text:
The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.
23 Related JoVE Articles!
Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
Institutions: Loyola University Chicago.
Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine ΔΨm and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria.
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine Δψm in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2
DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess ΔΨm or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of ΔΨm or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of ΔΨm or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2
DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2
. This protocol (with minor modifications) can be also used to determine changes in ∆Ψm and ROS in different cell types and in neurons isolated from other brain regions.
Neuroscience, Issue 51, Mitochondrial membrane potential, reactive oxygen species, neuroscience, cortical neurons
Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria
Institutions: University of Washington, Seattle.
The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1
, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+
, bioenergetics and redox signaling 3
mtPTP opening is triggered by matrix Ca2+
but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4
. In vitro
, mtPTP opening can be achieved by increasing Ca2+
concentration inside the mitochondrial matrix through exogenous additions of Ca2+
(calcium retention capacity). When Ca2+
levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+
release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+
handling (uptake and release, as measured by Ca2+
concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+
measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+
, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.
Cellular Biology, Issue 67, Mitochondria, respiration, mitochondrial permeability transition pore (mPTP), membrane potential, swelling, calcium, spectrofluorometer
F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes
Institutions: Yale University.
Mitochondria are involved in many important cellular functions including metabolism, survival1
, development and, calcium signaling2
. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5
. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6
. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7
interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8
and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al
. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.
Neuroscience, Issue 75, Medicine, Biomedical Engineering, Molecular Biology, Cellular Biology, Biochemistry, Neurobiology, Anatomy, Physiology, F1FO ATPase, mitochondria, patch clamp, electrophysiology, submitochondrial vesicles, Bcl-xL, cells, rat, animal model
Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence
Institutions: Yale University School of Medicine, NatureMost Laboratories, Bruker Preclinical Imaging.
Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.
Cancer Biology, Issue 93, ovarian cancer, recurrence, in vivo imaging, tumor burden, cancer stem cells, chemotherapy
Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines
Institutions: Indiana University School of Medicine.
Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 µM cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.
Medicine, Issue 91, cancer stem cells, stem cell markers, ovarian cancer, chemoresistance, cisplatin, cancer progression
Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time
Institutions: MIMR-PHI Institute of Medical Research, Monash University.
Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo
, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.
Medicine, Issue 87, Ovarian cancer, metastasis, invasion, mesothelial cells, spheroids, real time analysis
gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair
Institutions: Centre Georges-François Leclerc.
The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM
, and TP53
) from promoter to 3'-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.
Genetics, Issue 92, gDNA enrichment, Nextera, NGS, DNA damage, BRCA1, BRCA2
Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression
Institutions: University of Southern California, University College London.
Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2
. Whilst stage I tumors have 5-year survival rates >85%, survival rates for stage III/IV disease are <40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5
. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g.
age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF) cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT
) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC
oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.
Cancer Biology, Issue 66, Medicine, Tissue Engineering, three-dimensional cultures, stromal-epithelial interactions, epithelial ovarian cancer, ovarian surface epithelium, ovarian fibroblasts, tumor initiation
Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens
Institutions: University of Minnesota, Maricopa Medical Center and St Josephs Hospital and Medical Center, University of Minnesota.
Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro
passaging. Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.
Medicine, Issue 84, Neoplasms, Ovarian Cancer, Primary cell lines, Clinical Specimens, Downstream Applications, Targeted Therapies, Epithelial Cultures
Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c
Institutions: University of North Carolina , University of North Carolina .
Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1
. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c
from the mitochondrial intermembrane space to the cytoplasm2
. The release of cytochrome c
from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c
release from mitochondria5
. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c
(heme-attached) protein into cells6-9
. Cytochrome c
is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11
. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c
protein instead of its cDNA, because while the expression of cytochrome c
from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c
protein is a useful tool to mimic mitochondrial cytochrome c
release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c
protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c
for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.
Cellular Biology, Issue 52, Microinjection, apoptosis, cytochrome c, fibroblasts, neurons
In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis
Institutions: Harvard Medical School.
Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1
. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2
. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3
(Figure 1, Movie 1).
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6
(Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7
(Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process.
Previous in vitro
experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8
, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10
. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown.
Here we describe the methodology for an in vitro
assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo
(Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16
, and was first described in a report showing that ovarian tumor cells utilize an integrin –dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17
. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.
Medicine, Issue 60, Ovarian Cancer, Metastasis, In vitro Model, Mesothelial, Spheroid
Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria
Institutions: Université de Montréal, CRCHUM, Université de Montréal, CRCHUM.
Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.
Cellular Biology, Issue 91,
Mitochondria, flow cytometry, organelle isolation, immunolabeling, spinal cord, TMRM
A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy
Institutions: Tufts School of Medicine, Wake Forest Baptist Medical Center, Boston University Medical Center.
Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13
. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13
. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18
. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14
. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9
. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7
, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5
. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5
. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11
. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6
. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15
. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8
in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2
, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.
Molecular Biology, Issue 65, Genetics, Cellular Biology, Physics, confocal microscopy, mitochondria, fusion, TMRE, mtPAGFP, INS1, mitochondrial dynamics, mitochondrial morphology, mitochondrial network
Methods to Assess Subcellular Compartments of Muscle in C. elegans
Institutions: University of Nottingham.
Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans
is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans
is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo
. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo
. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans
provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo
in any other organism.
Developmental Biology, Issue 93, Physiology, C. elegans, muscle, mitochondria, sarcomeres, ageing
Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells
Institutions: Columbia University, Columbia University.
Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the Fo
-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.
Bioengineering, Issue 77, Microbiology, Cellular Biology, Molecular Biology, Biochemistry, life sciences, roGFP, redox-sensitive green fluorescent protein, GO-ATeam, ATP, FRET, ROS, mitochondria, biosensors, GFP, ImageJ, microscopy, confocal microscopy, cell, imaging
Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen
Institutions: University of Windsor, Brock University.
Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.
Cancer Biology, Issue 63, Medicine, Biochemistry, Breast adenocarcinoma, neuroblastoma, tamoxifen, combination therapy, apoptosis, autophagy
Analysis of Oxidative Stress in Zebrafish Embryos
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo
system to perform such studies and present a protocol to measure in vivo
oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo
: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas
Institutions: University of Tennessee, University of Tennessee.
Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation - is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.
In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.
In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.
The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.
Bioengineering, Issue 86, Lipid droplet, lipid body, fat body, oil body, Yeast, placenta, placental villous cells, isolation, purification, density gradient centrifugation
From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g.
, signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation
Institutions: Technion - Israel Institute of Technology.
Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes’ association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.
Biochemistry, Issue 85, mitochondria, mRNA localization, Yeast, S. cerevisiae, microarray, localized translation, biochemical fractionation
Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor
Institutions: University of Texas Medical Branch.
Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1
. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2
. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ
during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3
. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4
targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4
. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro
of ~1.7 μM4
. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.
Cellular Biology, Issue 50, Ratiometric pericam, mitochondria, calcium, apoptosis, staurosporine, live cell imaging
Preparation of Highly Coupled Rat Heart Mitochondria
Institutions: University of Manchester, Queen's University Belfast.
The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.
Cellular Biology, Issue 43, Bioenergetics, Mitochondriology, Mitochondria Purification, Oxidative Phosphorylation, Cellular Respiration, Cardiology
Purification of Mitochondria from Yeast Cells
Institutions: Concordia University.
Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1
. These complex organelles also play essential roles in apoptotic cell death2
, cell survival3
, mammalian development4
, neuronal development and function4
, intracellular signalling5
, and longevity regulation6
. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae
, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.
Cellular Biology, Issue 30, subcellular fractionation, organelles, organelle purification, mitochondria

