerular capillaries (GC) and sclerosis areas (SA). Clinical and biochemical variables were recorded. The cases were divided into C4d positive and C4d negative groups and compared. The correlation between C4d staining scores in CG and SA with clinical and biochemical variables were analyzed. RESULTS: Twenty samples were analyzed, 4 for each subtype. At the time of biopsy average age 38.8 ±â€¯18.6 years, 65% male, 8.7% were hypertension. The percentage of positivity for C4d was 40% in GC, 30% SA and 35% in mesangium. The highest expression was for cellular and collapsing subtypes. C4d positivity cases had increased proteinuria (p = 0.035). A significant correlation was found between percentage of C4d expression in CG with SA (p = 0.012) and SA with tubular atrophy and interstitial fibrosis (p < 0.05). CONCLUSIONS: C4d expression in FSGS predominated in the cellular and collapsing subtypes, which translates complement activation. C4d is a possible surrogate marker in GSFS.

[Diagnostic significance of C4d in membranous nephropathy]. / Importancia diagnóstica del C4d en la nefropatía membranosa.

INTRODUCTION: Membranous nephropathy (MN) is the most common cause of primary nephrotic syndrome in adults (20-30%). Light microscopy shows thickening of glomerular basement membrane with appearance of spikes. These histological findings are not evident in early forms, in which case the granular deposition pattern of IgG and/or C3 in the basement membrane by immunofluorescence (IF) constitutes the diagnostic tool that allows to differentiate it from minimal change disease (MCD). Complement system plays a key role in the pathophysiology of MN. C4d is a degradation product and a marker of the complement system activation. C4d labelling by immunohistochemical (HI) technique can help in the differential diagnosis between both glomerulopathies NM and MCD when the material for IF is insufficient and light microscopy is normal. Our objective was to explore the discrimination power of C4d to differentiate between MN and MCD in renal biopsy material. METHODS: Paraffin-embedded samples were recovered from renal biopsies with a diagnosis of MN and MCD performed between 1/1/2008 and 4/1/2019. IH staining was performed by immunoperoxidase technique using a rabbit anti-human C4d polyclonal antibody. RESULTS: In all cases with MN (n = 27, 15 males) with a median age of 63 (range: 18-87) years, C4d deposits were detected. In 21 cases with MCD (12 males) with a median age of 51 (range: 18-87) years, the C4d marking was negative in every samples. CONCLUSION: The results indicate that the marking of the renal biopsy with C4d is a useful tool for the differential diagnosis between NM and MCD.

Introducción: La nefropatía membranosa (NM) es la causa más frecuente de síndrome nefrótico primario en adultos (20-30%). En la microscopia óptica se observa engrosamiento de membrana basal glomerular con aparición de espigas. Estos hallazgos histológicos no son evidentes en formas tempranas, en cuyo caso el patrón de depósito granular de IgG y/o C3 en la membrana basal por inmunofluorescencia (IF) permite diferenciarla de enfermedad por cambios mínimos (ECM). El sistema del complemento juega un papel central en la fisiopatología de la NM. C4d es producto de degradación y un marcador de la activación del complemento. La marcación con C4d en muestras de biopsias renales, por técnica de inmunohistoquímica (IH) puede colaborar en el diagnóstico diferencial entre ambas glomerulopatías. Nuestro objetivo fue explorar el poder de discriminación del C4d para diferenciar NM de ECM en material de biopsias renales. Métodos: Se recuperaron muestras en parafina de biopsias renales con diagnóstico de NM y ECM realizados entre 1/1/2008 y 1/4/2019. Se realizaron tinciones de IH por técnica de inmunoperoxidasa con C4d usando un anticuerpo policlonal antihumano de conejo. Resultados: En todos los casos con NM (n = 27, 15 hombres) con mediana de edad de 63 (rango: 18-86) años se detectaron depósitos de C4d. En los 21 casos con ECM (12 hombres) con mediana de edad de 51 (rango: 18-87) años la marcación de C4d fue negativa. Conclusión: Los resultados indican que la marcación de la biopsia renal con C4d es una herramienta útil para el diagnóstico diferencial entre NM y ECM.

