Mechanosensitive miR-100 coordinates TGFß and Wnt signaling in osteocytes during fluid shear stress.

Organism scale mechanical forces elicit cellular scale changes through coordinated regulation of multiple signaling pathways. The mechanisms by which cells integrate signaling to generate a unified biological response remains a major question in mechanobiology. For example, the mechanosensitive response of bone and other tissues requires coordinated signaling by the transforming growth factor beta (TGFß) and Wnt pathways through mechanisms that are not well-defined. Here we report a new microRNA-dependent mechanism that mediates mechanosensitive crosstalk between TGFß and Wnt signaling in osteocytes exposed to fluid shear stress (FSS). From 60 mechanosensitive microRNA (miRs) identified by small-RNAseq, miR100 expression is suppressed by in vivo hindlimb loading in the murine tibia and by cellular scale FSS in OCY454 cells. Though FSS activates both TGFß and Wnt signaling in osteocytes, only TGFß represses miR-100 expression. miR-100, in turn, antagonizes Wnt signaling by targeting and inhibiting expression of Frizzled receptors (FZD5/FZD8). Accordingly, miR-100 inhibition blunts FSS- and TGFß-inducible Wnt signaling. Therefore, our results identify FSS-responsive miRNAs in osteocytes, including one that integrates the mechanosensitive function of two essential signaling pathways in the osteoanabolic response of bone to mechanical load.

Fluid shear stress generates a unique signaling response by activating multiple TGFß family type I receptors in osteocytes.

Bone is a dynamic tissue that constantly adapts to changing mechanical demands. The transforming growth factor beta (TGFß) signaling pathway plays several important roles in maintaining skeletal homeostasis by both coupling the bone-forming and bone-resorbing activities of osteoblasts and osteoclasts and by playing a causal role in the anabolic response of bone to applied loads. However, the extent to which the TGFß signaling pathway in osteocytes is directly regulated by fluid shear stress (FSS) is unknown, despite work suggesting that fluid flow along canaliculi is a dominant physical cue sensed by osteocytes following bone compression. To investigate the effects of FSS on TGFß signaling in osteocytes, we stimulated osteocytic OCY454 cells cultured within a microfluidic platform with FSS. We find that FSS rapidly upregulates Smad2/3 phosphorylation and TGFß target gene expression, even in the absence of added TGFß. Indeed, relative to treatment with TGFß, FSS induced a larger increase in levels of pSmad2/3 and Serpine1 that persisted even in the presence of a TGFß receptor type I inhibitor. Our results show that FSS stimulation rapidly induces phosphorylation of multiple TGFß family R-Smads by stimulating multimerization and concurrently activating several TGFß and BMP type I receptors, in a manner that requires the activity of the corresponding ligand. While the individual roles of the TGFß and BMP signaling pathways in bone mechanotransduction remain unclear, these results implicate that FSS activates both pathways to generate a downstream response that differs from that achieved by either ligand alone.

Cardiac abnormalities in Long COVID 1-year post-SARS-CoV-2 infection.

BACKGROUND: Long COVID is associated with multiple symptoms and impairment in multiple organs. Cross-sectional studies have reported cardiac impairment to varying degrees by varying methodologies. Using cardiac MR (CMR), we investigated a 12-month trajectory of abnormalities in Long COVID. OBJECTIVES: To investigate cardiac abnormalities 1-year post-SARS-CoV-2 infection. METHODS: 534 individuals with Long COVID underwent CMR (T1/T2 mapping, cardiac mass, volumes, function and strain) and multiorgan MRI at 6 months (IQR 4.3-7.3) since first post-COVID-19 symptoms. 330 were rescanned at 12.6 (IQR 11.4-14.2) months if abnormal baseline findings were reported. Symptoms, questionnaires and blood samples were collected at both time points. CMR abnormalities were defined as ≥1 of low left or right ventricular ejection fraction (LVEF), high left or right ventricular end diastolic volume, low 3D left ventricular global longitudinal strain (GLS), or elevated native T1 in ≥3 cardiac segments. Significant change over time was reported by comparison with 92 healthy controls. RESULTS: Technical success of multiorgan and CMR assessment in non-acute settings was 99.1% and 99.6% at baseline, and 98.3% and 98.8% at follow-up. Of individuals with Long COVID, 102/534 (19%) had CMR abnormalities at baseline; 71/102 had complete paired data at 12 months. Of those, 58% presented with ongoing CMR abnormalities at 12 months. High sensitivity cardiac troponin I and B-type natriuretic peptide were not predictive of CMR findings, symptoms or clinical outcomes. At baseline, low LVEF was associated with persistent CMR abnormality, abnormal GLS associated with low quality of life and abnormal T1 in at least three segments was associated with better clinical outcomes at 12 months. CONCLUSION: CMR abnormalities (left entricular or right ventricular dysfunction/dilatation and/or abnormal T1mapping), occurred in one in five individuals with Long COVID at 6 months, persisting in over half of those at 12 months. Cardiac-related blood biomarkers could not identify CMR abnormalities in Long COVID. TRIAL REGISTRATION NUMBER: NCT04369807.