domain: biology


text:
I am off back to Spain tomorrow to start knocking our garden back into shape. My friend Karl has kindly looked after it for two weeks and my wife has been looking after it for the last week. Who knows what I will find, my wife’s only hint of what is to come was to ask me whether the Beef tomatoes that I had planted were Cherry tomatoes.
Anyway, just time for a little bit of English gardening. Our daughter Sarah has a small town style walled garden which has artificial lawn some trellis for climbers and a stone filled small border under an Acer. The garden has been developed in this way to cope with the predations of her two dogs Harley and Alfie, who have been known to eat the odd plant or two.
17 June: Things I have been doing today
👀 Reviewing how Sarah’s garden works. From the first photo below you can see that the garden has two clematis on trellis that are both overgrown and are collapsing the wooden trellis structure beneath them. Also, the pots on the wall at the back do not do enough to draw your eye down this narrow part of the garden. The second photo shows the stoned bed under the Acer which has been left unplanted since the last time the dogs ate all the plants.
🔨 Refitting the trellis. When you have heavy fast growing plants growing up trellis it is a fair bet that at some time they are going to pull it down; unless you keep them tightly trimmed (which means losing flowers). To overcome this in Sarah’s garden we trimmed the plants so heavily they looked like Elvis joining the army in GI Blues. Then instead of trellis we used strong wire attached to hooks and vine ties that are tightened to hold the wire taut. The wires were spaced at 30cm apart running across the wall for 2.5 metres. As the plants recover from their scalping they can then be encouraged up and along the wires to spread out over a growing space of roughly 2.5 metres wide by 2 metres high, thereby giving them more space to display themselves and producing a more even weight distribution.
🌺 Drawing your eye down the garden. My original thought was to put Virginia Creeper or Boston Ivy at the back which would have given lovely greenery turning to deep red in the Autumn. The downside of this is that both plants are poisonous to dogs. The upside would have been no more plant chewing. After discussion and Sarah threatening to report me to the RSPCA we decided against this. Instead we removed the pots, replanted them with fuchsias and re-sited them down the patio stairs. In their place we put a Cobaea Scandens which may sound like a scandal involving Jeremy Corbyn in a Norwegian newspaper, but is in fact a fast growing climber which has lots of flowers. Again we placed this on wire with vine ties.
The first photo below shows the newly trimmed climbers and the Cobea on the back wall. This looks nothing at the moment, but before the end of summer it will all start to fill out and lead your eye down this part of the garden. The re-sited pots can be seen in the second photograph, notice that they follow the fall of the stairs; if they did not do this your eye would constantly be drawn to them for all the wrong reasons.
🌿 Planting the small stone bed. The stone bed had to fulfill two functions. Firstly, it had to be interesting all year. Secondly, it had to be not too interesting that the dogs would fancy it for salad. Here we planted Euonymous golden, a small slow growing variegated evergreen shrub that will provide interest all year and in a few years can be cut into topiary shapes (a bone perhaps).
The photograph below shows the finished garden with Harley the dog checking out which plants are potentially the tastiest.
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domain: hobby


