Search

The preservation of pure fungal cultures is considered as an important part of applied and industrial microbiology applications. In this study, we are focusing on filamentous fungi preservation as these species are very useful in industrial applications such as wastewater treatment, biofuel production etc. We are presenting an evaluation of three methods to maintain and preserve filamentous fungi: refrigeration on filter paper, refrigeration with glycerol and cryopreservation with DMSO. The three methods were tested on the pure cultures of Aspergillus versicolor and Aspergillus niger. The three methods were successful where the viability measured by colony forming unit CFU counts was confirmed. We suggest that for the short-term preservation of filamentous fungi, the filter paper method could be preferred as it is very simple, quick and effective.

Targeting cell cycle and inducing DNA damage by activating cell death pathways are considered as effective therapeutic strategy for combating breast cancer progression. Many of the naturally known small molecules target these signaling pathways and are effective against resistant and/or aggressive types of breast cancers. Here, we investigated the effect of catechol, a naturally occurring plant compound, for its specificity and chemotherapeutic efficacies in breast cancer (MCF-7 and MDA-MB-231) cells. Catechol treatment showed concentration-dependent cytotoxicity and antiproliferative growth in both MCF-7 and MDA-MB-231 cells while sparing minimal effects on noncancerous (F-180 and HK2) cells. Catechol modulated differential DNA damage effects by activating ATM/ATR pathways and showed enhanced γ-H2AX expression, as an indicator for DNA double-stranded breaks. MCF-7 cells showed G1 cell cycle arrest by regulating p21-mediated cyclin E/Cdk2 inhibition. Furthermore, activation of p53 triggered a caspase-mediated cell death mechanism by inhibiting regulatory proteins such as DNMT1, p-BRCA1, MCL-1, and PDCD6 with an increased Bax/Bcl-2 ratio. Overall, our results showed that catechol possesses favorable safety profile for noncancerous cells while specifically targeting multiple signaling cascades to inhibit proliferation in breast cancer cells.

OBJECTIVE To illustrate changes of untargeted metabolomics in patients with glioblastoma IDH wildtype undergoing concurrent radiation therapy (RT) with temozolomide (TMZ). This study implemented machine learning (ML) algorithms to predict treatment phase: pre-surgery, post-surgery, pre-radiation, and post radiation based on untargeted metabolomics data. METHODS Thirty-six patients with glioblastoma IDH wildtype (18 methylguanine methyltransferase [MGMT] methylated, 16 MGMT unmethylated, 2 MGMT status unknown) were enrolled into this study. Serum samples obtained from patients on the same day before surgery, 2 days after surgery, before starting their concurrent chemoradiation, and after concluding this phase of treatment. Blood samples were obtained via antecubital phlebotomy without regard for time of the day, diet, or fasting status. Untargeted metabolomics by GC-TOF mass spectrometry were obtained and compared. The proposed ML models analyzed 105 samples from 36 patients utilizing 157 structurally identified blood metabolites. Multinomial Logistic Regression (MLR) and GradientBoostingClassifier (GB Classifier) were used to classify patient samples based on detected changes in blood metabolites. The classification performance of these models was evaluated using performance metrics and AUC scores. RESULTS Post radiation; significant increase in the following metabolites: glycine, serine, threonine, oxoproline, 6-deoxyglucose, gluconic acid, glycerol-alpha-phosphate, ethanolamine, propyleneglycol, triethanolamine, xylitol, and fumaric acid were noted while significant decrease in 3-aminopiperidine 2,6-dione was noted post radiation. MLR produced 78% accuracy, 75% precision, and AUC = 0.89, and GB Classifier produced 75% accuracy, 77% precision and AUC = 0.91. Finally, we presented a pattern of metabolites changes per clinical stage based on pairwise correlations. CONCLUSIONS This study represent the first serum metabolic signature associated with RT in patients with glioblastoma. The results from the classification algorithms and pairwise correlations showed that blood metabolites have the potential to predict phase of treatment and potentially enable to evaluate response to treatment in patients with glioblastoma in a relatively small cohort.