Two Congeneric Shrubs from the Atacama Desert Show Different Physiological Strategies That Improve Water Use Efficiency under a Simulated Heat Wave.

Desert shrubs are keystone species for plant diversity and ecosystem function. Atriplex clivicola and Atriplex deserticola (Amaranthaceae) are native shrubs from the Atacama Desert that show contrasting altitudinal distribution (A. clivicola: 0-700 m.a.s.l.; A. deserticola: 1500-3000 m.a.s.l.). Both species possess a C4 photosynthetic pathway and Kranz anatomy, traits adaptive to high temperatures. Historical records and projections for the near future show trends in increasing air temperature and frequency of heat wave events in these species' habitats. Besides sharing a C4 pathway, it is not clear how their leaf-level physiological traits associated with photosynthesis and water relations respond to heat stress. We studied their physiological traits (gas exchange, chlorophyll fluorescence, water status) before and after a simulated heat wave (HW). Both species enhanced their intrinsic water use efficiency after HW but via different mechanisms. A. clivicola, which has a higher LMA than A. deserticola, enhances water saving by closing stomata and maintaining RWC (%) and leaf Ψmd potential at similar values to those measured before HW. After HW, A. deserticola showed an increase of Amax without concurrent changes in gs and a significant reduction of RWC and Ψmd. A. deserticola showed higher values of Chla fluorescence after HW. Thus, under heat stress, A. clivicola maximizes water saving, whilst A. deserticola enhances its photosynthetic performance. These contrasting (eco)physiological strategies are consistent with the adaptation of each species to their local environmental conditions at different altitudes.

Reflectance Spectroscopy for the Classification and Prediction of Pigments in Agronomic Crops.

Reflectance spectroscopy, in combination with machine learning and artificial intelligence algorithms, is an effective method for classifying and predicting pigments and phenotyping in agronomic crops. This study aims to use hyperspectral data to develop a robust and precise method for the simultaneous evaluation of pigments, such as chlorophylls, carotenoids, anthocyanins, and flavonoids, in six agronomic crops: corn, sugarcane, coffee, canola, wheat, and tobacco. Our results demonstrate high classification accuracy and precision, with principal component analyses (PCAs)-linked clustering and a kappa coefficient analysis yielding results ranging from 92 to 100% in the ultraviolet-visible (UV-VIS) to near-infrared (NIR) to shortwave infrared (SWIR) bands. Predictive models based on partial least squares regression (PLSR) achieved R2 values ranging from 0.77 to 0.89 and ratio of performance to deviation (RPD) values over 2.1 for each pigment in C3 and C4 plants. The integration of pigment phenotyping methods with fifteen vegetation indices further improved accuracy, achieving values ranging from 60 to 100% across different full or range wavelength bands. The most responsive wavelengths were selected based on a cluster heatmap, ß-loadings, weighted coefficients, and hyperspectral vegetation index (HVI) algorithms, thereby reinforcing the effectiveness of the generated models. Consequently, hyperspectral reflectance can serve as a rapid, precise, and accurate tool for evaluating agronomic crops, offering a promising alternative for monitoring and classification in integrated farming systems and traditional field production. It provides a non-destructive technique for the simultaneous evaluation of pigments in the most important agronomic plants.

Adaptive diversity in structure and function of C4 photosynthetic components.

Many tropical and subtropical plant lineages have independently evolved C4 photosynthesis. The convergent evolution of this complex functional trait from different ancestors is reflected in variations in the structural and biochemical characteristics of C4 components such as enzymes and cellular specializations. The mechanism of C4 carbon concentration mostly involves coordinated function of mesophyll and bundle sheath cells. Important adaptations of the C4 syndrome include increased vein density and the development of photosynthetic bundle sheath cells with low gas conductance. In addition, the enzymes and transporters of the C4 pathway evolved via the co-option of multiple genes, each derived from a specific lineage of isoforms present in nonC4-ancestors. In particular, the adaptation of C4 enzymes resulted in a variety of structural and biochemical modifications, generally leading to increased catalytic efficiency and regulation by metabolites and post-translational modifications. Differences in these adaptations are particularly evident in the C4-acid decarboxylation step, which can be catalyzed by three decarboxylases that define the C4 subtypes. Associated with the biochemical subtypes, there are also differences in the extend of grana staking and localization of bundle sheath cells chloroplasts. The presence of a suberin layer and symplastic connections also likely vary among the different C4-subtypes. This review examines the current understanding of the diversity of structural and functional changes in key components of the C4 carbon concentration mechanism. This knowledge is necessary not only to identify divergent solutions for convergent optimization of C4 components in different C4 lineages, but also to guide their creation for rational synthetic biology approaches.