text:
Yankees have faith they can keep Mo, Kuroda and Pettitte
New York isn't quite as confident when it comes to Rafael Soriano, who like Kuroda just rejected the $13.3-million qualifying offer.
|Mariano Rivera has already said he wants to return, but Andy Pettitte has yet to reveal his plans. (Getty Images)|
The Yankees expect to try to work something out with Rivera in the coming week, especially now that Soriano has rejected the $13.3-million qualifying offer. The club's hope is to get Rivera done at a bit of a pay cut from his $15-million 2012 salary, as he is 43 and coming back from a knee injury.
The Yankees seem to have a fair amount of hope, too, that Kuroda will stay with them on a one-year deal, especially if he desides to stay in the states. The team sees Japan as its major competition, not the Red Sox or even nouveau riche Dodgers, who are among the interested teams in the majors. The Yankees would enhance the $13.3-million offer Kuroda rejected a bit if it meant keeping Kuroda, who is said to have enjoyed his year with the Yankees and is very willing to do a one-year deal.
Word is, Kuroda has promised to finish his terrific career with a final year back in Japan, but the Yankees believe and hope the chances are that he delays that plan a year to give them another fine season in the Bronx.
Pettitte hasn't "officially'' told the Yankees he's returning, but they seem to believe he will. He'll get a raise from the $2.5 million he took to come out of retirement last year should he pitch, as they expect.
As for Soriano, the Yankees are amenable to talking about a two-year deal with the man who stepped in for Rivera and did a terrific job in 2012. However, they don't seem to feel the need to present one at the moment, not after Soriano opted out, then joined the other seven star players who turned down qualifying offers.
The Yankees made that same qualifying offer to Nick Swisher, whom they had no intention to re-sign, and will bank another draft choice for doing so since he has at least five or six teams interested in signing him to multiyear deals.
The Yankees will need to fill right field but don't seem inclined to go to two years on Torii Hunter, who is drawing immense interest and will surely get a multiyear deal elsewhere if he so desires. The Rangers, Tigers, Red Sox and Phillies are among the logical teams for Hunter.
Colon wants to break a Dennis Martinez record
The Twins have been rumored to be interested in Darvish all winter
Rolen is one of the best third basemen of all time, but you wouldn't know it based on this...
Neither Josh Donaldson nor Brian Dozier have talked about a new contract extension
Francisco leads the Dominican Winter League with seven home runs
Thome deserves enshrinement and here's why the case against him does not hold up

domain: sports


text:
Harnessing Our Digital Future
In The Second Machine Age, Andrew McAfee and Erik Brynjolfsson predicted some of the far-reaching effects of digital technologies on our lives and businesses. Now they’ve written a guide to help readers make the most of our collective future. Machine | Platform | Crowd outlines the opportunities and challenges inherent in the science fiction technologies that have come to life in recent years, like self-driving cars and 3D printers, online platforms for renting outfits and scheduling workouts, or crowd-sourced medical research and financial instruments.
About the Authors
Andrew McAfee is the co-director of the MIT Initiative on the Digital Economy and co-author of the best-selling The Second Machine Age and Machine, Platform, Crowd: Harnessing Our Digital Future. He and co-author Erik Brynjolfsson are the only people named to both the Thinkers 50 list of the world’s top management thinkers and the Politico 50 group of people transforming American politics.
Erik Brynjolfsson is the director of the MIT Initiative on the Digital Economy and Schussel Family Professor of Management Science at the MIT Sloan School. He is the author of several best-selling books with co-author Andrew McAfee, and one of the world’s most cited scholars in information systems and economics.
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domain: library


Column | Sep 3, 2013 | 9:04 PM
A collection of news and information related to Los Angeles County Sheriff's Department published by this site and its partners.
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Blog | Apr 6, 2009 | 4:30 PMThis wire story crossed a few hours ago, but just in case you haven't yet seen...The producer who's working on Farrah Fawcett's documentary says she is "not on death's door," per indications earlier today, while she was hospitalized for...
Column | Apr 3, 2013
Column | Dec 3, 2013 | 6:19 PM
L.A. County sheriff's investigators must devote more time to patrols, administrative tasks. Make sense?
Story | Oct 2, 2010 | 4:34 PM
Most of the Los Angeles County Sheriff's Department's sworn personnel are being told to put aside their regular duties and devote several hours a month filling in on routine patrols and low-level administrative tasks, according to a report by The...


text:
Irish Coffee Affogato
On World Chocolate Day, chocolate lovers from all over the world unite to celebrate perhaps the most delicious discovery made by man in centuries. Celebrated on 7th July each year, World Chocolate Day is commemorated by consuming chocolate in whatever form you like it.
Of course none of us really require a specific day to express our love for the things we treasure the most, whether it’s chocolate or whisky but it is always nice to have a reason to do so.
For our community of whiskey lovers that also happen to share a penchant for the delicious chocolaty goodness, we have the perfect whiskey cocktails that combine your favourite sinful indulgences.
The is a unique twist on a traditional Italian coffee-based dessert that comprises of a shot of espresso and vanilla ice-cream. Although you can see why this combination can be made significantly better with some good ol’ Irish whiskey wouldn’t you say?
- Jameson Irish whiskey
- Kahlua Cream – 15 ML
- Vanilla/Coffee Flavoured Ice-Cream – 1 Scoop
- Cold Brew Coffee – 60 ML
- Whipped Cream – For Garnish
Step 1: Combine the Cold Brew Coffee, Jameson Irish whiskey and Kahlua Cream in a tumbler glass, or coffee mug of your choice. Gently stir with a bar spoon.
Step 2: Scoop out your favourite vanilla or coffee flavoured ice-cream and add to the container.
Step 3: Add a dollop of whipped cream for garnish.
Enjoy the Irish Coffee Affogato, one of the easiest whiskey cocktails, and most definitely the most mouth-watering one.
Whisky Smash using Blenders Pride [Whisky Smash with a Twist]
A closely related descendent of many a whisky cocktail, the Whisky Smash is a clever, slightly more tangy version of the Mint Julep and the...Learn Recipe
Whisky Sour Cocktail with Blenders Pride
First mentions of the very preparations of this delectable indulgence date back as far as 1870, but the Whisky Sour continues to enjoy its status...Read More

domain: food


text:
Charles Grooms Report is an exceptionally thought-provoking English book that delves into the intriguing concepts of identity, race, and social dynamics. Written by the talented author, Charles Grooms, this report presents a stimulating analysis of these themes within a captivating narrative.
The book follows the life of its protagonist, Charles Grooms, a biracial man navigating his way through a complex society. Grooms unflinchingly confronts the challenges posed by his dual heritage, exploring the intricate notions of self-acceptance and the struggle to fit in. His report candidly discusses the prevailing racial disparities and injustices experienced by individuals like himself. Through his personal experiences and insightful observations, Grooms examines the societal constructs and stereotypes that continue to shape perceptions of race.
As the narrative unfolds, Grooms delves deeper into the complexities of identity, highlighting how one’s racial background can influence their interactions, relationships, and opportunities. His report also delves into the larger context of race in society, exploring the effects of systemic discrimination and the far-reaching consequences it has for marginalized communities.
What sets Charles Grooms Report apart is its candid and thought-provoking approach to the subject matter. Grooms’ writing style is both engaging and profound, inviting readers to critically reflect on their own biases and assumptions. The book not only sheds light on the struggles faced by biracial individuals but the broader issues of race and discrimination that persist today.
With its compelling narrative and insightful analysis, Charles Grooms Report is a must-read for anyone interested in exploring the complexities of race, identity, and social dynamics. Grooms’ report has the power to challenge readers’ perspectives and promote meaningful conversations about the ongoing struggle for racial equality.
|Charles Grooms Report
|No Of pages
|Orrisa State Archive

domain: sociology


text:
The Netherlands has its own island in the Caribbean called Mount Scenery, a volcano owned by the Netherlands in the Caribbean.
It is on the north side of the line of islands going from the Dominican Republic down to the north end of South America. Mount Scenery is a lava dome and is considered an active volcano with the potential to still erupt.
Its last eruption, however, was in 1640. Its lava dome forms the summit of the Saba island stratovolcano.
It is the largest mountain in the Kingdom of the Netherlands and the Netherlands proper with an elevation of 877 meters. Even though it hasn’t erupted since 1640, it included explosions and pyroclastic flow, or lava.

domain: travel


text:
Mother has been appointed to handle a multimedia marketing campaign for Peoplesound.com, the free music internet download service.
Peoplesound.com, which claims to have a budget of £10 million to spend on a pan-European campaign this year, appointed Mother without a pitch.
Mike Levine, Peoplesound.com's marketing director, said: "I really like the approach Mother took to our business and the enthusiasm they showed for our brand. Their creative work speaks for itself."
Levine, a former Procter & Gamble brand manager for Lenor, joined Peoplesound.com from Polygram/Universal, where he was senior marketing manager, in September.
Andrew Medd, a partner at Mother, said: "Mike inspired us with his vision of how Peoplesound.com could revolutionise the music industry through the internet and the company has the resources to do it. We liked everyone we met and their commitment to making a difference, something we admire in all our clients."
Peoplesound.com aims to provide unsigned artists with a well-known, well-visited and global site to showcase their work. The company, which has 500 talent scouts in the UK, signed 1,500 artists -- representing over 40 genres of music -- within four weeks of going live. Consumers can download tracks free of charge or buy the artists' music on CDs manufactured by Peoplesound.
Artists represented on Peoplesound.com are paid a £100 advance on royalties and are not held to an exclusivity deal. They also get 50 per cent of royalties, compared to record labels' traditional 16 to 19 per cent rate.

domain: news


When encountering public services, it can be tempting to make the analogy with a supply chain, where products make a journey from factory, to warehouse, to distributor, to retailer, to customer and as shown in earlier Chapters this can be helpful in health and social care. However, sometimes in human systems things are less straightforward and the health and social care system is arguably much more complex than any industrial process.
Unlike production, where materials mostly flow in one direction,
in health and social care people flow in multiple directions, miss out some stages and perhaps repeat others. The same service capacity might be used for different purposes; for example in Fig.
, an ‘intermediate care’ service which has a single capacity, might be used BOTH ‘upstream’, as an alternative to hospital admission, AND ‘downstream’ to facilitate hospital discharge of people who have recovered somewhat, no longer need to be in hospital, but are not quite ready to go home. In the same system, some people might be waiting in hospital to be discharged to an intermediate care setting at the same time as other people in an intermediate care setting now need hospital admission, perhaps because their condition has deteriorated. Alternatively, people might be admitted to hospital having been users of a home care package, losing that package on or shortly after admission, then, several weeks later, cannot be discharged from hospital until another, identical or more intensive, home care package can be procured. Such eventualities are not only possible, but common.
(same as Fig.
) High level view of integrated care model 8.1
Therefore, at any point in time, people are waiting to be transferred between services, when capacity allows, in various combinations:
(waiting in A and needing B)
(waiting in A and needing C)
(waiting in A and needing D)
(waiting in B and needing A)
(waiting in B and needing C)
Etc. (but some pairings are not possible or likely).
This degree of disaggregation creates a challenge to the transparency of a stock/flow diagram. Depending on the software used, the model can be constructed with each service being represented as a single stock. These stocks are further disaggregated elsewhere on the model screen, using a co-flow structure and, in the case of Stella Architect software, using its modular capabilities.
The disaggregated representation of each service will look something like the diagram in Fig.
Disaggregated view of one service—note that the stocks drawn with dotted lines are copies of stocks that appear elsewhere in the model diagram
Each service might have people currently waiting for a place, of whom some are not currently using a service, and others are using other services. To make this diagram clearer, we have drawn ‘sector frames’ round the people who are ‘waiting for A’ and those who are using ‘Service A’. Note that, in the diagram, those waiting but currently using other services are shown as ‘ghosted’ stocks (dotted lines), copied from elsewhere on the model. The stock called ‘using service A’ represents the number of people currently using service A and are appropriately placed. Service A is also being used by people who need, variously, services B, C, and E. And, in this example, nobody ever goes from A to D. The total number of people using service A is therefore the sum of the four stocks.
Note that another way of representing this degree of disaggregation more simply, would be to ‘array’ (or subscript) the ‘waiting in’ stocks.
Allocation of People Waiting to Capacity Restricted Services
The key question that arises, for each service, is ‘what is the policy for allocating spare places across the various waiting stocks?’ Various possibilities apply, all of which can be modelled. For example:
Spare places might simply be allocated in proportion to the number of people in each waiting stock
Places might be allocated based on a very strict order of priority: we allocate spare places first to people waiting here, then to people waiting there, then there, and so on; one problem with this approach is that if the whole system is under pressure, some people ‘waiting here, needing a place there’ might never be allocated a place, in which case, the modeller must also bear in mind ‘what happens to people who wait for a very long time?’ because their state of health/or need for care is likely to change
Places could be allocated according to some kind of weighting system (we admit relatively more people from here but always some from there), which gets round the problem of some people never moving
A variation might be to base that weighting on some kind of ‘floating goal’ derived from some other dynamic in the model, such as ‘the length of time for which people have been waiting to move from x to A’; for example, people waiting in B should wait no more than twice as long as people in C.
Obviously, the modeller should create an allocation policy that most closely matches the actual policies that are deployed (Sterman
, Chap. 15), assuming that these are known and can be codified. It is perfectly possible that what actually happens is the result of various informal decision-making heuristics that are brought to light by the modelling process itself and are poorly understood by key people across the whole system. 2000
A further important consideration with this kind of model is the question of ‘what happens to people who wait for a long time in any one state?’ Depending on where they are in the system, their condition might improve or deteriorate.
Where people are improving/recovering, it is common for people in hospital on reaching the status of ‘no longer needing to be in hospital but needing something else’ to ‘step down’ to some kind of intermediate care setting, the purpose of which might either be to provide care whilst a permanent home care package is organised, and/or to provide additional rehabilitation. In these cases, if intermediate care is NOT provided, they will remain in hospital for the duration of their recovery, then be discharged to a home care package when it is ready. Going back to Fig.
, people waiting to transfer from A to B might actually transfer from A to C if B is not available in time (detail not shown in the diagram). 8.15
The opposite effect might happen in the case where the existing level of support for someone waiting is less than is needed (or completely absent). People waiting at home for a home care package to be arranged might be admitted to hospital. People on an elective waiting list might be admitted to hospital as an emergency.
To summarise, it is quite common in these structures to have at least two outflows from a waiting stock, the outflow to the service that is desired, and the outflow to the destination that will result if the desired service is not provided in time.