Left ventricular reverse remodeling and function by strain analysis in aortic stenosis: A CMR analysis of the EPICHEART study.

INTRODUCTION AND OBJECTIVES: In severe aortic stenosis (AS), the impact of aortic valve replacement (AVR) on left ventricular (LV) systolic function assessed by strain and measured by echocardiography or cardiac magnetic resonance (CMR) has been controversial. We aimed to investigate LV systolic myocardial function changes six months after AVR using global longitudinal (GLS), circumferential (GCS) and radial (GRS) strain derived from CMR imaging. METHODS: We included 39 severe AS patients (69.3±7.8 years; 61.5% male) with preserved LV ejection fraction (LVEF) who were recruited as part of the EPICHEART study and underwent successful AVR (aortic valvular area: 0.8 cm2 (IQR: 0.2) pre- to 1.8 cm2 (IQR:0.5) post-AVR). Structural and functional parameters were assessed at baseline and six months after AVR, including LV GRS, GCS and GLS analysis by CMR, using cine short-axial and two-, three-, and four-chamber long-axial view. Comparison between baseline and postoperative LV remodeling was performed using Student t-test and Wilcoxon test. RESULTS: At six-month follow-up, LV mass, end-diastolic and end-systolic volumes, stroke volume, cardiac output, lateral E/e', tricuspid annular plane systolic excursion, right ventricular (RV) S wave velocity, GLS [-15.6% (IQR: 4.39) to -13.7% (IQR: 4.62)] and GCS [-17.8±3.58% to -16.1±2.94%] reduced significantly, while LVEF and GRS remained unchanged and lateral e' velocity increased. CONCLUSIONS: Despite favorable reverse LV structural and diastolic functional remodeling six months following AVR, GLS and GCS assessed by CMR reduced compared to baseline, LVEF remained unchanged. The clinical utility and timing of assessment of postoperative strain changes as a marker of systolic function progression needs further research.

Fatores que predispõem o uso inadequado de insulinas em diabéticos em uma unidade de saúde no município de Jaguaruana-Ceará / Factors that predispose to the inappropriate use of insulin in diabetics in a health unit in the city of Jaguaruana- Ceará / Factores que predisponen al uso inadecuado de insulina en diabéticos de una unidad de salud de la ciudad de Jaguaruana-Ceará

Introdução: O diabetes mellitus é um distúrbio metabólico crônico que afeta grande parcela da população mundial, no Brasil o número de diabéticos vem crescendo significativamente com o passar dos anos. A qualidade de vida destes pacientes está intimamente relacionada ao tratamento adequado que pode reduzir as complicações decorrentes da doença. O estudo teve como objetivo identificar dificuldades no uso de insulinas em pacientes da unidade de saúde do município de Jaguaruana-CE que possam estar relacionados a falhas no tratamento. Métodos: Tratou-se de uma análise do tipo descritivo, transversal e observacional com uma abordagem quantitativa, o questionário foi aplicado aos pacientes no momento da consulta na Unidade Básica de Saúde (UBS). A pesquisa ocorreu entre os meses de setembro a outubro de 2022 com mulheres e homens insulinodependentes acima de 18 anos. Resultados: Os dados mostraram maior prevalência de mulheres, 20 (67%), a maior faixa etária foi acima de 40 anos, 19 (63%) e a renda familiar prevaleceu de 1 à 2 salários mínimos, 14 (47%). O profissional de prestou maior orientação quanto ao uso da insulina foi o enfermeiro, citado por 12 (40%) pacientes, 20 (67%) relataram armazenar a insulina na porta da geladeira, 22 (74%) relataram desconhecer o motivo das aplicações dolorosas e 9 (30%) confirmam perder ou pular uma das aplicações diárias. Conclusão: Nota-se, portanto, a necessidade da atuação do farmacêutico na saúde da família, na avaliação e orientação quanto ao uso correto das insulinas, garantindo assim o uso racional e com maior eficácia.

