Innovative sustainable bioreactor-in-a-granule formulation of Trichoderma asperelloides.

The advancement of fungal biocontrol agents depends on replacing cereal grains with low-cost agro-industrial byproducts for their economical mass production and development of stable formulations. We propose an innovative approach to develop a rice flour-based formulation of the beneficial biocontrol agent Trichoderma asperelloides CMAA1584 designed to simulate a micro-bioreactor within the concept of full biorefinery process, affording in situ conidiation, extended shelf-life, and effective control of Sclerotinia sclerotiorum, a devastating pathogen of several dicot agricultural crops worldwide. Rice flour is an inexpensive and underexplored byproduct derived from broken rice after milling, capable of sustaining high yields of conidial production through our optimized fermentation-formulation route. Conidial yield was mainly influenced by nitrogen content (0.1% w/w) added to the rice meal coupled with the fermentor type. Hydrolyzed yeast was the best nitrogen source yielding 2.6 × 109 colony-forming units (CFU)/g within 14 days. Subsequently, GControl, GLecithin, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru formulations were obtained by extrusion followed by air-drying and further assessed for their potential to induce secondary sporulation in situ, storage stability, and efficacy against Sclerotinia. GControl, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru stood out with the highest number of CFU after sporulation upon re-hydration on water-agar medium. Shelf-life of formulations GControl and GBentonite remained consistent for > 3 months at ambient temperature, while in GBentonite and GOrganic compost+Break-Thru formulations remained viable for 24 months during refrigerated storage. Formulations exhibited similar efficacy in suppressing the myceliogenic germination of Sclerotinia irrespective of their concentration tested (5 × 104 to 5 × 106 CFU/g of soil), resulting in 79.2 to 93.7% relative inhibition. Noteworthily, all 24-month-old formulations kept under cold storage successfully suppressed sclerotia. This work provides an environmentally friendly bioprocess method using rice flour as the main feedstock to develop waste-free granular formulations of Trichoderma conidia that are effective in suppressing Sclerotinia while also improving biopesticide shelf-life. KEY POINTS: • Innovative "bioreactor-in-a-granule" system for T. asperelloides is devised. • Dry granules of aerial conidia remain highly viable for 24 months at 4 °C. • Effective control of white-mold sclerotia via soil application of Trichoderma-based granules.

Functional properties of starch cultivars of two Andean grains grown in Bolivia: Amaranth (Amaranthus caudatus) and canihua (Chenopodium pallidicaule).

The functional properties of Andean grain starches of two species, amaranth (Amaranthus caudatus) and canihua (Chenopodium pallidicaule), three cultivars each, were studied. The study focused on chemical composition, pasting properties, thermal properties, water solubility index (WSI), swelling power (SP), and granule morphology. All amaranth starches were waxy starches, with amylose content less than 5 %, which had some differences in chemical composition (p < 0.05). The pasting properties differed between the species, canihua showed more resistance, than amaranth, to heat and shear stress (higher cool paste (CPV) and lower breakdown (BD), ranged between 1250 and 1600 cP and -30 - 10 cP respectively. The amaranth starches presented only similar CPV with 800-1000 cP, while canihua cultivars presented similar PT and BD, and both species presented similar PV, around 1000 cP. Thermal properties (To, Tp, Tc, ΔH, and ΔT) differed among cultivars and species. These differences could be related to the homogeneity molecular structure and content of amylose in canihua cultivars and possibly to genotype factor. Polygonal shapes were the predominant shape of starch granules, ranged 1.0-1.4 µm and 0.8-0.9 µm, for amaranth and canihua starches respectively. Amaranth starches swelled quickly to disintegrate partially at the end, contrary to canihua starches. The thermal and pasting properties were correlated between them. SB, CPV, HPV, CS, were correlated to the content of amylose in canihua starches. One amaranth cultivar was significantly different from the others. Thus, according the functional properties differenced both species and some cultivars in each species. Additionally, the amaranth starch has the potential to be used in the food industry where heat and stress are applied such as extrusion, while canihua starches can be used in desserts or in cosmetic uses, based on their functional properties.

ROS produced by NOX promote the neurite growth in a PI3K/Akt independent manner.