De novo transcriptome assemblies of C3 and C4 non-model grass species reveal key differences in leaf development.

BACKGROUND: C4 photosynthesis is a mechanism that plants have evolved to reduce the rate of photorespiration during the carbon fixation process. The C4 pathway allows plants to adapt to high temperatures and light while more efficiently using resources, such as water and nitrogen. Despite decades of studies, the evolution of the C4 pathway from a C3 ancestor remains a biological enigma. Interestingly, species with C3-C4 intermediates photosynthesis are usually found closely related to the C4 lineages. Indeed, current models indicate that the assembly of C4 photosynthesis was a gradual process that included the relocalization of photorespiratory enzymes, and the establishment of intermediate photosynthesis subtypes. More than a third of the C4 origins occurred within the grass family (Poaceae). In particular, the Otachyriinae subtribe (Paspaleae tribe) includes 35 American species from C3, C4, and intermediates taxa making it an interesting lineage to answer questions about the evolution of photosynthesis. RESULTS: To explore the molecular mechanisms that underpin the evolution of C4 photosynthesis, the transcriptomic dynamics along four different leaf segments, that capture different stages of development, were compared among Otachyriinae non-model species. For this, leaf transcriptomes were sequenced, de novo assembled, and annotated. Gene expression patterns of key pathways along the leaf segments showed distinct differences between photosynthetic subtypes. In addition, genes associated with photorespiration and the C4 cycle were differentially expressed between C4 and C3 species, but their expression patterns were well preserved throughout leaf development. CONCLUSIONS: New, high-confidence, protein-coding leaf transcriptomes were generated using high-throughput short-read sequencing. These transcriptomes expand what is currently known about gene expression in leaves of non-model grass species. We found conserved expression patterns of C4 cycle and photorespiratory genes among C3, intermediate, and C4 species, suggesting a prerequisite for the evolution of C4 photosynthesis. This dataset represents a valuable contribution to the existing genomic resources and provides new tools for future investigation of photosynthesis evolution.

Trehalose 6-phosphate metabolism in C4 species.

Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signal metabolite in plants, linking growth and development to carbon status. Our current understanding of Tre6P metabolism and signaling pathways in plants is based almost entirely on studies performed with Arabidopsis thaliana, a model plant that performs C3 photosynthesis. Conversely, our knowledge on the molecular mechanisms involved in Tre6P regulation of carbon partitioning and metabolism in C4 plants is scarce. This topic is especially relevant due to the agronomic importance of crops performing C4 photosynthesis, such as maize, sorghum and sugarcane. In this review, we focused our attention on recent developments related to Tre6P metabolism in C4 species and raised some open questions that should be addressed in the near future to improve the yield of economically important crops.

High Recovery from Either Waterlogging or Drought Overrides Any Beneficial Acclimation of Chloris gayana Facing a Subsequent Round of Stress.

Climate models predict that plants will face extreme fluctuations in water availability in future global change scenarios. Then, forage production will be more frequently subjected to the destabilizing pressure of sequentially occurring waterlogging and drought events. While the isolated effects of drought (D) and waterlogging (WL) are well characterized, little is known about the consequences when both stresses occur sequentially. We hypothesized that plants sequentially subjected to opposite water scenarios (D followed by WL or vice versa) are less stress tolerant than plants experiencing repetitions of the same type of water stress (i.e., D + D or WL + WL) due to contrasting acclimation and allocation to either shoots (WL) or roots (D). Chloris gayana (a tropical forage grass capable of tolerating either D and WL) plants were randomly assigned to nine treatments (a sequence of two stress rounds-WL or D-each followed by a recovery phase at field capacity). Relative growth rates and allometric responses were measured after each stress round and recovery period. In the first round of stress, both WL and D reduced plant RGR similarly, despite their allocation being opposite-prioritizing shoots or roots under WL and D, respectively. The high recovery displayed after either WL or D overrode any possible acclimation of the plants facing a second round of water stress. We conclude that the tolerance of C. gayana to sequential water stress (either for WL or D) is likely to depend more heavily on its recovery ability than on its previous adjustment to any stress scenario that may evoke memory responses. Knowledge like this could help improve forage grass breeding and the selection of cultivars for poorly drained soils subject to sequential stress events.