Introduction: Diabetes mellitus is a chronic metabolic disorder that affects a large portion of the world population. In Brazil, the number of diabetics has been growing significantly over the years. The quality of life of these patients is closely related to adequate treatment that can reduce the complications arising from the disease. This study aimed to identify difficulties in the use of insulin in patients at the health care facility in the city of Jaguaruana-CE that may be related to treatment failure. Methods: This was a descriptive, cross-sectional and observational analysis with a quantitative approach, the questionnaire was applied to patients at the time of consultation in the Basic Health Unit (BHU). The research occurred between the months of September to October 2022 with insulin-dependent women and men over 18 years. Results: The data showed higher prevalence of women, 20 (67%), the highest age group was above 40 years, 19 (63%) and family income prevailed from 1 to 2 minimum wages, 14 (47%). The professional who provided the most orientation regarding insulin use was the nurse, mentioned by 12 (40%) patients, 20 (67%) reported storing insulin in the refrigerator door, 22 (74%) reported not knowing the reason for the painful applications, and 9 (30%) confirmed missing or skipping one of the daily applications. Conclusion: Therefore, it is noted the need for the role of the pharmacist in family health, in the evaluation and guidance on the correct use of insulin, thus ensuring the rational use and with greater efficacy.

Introducción: La diabetes mellitus es un trastorno metabólico crónico que afecta a gran parte de la población mundial, en Brasil el número de diabéticos viene creciendo significativamente a lo largo de los años. A qualidade de vida destes pacientes está estreitamente relacionada com um tratamento adequado que permita reduzir as complicações decorrentes da doença. O estudo teve como objetivo identificar dificuldades no uso de insulinas em pacientes da unidade de saúde do município de Jaguaruana-CE que possam estar relacionadas a falhas no tratamento. Método: Se trató de un análisis descriptivo, transversal y observacional con abordaje cuantitativo, el cuestionario fue aplicado a los pacientes en el momento de la consulta en la Unidad Básica de Salud (UBS). La investigación ocurrió entre los meses de septiembre a octubre de 2022 con mujeres y hombres insulinodependientes mayores de 18 años. Resultados: Los datos mostraron mayor prevalencia de mujeres, 20 (67%), el grupo de mayor edad fue superior a 40 años, 19 (63%) y la renta familiar prevaleció de 1 a 2 salarios mínimos, 14 (47%). El profesional que más orientó sobre el uso de la insulina fue la enfermera, mencionada por 12 (40%) pacientes, 20 (67%) relataron haber guardado la insulina en la puerta del refrigerador, 22 (74%) relataron no saber el motivo de las aplicaciones dolorosas y 9 (30%) confirmaron haber omitido o saltado una de las aplicaciones diarias. Conclusión: Se constata, por lo tanto, la necesidad del papel del farmacéutico en la salud familiar, en la evaluación y orientación en cuanto al uso correcto de las insulinas, garantizando así el uso racional y con mayor eficacia.

Análise de intoxicação por medicamentos / Analysis of drug intoxication / Análisis de intoxicaciones medicamentosas

Os Centros de Assistência Toxicológica possuem uma importância relevante na promoção, prevenção e controle de agravos à saúde em casos de intoxicação, pois fornece um serviço de orientação e atendimento especializado para lidar com essas situações e para minimizar os prejuízos causados pelos agentes tóxicos. Diante disso, o objetivo do estudo foi analisar o perfil epidemiológico de intoxicações por medicamento, no período de 2017 a 2021, no estado do Ceará. Este estudo está caracterizado como epidemiológico exploratório- descritivo, de abordagem quantitativa, em que serão analisados os dados secundários do registro de casos de intoxicações por medicamento, registrados no Sistema Nacional de Agravos de Notificação, entre os anos de 2017 e 2021. Os dados foram coletados no período de março a abril de 2022. As variáveis levantadas são faixa etária, sexo, circunstâncias das intoxicações, óbito. Nas análises epidemiológicas foi feito o cálculo dos registros de intoxicação por medicamentos coletados no SINAN-TABNET. Resultados: Os resultados desse estudo no período de 2017 a 2021 permitiram a identificação do perfil de intoxicações medicamentosas no Estado do Ceará. Observamos que dentre as variáveis estudadas houve uma prevalência do sexo feminino, faixa etária entre 20-39 anos, e dentre as circunstâncias demostradas, a tentativa de suicídio apresentou dados mais alarmantes. Conclusão: Verificamos a necessidade de medidas efetivas no combate ao uso incorreto dos medicamentos, com ações sociais para conscientizar a população para o uso racional dos medicamentos e orientação para os riscos apresentados por medicamentos devido seu abuso intencional ou não intencional, e uma reavaliação do perfil das pessoas com alta prevalência de intoxicações.

The Toxicological Assistance Centers are relevant in promoting, preven- tion, and control of health aggravations in cases of poisoning, as it provides a specialized guidance and assistance service to deal with such situations and to minimize the damage caused by toxic agents. Therefore, the aim of the study was to analyze the epidemiolog- ical profile of medication poisonings in the period from 2017 to 2021 in the state of Ceará. This study is characterized as an exploratory-descriptive epidemiological study, with a quantitative approach, in which secondary data from the record of drug poisoning cases, registered in the National System of Notifiable Diseases, registered in the years 2017 to 2021 will be analyzed. collected from March to April 2022. The variables are age group, sex, circumstances of poisoning, death. In the epidemiological analyses, the calculation of the records of intoxication by drugs collected in SINAN-TABNET was performed. Results: The results of this study in the period from 2017 to 2021 allowed the identification of the profile of drug poisoning in the State of Ceará. We observed that among the variables studied, there was a prevalence of females, the age group is 20-39 years, and among the circumstances, suicide attempt presented the most alarming data. Conclusion: We verified the need for effective measures to combat the misuse of medi- cine, with social actions to make the population aware about the rational use of medi- cines and guidance for the risks presented by medicines due to their intentional or unin- tentional abuse, and a reassessment of the profile of the people with a high prevalence of poisoning.

Los Centros de Asistencia Toxicológica son relevantes en la promoción, prevención y control de los agravamientos de la salud en casos de intoxicación, ya que proporciona un servicio especializado de orientación y asistencia para hacer frente a es- tas situaciones y minimizar los daños causados por los agentes tóxicos. Por lo tanto, el objetivo del estudio fue analizar el perfil epidemiológico de las intoxicaciones medica- mentosas en el período de 2017 a 2021 en el estado de Ceará. Este estudio se caracteriza por ser un estudio epidemiológico exploratorio-descriptivo, con enfoque cuantitativo, en el que se analizarán datos secundarios del registro de casos de intoxicación medicamentosa, inscritos en el Sistema Nacional de Enfermedades de Declaración Obli- gatoria, registrados en los años 2017 a 2021. recogidos de marzo a abril de 2022. Las variables son grupo de edad, sexo, circunstancias de la intoxicación, muerte. En los aná- lisis epidemiológicos se ha realizado el cálculo de los registros de intoxicaciones por drogas recogidos en SINAN-TABNET. Resultados: Los resultados de este estudio en el período de 2017 a 2021 permitieron identificar el perfil de las intoxicaciones por drogas en el Estado de Ceará. Observamos que entre las variables estudiadas, hubo una preva- lencia de fe- varones, el grupo de edad es de 20-39 años, y entre las circunstancias, el intento de suicidio pre-sentó los datos más alarmantes. Conclusiones: Verificamos la necesidad de medidas eficaces para combatir el uso indebido de medicamentos, con ac- ciones sociales de concienciación de la población sobre el uso racional de medicamentos y orientación sobre los riesgos que presentan los medicamentos por su abuso intencional o no intencional, y una reevaluación del perfil de las personas con alta prevalencia de intoxicaciones.

Phage-Based Structural Color Sensors and Their Pattern Recognition Sensing System.