Reactive oxygen species (ROS) function as signaling molecules in several physiologic and pathologic processes. In central nervous system, ROS are critical for differentiation, migration, polarization, and neurite growth. These actions are mediated by reversible oxidation of target proteins. On the other hand, PI3K/Akt signaling pathway is susceptible to be modulated by ROS and it has been implicated in neurite growth. In this study, we evaluated the participation of ROS in the neurite growth of cultured rat cerebellar granule neurons (CGN), as well as the possible regulation of the PI3K/Akt pathway by ROS during neurite outgrowth. For this purpose, CGN were treated with cellular or mitochondrial antioxidants, or an NOX inhibitor and neurite growth was evaluated. Moreover, to assess the participation Akt in this process, the p-Akt levels were measured in CGN treated with antioxidants or a NOX inhibitor. The effect of antioxidants on the neurite growth in the presence of a PI3K inhibitor was also measured. We found that cellular antioxidants and the NOX inhibitor decreased the neurite growth, but not the mitochondrial antioxidant. Interestingly, the antioxidants increased the p-Akt levels; however, the effect of antioxidants on neurite growth was no dependent on the Akt activity since the inhibitor of PI3K did not modify the antioxidant action on neurite growth. Our results show that the PI3K/Akt pathway participates in neurite growth and that ROS produced by NOX could function as signals in this process; however, this action is not mediated by a redox regulation of Akt activity.

Acute and sub-acute toxicity study of anti-obesity herbal granules in Sprague Dawley rats / Estudo de toxicidade aguda e subaguda de grânulos de ervas antiobesidade em ratos Sprague Dawley

Toxicological studies are essential for developing novel medications in pharmaceutical industries including ayurvedic preparation. Hence, the present study is aimed to evaluate acute and 28-days repeated dose oral toxicity of anti-obesity polyherbal granules (PHG) in Sprague Dawley rats by OECD guidelines No 425 and 407, respectively. In an acute oral toxicity study, a single dose of 2 g/kg PHG was administered to rats and mortality, body weight, and clinical observations were noted for fourteen days. However, in the subacute oral toxicity study, the PHG was administered orally at doses of 0.3, 0.5 and 1 g/kg daily for 28 days to rats. Food intake and body weight were recorded weekly. On the 29th day, rats were sacrificed and subjected to haematological, biochemical, urine, necropsy, and histopathological analysis. In an acute oral toxicity study, no treatment-related, mortality, behavioral changes, and toxicity were found throughout fourteen days. Likewise, in the sub-acute toxicity study, no mortality and toxic effects were found in haematology, biochemical, urine, necropsy and histopathological analysis in rats for 28 days of treatment with PHG. Based on these results, the LD50 of PHG was found to be greater than 2 g/kg and the no-observed-adverse-effect level (NOAEL) of PHG for rats was found to be 0.5 g/kg/day. Thus, anti-obesity polyherbal granules showed a good safety profile in animal studies and can be considered an important agent for the clinical management of obesity.

Estudos toxicológicos são essenciais para o desenvolvimento de novos medicamentos nas indústrias farmacêuticas, incluindo a preparação aiurvédica. Assim, o presente estudo tem como objetivo avaliar a toxicidade oral aguda e de dose repetida de 28 dias de grânulos de polierva (PHG) antiobesidade em ratos Sprague Dawley pelas diretrizes da OCDE nº 425 e 407, respectivamente. Em um estudo de toxicidade oral aguda, uma dose única de 2 g/kg de PHG foi administrada a ratos, e mortalidade, peso corporal e observações clínicas foram observadas por 14 dias. No entanto, no estudo de toxicidade oral subaguda, o PHG foi administrado oralmente em doses de 0,3, 0,5 e 1 g/kg diariamente por 28 dias em ratos. A ingestão alimentar e o peso corporal foram registrados semanalmente. No 29º dia, os ratos foram sacrificados e submetidos a análises hematológicas, bioquímicas, de urina, necropsia e histopatológica. Em um estudo de toxicidade oral aguda, nenhuma mortalidade, alterações comportamentais e toxicidade relacionadas ao tratamento foram encontradas ao longo de 14 dias. Da mesma forma, no estudo de toxicidade subaguda, não foram encontrados mortalidade e efeitos tóxicos em análises hematológicas, bioquímicas, de urina, necropsia e histopatológica em ratos durante 28 dias de tratamento com PHG. Com base nesses resultados, verificou-se que a DL50 de PHG era superior a 2 g/kg e o nível de efeitos adversos não observados (NOAEL) de PHG para ratos foi de 0,5 g/kg/dia. Assim, os grânulos poliervais antiobesidade apresentaram um bom perfil de segurança em estudos com animais e podem ser considerados um importante agente para o manejo clínico da obesidade.

