The Miniaturized Isolation of Neutrophil Granules (MING) method allowed a deep proteome mapping of human neutrophil granules.

Neutrophils are the innate immune system's first line of defense, and their storage organelles are essential to their function. The storage organelles are divided into three different granule types named azurophilic, specific, and gelatinase granules, besides a fourth component called secretory vesicles. The isolation of neutrophil's granules is challenging, and the existing procedures rely on large sample volumes, about 400 mL of peripheral blood, precluding the use of multiple biological and technical replicates. Therefore, the aim of this study was to develop a miniaturized isolation of neutrophil granules (MING) method, using biochemical assays, mass spectrometry-based proteomics and a machine learning approach to investigate the protein content of these organelles. Neutrophils were isolated from 40 mL of blood collected from three apparently healthy volunteers and disrupted using nitrogen cavitation; the organelles were fractionated with a discontinuous 3-layer Percoll density gradient. The isolation was proven successful and allowed for a reasonable separation of neutrophil's storage organelles using a gradient approximately 37 times smaller than the methods described in the literature. Moreover, mass spectrometry-based proteomics identified 368 proteins in at least 3 of the 5 analyzed samples, and using a machine learning strategy aligned with markers from the literature, the localization of 50 proteins was predicted with confidence. When using markers determined within our dataset by a clusterization tool, the localization of 348 proteins was confidently determined. Importantly, this study was the first to investigate the proteome of neutrophil granules using technical and biological replicates, creating a reliable database for further studies.

The landscape of lncRNAs in cell granules: Insights into their significance in cancer.

Cellular compartmentalization, achieved through membrane-based compartments, is a fundamental aspect of cell biology that contributes to the evolutionary success of cells. While organelles have traditionally been the focus of research, membrane-less organelles (MLOs) are emerging as critical players, exhibiting distinct morphological features and unique molecular compositions. Recent research highlights the pivotal role of long noncoding RNAs (lncRNAs) in MLOs and their involvement in various cellular processes across different organisms. In the context of cancer, dysregulation of MLO formation, influenced by altered lncRNA expression, impacts chromatin organization, oncogenic transcription, signaling pathways, and telomere lengthening. This review synthesizes the current understanding of lncRNA composition within MLOs, delineating their functions and exploring how their dysregulation contributes to human cancers. Environmental challenges in tumorigenesis, such as nutrient deprivation and hypoxia, induce stress granules, promoting cancer cell survival and progression. Advancements in biochemical techniques, particularly single RNA imaging methods, offer valuable tools for studying RNA functions within live cells. However, detecting low-abundance lncRNAs remains challenging due to their limited expression levels. The correlation between lncRNA expression and pathological conditions, particularly cancer, should be explored, emphasizing the importance of single-cell studies for precise biomarker identification and the development of personalized therapeutic strategies. This article is categorized under: RNA Export and Localization > RNA Localization RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

The joint action of yeast eisosomes and membraneless organelles in response to ethanol stress.

Elevated ethanol concentrations in yeast affect the plasma membrane. The plasma membrane in yeast has many lipid-protein complexes, such as Pma1 (MCP), Can1 (MCC), and the eisosome complex. We investigated the response of eisosomes, MCPs, and membraneless structures to ethanol stress. We found a correlation between ethanol stress and proton flux with quick acidification of the medium. Moreover, ethanol stress influences the symporter expression in stressed cells. We also suggest that acute stress from ethanol leads to increases in eisosome size and SG number: we hypothesized that eisosomes may protect APC symporters and accumulate an mRNA decay protein in ethanol-stressed cells. Our findings suggest that the joint action of these factors may provide a protective effect on cells under ethanol stress.