The mammalian olfactory system provides great inspiration for the design of intelligent sensors. To this end, we have developed a bioinspired phage nanostructure-based color sensor array and a smartphone-based sensing network system. Using a M13 bacteriophage (phage) as a basic building block, we created structural color matrices that are composed of liquid-crystalline bundled nanofibers from self-assembled phages. The phages were engineered to express cross-responsive receptors on their major coat protein (pVIII), leading to rapid, detectable color changes upon exposure to various target chemicals, resulting in chemical- and concentration-dependent color fingerprints. Using these sensors, we have successfully detected 5-90% relative humidity with 0.2% sensitivity. In addition, after modification with aromatic receptors, we were able to distinguish between various structurally similar toxic chemicals including benzene, toluene, xylene, and aniline. Furthermore, we have developed a method of interpreting and disseminating results from these sensors using smartphones to establish a wireless system. Our phage-based sensor system has the potential to be very useful in improving national security and monitoring the environment and human health.

Osteocyte-Intrinsic TGF-ß Signaling Regulates Bone Quality through Perilacunar/Canalicular Remodeling.

Poor bone quality contributes to bone fragility in diabetes, aging, and osteogenesis imperfecta. However, the mechanisms controlling bone quality are not well understood, contributing to the current lack of strategies to diagnose or treat bone quality deficits. Transforming growth factor beta (TGF-ß) signaling is a crucial mechanism known to regulate the material quality of bone, but its cellular target in this regulation is unknown. Studies showing that osteocytes directly remodel their perilacunar/canalicular matrix led us to hypothesize that TGF-ß controls bone quality through perilacunar/canalicular remodeling (PLR). Using inhibitors and mice with an osteocyte-intrinsic defect in TGF-ß signaling (TßRIIocy-/-), we show that TGF-ß regulates PLR in a cell-intrinsic manner to control bone quality. Altogether, this study emphasizes that osteocytes are key in executing the biological control of bone quality through PLR, thereby highlighting the fundamental role of osteocyte-mediated PLR in bone homeostasis and fragility.

Mechanobiology of TGFß signaling in the skeleton.

Physical and biochemical cues play fundamental roles in the skeleton at both the tissue and cellular levels. The precise coordination of these cues is essential for skeletal development and homeostasis, and disruption of this coordination can drive disease progression. The growth factor TGFß is involved in both the regulation of and cellular response to the physical microenvironment. It is essential to summarize the current findings regarding the mechanisms by which skeletal cells integrate physical and biochemical cues so that we can identify and address remaining gaps that could ultimately improve skeletal health. In this review, we describe the role of TGFß in mechanobiological signaling in bone and cartilage at the tissue and cellular levels. We provide detail on how static and dynamic physical cues at the macro-level are transmitted to the micro-level, ultimately leading to regulation at each level of the TGFß pathway and to cell differentiation. The continued integration of engineering and biological approaches is needed to answer many remaining questions, such as the mechanisms by which cells generate a coordinated response to physical and biochemical cues. We propose one such mechanism, through which the combination of TGFß and an optimal physical microenvironment leads to synergistic induction of downstream TGFß signaling.

Discrete spatial organization of TGFß receptors couples receptor multimerization and signaling to cellular tension.

Cell surface receptors are central to the cell's ability to generate coordinated responses to the multitude of biochemical and physical cues in the microenvironment. However, the mechanisms by which receptors enable this concerted cellular response remain unclear. To investigate the effect of cellular tension on cell surface receptors, we combined novel high-resolution imaging and single particle tracking with established biochemical assays to examine TGFß signaling. We find that TGFß receptors are discretely organized to segregated spatial domains at the cell surface. Integrin-rich focal adhesions organize TßRII around TßRI, limiting the integration of TßRII while sequestering TßRI at these sites. Disruption of cellular tension leads to a collapse of this spatial organization and drives formation of heteromeric TßRI/TßRII complexes and Smad activation. This work details a novel mechanism by which cellular tension regulates TGFß receptor organization, multimerization, and function, providing new insight into the mechanisms that integrate biochemical and physical cues.

Inkjet printed antibiotic- and calcium-eluting bioresorbable nanocomposite micropatterns for orthopedic implants.

Inkjet printing of antibiotic- and calcium-eluting micropatterns was explored as a novel means of preventing the formation of biofilm colonies and facilitating osteogenic cell development on orthopedic implant surfaces. The micropatterns consisted of a periodic array of ∼50 µm circular dots separated by ∼150 µm. The composition of the micropatterns was controlled by formulating inks with rifampicin (RFP) and poly(D,L-lactic-co-glycolic) acid (PLGA) dissolved in an organic solvent with ∼100 nm biphasic calcium phosphate (BCP) nanoparticles suspended in the solution. During printing RFP and PLGA co-precipitated to form a nanocomposite structure with ∼10-100 nm RFP and the BCP particles dispersed in the PLGA matrix. The rate of RFP release was strongly influenced by the RFP loading in the micropattern, particularly on the first day. The RFP-containing micropatterns effectively prevented the formation of Staphylococcus epidermidis biofilm colonies due to their ability to kill bacteria prior to forming colonies on the patterned surfaces. The BCP-containing micropatterns printed on the surface of the alloy TiAl6V4 significantly accelerated osteoblast cell differentiation, as measured by alkaline phosphatase expression and calcium deposition, without compromising cell proliferation.