Electroacupuncture promotes neurogenesis in the dentate gyrus and improves pattern separation in an early Alzheimer's disease mouse model.

BACKGROUND: Impaired pattern separation occurs in the early stage of Alzheimer's disease (AD), and hippocampal dentate gyrus (DG) neurogenesis participates in pattern separation. Here, we investigated whether spatial memory discrimination impairment can be improved by promoting the hippocampal DG granule cell neogenesis-mediated pattern separation in the early stage of AD by electroacupuncture (EA). METHODS: Five familial AD mutations (5 × FAD) mice received EA treatment at Baihui and Shenting points for 4 weeks. During EA, mice were intraperitoneally injected with BrdU (50 mg/kg) twice a day. rAAV containing Wnt5a shRNA was injected into the bilateral DG region, and the viral efficiency was evaluated by detecting Wnt5a mRNA levels. Cognitive behavior tests were conducted to assess the impact of EA treatment on cognitive function. The hippocampal DG area Aß deposition level was detected by immunohistochemistry after the intervention; The number of BrdU+/CaR+ cells and the gene expression level of calretinin (CaR) and prospero homeobox 1(Prox1) in the DG area of the hippocampus was detected to assess neurogenesis by immunofluorescence and western blotting after the intervention; The gene expression levels of FZD2, Wnt5a, DVL2, p-DVL2, CaMKII, and p-CaMKII in the Wnt signaling pathway were detected by Western blotting after the intervention. RESULTS: Cognitive behavioral tests showed that 5 × FAD mice had impaired pattern separation (P < 0.001), which could be improved by EA (P < 0.01). Immunofluorescence and Western blot showed that the expression of Wnt5a in the hippocampus was decreased (P < 0.001), and the neurogenesis in the DG was impaired (P < 0.001) in 5 × FAD mice. EA could increase the expression level of Wnt5a (P < 0.05) and promote the neurogenesis of immature granule cells (P < 0.05) and the development of neuronal dendritic spines (P < 0.05). Interference of Wnt5a expression aggravated the damage of neurogenesis (P < 0.05), weakened the memory discrimination ability (P < 0.05), and inhibited the beneficial effect of EA (P < 0.05) in AD mice. The expression level of Wnt pathway related proteins such as FZD2, DVL2, p-DVL2, CAMKII, p-CAMKII increased after EA, but the effect of EA was inhibited after Wnt5a was knocked down. In addition, EA could reduce the deposition of Aß plaques in the DG without any impact on Wnt5a. CONCLUSION: EA can promote hippocampal DG immature granule cell neogenesis-mediated pattern separation to improve spatial memory discrimination impairment by regulating Wnt5a in 5 × FAD mice.

Corrigendum: Txnip expression promotes JNK-mediated neuronal death in response to reactive oxygen species.

[This corrects the article DOI: 10.3389/fnmol.2023.1210962.].

Txnip expression promotes JNK-mediated neuronal death in response to reactive oxygen species.