Transcriptional and metabolic changes associated with internode development and reduced cinnamyl alcohol dehydrogenase activity in sorghum.

The molecular mechanisms associated with secondary cell wall (SCW) deposition in sorghum remain largely uncharacterized. Here, we employed untargeted metabolomics and large-scale transcriptomics to correlate changes in SCW deposition with variation in global gene expression profiles and metabolite abundance along an elongating internode of sorghum, with a major focus on lignin and phenolic metabolism. To gain deeper insight into the metabolic and transcriptional changes associated with pathway perturbations, a bmr6 mutant [with reduced cinnamyl alcohol dehydrogenase (CAD) activity] was analyzed. In the wild type, internode development was accompanied by an increase in the content of oligolignols, p-hydroxybenzaldehyde, hydroxycinnamate esters, and flavonoid glucosides, including tricin derivatives. We further identified modules of genes whose expression pattern correlated with SCW deposition and the accumulation of these target metabolites. Reduced CAD activity resulted in the accumulation of hexosylated forms of hydroxycinnamates (and their derivatives), hydroxycinnamaldehydes, and benzenoids. The expression of genes belonging to one specific module in our co-expression analysis correlated with the differential accumulation of these compounds and contributed to explaining this metabolic phenotype. Metabolomics and transcriptomics data further suggested that CAD perturbation activates distinct detoxification routes in sorghum internodes. Our systems biology approach provides a landscape of the metabolic and transcriptional changes associated with internode development and with reduced CAD activity in sorghum.

A matter of time: regulatory events behind the synchronization of C4 and crassulacean acid metabolism in Portulaca oleracea.

Portulaca species can switch between C4 and crassulacean acid metabolism (CAM) depending on environmental conditions. However, the regulatory mechanisms behind this rare photosynthetic adaptation remain elusive. Using Portulaca oleracea as a model system, here we investigated the involvement of the circadian clock, plant hormones, and transcription factors in coordinating C4 and CAM gene expression. Free-running experiments in constant conditions suggested that C4 and CAM gene expression are intrinsically connected to the circadian clock. Detailed time-course, drought, and rewatering experiments revealed distinct time frames for CAM induction and reversion (days versus hours, respectively), which were accompanied by changes in abscisic acid (ABA) and cytokinin metabolism and signaling. Exogenous ABA and cytokinins were shown to promote and repress CAM expression in P. oleracea, respectively. Moreover, the drought-induced decline in C4 transcript levels was completely recovered upon cytokinin treatment. The ABA-regulated transcription factor genes HB7, NFYA7, NFYC9, TT8, and ARR12 were identified as likely candidate regulators of CAM induction following this approach, whereas NFYC4 and ARR9 were connected to C4 expression patterns. Therefore, we provide insights into the signaling events controlling C4-CAM transitions in response to water availability and over the day/night cycle, highlighting candidate genes for future functional studies in the context of facultative C4-CAM photosynthesis.

A comparative genomics examination of desiccation tolerance and sensitivity in two sister grass species.