Phosphoinositides and phosphatidic acid regulate pollen tube growth and reorientation through modulation of [Ca2+]c and membrane secretion.

The maintenance of a calcium gradient and vesicle secretion in the apex of pollen tubes is essential for growth. It is shown here that phosphatidylinositol-4,5-bisphosphate (PIP2) and D-myo-inositol-1,4,5-trisphosphate (IP3), together with phosphatidic acid (PA), play a vital role in the regulation of these processes. Changes in the intracellular concentration of both PIP2 and IP3 (induced by photolysis of caged-probes), modified growth and caused reorientation of the growth axis. However, measurements of cytosolic free calcium ([Ca2+]c) and apical secretion revealed significant differences between the photo-release of PIP2 or IP3. When released in the first 50 mum of the pollen tube, PIP2 led to transient growth perturbation, [Ca2+]c increases, and inhibition of apical secretion. By contrast, a concentration of IP3 which caused a [Ca2+]c transient of similar magnitude, stimulated apical secretion and caused severe growth perturbation. Furthermore, the [Ca2+]c transient induced by IP3 was spatially different causing a pronounced elevation in the sub-apical region. These observations suggest different targets for the two phosphoinositides. One of the targets is suggested to be PA, a product of PIP2 hydrolysis via phospholipase C (PLC) or phospholipase D (PLD) activity. It was found that antagonists of PA accumulation (e.g. butan-1-ol) and inhibitors of PLC and PLD reversibly halted polarity. Reduction of PA levels caused the dissipation of the [Ca2+]c gradient and inhibited apical plasma membrane recycling. It was also found to cause abolition of the apical zonation. These data suggest that phosphoinositides and phospholipids regulate tip growth through a multiple pathway system involving regulation of [Ca2+]c levels, endo/exocytosis, and vesicular trafficking.

Calmodulin activity and cAMP signalling modulate growth and apical secretion in pollen tubes.

Our present understanding implicates both calmodulin (CaM) and 3',5'-cyclicAMP (cAMP) in the regulation of pollen tube growth. However, downstream molecules of these signalling pathways and the cellular processes they modulate remain largely unknown. In order to elucidate the role of CaM, we mapped its activity in growing pollen tubes. 2-chloro-(epsilon-amino-Lys(75))-[6-4-(N,N'-diethylaminophenyl)-1,3,5-triazin-4-yl]-calmodulin (TA-CaM) and fluorescein-calmodulin (FL-CaM), fluorescent analogues of CaM, were loaded into pollen tubes and CaM activity was mapped by fluorescence ratio imaging. It was found that CaM activity exhibits a tip-focused gradient, similar to the distribution of cytosolic-free calcium ([Ca(2+)](c)). In long pollen tubes, apical CaM activity was also found to oscillate with a period similar to [Ca(2+)](c) (40-80 sec). This oscillatory behaviour was not observed in small pollen tubes or in tubes that had stopped growing. Changes in CaM activity within the dome of the pollen tube apex resulting from the photolysis of caged photolysis of RS-20 (a peptide antagonist of CaM) induced re-orientation of the growth axis, suggesting that CaM is also involved in the guidance mechanism. CaM activity was strongly modulated by intracellular changes in cAMP (induced by activators and antagonists of adenylyl cyclase). These results indicate that the action of this protein is dependent not solely on [Ca(2+)](c) but also on a cross-talk with other signalling pathways. A putative target of this cross-talk is the secretory machinery as observed in pollen tubes loaded with the FM (N-(3-triethylammoniumpropyl)-4-(4-dibutylamino)styryl)pyridinium dibromide 1-43 dye and exposed to different antagonists and activators of these molecules. Our data thus suggest that pollen tube growth and orientation depend on an intricate cross-talk between multiple signalling pathways in which CaM is a key element.