TXNIP is a protein sensitive to oxidant conditions whose expression is related to the progression of death in cancer, diabetes, ischemia, and neurodegenerative diseases, among others. Because of this, many studies propose TXNIP as a therapeutic target in several diseases. Exposure of cerebellar granule neurons to staurosporine or low potassium leads to apoptotic death. Both conditions generate an early production of reactive oxygen species (ROS) that induces the activation of the ASK1 pathway and the apoptotic machinery. In these models, it has been shown an increase in TXNIP protein mediated by ROS. Here, we evaluated the molecular mechanisms involved in the regulation of the Txnip expression during neuronal death, as well as the role of the protein in the progression of cell death induced by these two apoptotic conditions. In cultured cerebellar granule neurons, we observed that low potassium and staurosporine induced an early increase in ROS that correlated with an increase in Txnip mRNA. When we evaluated the promoter of the gene, we found that the JASPAR-reported FOXO1/3 transcription factor motifs are close to the transcription start site (TSS). We then verified through the Chromatin immunoprecipitation technique (ChIP) that FOXO3 interacts with the Txnip promoter after 1 h of low potassium treatment. We also detected FOXO3 nuclear translocation by low potassium and staurosporine treatments. Finally, by using shRNA in the neuroblastoma MSN cell line, we found that Txnip downregulation decreased neuronal death induced by staurosporine stimulus. Together, these results suggest that ROS promotes the expression of Txnip through the activation of the FOXO3 transcription factor mediated by Akt inhibition. We also demonstrated that TXNIP is necessary for neuronal death progression.

Condensation of the ß-cell secretory granule luminal cargoes pro/insulin and ICA512 RESP18 homology domain.

ICA512/PTPRN is a receptor tyrosine-like phosphatase implicated in the biogenesis and turnover of the insulin secretory granules (SGs) in pancreatic islet beta cells. Previously we found biophysical evidence that its luminal RESP18 homology domain (RESP18HD) forms a biomolecular condensate and interacts with insulin in vitro at close-to-neutral pH, that is, in conditions resembling those present in the early secretory pathway. Here we provide further evidence for the relevance of these findings by showing that at pH 6.8 RESP18HD interacts also with proinsulin-the physiological insulin precursor found in the early secretory pathway and the major luminal cargo of ß-cell nascent SGs. Our light scattering analyses indicate that RESP18HD and proinsulin, but also insulin, populate nanocondensates ranging in size from 15 to 300 nm and 10e2 to 10e6 molecules. Co-condensation of RESP18HD with proinsulin/insulin transforms the initial nanocondensates into microcondensates (size >1 µm). The intrinsic tendency of proinsulin to self-condensate implies that, in the ER, a chaperoning mechanism must arrest its spontaneous intermolecular condensation to allow for proper intramolecular folding. These data further suggest that proinsulin is an early driver of insulin SG biogenesis, in a process in which its co-condensation with RESP18HD participates in their phase separation from other secretory proteins in transit through the same compartments but destined to other routes. Through the cytosolic tail of ICA512, proinsulin co-condensation with RESP18HD may further orchestrate the recruitment of cytosolic factors involved in membrane budding and fission of transport vesicles and nascent SGs.

Isolation and Purification of Chromaffin Granules from Adrenal Glands and Cultured Neuroendocrine Cells.

Chromaffin granules isolated from adrenal glands constitute a powerful experimental tool to the study of secretory vesicle components and their participation in fusion and docking processes, vesicle aggregation, and interactions with cytosolic components. Although it is possible to isolate and purify chromaffin granules from adrenal glands of different species, bovine adrenal glands are the most used tissue source due to its easy handling and the large amount of granules that can be obtained from this tissue. In this chapter, we describe an easy-to-use and short-term protocol for efficiently obtaining highly purified chromaffin granules from bovine adrenal medulla. We additionally include protocols to isolate granules from cultured bovine chromaffin cells and PC12 cells, as well as a section to obtain chromaffin granules from mouse adrenal glands.

Exogenous amylase increases gas production and improves in vitro ruminal digestion kinetics of sorghum and corn grains / Amilase exógena aumenta a produção de gases e melhora a cinética de digestão ruminal in vitro de grãos de sorgo e milho

The aim of this study was to evaluate the effect of exogenous amylase on gas production, in vitro dry matter digestibility (IVDMD), and in vitro digestion kinetics of sorghum (Sorghum vulgaris) and two corn hybrids of different grain textures. Ruminal fluid was collected from two rumen-fistulated cows receiving or not exogenous amylase (0.7g kg-1 of dry matter (DM basis)), provided to achieve 396 kilo Novo units kg-1 for amylase activity (DM basis). Gas production was measured after 1, 3, 6, 9, 12, 15, 18, 21, 24, 30, 36, 42 e 48 hours of incubation. Amylase increased gas production (mL) by 5.4%. Corn hybrids have higher in vitro dry matter digestibility than sorghum. Exogenous amylase increased the potential of gas production (A) (P=0.01). There was an effect of hybrid for IVDMD (P<0.01). The addition of exogenous amylase increases the in vitro gas production, improves fermentation kinetics, and increases the production of the ammonia nitrogen of corn and sorghum grains, but does not affect in vitro and dry matter digestibility or the short-chain fatty acids production.