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The Hawthorne Studies: An Introduction
The Hawthorne Studies refer to a series of experiments conducted by Elton Mayo and his colleagues at the Western Electric Hawthorne Works manufacturing plant in Cicero, Illinois from 1924 to 1932. The experiments aimed to investigate the relationship between workers’ productivity and their physical and social environment at work.
The Hawthorne Works plant was chosen because it had an experienced workforce, with diverse skills and backgrounds, and was representative of industrial plants in the United States at the time. The study involved several phases, each focusing on different aspects of the work environment such as lighting, rest periods, and incentives.
The experiments started with a test of the lighting conditions in the workplace. Mayo and his team wanted to see if increased lighting levels would improve workers’ productivity. The researchers increased the light in one section of the plant while leaving another section with the normal lighting conditions as a control group. However, the results showed that productivity increased in both sections regardless of the lighting conditions.
The next phase of the study involved the manipulation of rest periods. Mayo and his colleagues wanted to see if workers’ productivity would be improved by changing their rest periods. They experimented with different durations and timings of rest breaks but found no significant difference in productivity. Interestingly, the workers became more productive when they were allowed to take breaks together. This finding led to the third phase of the study where the researchers examined the impact of group work on productivity.
The group work experiments were the most significant of the series. Mayo and his team created small groups of workers who were responsible for completing a specific task. The researchers observed that the workers’ productivity increased significantly when they were working in a group. Furthermore, the workers developed a unique sense of unity, cooperation, and motivation which the researchers called the “Hawthorne effect.”
The researchers realized that the workers’ productivity was influenced not only by physical and environmental factors but also by social interactions and the management style. The study revealed that workers’ productivity was influenced by the feelings of recognition and attention they received from their supervisors and colleagues. Consequently, management’s attitude and communication became an essential factor affecting worker productivity and job satisfaction.
The Hawthorne Studies changed the way researchers and managers thought about the relationship between workers and productivity. Before the experiments, many experts believed that productivity was mainly driven by physical factors such as lighting, heating, and ventilation. However, the studies demonstrated that the human factor, such as social interaction, motivation, and management style, played an essential role in workers’ productivity.
Despite the critique and controversy that surrounded the Hawthorne Studies, their findings remain a significant contribution to the field of management studies. The experiments provided a basis for the development of theories such as human relations theory, which highlights the importance of understanding human behavior and motivation in the workplace. The studies have also influenced practices such as job enrichment and participative management, which aim to enhance worker satisfaction and productivity.
The Impact on Industrial Psychology
The Hawthorne Studies, conducted between 1924 and 1932, were a series of experiments conducted by Elton Mayo and a team of researchers at the Hawthorne Works plant in Cicero, Illinois. These studies aimed to understand the relationship between working conditions and productivity. The research findings, which revealed that improvements in working conditions did not always lead to increased productivity, had a significant impact on industrial psychology.
Before the Hawthorne Studies, industrial psychology was primarily concerned with improving worker efficiency through scientific management techniques, which focused on maximizing output through standardized work methods. However, the Hawthorne Studies challenged this approach by showing that non-financial incentives, such as social relationships and a sense of job satisfaction, could also have a significant impact on productivity. This led to a shift in industrial psychology research, which began to focus on the social and psychological factors that influence worker behavior.
The Hawthorne Studies also had a profound impact on the field of organizational behavior. Prior to these studies, it was assumed that workers were motivated solely by financial incentives and that their behavior could be effectively controlled through job design and other management techniques. However, the Hawthorne Studies exposed the complexity of human motivation and behavior, highlighting the importance of non-financial factors in shaping human behavior.
Furthermore, the Hawthorne Studies led to the development of the human relations movement, which emphasized the importance of treating workers as individuals with unique needs and motivations. This movement promoted the use of participative management and other democratic approaches to organizational decision-making, which aimed to empower workers and involve them in the decision-making process.
Overall, the Hawthorne Studies had a lasting impact on industrial psychology, organizational behavior, and management theory. Their findings challenged prevailing assumptions about worker motivation and behavior, highlighting the importance of social and psychological factors in shaping human behavior. This shift in focus has led to significant advancements in our understanding of how to create productive and fulfilling work environments.
The Four Phases of the Hawthorne Studies
The Hawthorne studies were a series of studies conducted in the 1920s and 1930s at the Hawthorne Works of the Western Electric Company in Chicago with the aim of exploring ways to increase productivity in the workplace. Led by Elton Mayo, the studies, which lasted for almost a decade, involved changing work conditions, taking measurements, and monitoring the workers. The studies were conducted in four phases, each with its unique objective. The phases include the illumination experiment, the relay assembly test room experiment, the interviewing program, and the bank wiring observation room experiment. In this article, we will focus on the third phase, the interviewing program, and explore how it contributed to the overall outcomes of the Hawthorne studies.
The Interviewing Program
The interviewing program was conducted between May 1928 and September 1929 and involved interviewing workers at the Hawthorne Works. The objective of the program was to explore the social and psychological factors that influenced the workers’ behavior, attitudes, and productivity. The program covered different topics such as the workers’ work experiences, the social relations at work, and their personal lives outside work. The interviews were conducted in a non-structured way, allowing the workers to express their opinions and feelings freely without any predetermined questions from the interviewers. The interviewers were trained to listen to the workers without interrupting or imposing their own views on the workers.
The interviewing program led to some surprising results that challenged the orthodox view of management. The workers, who were previously seen as motivated solely by financial incentives, displayed strong social and psychological needs that influenced their behavior at work. For example, the workers’ desire for job security, a sense of belonging, and recognition from their peers were found to be more important motivators than financial incentives. Additionally, the interviews revealed that the workers’ behavior was influenced by the social norms and values of the workgroup, which created a sense of camaraderie and loyalty among the workers.