Antibiotic resistance (AR) is a significant threat to the health of many worldwide. The emergence of AR has become the main source of morbidity and mortality from infections that would otherwise have been treatable. AR is mainly caused by the inappropriate use of antibiotics. In the United Arab Emirates (UAE), bacterial resistance to antibiotics is escalating due to: a) practice of self-medication with antibiotics among the residents of the UAE; b) the easy procurement of antibiotics from local pharmacies without prescription; and c) expatriates’ importation of antibiotics from their home country. The present study focused on the usage of antibiotics among the population of Ras Al Khaimah. This study surveyed 306 residents of Ras Al Khaimah to understand their knowledge, awareness, attitude, practices, and perceptions on antibiotics usage. The major pharmacies in Ras Al Khaimah were also surveyed to identify whether the policies on dispensing antibiotics are followed. Of the 306 respondents, %49 have practiced self-medication using antibiotics at least once. The study also finds that pharmacies dispense antibiotics without prescription due to customer demand for antibiotics, lack of patients’ time, patients’ needs, lack of medical insurance, and money concerns. In conclusion, this study provides insight into the level of AR awareness and usage among the Ras Al Khaimah population and emphasizes the need to implement best practices at the grassroots level to overcome the crisis.

In medical and health sciences, the detection of cell injury plays an important role in diagnosis, personal treatment and disease prevention. Despite recent advancements in tools and methods for image classification, it is challenging to classify cell images with higher precision and accuracy. Cell classification based on computer vision offers significant benefits in biomedicine and healthcare. There have been studies reported where cell classification techniques have been complemented by Artificial Intelligence-based classifiers such as Convolutional Neural Networks. These classifiers suffer from the drawback of the scale of computational resources required for training and hence do not offer real-time classification capabilities for an embedded system platform. Field Programmable Gate Arrays (FPGAs) offer the flexibility of hardware reconfiguration and have emerged as a viable platform for algorithm acceleration. Given that the logic resources and on-chip memory available on a single device are still limited, hardware/software co-design is proposed where image pre-processing and network training were performed in software, and trained architectures were mapped onto an FPGA device (Nexys4DDR) for real-time cell classification. This paper demonstrates that the embedded hardware-based cell classifier performs with almost 100% accuracy in detecting different types of damaged kidney cells.

The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP–Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium.

Regulation of Ca2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca2+ influx pathway in oocytes is Store-Operated Ca2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260–275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.Other Information Published in: Scientific Reports License: https://creativecommons.org/licenses/by/4.0See article on publisher's website: http://dx.doi.org/10.1038/s41598-021-82048-z

Siglecs (Sialic acid-binding immunoglobulin-type lectins) are a I-type lectin that typically binds sialic acid. Siglecs are predominantly expressed in immune cells and generate activating or inhibitory signals. They are also shown to be expressed on the surface of cells in the nervous system and have been shown to play central roles in neuroinflammation. There has been a plethora of reviews outlining the studies pertaining to Siglecs in immune cells. However, this review aims to compile the articles on the role of Siglecs in brain function and neurological disorders. In humans, the most abundant Siglecs are CD33 (Siglec-3), Siglec-4 (myelin-associated glycoprotein/MAG), and Siglec-11, Whereas in mice the most abundant are Siglec-1 (sialoadhesin), Siglec-2 (CD22), Siglec-E, Siglec-F, and Siglec-H. This review is divided into three parts. Firstly, we discuss the general biological aspects of Siglecs that are expressed in nervous tissue. Secondly, we discuss about the role of Siglecs in brain function and molecular mechanism for their function. Finally, we collate the available information on Siglecs and neurological disorders. It is intriguing to study this family of proteins in neurological disorders because they carry immunoinhibitory and immunoactivating motifs that can be vital in neuroinflammation.

The steroid hormone progesterone (P4) mediates many physiological processes through either nuclear receptors that modulate gene expression or membrane P4 receptors (mPRs) that mediate nongenomic signaling. mPR signaling remains poorly understood. Here we show that the topology of mPRβ is similar to adiponectin receptors and opposite to that of G-protein-coupled receptors (GPCRs). Using Xenopus oocyte meiosis as a well-established physiological readout of nongenomic P4 signaling, we demonstrate that mPRβ signaling requires the adaptor protein APPL1 and the kinase Akt2. We further show that P4 induces clathrin-dependent endocytosis of mPRβ into signaling endosome, where mPR interacts transiently with APPL1 and Akt2 to induce meiosis. Our findings outline the early steps involved in mPR signaling and expand the spectrum of mPR signaling through the multitude of pathways involving APPL1.