O objetivo deste estudo foi avaliar o efeito da amilase exógena na produção de gases, a digestibilidade in vitro da matéria seca (DIVMS) e a cinética de digestão in vitro de sorgo (Sorghum vulgaris) e de dois híbridos de milho de diferentes texturas de grãos. O líquido ruminal foi coletado de duas vacas fistuladas no rúmen recebendo ou não amilase exógena (0,7g kg-1 de matéria seca (MS)), fornecida para atingir 396 kg Novo unidades kg-1 para atividade de amilase (base na MS). A produção de gás foi medida após uma, três, seis, nove, 12, 15, 18, 21, 24, 30, 36, 42 e 48 horas de incubação. A amilase aumentou a produção de gás (mL) em 5,4%. Híbridos de milho apresentam maior DIVMS que o sorgo. A amilase exógena aumentou o potencial de produção de gás (A) (P=0,01). Houve efeito de híbrido para DIVMS (P<0,01). Amilase exógena aumenta a produção de gás in vitro, melhora a cinética da fermentação e aumenta a produção de nitrogênio amoniacal de grãos de milho e sorgo, mas não afeta a digestibilidade in vitro da matéria seca ou a produção de ácidos graxos de cadeia curta.

TDP-43 CSF Concentrations Increase Exponentially with Age in Metropolitan Mexico City Young Urbanites Highly Exposed to PM2.5 and Ultrafine Particles and Historically Showing Alzheimer and Parkinson's Hallmarks. Brain TDP-43 Pathology in MMC Residents Is Associated with High Cisternal CSF TDP-43 Concentrations.

Environmental exposures to fine particulate matter (PM2.5) and ultrafine particle matter (UFPM) are associated with overlapping Alzheimer's, Parkinson's and TAR DNA-binding protein 43 (TDP-43) hallmark protein pathologies in young Metropolitan Mexico City (MMC) urbanites. We measured CSF concentrations of TDP-43 in 194 urban residents, including 92 MMC children aged 10.2 ± 4.7 y exposed to PM2.5 levels above the USEPA annual standard and to high UFPM and 26 low pollution controls (11.5 ± 4.4 y); 43 MMC adults (42.3 ± 15.9 y) and 14 low pollution adult controls (33.1 ± 12.0 y); and 19 amyotrophic lateral sclerosis (ALS) patients (52.4 ± 14.1 y). TDP-43 neuropathology and cisternal CSF data from 20 subjects­15 MMC (41.1 ± 18.9 y) and 5 low pollution controls (46 ± 16.01 y)­were included. CSF TDP-43 exponentially increased with age (p < 0.0001) and it was higher for MMC residents. TDP-43 cisternal CSF levels of 572 ± 208 pg/mL in 6/15 MMC autopsy cases forecasted TDP-43 in the olfactory bulb, medulla and pons, reticular formation and motor nuclei neurons. A 16 y old with TDP-43 cisternal levels of 1030 pg/mL exhibited TDP-43 pathology and all 15 MMC autopsy cases exhibited AD and PD hallmarks. Overlapping TDP-43, AD and PD pathologies start in childhood in urbanites with high exposures to PM2.5 and UFPM. Early, sustained exposures to PM air pollution represent a high risk for developing brains and MMC UFPM emissions sources ought to be clearly identified, regulated, monitored and controlled. Prevention of deadly neurologic diseases associated with air pollution ought to be a public health priority and preventive medicine is key.

A Direct Interaction between Cyclodextrins and TASK Channels Decreases the Leak Current in Cerebellar Granule Neurons.