The interviewing program also uncovered the importance of informal communication in the workplace. The workers often communicated with each other to exchange information, support each other, and resolve conflicts. The informal communication networks were found to be crucial in maintaining the social cohesion of the workgroup and promoting productivity.
Overall, the interviewing program contributed significantly to the Hawthorne studies’ outcomes by uncovering the social and psychological factors that influenced workers’ behavior, attitudes, and productivity. The program challenged the traditional view of workers as purely rational beings motivated solely by financial incentives and emphasized the importance of social and psychological factors in the workplace.
In conclusion, the Hawthorne studies, particularly the interviewing program, paved the way for the human relations approach to management, which emphasized the importance of understanding workers’ social and psychological needs to increase productivity. The program revealed that creating an environment that promotes job security, social cohesion, and recognition from peers can be more effective in motivating workers than financial incentives. The interviewing program also provided insights into the importance of informal communication networks in the workplace and how they can enhance productivity. The Hawthorne studies continue to be relevant today as they provide valuable insights into the complex interactions between social and psychological factors that influence human behavior in the workplace.
The Hawthorne Effect: Myth or Reality?
The Hawthorne studies, conducted between 1924 and 1932 at the Western Electric Company’s Hawthorne Works in Chicago, Illinois, have been the subject of a great deal of controversy over the years. The experiments were intended to study how variations in lighting conditions affected employee productivity. However, what the researchers discovered was something much more complex: that social factors, such as peer pressure and belonging to a group, could have a significant impact on workers’ attitudes and behavior.
One of the most famous aspects of the Hawthorne studies is the concept of the Hawthorne Effect, which refers to the idea that people who know they are being observed will alter their behavior accordingly. In the case of the Hawthorne studies, the workers who were being studied seemed to be more productive simply because they knew they were part of an experiment.
However, some researchers have argued that the Hawthorne Effect is not, in fact, a real phenomenon, but rather a myth that has been perpetuated over the years. They point out that there is little evidence to support the idea that people actually change their behavior simply because they are being observed.
So, is the Hawthorne Effect real, or is it just a myth? The answer is not entirely clear. While some researchers have claimed that the effect is nothing more than a statistical anomaly, others have insisted that it is a very real phenomenon that can significantly influence the results of research studies.
One major critique of the Hawthorne Effect is that it is difficult to measure. Researchers have struggled to find accurate ways of gauging how much people’s behavior is influenced by being observed. In addition, the degree to which people are affected by the Hawthorne Effect may vary depending on the situation and the individuals involved.
Despite these challenges, many researchers still believe that the Hawthorne Effect is a legitimate concept. They argue that people’s behavior can be impacted by a variety of social and psychological factors, including the knowledge that they are being studied. This, in turn, can influence the results of scientific experiments.
The controversy surrounding the Hawthorne Effect only underscores the complex nature of human behavior. While it may be difficult to definitively prove the existence of the effect, the debates surrounding it have led to a deeper understanding of the complex interplay between social factors and individual behavior.
At the end of the day, whether the Hawthorne Effect is real or not is less important than its broader implications. The studies conducted at Hawthorne Works have shown us that factors such as group dynamics, workplace culture, and employee morale can have a profound impact on productivity and success.
Continued Relevance of the Hawthorne Studies in Modern Organizations
The Hawthorne Studies are a set of experiments that took place in the late 1920s and early 1930s at the Hawthorne Works Plant in Cicero, Illinois, where researchers observed the behavior of factory workers and studied the effects of various working conditions on their productivity. The studies have since become well-known due to their significant contributions to the field of management and organizational behavior. Nearly a century later, it is clear that the Hawthorne Studies still hold value for modern organizations in several ways.
Improved Working Conditions
The Hawthorne Studies demonstrated that an employee’s working conditions are critical to their performance. The studies showed that workers had higher productivity when their managers paid attention to their needs, communicated more frequently, showed care, and provided comfortable working conditions. Nowadays, organizations still need to be conscious of their employees’ needs and well-being, which necessitates providing a safe and comfortable working environment, as well as establishing a positive work culture. By doing this, employees can be more efficient and committed to their work.
The findings of the Hawthorne Studies underscored the need for recognition of employee work and achievements. The study showed that the workers’ satisfaction improved with the presence of recognition from management. Therefore, organizations that know how to appreciate and reward their employees’ efforts are more likely to increase job satisfaction, morale, and commitment. Consequently, leading to an improved performance level of the employees’ towards the organization and towards the work as well. Each employee has their own contribution that helps the organization achieve its goals, and recognition is a way to ensure that they know their efforts are not taken for granted.
The Hawthorne Studies identified that employees demonstrated a higher level of productivity level when they were engaged, relaxed, and had positive feelings about their work. Therefore, with this lesson in mind, organizations should prioritize employee engagement. They should try to make work engaging and enjoyable while enabling their employees to grow and advance. As a result, they can motivate their workforce to commit to the company and perform at their best levels.
Effective communication is a vital component of organizational success. The Hawthorne Studies showed that communication supports a healthy and efficient workforce. They found that when management communicated effectively with their employees, it positively impacted the productivity of the entire organization. Nowadays, companies must focus on good communication practices. Leaders must have clarity and express themselves correctly, and employees need to feel comfortable sharing their views and perspectives. Efficient communication can avoid misunderstandings and build trust, which is crucial to the success of any organization.
In conclusion, the Hawthorne Studies have demonstrated that companies that look after their employees, guarantee recognition and communication, prioritize engagement, and are mindful of their working environment, ultimately, reap rewards with improved performance and increased productivity. By incorporating such lessons into their organizations, companies can create a culture of engagement, productivity, and growth that benefits everyone involved.

domain: psychology