Combining metabolomics and network pharmacology to investigate the protective effect of Jiawei Xinglou Chengqi Granules in ischemic stroke

Jiawei Xinglou Chengqi Granule (JXCG) is an effective herbal medicine for the treatment of ischemic stroke (IS). JXCG has been shown to effectively ameliorate cerebral ischemic symptoms in clinical practice, but the underlying mechanisms are unclear. In this study, we investigated the mechanisms of action of JXCG in the treatment of IS by combining metabolomics with network pharmacology. The chemical composition of JXCG was analyzed using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry (UHPLC-Q-TOF MS) untargeted metabolomics were used to identify differential metabolites within metabolic pathways. Network pharmacology was applied to mine potential targets of JXCG in the treatment of IS. The identified key targets were validated by constructing an integrated network of metabolomics and network pharmacology and by molecular docking using Cytoscape. The effect of JXCG on IS was evaluated in vivo, and the predicted targets and pathways of JXCG in IS therapy were assessed using immunoblotting. Combining metabolomics and network pharmacology, we identified the therapeutic targets of JXCG for IS. Notably, JXCG lessened neuronal damage and reduced cerebral infarct size in rats with IS. Western blot analysis showed that JXCG upregulated PRKCH and downregulated PRKCE and PRKCQ proteins. Our combined network pharmacology and metabolomics findings showed that JXCG may have therapeutic potential in the treatment of IS by targeting multiple factors and pathways.

The light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibits cortical reaction in mouse oocytes.

Introduction: Cortical reaction is a secretory process that occurs after a spermatozoon fuses with the oocyte, avoiding the fusion of additional sperm. During this exocytic event, the cortical granule membrane fuses with the oocyte plasma membrane. We have identified several molecular components involved in this process and confirmed that SNARE proteins regulate membrane fusion during cortical reaction in mouse oocytes. In those studies, we microinjected different nonpermeable reagents to demonstrate the participation of a specific protein in the cortical reaction. However, the microinjection technique has several limitations. In this work, we aimed to assess the potential of cell-penetrating peptides (CPP) as biotechnological tools for delivering molecules into oocytes, and to evaluate the functionality of the permeable tetanus toxin (bound to CPP sequence) during cortical reaction. Methods: Arginine-rich cell-penetrating peptides have demonstrated the optimal internalization of small molecules in mammalian cells. Two arginine-rich CPP were used in the present study. One, labeled with 5-carboxyfluorescein, to characterize the factors that can modulate its internalization, and the other, the permeable light chain of tetanus toxin, that cleaves the SNAREs VAMP1 and VAMP3 expressed in mouse oocytes. Results: Results showed that fluorescent CPP was internalized into the oocyte cytoplasm and that internalization was dependent on the concentration, time, temperature, and maturation stage of the oocyte. Using our functional assay to study cortical reaction, the light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibited cortical granules exocytosis. Discussion: Results obtained from the use of permeable peptides demonstrate that this CPP is a promising biotechnological tool to study functional macromolecules in mouse oocytes.

Ultrastructure of coarse granules in the perivitelline space and association with ovulation induction protocols.

OBJECTIVE: To evaluate the origin and ultrastructure of the coarse granules in the perivitelline space (PVS) of oocytes of a group of couples attending assisted reproduction treatment. METHODS: The ultrastructure of five oocytes with coarse granulues in the PVS obtained from three patients were evaluated by transmission electron microscopy (TEM). The influence of the ovulation induction regimen on the formation of granules in the PVS of the oocytes of 214 couples and the developmental capacity of these oocytes presenting granules in the PVS was analyzed retrospectively. RESULTS: In TEM analysis, the microvilli structure was irregular, short, and loosely scattered through the oolemma in the oocytes presenting coarse granules in the PVS. Furthermore, dense lipid droplets were identified within the PVS and the surrounding cumulus cells. In retrospective analysis, the number of oocytes with coarse granules in the PVS was positively correlated with the duration of antagonist administration (r=0.23, p=0.013). Regardless of the type of granule, the presence of coarse or moderately coarse granules in the PVS was positively correlated with low-quality embryos on D3 (r=0.29, p=0.005) and the total number of arrested embryos up to D3 (r=0.33, p<0.001). Furthermore, the presence of coarse granules in the PVS severely exacerbated miscarriage rates. CONCLUSIONS: Our findings suggest that the presence of especially coarse granules in the PVS is correlated with the reduction of further embryonic developmental capacity in post-implantation stages of embryonic development, indicating a negative impact from aggressive ovulation induction protocols on developing oocytes.

Altered Chief Cell Morphology in the Gastric Gland of Streptozotocin-Diabetic Rats

SUMMARY: Diabetes mellitus (DM) is a metabolic disorder with rising incidences worldwide. Gastric symptoms of DM have been reported, including nausea, vomiting, bloating, and epigastric pain. Moreover, acute to chronic gastritis and atrophic gastritis occur in DM can affect the chief cells of the gastric gland. Chief cells are vital because of their ability to digest and separate vitamin B12 from protein. Lack of vitamin B12 leads to impaired DNA synthesis and abnormal metabolism in red blood cells, and eventually leading to pernicious anemia. Furthermore, decreased vibratory and positional senses, numbness, ataxia with subacute combined degeneration, and dementia are present in pernicious anemic patients. Twenty-four male adult Sprague-Dawley rats were used in this study. The rats were divided into control (n = 12) and diabetic (n = 12) groups. The rats were further separated into two categories: short-term (4 weeks) and long-term (24 weeks) groups. DM model was induced by manually injecting intraperitoneally with streptozotocin in citrate buffer at a dose of 60 mg/kg body weight. The same amount of buffer was injected into the control group. After sacrifice, three regions of the stomach (the cardia, body, and pylorus) were dissected. Histopathology was performed by staining with toluidine blue. Image analysis was used to quantify the zymogen granule accumulation in chief cells. The data were compared between the control and DM rats in each period using Student's t-test. In addition, transmission electron microscopy (TEM) was also used to examine the ultrastructures. There was a significant decrease in the percentage of zymogen granules in DM rats. Under TEM, the destructions of mitochondria, rough endoplasmic reticulum, and Golgi apparatus in the DM rat were observed in the chief cells. In rats with uncontrolled diabetes, there is damage to the chief cells all over the area of the stomach, affecting digestion and malabsorption of vitamin B12. Therefore, this result helps clinicians recognize that diabetic patients with gastric symptoms may have hidden pernicious anemia.

La diabetes mellitus (DM) es un trastorno metabólico con incidencia creciente a nivel mundial. Se han informado síntomas gástricos de DM, que incluyen náuseas, vómitos, distensión abdominal y dolor epigástrico. Además, la gastritis aguda a crónica y la gastritis atrófica que ocurren en la DM pueden afectar las células principales de la glándula gástrica. Las células principales son vitales debido a su capacidad para digerir y separar la vitamina B12 de las proteínas. La falta de vitamina B12 conduce a una síntesis de ADN deteriorada y un metabolismo anormal en los glóbulos rojos, lo que eventualmente conduce a una anemia perniciosa. Además, los pacientes con anemia perniciosa presentan disminución de los sentidos vibratorio y posicional, entumecimiento, ataxia con degeneración combinada subaguda y demencia. En este estudio se usaron 24 ratas Sprague-Dawley macho adultas. Las ratas se dividieron en grupos control (n = 12) y diabéticas (n = 12). Las ratas se separaron además en dos categorías: grupos a corto plazo (4 semanas) y a largo plazo (24 semanas). El modelo de DM se indujo inyectando manualmente por vía intraperitoneal estreptozotocina en tampón de citrato a una dosis de 60 mg/kg de peso corporal. Se inyectó la misma cantidad de tampón en el grupo control. Después del sacrificio, se disecaron tres regiones del estómago (cardias, cuerpo y píloro). La histopatología se realizó mediante tinción con azul de toluidina. El análisis de imágenes se utilizó para cuantificar la acumulación de gránulos de zimógeno en las células principales. Los datos se compararon entre las ratas control y DM en cada período utilizando la prueba t de Student. Además, se utilizó microscopía electrónica de transmisión (TEM) para examinar la ultraestructura celular. Hubo una disminución significativa en el porcentaje de gránulos de zimógeno en ratas DM. Bajo TEM, se observaron en las células principales las destrucción de las mitocondrias, del retículo endoplásmico rugoso y del complejo golgiense en la rata DM. En ratas con diabetes no controlada, hay daño en las células principales de toda el área del estómago, lo que afecta la digestión y la malabsorción de vitamina B12. Por lo tanto, este resultado ayuda a los médicos a reconocer que los pacientes diabéticos con síntomas gástricos pueden tener una anemia perniciosa oculta.

Sustancia Cromatofílica: Una Propuesta de Cambio a la Terminologia Histologica y Terminologia Neuroanatomica / Chromatophilic Substance: a Proposal to Change the Terminologia Histologica and Terminologia Neuroanatomica

En Terminologia Histologica y Terminologia Neuroanatomica está registrado el término Substantia chromatophilica de origen grecolatino, con los códigos H2.00.06.1.00009 y 78, respectivamente. Dicho término ha sido empleado para referirse a un conglomerado de estructuras que en unión fungen como maquinaria de síntesis proteica y que son característicos de las células nerviosas. Teniendo en cuenta los lineamientos de la Federative International Programme for Anatomical Terminology (FIPAT) referentes a la denominación de nombres estructurales con un valor descriptivo e informativo, creemos que el término en cuestión no es el más adecuado. Por lo anterior, el objetivo del presente estudio fue analizar y evaluar la concordancia de las raíces grecolatinas que componen el término. Para ello, se llevó a cabo una búsqueda en el Diccionario de la lengua española, Diccionario de Términos Médicos, diccionario VOX Griego-Español y el diccionario VOX Ilustrado Latino-Español Español-Latino. Los resultados obtenidos indican que la palabra chromatophilica no presenta registro en español, sin embargo, sus sinónimos hacen referencia a material biológico afín por los colorantes. En base a lo anterior, proponemos el término Ribocumulus corponeuralis en función de su estructura y ubicación, en reemplazo de Substantia chromatophilica.

SUMMARY: In Terminologia Histologica and Terminologia Neuroanatomica the term Substantia chromatophilica of Greco-Latin origin is registered with the codes H2.00.06.1.00009 and 78, respectively. This term has been used to refer to a conglomerate of structures that together function as protein synthesis machinery and are characteristic of nerve cells. Considering the guidelines of the Federative International Programme for Anatomical Terminology (FIPAT) regarding the denomination of structural names with a descriptive and informative value, we believe that the term in question is not the most appropriate. Therefore, this study aimed to analyze and evaluate the concordance of the Greco-Latin roots that compose the term. For this purpose, a search was conducted in the Diccionario de la Lengua Española, Diccionario de Términos Médicos, Diccionario VOX Griego-Español and the Diccionario VOX Ilustrado Latino-Español Español-Latino. The results obtained indicate that the word chromatophilica is not registered in Spanish, however, its synonyms refer to biological material related to dyes. Based on the aforementioned, we propose the term Ribocumulus corponeuralis based on its structure and location, as a replacement for Substantia chromatophilica.

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.

Two predicted α-helices within the prion-like domain of TIAR-1 play a crucial role in its association with stress granules in Caenorhabditis elegans.

Stress granules (SGs) are sites for mRNA storage, protection, and translation repression. TIA1 and TIAR1 are two RNA-binding proteins that are key players in SGs formation in mammals. TIA1/TIAR have a prion-like domain (PrD) in their C-terminal that promotes liquid-phase separation. Lack of any TIA1/TIAR has severe consequences in mice. However, it is not clear whether the failure to form proper SGs is the cause of any of these problems. We disrupted two predicted α-helices within the prion-like domain of the Caenohabditis elegans TIA1/TIAR homolog, TIAR-1, to test whether its association with SGs is important for the nematode. We found that tiar-1 PrD mutant animals continued to form TIAR-1 condensates under stress in the C. elegans gonad. Nonetheless, TIAR-1 condensates appeared fragile and disassembled quickly after stress. Apparently, the SGs continued to associate regularly as observed with CGH-1, an SG marker. Like tiar-1-knockout nematodes, tiar-1 PrD mutant animals exhibited fertility problems and a shorter lifespan. Notwithstanding this, tiar-1 PrD mutant nematodes were no sensitive to stress. Our data demonstrate that the predicted prion-like domain of TIAR-1 is important for its association with stress granules. Moreover, this domain may also play a significant role in various TIAR-1 functions unrelated to stress, such as fertility, embryogenesis and lifespan.

Development of metformin HCl granules using brown flaxseed mucilage as a retardant polymer: effects of polymer and drug ratio

Abstract Flaxseed (Linum usitatissimum L.) is the seed of a multipurpose plant of pharmaceutical interest, as its mucilage can be used as a natural matrix to develop extended-release dosage forms and potentially replace synthetic polymers. In this study, a 3² factorial design with two replicates of the central point was applied to optimize the development of extended-release granules of metformin HCl. The total fiber content of the mucilage as well as the friability and dissolution of the formulations were evaluated. The lyophilized mucilage presented a high total fiber content (42.63%), which suggests a high efficiency extraction process. Higher concentrations of the mucilage and metformin HCl yielded less friable granules. In addition, lower concentrations of metformin HCl and higher concentrations of the mucilage resulted in slower drug release during the dissolution assays. The release kinetics for most formulations were better represented by the Hixson-Crowell model, while formulations containing a higher concentration of the mucilage were represented by the Korsmeyer-Peppas model. Nonetheless, five formulations showed a longer release than the reference HPMC formulation. More desirable results were obtained with a higher concentration of the mucilage (13-18%) and a lower concentration of metformin (40%).

Engineered Fluorescent Strains of Cryptococcus neoformans: a Versatile Toolbox for Studies of Host-Pathogen Interactions and Fungal Biology, Including the Viable but Nonculturable State.

Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hygr] or neomycin resistance [Neor]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans. IMPORTANCE Cryptococcus neoformans is a human-pathogenic yeast that can undergo a dormant state and is responsible for over 180,000 deaths annually worldwide. We engineered a set of fluorescent transformants to aid in research on C. neoformans. A mutant with GFP-tagged Pab1 improved fluorescence-based techniques used in host interaction studies. Moreover, this mutant induced a viable but nonculturable phenotype and uncovered posttranscriptional mechanisms associated with dormant C. neoformans. The experimental use of fluorescent mutants may shed light on C. neoformans-host interactions and fungal biology, including dormant phenotypes.

Pediatric inborn errors of immunity causing hemophagocytic lymphohistiocytosis: Case report and review of the literature.

Inborn errors of immunity are a group of genetic disorders caused by mutations that affect the development and/or function of several compartments of the immune system, predisposing patients to infections, autoimmunity, allergy and malignancies. In this regard, mutations that affect proteins involved in trafficking, priming, docking, or membrane fusion will impair the exocytosis of lytic granules of effector NK and cytotoxic T lymphocytes. This may predispose patients to hemophagocytic lymphohistiocytosis, a life-threatening immune disorder characterized by systemic lymphocyte and macrophage activation, and increased levels of cytokines, which lead to an uncontrolled hyperinflammation state and progressive multiorgan damage. In this review, we will describe a clinical case and recent advances in inborn errors of immunity predisposing to hemophagocytic lymphohistiocytosis. Summary sentence: Review of recent advances in inborn errors of immunity predisposing to hemophagocytic lymphohistiocytosis.

Is IFN expression by NK cells a hallmark of severe COVID-19?

Natural Killer cells (NK) are crucial in host defense against viruses. There are many unanswered questions about the immune system in COVID-19, especially the mechanisms that contribute to the development of mild or severe forms of the disease. Although NK cells may have an essential role in the pathogenesis of COVID-19, the mechanisms involved in this process are not yet fully elucidated. Here, we demonstrate that CD3-CD56+ NK cells frequency in the volunteers who recovered from mild COVID-19 (Mild CoV) presented a significant increase compared to the healthy control (HC) and individuals recovering from severe COVID-19 (Severe CoV) groups. Furthermore, distinct IFN profiles in recovered COVID-19 patients with mild or severe clinical forms of the disease were observed in the total NK cells (CD3-CD56+). In the first group, NK cells express increased levels of IFN-α compared to the severe CoV, while higher production of IFN-γ in severe CoV was found. Moreover, NK cells in mild CoV express more cytolytic granules depicted by granzyme B and perforin. Compared to HC, PBMCs from mild CoV presented higher Ki-67 and TIM-3 production after Pool CoV-2 and Pool Spike CoV-2 peptides stimulus. In addition, non-stimulated PBMCs in the mild CoV group had higher NK TIM-3+ frequency than severe CoV. In the mild CoV group, Pool Spike CoV-2 and Pool CoV-2 peptides stimuli elicited higher granzyme B and perforin coexpression and IFN-α production by PBMCs. However, in severe CoV, Pool Spike CoV-2 reduced the coexpression of granzyme B, perforin, and CD107a suggesting a decrease in the cytotoxic activity of NK cells. Therefore, our study shows that NK cells may have a crucial role in COVID-19 with the involvement of IFN-α and cytotoxic properties that aid in developing qualified immune responses. Furthermore, the data suggest that higher amounts of IFN-γ may be linked to the severity of this disease.