Two pore domain potassium channels (K2P) are strongly expressed in the nervous system (CNS), where they play a central role in excitability. These channels give rise to background K+ currents, also known as IKSO (standing-outward potassium current). We detected the expression in primary cultured cerebellar granule neurons (CGNs) of TWIK-1 (K2P1), TASK-1 (K2P3), TASK-3 (K2P9), and TRESK (K2P18) channels by immunocytochemistry and their association with lipid rafts using the specific lipids raft markers flotillin-2 and caveolin-1. At the functional level, methyl-ß-cyclodextrin (MßCD, 5 mM) reduced IKSO currents by ~40% in CGN cells. To dissect out this effect, we heterologously expressed the human TWIK-1, TASK-1, TASK-3, and TRESK channels in HEK-293 cells. MßCD directly blocked TASK-1 and TASK-3 channels and the covalently concatenated heterodimer TASK-1/TASK-3 currents. Conversely, MßCD did not affect TWIK-1- and TRESK-mediated K+ currents. On the other hand, the cholesterol-depleting agent filipin III did not affect TASK-1/TASK-3 channels. Together, the results suggest that neuronal background K+ channels are associated to lipid raft environments whilst the functional activity is independent of the cholesterol membrane organization.

Platelet VPS16B is dependent on VPS33B expression, as determined in two siblings with arthrogryposis, renal dysfunction, and cholestasis syndrome.

BACKGROUND: Platelet α-granule biogenesis in precursor megakaryocytes is critically dependent on VPS33B and VPS16B, as demonstrated by the platelet α-granule deficiency seen in the rare multisystem disorder arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome associated with biallelic pathogenic variants in VPS33B and VIPAS39 (encoding VPS16B). VPS33B and VPS16B are ubiquitously expressed proteins that are known to interact and play key roles in protein sorting and trafficking between subcellular locations. However, there remain significant gaps in our knowledge of the nature of these interactions in primary cells from patients with ARC syndrome. OBJECTIVES: To use primary cells from patients with ARC syndrome to better understand the interactions and roles of VPS33B and VPS16B in platelets and precursor megakaryocytes. PATIENTS/METHODS: The proband and his male sibling were clinically suspected to have ARC syndrome. Confirmatory genetic testing and platelet phenotyping, including electron microscopy and protein expression analysis, was performed with consent in a research setting. RESULTS: We describe the first case of ARC syndrome identified in Costa Rica, associated with a novel homozygous nonsense VPS33B variant that is linked with loss of expression of both VPS33B and VPS16B in platelets. CONCLUSION: These results indicate that stable expression of VPS16B in platelets, their precursor megakaryocytes, and other cells is dependent on VPS33B. We suggest that systematic evaluation of primary cells from patients with a range of VPS33B and VIPAS39 variants would help to elucidate the interactions and functions of these proteins.

NMR characterization of structure and moisture sorption dynamics of damaged starch granules.

Among the many biopolymers that constitute food products, starch is one of the most common. Starch granules are often damaged in the milling process, which affects the final product quality, mainly due to changes in water adsorption properties. In this work, the crystallinity degree of wheat starch samples as a function of the mechanical damage is determined by low field 1H NMR. We also introduce the use of single-sided NMR to determine granular swelling, water distribution and sorption dynamics of the samples. Results show that the crystallinity of the samples decreases with the milling. We also observed that swelling index and sorption capacity values are higher in the milled samples than in the native starch. Our experiments show how single-sided NMR is a valuable tool to provide information on dynamic processes not only in starch, but also in many carbohydrate polymeric samples with the additional benefit of spatial resolution.

Starch synthesis and gelatinization properties of potato tubers / Síntese de amido e propriedades de gelatinização de tubérculos de batata com diferentes teores de amido

Biosynthesis is the only source of potato starch which is an important raw material for food processing, modified starch and biomass energy. However, it is not clear about the evolution of starch synthesis with tuber development in potato. The present study evaluated the differences of starch synthesis and gelatinization properties of potato tubers with different starch content. Relative to cultivars of medium and low starch content, cultivars of high starch content showed significantly higher SBEII gene expression, AGPase and SSS enzyme activity, and total starch content after middle stage of starch accumulation, and had smaller average starch granule size during whole process of tuber development, and had higher pasting temperature before late stages of tuber growth, and had lower pasting temperature after middle stage of starch accumulation. Path analysis showed that, after middle stage of starch accumulation, effects on starch gelatinization of cultivars with high, medium and low starch content represented starch synthesis enzyme activity > starch accumulation > starch granule distribution > starch synthesis enzyme gene expression, starch synthesis enzyme gene expression > starch synthesis enzyme activity > starch accumulation > starch granule distribution, starch synthesis enzyme gene expression > starch granule distribution > starch synthesis enzyme activity > starch accumulation, respectively. In the study, phases existed in the starch biosynthesis of potato tuber, and the starch quality and its formation process were different among varieties with different starch content. The findings might contribute to starch application and potato industries.