Store-operated Ca 2+ entry (SOCE) encodes a range of cellular responses downstream of Ca 2+ influx through the SOCE channel Orai1. Orai1 recycles at the plasma membrane (PM), with ~40% of the total Orai1 pool residing at the PM at steady state. The mechanisms regulating Orai1 recycling remain poorly understood. We map the domains in Orai1 that are required for its trafficking to and recycling at the PM. We further identify, using biochemical and proteomic approaches, the CCT [chaperonin-containing TCP-1 (T-complex protein 1)] chaperonin complex as a novel regulator of Orai1 recycling by primarily regulating Orai1 endocytosis. We show that Orai1 interacts with CCT through its intracellular loop and that inhibition of CCT-Orai1 interaction increases Orai1 PM residence. This increased residence is functionally significant as it results in prolonged Ca 2+ signaling, early formation of STIM1-Orai1 puncta, and more rapid activation of NFAT (nuclear factor of activated T cells) downstream of SOCE. Therefore, the CCT chaperonin is a novel regulator of Orai1 trafficking and, as such, a modulator of Ca 2+ signaling and effector activation kinetics.

By analyzing the abundant microorganisms in the naturally occurring mangroves of Ras Al Khaimah in the United Arab Emirates (UAE), this policy paper discusses the feasibility of a future where the emirate invests in biofuels sourced from its mangroves as opposed to using conventional methods of fuel production and usage, which can have negative impacts on the environment and economy. It explores the successes and challenges of each phase of biofuel technology innovation, so that the previous trials and errors can help decision makers critically assess the potentials of utilizing biofuels in current technology as well as in future projects. This paper finds that currently it is possible to use the existing mangroves as a source of fungi for biofuel production, but more investment is needed to support industrial-level production. It concludes with policy recommendations for supporting future research, implementing long-term energy solutions that address the UAE’s environmental and economic concerns, and encouraging future leaders and innovators to think local in addressing global problems.

Store-operated Ca

Progesterone mediates its physiological functions through activation of both transcription-coupled nuclear receptors and seven-pass-transmembrane progesterone receptors (mPRs), which transduce the rapid non-genomic actions of progesterone by coupling to various signaling modules. However, the immediate mechanisms of action downstream of mPRs remain in question. Herein, we use an untargeted quantitative proteomics approach to identify mPR interactors to better define progesterone non-genomic signaling. Surprisingly, we identify the very-low-density lipoprotein receptor (VLDLR) as an mPRβ (PAQR8) partner that is required for mPRβ plasma membrane localization. Knocking down VLDLR abolishes non-genomic progesterone signaling, which is rescued by overexpressing VLDLR. Mechanistically, we show that VLDLR is required for mPR trafficking from the endoplasmic reticulum to the Golgi. Taken together, our data define a novel function for the VLDLR as a trafficking chaperone required for the mPR subcellular localization and, as such, non-genomic progesterone-dependent signaling.This article has an associated First Person interview with the first author of the paper.

Ca2+ is a ubiquitous intracellular messenger that transduces a variety of cellular responses downstream of the activation of G-protein-coupled or tyrosine kinase receptors. Depending on the agonist and cellular context, Ca2+ can mediate different responses in the same cell [1]. The specific cellular response transduced downstream of the particular Ca2+ transient is encoded in the spatial and temporal dynamics of the Ca2+ signal, leading to the activation of a subset of Ca2+-dependent effectors and the ensuing cellular response. As such, the duration, amplitude, frequency, and spatial localization of Ca2+ signals encode targeted signals that activate Ca2+-sensitive effectors to define a particular cellular response. To generate and fine-tune those Ca2+ signals, cells use two main Ca2+ sources: entry of extracellular Ca2+ and Ca2+ release from intracellular stores. The primary intracellular Ca2+ store is the endoplasmic reticulum (ER), which can concentrate Ca2+ in the hundreds of μM range [2]. In contrast, cytoplasmic Ca2+ concentration is kept at rest at extremely low levels (∼100 nM or lower), thus providing a low-noise background for detection of complex Ca2+ dynamics [3].The Ca2+-signaling machinery includes Ca2+ entry and extrusion pathways in the plasma membrane (PM), ER membrane Ca2+ release channels, and Ca2+ reuptake ATPases within the ER membrane [4]. These Ca2+ transport pathways, in addition to intracellular Ca2+ buffers and Ca2+ uptake and release through other intracellular organelles, primarily the mitochondria, combine to shape highly tuned and dynamic Ca2+ transients that regulate cellular functions [5].Under physiological conditions in non-excitable cells, Ca2+ transients are typically initiated downstream of agonist stimulation through the activation of the PLC-IP3 signal transduction cascade, which leads to the opening of intracellular Ca2+ channel inositol 1,4,5-trisphosphate receptors (IP3Rs) to release Ca2+ from intracellular stores [6]. Ca2+ release depletes the stores and activates a Ca2+ influx pathway in the PM termed store-operated Ca2+ entry (SOCE). SOCE is mediated by two key players: ER transmembrane Ca2+ sensors represented by the STIM family of proteins and PM Ca2+ channels of the Orai family that link directly to STIMs (see Chapters 1 through 3). The N-terminus of STIM1 faces the ER lumen and consists of two EF-hand domains that detect luminal Ca2+ concentration. The loss of STIM1 Ca2+ binding upon store depletion leads to conformational changes in the protein and its aggregation into clusters that translocate and stabilize into ER-PM junctions with very close apposition (∼20 nm) [7]. STIM1 within these ER-PM junctions binds to and recruits Orai1 through a diffusional trap mechanism, resulting in opening Orai1 channels and Ca2+ entry [8]. As such, the STIM-Orai clusters at ER-PM junctions define a specific microdomain at ER-PM junctions that also include the ER Ca-ATPase (SERCA) [9,10].The tightly regulated remodeling of the Ca2+-signaling machinery upon store depletion allows for specific Ca2+ signaling in the midrange between Ca2+ microdomains and global Ca2+ waves [10] (see Chapter 5). Spatially, Ca2+ signaling can occur in localized spatially restricted elementary Ca2+ release events that activate effectors located in the immediate proximity of the Ca2+ channel. Alternatively, Ca2+ signals/waves occur/spread through the entire cell resulting in a global spatially unrestricted signal.We have recently described a SOCE-dependent Ca2+-signaling modularity that signals in the midrange between these two spatial extremes [10]. Store depletion downstream of receptor activation and IP3 generation results in a localized Ca2+ entry point source at the SOCE clusters that induces Ca2+ entry into the cytoplasm, which is readily taken up into the ER lumen through SERCA activity only to be released again through open IP3Rs distally to the SOCE entry site and gate Ca2+-activated Cl- channels as downstream Ca2+ effectors. This mechanism, referred to as “Ca2+ teleporting,” allows for specific activation of Ca2+ effectors that are distant from the point source Ca2+ channel without inducing a global Ca2+ wave, thus providing a novel module in the Ca2+-signaling repertoire. A cartoon summary of Ca2+ teleporting is found in Figure 12.1.The relationship between Ca2+ signaling and cellular proliferation is complex with Ca2+ transients detected at various stages of the cell cycle [2]. These transients are thought to activate a multitude of Ca2+ effectors downstream of the initial Ca2+ signal, which were shown to be important for cellular proliferation, including, for example, calmodulin (CaM) and Ca2+-CaM-dependent protein kinase II (CaMKII).However, there are a few cases where Ca2+ signals have been shown directly to be critical for cell cycle progression, including in mitosis for nuclear envelope breakdown and for chromosome disjunction [11]. In contrast, nuclear envelope breakdown during meiosis in vertebrate oocytes occurs independently of Ca2+, but Ca2+ is required for the completion of meiosis I in vertebrate oocytes [12–14]. Interestingly, multiple Ca2+ signaling pathways are modified during M-phase of the cell cycle, with the best defined example being Xenopus oocyte maturation [15].Several Ca2+ influx pathways have been implicated in cell proliferation and cell cycle progression, including TRP channels, voltage-gated Ca2+ channels (CaV), purinergic P2X receptors, ionotropic glutamate receptors, and SOCE [16]. Blockers of voltage-gated Ca2+ channels were shown to slow down cell growth, arguing for a role for these channels in cell cycle progression [16–18]. Experimental manipulation of the expression levels of members of the TRPC, TRPV, and TRPM families of cation channels, which are Ca2+ permeable, was linked to cell proliferation with differential effects depending on the particular channel studied [16]. However, some of these studies are difficult to interpret because channel knockdown or overexpression could have significantly broader effects on Ca2+ signaling than affecting Ca2+ influx through the specific channel in question, as it may lead to changes in expression of other Ca2+-signaling pathways as a compensatory mechanism. Furthermore, the majority of TRP channels conduct cations with some Ca2+ permeability and are not Ca2+ selective like Orai1 or CaV channels, with the exception of TRPV5 and TRPV6 (see Chapter 13). Hence, changes in their expression is likely to affect the ionic balance across the cell membrane with effects on resting membrane potential, which may in turn affect cell proliferation.The relationship between SOCE and cell proliferation is an intimate one that goes beyond the well-recognized roles of Ca2+ signaling in cellular growth and proliferation. SOCE is dramatically downregulated during the division phase of the cell cycle through mechanisms that have not been fully elucidated. This is in line with the significant remodeling of the Ca2+-signaling machinery during M-phase, which has been well characterized during oocyte meiosis. Furthermore, there is mounting evidence from multiple neoplasms for an important role for SOCE in metastasis.This chapter presents a brief overview of our current knowledge as to the mechanisms regulating SOCE during cell cycle from cellular proliferation to metastasis with an emphasis on SOCE regulation during cell division (mitosis and meiosis).

Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca2+signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+signaling, including most prominently an inhibition of the passive ER Ca2+leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+signaling machinery are different.

Mitochondria are unique organelles carrying their own genetic material, independent from that in the nucleus. This review will discuss the nature of mitochondrial DNA (mtDNA) and its levels in the cell, which are the key elements to consider when trying to achieve molecular identification in ancient and degraded samples. mtDNA sequence analysis has been appropriately validated and is a consistent molecular target for the examination of biological evidence encountered in forensic cases—and profiling, in certain conditions—especially for burnt bodies and degraded samples of all types. Exceptional cases and samples will be discussed in this review, such as mtDNA from leather in Beethoven’s grand piano, mtDNA in mummies, and solving famous historical criminal cases. In addition, this review will be discussing the use of ancient mtDNA to understand past human diet, to trace historical civilizations and ancient trade routes, and to uncover geographical domestication origins and lineage relationships. In each topic, we will present the power of mtDNA and how, in many cases, no nuclear DNA was left, leaving mitochondrial DNA analysis as a powerful alternative. Exploring this powerful tool further will be extremely useful to modern science and researchers, due to its capabilities in providing us with previously unattainable knowledge.

Ca(2+)-activated Cl(-) channels (CaCCs) play important physiological functions in epithelia and other tissues. In frog oocytes the CaCC Ano1 regulates resting membrane potential and the block to polyspermy. Here, we show that Ano1 expression increases the oocyte surface, revealing a novel function for Ano1 in regulating cell morphology. Confocal imaging shows that Ano1 increases microvilli length, which requires ERM-protein-dependent linkage to the cytoskeleton. A dominant-negative form of the ERM protein moesin precludes the Ano1-dependent increase in membrane area. Furthermore, both full-length and the truncated dominant-negative forms of moesin co-localize with Ano1 to the microvilli, and the two proteins co-immunoprecipitate. The Ano1-moesin interaction limits Ano1 lateral membrane mobility and contributes to microvilli scaffolding, therefore stabilizing larger membrane structures. Collectively, these results reveal a newly identified role for Ano1 in shaping the plasma membrane during oogenesis, with broad implications for the regulation of microvilli in epithelia.

Apoptosis is a regulated form of cell death that proceeds by defined biochemical pathways. Most apoptosis is controlled by interactions between pro-survival and pro-apoptotic Bcl-2 family proteins in which death is often the consequence of permeabilization of the mitochondrial outer membrane. Many drugs affect this equilibrium to favor apoptosis but this process is not completely understood. We show that the chemotherapeutic drug cisplatin initiates an apoptotic pathway by phosphorylation of a pro-survival Bcl-2 family member, Bcl-xL, by cyclin-dependent kinase 2. The phosphorylation occurred at a previously unreported site and its biologic significance was demonstrated by a phosphomimetic modification of Bcl-xL that was able to induce apoptosis without addition of cisplatin. The mechanism of cell death induction was similar to that initiated by pro-apoptotic Bcl-2 family proteins, that is, phosphorylated Bcl-xL translocated to the mitochondrial membrane, and formed pores in the membrane. This initiated cytochrome c release and caspase activation that resulted in cell death.

Store-operated calcium entry (SOCE) is a ubiquitous Ca2+ influx pathway essential for many physiological functions and failure to maintain normal calcium homeostasis is one of the leading causes of cellular dysfunction in a wide variety of pathological conditions. Orai1, a key regulator of SOCE, constitutively recycles at steady state in the frog oocyte and internalizes into intracellular vesicular compartments during meiosis, leading to inactivation of SOCE. Such mechanism provides proper regulation of Ca2+ signaling in preparation for fertilization and embryonic development. Previous data showed a role for Orai1 C-terminus in its internalization during meiosis. However, the minimal region required for Orai1 internalization at steady state and during meiosis is not known.We began the study of the molecular determinants of Orai1 trafficking in Xenopus oocytes by comparing the localization of multiple GFP-Orai1 C-terminal mutants 1-266, 1-275, and 1-285 intracellularly. Orai1 mutants 1-275 and 1-285 were both internalized during meiosis behaving similarly to WT, however, Orai1 1-266 was significantly enriched at the plasma membrane and did not internalize during oocyte maturation. To further map the region required for Orai1 internalization, we will generate specific mutants in the region 267-275 and evaluate the contribution of these signals in Orai1 internalization during meiosis. Mutants showing defect in internalization will be tested for phenotype rescue by co-injecting Orai1 with different potential candidates. These proteins include: 1) Rab5, a member of Rab family of GTPases has been shown to play a role as a regulator of intracellular trafficking during endocytosis. Previous data from our group has shown that Rab5 colocalizes with Orai1 in both oocytes and eggs. 2) Caveolin-1, a coat protein mediating caveolin-dependent endocytosis. 3) Flotilin-1, a membrane-associated protein involved in flotilin-dependent endocytosis.As a second step in exploring the molecular and biochemical mechanisms underlying Orai1 internalization during meiosis, we will identify Orai1 associated proteins by co-immunoprecipitation of Orai1 complexes followed by quantitative proteomic analysis using dimethyl labeling coupled with tandem liquid chromatography-mass spectrometry (LC-MS). Candidates that are enriched in wild-type Orai1 immunoprecipitation but missing in internalization-deficient Orai1 mutants are suggested to play role in Orai1 endocytosis.In conclusion, our results here suggest that residues 266–275 at C-terminus of Orai1 are essential for its internalization during meiosis. Using specific mutant(s) within this region, we will test the rescue of mutant(s) phenotype with potential candidates, and by quantitative proteomic analysis identify proteins associated with Orai1 and potentially important in its internalization.

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. We found that an 18-kDa protein identified by mass spectrometry as p21WAF1/Cip1was phosphorylated by Cdk2 starting 12 h after cisplatin exposure. The analysis showed it was phosphorylated at serine 78, a site not previously identified. The adenoviral transduction of p21 before cisplatin exposure protects from cytotoxicity by inhibiting Cdk2. Although cisplatin causes induction of endogenous p21, the protection is inefficient. We hypothesized that phosphorylation of p21 at serine 78 could affect its role as a Cdk inhibitor, and thereby lessen its ability to protect from cisplatin cytotoxicity. To investigate the effect of serine 78 phosphorylation on p21 activity, we replaced serine 78 with aspartic acid, creating the phosphomimic p21S78D. Mutant p21S78Dwas an inefficient inhibitor of Cdk2 and was inefficient at protecting TKPTS cells from cisplatin-induced cell death. We conclude that phosphorylation of p21 by Cdk2 limits the effectiveness of p21 to inhibit Cdk2, which is the mechanism for continued cisplatin cytotoxicity even after the induction of a protective protein.

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. A Cdk2 mutant (Cdk2-F80G) was designed in which the ATP-binding pocket was altered. When expressed in mouse kidney cells, this protein was kinase inactive, did not inhibit endogenous Cdk2, but protected from cisplatin. The mutant was localized in the cytoplasm, but when coexpressed with cyclin A, it was activated, localized to the nucleus, and no longer protected from cisplatin cytotoxicity. Cells exposed to cisplatin in the presence of the activated mutant had an apoptotic phenotype, and endonuclease G was released from mitochondria similar to that mediated by endogenous Cdk2. But unlike apoptosis mediated by wild-type Cdk2, cisplatin exposure of cells expressing the activated mutant did not cause cytochrome c release or significant caspase-3 activation. We conclude that cisplatin likely activates both caspase-dependent and -independent cell death, and Cdk2 is required for both pathways. The mutant-inactive Cdk2 protected from both death pathways, but after activation by excess cyclin A, caspase-independent cell death predominated.

In contrast to the well-defined role of Ca 2+ signals during mitosis, the contribution of Ca 2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca 2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca 2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca 2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca 2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca 2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca 2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca 2+ deprivation. These findings argue that Ca 2+ homeostasis is important in establishing the oocyte’s competence to undergo maturation in preparation for fertilization and embryonic development.

An effect of PGE2 on water and chloride absorption was already established in a previous work. This study is an attempt to find the mechanism of action of the prostaglandin by investigating the involvement of three major transporters namely the Na+ -K+ ATPase, the Na+/H+ exchanger and the Na+ K+ 2Cl- cotransporter. Rats were injected with PGE2 and 15 min later, the colon was perfused in situ with Krebs Ringer buffer, and net water and chloride absorption were determined. When the involvement of the cotransporter and/or the exchanger was investigated, animals were injected with, respectively, furosemide and amiloride 10 min before PGE2. Superficial and crypt colonocytes were then isolated and the protein expression of the Na+ -K+ ATPase and the Na+ K+ 2Cl- was determined by western blot analysis. The effect of PGE2 on the pump activity in presence or absence of the transporters' inhibitors was also studied. PGE2 decreased net water and chloride absorption from the colon, increased the Na+ -K+ ATPase activity in superficial cells and reduced it in crypt cells. The prostaglandin was found to stimulate secretion in superficial cells by targeting the Na+ K+ 2Cl- symporter, and reduce absorption in crypt cells by targeting the Na+/H+ antiporter. Changes in the activity of the pump are secondary to changes in the activity of the other transporters.

The key proteins mediating store-operated Ca(2+) entry (SOCE) are the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and the plasma membrane Ca(2+)-selective channel Orai1. Here, we quantitatively dissect Orai1 trafficking dynamics and show that Orai1 recycles rapidly at the plasma membrane (Kex≃0.1 min(-1)), with ∼40% of the total Orai1 pool localizing to the plasma membrane at steady state. A subset of intracellular Orai1 localizes to a sub-plasmalemal compartment. Store depletion is coupled to Orai1 plasma membrane enrichment in a STIM1-dependent fashion. This is due to trapping of Orai1 into cortical ER STIM1 clusters, leading to its removal from the recycling pool and enrichment at the plasma membrane. Interestingly, upon high STIM1 expression, Orai1 is trapped into STIM1 clusters intracellularly, thus preventing its plasma membrane enrichment following store depletion. Consistent with this, STIM1 knockdown prevents trapping of excess Orai1 into limiting STIM1 clusters in the cortical ER. SOCE-dependent Ca(2+) influx shows a similar biphasic dependence on the Orai1:STIM1 ratio. Therefore, a STIM1-dependent Orai1 'trafficking trap' mechanism controls Orai1 plasma membrane enrichment and SOCE levels, thus modulating the SOCE 'bandwidth' for downstream signaling.

The key proteins mediating store-operated Ca2+ entry (SOCE) are the endoplasmic reticulum (ER) Ca2+ sensor STIM1 and the plasma membrane Ca2+-selective channel Orai1. Here, we quantitatively dissect Orai1 trafficking dynamics and show that Orai1 recycles rapidly at the plasma membrane (Kex≃0.1 min−1), with ∼40% of the total Orai1 pool localizing to the plasma membrane at steady state. A subset of intracellular Orai1 localizes to a sub-plasmalemal compartment. Store depletion is coupled to Orai1 plasma membrane enrichment in a STIM1-dependent fashion. This is due to trapping of Orai1 into cortical ER STIM1 clusters, leading to its removal from the recycling pool and enrichment at the plasma membrane. Interestingly, upon high STIM1 expression, Orai1 is trapped into STIM1 clusters intracellularly, thus preventing its plasma membrane enrichment following store depletion. Consistent with this, STIM1 knockdown prevents trapping of excess Orai1 into limiting STIM1 clusters in the cortical ER. SOCE-dependent Ca2+ influx shows a similar biphasic dependence on the Orai1:STIM1 ratio. Therefore, a STIM1-dependent Orai1 ‘trafficking trap’ mechanism controls Orai1 plasma membrane enrichment and SOCE levels, thus modulating the SOCE ‘bandwidth’ for downstream signaling.

This work investigated the effect of different doses of PGE2 on net water and Cl− absorption from the rat colon, using an in situ perfusion technique. PGE2 exerted opposite effects at different concentrations. Net water and Cl− absorption was significantly reduced at low doses with a minimum at 0.4 μg/100 g BW, and significantly elevated at high doses with an observed maximal effect at 21 μg/100 g BW. At low doses, PGE2 increased in superficial cells, the activity of the Na+–K+ ATPase and the protein expression of the Na+K+2Cl− cotransporter, but reduced them in crypt cells. Thus, the reduction in net water and Cl− absorption was ascribed to an increase in secretion by surface cells that masked absorptive processes. At high doses, PGE2 increased significantly the activity of the Na+–K+ ATPase in superficial cells only, and was without any effect on the protein expression of the cotransporter and the pump in both surface and crypt cells. The observed increase in net water and Cl− absorption was attributed in this case to an increase in absorptive processes with no effect on secretion.