Expert opinion on the use of platelet secretion assay for the diagnosis of inherited platelet function disorders: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

Assessment of platelet secretion is crucial for diagnosing suspected inherited platelet function disorders (IPFD). A previous survey of the SSC on Platelet Physiology of the ISTH and a comprehensive review highlighted that most of the platelet secretion assays (PSAs) lack standardization and validation. The aim of this study was to provide expert consensus guidance on the use of PSAs for IPFD diagnosis. We surveyed 26 experts from 10 different countries using the RAND/UCLA methodology, to attain a consensus on sensitivity, specificity, feasibility, time to readout, and cost of most PSAs. Answers were then graded in three categories: appropriate, uncertain, and inappropriate. Equivocal or misinterpretable statements required a second and third round survey involving 14 of the original 26 experts. We report here the consolidated results of the entire procedure. There was uniform agreement on several general statements, including that PSAs should be performed in hemostasis laboratories as first line diagnostic tests even in patients with normal platelet aggregation, and should include a δ-granule secretion marker. Among the specific assays examined, lumiaggregometry, other luciferin/luciferase-based assays, high-performance liquid chromatography methods, radiolabeled-serotonin based assays, and whole-mount transmission electron microscopy were rated as appropriate for the measurement of δ-granule release, and platelet P-selectin expression by flow cytometry and released proteins by ELISA for α-granule release. For most of the other PSAs, the expert opinions were widely dispersed. Lack of expert consensus on many PSAs clearly indicates an unmet need for rigorous standardization, multicenter comparison of results, and validation of PSAs for clinical laboratory practice.

Formulation of a protocol to evaluate the aerobic granulation potential (AGP) of an inoculum.

This paper proposes and develops a protocol for measuring the aerobic granulation potential of sludge, aiming to provide an affordable and simple alternative that can facilitate the development of aerobic granulation technology. In this sense, the protocol comprises a set of parameters and considerations that interact to create a controlled environment and stimulate cell population clustering. All of this is done in the context of procedural simplicity, low cost, and the speed at which results are obtained. The protocol is essentially a three-stage method: preparation of the substrate, adaptation of the inoculum, and implementation of the protocol. Simple parameters were measured to evaluate the granulation process: SVI, settling velocity, and morphological parameters. The protocol was validated according to optimal ranges and criteria previously established in the literature. For this purpose, an activated sludge inoculum from a domestic wastewater treatment plant was submitted to the protocol, obtaining an optimal response of the biomass (SVI5 =13.90 mL g-1, settling velocity= 25,79 m h-1, Diameter > 0.2 mm) in a relatively short time (7 d). The results show that this protocol can constitute a tool for evaluation and decision-making using traditional laboratory equipment and is applicable at different scales.

Tryptophan regulates Drosophila zinc stores.

Zinc deficiency is commonly attributed to inadequate absorption of the metal. Instead, we show that body zinc stores in Drosophila melanogaster depend on tryptophan consumption. Hence, a dietary amino acid regulates zinc status of the whole insect­a finding consistent with the widespread requirement of zinc as a protein cofactor. Specifically, the tryptophan metabolite kynurenine is released from insect fat bodies and induces the formation of zinc storage granules in Malpighian tubules, where 3-hydroxykynurenine and xanthurenic acid act as endogenous zinc chelators. Kynurenine functions as a peripheral zinc-regulating hormone and is converted into a 3-hydroxykynurenine­zinc­chloride complex, precipitating within the storage granules. Thus, zinc and the kynurenine pathway­well-known modulators of immunity, blood pressure, aging, and neurodegeneration­are physiologically connected.

Potential mechanisms involved on how systemic IgG antibodies specific to vascular endothelial growth factor (VEGF) and induced by active immunotherapy decrease platelet derived free-VEGF.

CIGB-247 is a vascular endothelial growth factor (VEGF)-based active immunotherapy and it is currently under investigation for cancer treatment. This specific active immunotherapy encompasses two vaccine candidates that use a human VEGF variant molecule as antigen, in combination with two clinically tested adjuvants: VSSP or aluminum phosphate. CIGB-247 has been evaluated in patients with advanced solid tumors, recruited in two phase I clinical trials, and it has been shown to be safe and immunogenic by activating both cellular and humoral immune responses against human VEGF. The immunization induces specific IgG antibodies, and also shows as effect, the reduction of free-VEGF levels within platelets (platelet-derived free VEGF). The production of systemic IgG antibodies and the presence of VEGF in another compartment, almost exclusively within platelets, have arisen some questions about this effect detected in the vaccinated-cancer patients. Based on some relevant published works about platelet endocytosis and VEGF pharmacodynamics during bevacizumab treatment as well as the phase I clinical data of CIGB-247, this investigation aims to hypothesize and analyze the potential mechanisms involved in the reduction of platelet-derived free VEGF as a result of vaccination with CIGB-247.Abbreviations: FcγR: Fc gamma receptors; IC: immune complexes; VEGF: vascular endothelial growth factor; VEGFR1: vascular endothelial growth factor receptor 1; VEGFR2: vascular endothelial growth factor receptor 2.

Open questions on the functional biology of the yolk granules during embryo development.

Biogenesis and consumption of the yolk are well-conserved aspects of the reproductive biology in oviparous species. Most egg-laying animals accumulate yolk proteins within the oocytes thus creating the source of nutrients and energy that will feed embryo development. Yolk accumulation drives the generation of a highly specialized oocyte cytoplasm with maternal mRNAs, ribosomes, mitochondria, and, mainly, a set of organelles collectively referred to as yolk granules (Ygs). Following fertilization, the Ygs are involved in regulated mechanisms of yolk degradation to fuel the anabolic metabolism of the growing embryo. Thus, yolk accumulation and degradation are essential processes that allow successful development in many species. Nevertheless, the molecular machinery and mechanisms dedicated to the programmed yolk mobilization throughout development are still enigmatic and remain mostly unexplored. Moreover, while the Ygs functional biology as a nutritional source for the embryo has been acknowledged, several reports have suggested that Ygs cargoes and functions go far beyond yolk storage. Evidence of the role of Ygs in gene expression, microbiota harboring, and paracrine signaling has been proposed. In this study, we summarize the current knowledge of the Ygs functional biology pointing to open questions and where further investigation is needed.

X-ray diffraction and in vivo studies reveal the quinary structure of Trypanosoma cruzi nucleoside diphosphate kinase 1: a novel helical oligomer structure.

Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease, which represents a serious health problem in the Americas. Nucleoside diphosphate kinases (NDPKs) are key enzymes that are implicated in cellular energy management. TcNDPK1 is the canonical isoform in the T. cruzi parasite. TcNDPK1 has a cytosolic, perinuclear and nuclear distribution. It is also found in non-membrane-bound filaments adjacent to the nucleus. In the present work, X-ray diffraction and in vivo studies of TcNDPK1 are described. The structure reveals a novel, multi-hexameric, left-handed helical oligomer structure. The results of directed mutagenesis studies led to the conclusion that the microscopic TcNDPK1 granules observed in vivo in T. cruzi parasites are made up by the association of TcNDPK1 oligomers. In the absence of experimental data, analysis of the interactions in the X-ray structure of the TcNDPK1 oligomer suggests the probable assembly and disassembly steps: dimerization, assembly of the hexamer as a trimer of dimers, hexamer association to generate the left-handed helical oligomer structure and finally oligomer association in a parallel manner to form the microscopic TcNDPK1 filaments that are observed in vivo in T. cruzi parasites. Oligomer disassembly takes place on the binding of substrate in the active site of TcNDPK1, leading to dissociation of the hexamers. This study constitutes the first report of such a protein arrangement, which has never previously been seen for any protein or NDPK. Further studies are needed to determine its physiological role. However, it may suggest a paradigm for protein storage reflecting the complex mechanism of action of TcNDPK1.