A biossíntese é a única fonte de amido de batata que é uma importante matéria-prima para o processamento de alimentos, amido modificado e energia de biomassa. No entanto, não está claro sobre a evolução da síntese do amido com o desenvolvimento do tubérculo na batata. O presente estudo teve como objetivo avaliar as diferenças nas propriedades de síntese e gelatinização do amido de tubérculos de batata com diferentes teores de amido. Em relação às cultivares de médio e baixo teor de amido, as cultivares de alto teor de amido apresentaram expressão do gene SBEII, atividade enzimática AGPase e SSS e teor de amido total significativamente maiores após o estágio intermediário de acúmulo de amido, bem como menor tamanho médio dos grânulos de amido durante todo o processo de desenvolvimento do tubérculo, maior temperatura de colagem antes dos estágios finais de crescimento do tubérculo e menor temperatura de colagem após o estágio intermediário de acúmulo de amido. A análise de trilha mostrou que, após o estágio intermediário de acúmulo de amido, os efeitos na gelatinização do amido de cultivares com alto, médio e baixo teor de amido representaram a atividade da enzima de síntese de amido> acúmulo de amido> distribuição de grânulos de amido> expressão gênica de enzima de síntese de amido; expressão gênica de enzima de síntese de amido > atividade da enzima de síntese de amido> acúmulo de amido> distribuição de grânulos de amido; expressão gênica da enzima de síntese de amido> distribuição de grânulos de amido> atividade de síntese de amido> acúmulo de amido, respectivamente. No estudo, as fases existentes na biossíntese do amido do tubérculo de batata, e a qualidade do amido e seu processo de formação foram diferentes entre as variedades com diferentes teores de amido. As descobertas podem contribuir para a aplicação de amido e as indústrias de batata.

Optimization of the Extragranular Excipient Composition of Paracetamol Tablet formulation using the Quality by Design Approach

Abstract The purpose of this study is to optimize the composition of extragranular excipients (EGE) and mixing time of granules with EGE of paracetamol tablet formulation using Design of Experiments (DoE) approach. The effect of the composition of EGE and the mixing time of granules with EGE on granule and tableting properties of paracetamol tablet formulation was investigated using a combined model of mixture and process factors (Design-Expert 12). A total of 18 tablet formulations were manufactured by wet granulation using varying compositions of EGE and varying mixing time. Granule and tablet properties of each formulation were evaluated as response variables for the design, data generated were fitted into models and analysed to generate a design space that was used for optimization studies. The proposed EGE composition as predicted by the design was confirmed and validated after preparation and evaluation of the granule and tablet properties. The optimized composition for the EGE that yielded granules and tablets of desirable characteristics was found to be maize starch (5 %), talc (4.9 %) and magnesium stearate (0.1 %) with a mixing time of 2 min. The tablets produced with the optimized composition had better mechanical strength and disintegration time than the formulation prepared using an existing formula of maize starch (7.8 %), talc (2 %) and magnesium stearate (0.2 %) that were obtained using the One Variable at a Time (OVAT) approach. This study confirmed the relevance of quality by design in development of pharmaceutical formulations.

VAMPs sensitive to tetanus toxin are required for cortical granule exocytosis in mouse oocytes.