Desiccation tolerance is an ancient and complex trait that spans all major lineages of life on earth. Although important in the evolution of land plants, the mechanisms that underlay this complex trait are poorly understood, especially for vegetative desiccation tolerance (VDT). The lack of suitable closely related plant models that offer a direct contrast between desiccation tolerance and sensitivity has hampered progress. We have assembled high-quality genomes for two closely related grasses, the desiccation-tolerant Sporobolus stapfianus and the desiccation-sensitive Sporobolus pyramidalis Both species are complex polyploids; S. stapfianus is primarily tetraploid, and S. pyramidalis is primarily hexaploid. S. pyramidalis undergoes a major transcriptome remodeling event during initial exposure to dehydration, while S. stapfianus has a muted early response, with peak remodeling during the transition between 1.5 and 1.0 grams of water (gH2O) g-1 dry weight (dw). Functionally, the dehydration transcriptome of S. stapfianus is unrelated to that for S. pyramidalis A comparative analysis of the transcriptomes of the hydrated controls for each species indicated that S. stapfianus is transcriptionally primed for desiccation. Cross-species comparative analyses indicated that VDT likely evolved from reprogramming of desiccation tolerance mechanisms that evolved in seeds and that the tolerance mechanism of S. stapfianus represents a recent evolution for VDT within the Chloridoideae. Orthogroup analyses of the significantly differentially abundant transcripts reconfirmed our present understanding of the response to dehydration, including the lack of an induction of senescence in resurrection angiosperms. The data also suggest that failure to maintain protein structure during dehydration is likely critical in rendering a plant desiccation sensitive.

The genotype does not influence the establishment of elephantgrass (Pennisetum purpureum Schum)

This study investigated whether genotype influences the establishment of Pennisetum purpureum Schumach. The experimental design was a randomized complete blocks with four treatments and eight replications (n=8). The treatments were four genotypes of P. purpureum, two classified as tall sizes: P. purpureum cv. Elephant B and cv. IRI 381; and two as dwarf types: P. purpureum cv. Mott and Taiwan A-146 2.37. They were planted in a tropical wet and dry region of Brazil. Tall genotypes showed superior field sprouting rates (p < 0.05), ranging between 95-99%, while dwarfs varied between 88-90%, however, Elephant B and IRI 381 produced a much lower average number of tillers (31 and 32 linear m-1, respectively), than Taiwan A-146 2.37 and Mott (56 and 41 linear m-1, respectively) (p < 0.05). Dwarf genotypes produced lower biomass yields (p < 0.05), but this was genotype-dependent and did not impact on their establishment. The levels of non-structural carbohydrates (NSC) (>10%) in the planted stems were associated with satisfactory field sprouting of the elephantgrass genotypes. Despite some variations between the genotypes in terms of sprouting, tillering, and growth rates, the kind of genotype had no major significance on the establishment of the elephantgrass.(AU)

Angioedema Without Wheals: Challenges in Laboratorial Diagnosis.

Angioedema is a prevailing symptom in different diseases, frequently occurring in the presence of urticaria. Recurrent angioedema without urticaria (AE) can be hereditary (HAE) and acquired (AAE), and several subtypes can be distinguished, although clinical presentation is quite similar in some of them. They present with subcutaneous and mucosal swellings, affecting extremities, face, genitals, bowels, and upper airways. AE is commonly misdiagnosed due to restricted access and availability of appropriate laboratorial tests. HAE with C1 inhibitor defect is associated with quantitative and/or functional deficiency. Although bradykinin-mediated disease results mainly from disturbance in the kallikrein-kinin system, traditionally complement evaluation has been used for diagnosis. Diagnosis is established by nephelometry, turbidimetry, or radial immunodiffusion for quantitative measurement of C1 inhibitor, and chromogenic assay or ELISA has been used for functional C1-INH analysis. Wrong handling of the samples can lead to misdiagnosis and, consequently, mistaken inappropriate approaches. Dried blood spot (DBS) tests have been used for decades in newborn screening for certain metabolic diseases, and there has been growing interest in their use for other congenital conditions. Recently, DBS is now proposed as an efficient tool to diagnose HAE with C1 inhibitor deficiency, and its use would improve the access to outbound areas and family members. Regarding HAE with normal C1 inhibitor, complement assays' results are normal and the genetic sequencing of target genes, such as exon 9 of F12 and PLG, is the only available method. New methods to measure cleaved high-molecular-weight kininogen and activated plasma kallikrein have emerged as potential biochemical tests to identify bradykinin-mediated angioedema. Validated biomarkers of kallikrein-kinin system activation could be helpful in differentiating mechanisms of angioedema. Our aim is to focus on the capability to differentiate histaminergic AE from bradykinin-mediated AE. In addition, we will describe the challenges developing specific tests like direct bradykinin measurements. The need for quality tests to improve the diagnosis is well represented by the variability of results in functional assays.

Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme.

The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous kinetic results suggested functionally relevant different R-states of the maize enzyme (ZmPEPC-C4) elicited by PEP or its two kinds of activators, glucose 6-phosphate or glycine. To detect these different R-state conformations, we used as conformational probes the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS), near-UV circular dichroism (CD) spectroscopy, and limited proteolysis by trypsin. Phosphoenolpyruvate and malate binding caused distinct concentration-dependent fluorescence changes of ZmPEPC-C4/ANS, suggesting that they elicited conformational states different from that of the free enzyme, while glucose 6-phosphate or glycine binding did not produce fluorescence changes. Differences were also observed in the near UV CD spectra of the enzyme, free or complexed with its substrate or allosteric effectors. Additionally, differences in the trypsin-digestion fragmentation patterns, as well as in the susceptibility of the free and complexed enzyme to digestion and digestion-provoked loss of activity, provided evidence of several ZmPEPC-C4 conformations in solution elicited by the substrate and the allosteric effectors. Using the already reported ZmPEPC-C4 crystal structures and bioinformatics methods, we predicted that the most probable trypsin-cleavage sites are located in superficial flexible regions, which seems relevant for the protein dynamics underlying the function and allosteric regulation of this enzyme. Together, our findings agree with previous kinetic results, shed light on this enzyme's complex allosteric regulation, and place ZmPEPC-C4 in the growing list of allosteric enzymes possessing an ensemble of closely related R-state conformations.

The C4 cycle and beyond: diverse metabolic adaptations accompany dual-cell photosynthetic functions in Setaria.

C4 photosynthesis is typically characterized by the spatial compartmentalization of the photosynthetic reactions into mesophyll (M) and bundle sheath (BS) cells. Initial carbon fixation within M cells gives rise to C4 acids, which are transported to the BS cells. There, C4 acids are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle. This work is focused on the study of Setaria viridis, a C4 model plant, closely related to several major feed and bioenergy grasses. First, we performed the heterologous expression and biochemical characterization of Setaria isoforms for chloroplastic NADP-malic enzyme (NADP-ME) and mitochondrial NAD-malic enzyme (NAD-ME). The kinetic parameters obtained agree with a major role for NADP-ME in the decarboxylation of the C4 acid malate in the chloroplasts of BS cells. In addition, mitochondria-located NAD-ME showed regulatory properties that could be important in the context of the operation of the C4 carbon shuttle. Secondly, we compared the proteomes of M and BS compartments and found 825 differentially accumulated proteins that could support different metabolic scenarios. Most interestingly, we found evidence of metabolic strategies to insulate the C4 core avoiding the leakage of intermediates by either up-regulation or down-regulation of chloroplastic, mitochondrial, and peroxisomal proteins. Overall, the results presented in this work provide novel data concerning the complexity of C4 metabolism, uncovering future lines of research that will undoubtedly contribute to the expansion of knowledge on this topic.

Bioaccessibility and Cellular Uptake of Carotenoids Extracted from Bactris gasipaes Fruit: Differences between Conventional and Ionic Liquid-Mediated Extraction.

Currently, on an industrial scale, synthetic colorants are used in many fields, as well as those extracted with conventional organic solvents (COSs), leading to several environmental issues. Therefore, we developed a sustainable extraction and purification method mediated by ionic liquids (IL), which is considered an alternative high-performance replacement for COSs. Carotenoids are natural pigments with low bioaccessibility (BCT) and bioavailability (BV) but with huge importance to health. To investigate if the BCT and cellular uptake of the carotenoids are modified by the extraction method, we conducted a comparison assay between both extraction procedures (IL vs. COS). For this, we used the Amazonian fruit Bactris gasipaes, a rich source of pro-vitamin A carotenoids, to obtain the extract, which was emulsified and subjected to an in vitro digestion model followed by the Caco-2 cell absorption assay. The bioaccessibility of carotenoids using IL was better than those using COS (33.25%, and 26.84%, respectively). The cellular uptake of the carotenoids extracted with IL was 1.4-fold higher than those extracted using COS. Thus, IL may be a feasible alternative as extraction solvent in the food industry, replacing COS, since, in this study, no IL was present in the final extract.