Tissue fibrosis is a major cause of death in developed countries. It commonly occurs after either acute or chronic injury and affects diverse organs, including the heart, liver, lung, and kidney. Using the renal ablation model of chronic kidney disease, we previously found that the development of progressive renal fibrosis was dependent on p21WAF1/Cip1 expression; the genetic knockout of the p21 gene greatly alleviated this disease. In the present study, we expanded on this observation and report that fibrosis induced by two different acute injuries to the kidney is also dependent on p21. In addition, when p21 expression was restricted only to the proximal tubule, fibrosis after injury was induced in the whole organ. One molecular fibrogenic switch we describe is transforming growth factor-β induction, which occurred in vivo and in cultured kidney cells exposed to adenovirus expressing p21. Our data suggests that fibrosis is p21 dependent and that preventing p21 induction after stress could be a novel therapeutic target.

Store-operated calcium entry (SOCE) is a ubiquitous Ca2+ influx pathway essential for many physiological functions. Dysregulation of SOCE causes disruption in Ca2+ homeostasis leading to cellular pathology of several diseases. Orai1, a key regulator of SOCE, constitutively recycles at steady state in the frog oocyte and internalizes into an intracellular vesicular compartments during meiosis, leading to inactivation of SOCE. Previous data showed a role for Orai1 C-terminus in its internalization during meiosis. However, the minimal region required for Orai1 internalization at steady state and during meiosis is not known. In this study we investigate the molecular determinants of Orai1 trafficking in Xenopus oocytes by comparing the localization of multiple Orai1 C-terminal mutants 1-266, 1-275, and 1-285 intracellularly. Orai1 mutants 1-275 and 1-285 were both present in endosomes and were internalized during meiosis behaving similarly to WT. However, Orai1 1-266 was enriched at the plasma membrane and showed decreased localization to the endosomal compartment at steady state. Furthermore, in contrast to wild-type Orai1, it was not internalized during oocyte maturation. To identify proteins interacting with Orai1 and involved in its trafficking, we co-immunoprecipitated Orai1 complexes from oocytes expressing WT Orai1 and Orai1 1-266 and analyzed complexes by mass spectrometry. Our preliminary data identified several candidate proteins co-immunoprecipitating with WT Orai1 but not with Orai1 1-266 mutant. To summarize, our results show that the region of Orai1 containing residues 267-275 is likely essential for Orai1 localization to endosomes suggesting a role for this domain in Orai1 intracellular distribution. Mass spectrometry analysis on co-immunoprecipitation of Orai1 WT identified several candidates that are missing in Orai1 1-266 co-IPs, suggesting a role for these candidates in Orai1 trafficking.

Intracellular calcium ion is a universal messenger that plays a critical role in wide range of physiological functions and failure to maintain calcium homeostasis is linked to cellular dysfunctions in several diseases. Store operated calcium entry (SOCE) is a fundamental Ca2+ influx pathway at the cell membrane that is activated in response to intracellular store depletion. The SOCE machinery consists of the ER Ca2+ sensor STIM1, and Orai1 a Ca2+ selective channel at the plasma membrane (PM). Despite advancements in our understanding of the regulation of STIM1 and Orai1 very little is known about the biogenesis and trafficking of Orai1 and how that affects SOCE in mammalian cells. Here we describe the trafficking dynamics underlying Orai1subcelluar distribution at steady state and after store depletion. We show that at steady state 40% of total cellular Orai1 localizes to the PM of CHO cells. The other 60% of Orai1 localizes intracellularly. Time lapse imaging shows continuous shuttling of Orai1-positive vesicles between the PM and intracellular compartments. Quantitative measurements of the exocytosis and endocytosis rates at steady state show a rapid recycling rate of the protein between the intracellular pool and the plasma membrane. Following store depletion, ~80% of Orai1 is localized at PM. Interestingly, we localized Orai1 to novel sub-plasma membrane vesicular compartment of unknown molecular composition. To study the domain of Orai1 involved in its trafficking, we generated deletions of the N-(1-90) or C-(257-301)-terminus. Deletion of N-terminus did not change the surface to total distribution of Orai1, while C-terminus deletion significantly altered Orai1 distribution, with majority of Orai1-∆C localizes intracellularly. Similarly, deletion of residues 267-301 and residues 276-301 showed significantly decreased surface/total distribution compared to wild type (WT), but a less pronounced defect than Orai1-∆C. In contrast, 1-285 behaved like WT Orai1 showing that residues 286-301 are not required for Orai1 biogenesis and localization to the cell membrane. In conclusion, our data suggest that Orai1 recycles at high rate between PM and intracellular compartments at steady state and is enriched at PM after store depletion. Our results also suggest that the amino acid region (257-285) of Orai1 C-terminus is essential for its biogenesis and PM targeting.