Fusion of cortical granules with oocyte plasma membrane is one of the most significant secretory events to prevent polyspermy during oocyte activation. Cortical granule exocytosis (CGE) is distinct from most other exocytosis because cortical granules are not renewed after secretion. However, it is thought to be mediated by SNARE complex, which mediates membrane fusion in other exocytoses. SNAREs proteins are divided into Q (glutamine)- and R (arginine)-SNAREs. Q-SNAREs include Syntaxins and SNAP25 family, and R-SNAREs include VAMPs family. In mouse oocytes, Syntaxin4 and SNAP23 have been involved in CGE; nevertheless, it is unknown if VAMP is required. Here, we demonstrated by RT-PCR and immunoblotting that VAMP1 and VAMP3 are expressed in mouse oocyte, and they localized in the cortical region of this cell. Using a functional assay to quantify CGE, we showed that tetanus toxin -which specifically cleavages VAMP1, VAMP2 or VAMP3- inhibited CGE suggesting that at least one VAMP was necessary. Function blocking assays demonstrated that only the microinjection of anti-VAMP1 or anti-VAMP3 antibodies abolished CGE in activated oocytes. These findings demonstrate that R-SNAREs sensitive to tetanus toxin, VAMP1 and VAMP3 -but not VAMP2-, are required for CGE and demonstrate that CGE is mediated by the SNARE complex.

Relative humidity impacts development and activity against Aedes aegypti adults by granular formulations of Metarhizium humberi microsclerotia.

The impact of ambient relative humidity (RH) on conidial production of Metarhizium humberi IP 46 microsclerotia (MS) formulated in pellets or granules was investigated, and a promising granular formulation was tested against Aedes aegypti adults to confirm its efficacy. Microcrystalline cellulose (MC) and diatomaceous earth (DE) or a combination of vermiculite (VE), DE and silicon dioxide (SD) were tested as carriers in granular formulations containing MS. A range of 93-96.5% RH was critical for fungal development, and at least 96.5-98.5% RH was required for high conidial production on pellets or granules. Conidial production was clearly higher on pellets and granules prepared with VE than MC as the main carrier. VE granules containing MS were highly active against A. aegypti adults. Most mosquitoes were killed within 6 days after treatment regardless of the exposure time of adults to the formulation (1 min-24 h) or ambient humidity (75 or >98%). Production of conidia on dead adults varied between 7.3 × 106 and 2.2 × 107 conidia/individual, when exposed to MS granules for 12 h and 1 min, respectively. Granular formulations containing VE as the main carrier and MS as the active ingredient of M. humberi have strong potential for use against A. aegypti. KEY POINTS: • High conidial production on granular microsclerotial formulations at >96.5% RH • Vermiculite is more appropriate as a carrier than microcrystalline cellulose • Granules with IP 46 microsclerotia are highly active against Aedes aegypti adults.

Treatment of real domestic sewage in a pilot-scale aerobic granular sludge reactor: Assessing start-up and operational control.

Aerobic granular sludge (AGS) has been considered a breakthrough in the wastewater treatment sector given its key characteristics, such as excellent settleability, simultaneous removal of organic and nutrient pollutants, and compactness. However, the formation of granules often delays the start-up of granular-based systems, especially in large-scale settings. This study addressed the start-up of a pilot-scale AGS sequencing batch reactor (SBR) treating domestic sewage, monitored for over 280 days. The challenges faced during aerobic granulation using a mixture of activated sludge and anaerobic granular sludge as inoculum and the performance of the reactor on organic matter, nitrogen, and phosphorus removal were discussed. Results showed that robust and stable granules were formed after an initial period of around six months, with the settling time playing a key role on granules development. At least 80% of granules had a diameter greater than 0.2 mm and 60% >1 mm. In general, the reactor achieved high nitrogen removal efficiency, as well as satisfactory removal of soluble COD. However, total COD abatement was impaired by the various episodes of suspended solids loss with the effluent. Overall, this study demonstrated that the reactor was efficient in the treatment of domestic sewage, but its performance was adversely affected from sudden changes in the influent quality. PRACTITIONER POINTS: Aerobic granular sludge (AGS) applied to small-scale domestic sewage treatment. The control of sludge age in AGS can be a problem due to short sedimentation times. High DO to maintain aerobic granulation can economically make the process economically unfeasible in tropical countries. A sludge with excellent sedimentation properties was obtained. However, maintaining the granule over time is a challenge.

The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues.

Exocytosis is a fundamental process in physiology, that ensures communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. We report here a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, we found that Zda is required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen we identified the small GTPase RalA as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane.