Development of a nomogram based on the clinicopathological and CT features to predict the survival of primary pulmonary lymphoepithelial carcinoma patients.

BACKGROUND: The aim of this study was to develop a nomogram by combining chest computed tomography (CT) images and clinicopathological predictors to assess the survival outcomes of patients with primary pulmonary lymphoepithelial carcinoma (PLEC). METHODS: 113 patients with stage I-IV primary PLEC who underwent treatment were retrospectively reviewed. The Cox regression analysis was performed to determine the independent prognostic factors associated with patient's disease-free survival (DFS) and cancer-specific survival (CSS). Based on results from multivariate Cox regression analysis, the nomograms were constructed with pre-treatment CT features and clinicopathological information, which were then assessed with respect to calibration, discrimination and clinical usefulness. RESULTS: Multivariate Cox regression analysis revealed the independent prognostic factors for DFS were surgery resection and hilar and/or mediastinal lymphadenopathy, and that for CSS were age, smoking status, surgery resection, tumor site in lobe and necrosis. The concordance index (C­index) of nomogram for DFS and CSS were 0.777 (95% CI: 0.703-0.851) and 0.904 (95% CI: 0.847-0.961), respectively. The results of the time­dependent C­index were internally validated using a bootstrap resampling method for DFS and CSS also showed that the nomograms had a better discriminative ability. CONCLUSIONS: We developed nomograms based on clinicopathological and CT factors showing a good performance in predicting individual DFS and CSS probability among primary PLEC patients. This prognostic tool may be valuable for clinicians to more accurately drive treatment decisions and individualized survival assessment.

Whole-exome sequencing identifies cancer-associated variants of the endo-lysosomal ion transport channels in the Saudi population.

Background: Although national efforts are underway to document the genomic variability of the Saudi population relative to other populations, such variability remains largely unexplored. Genetic variability is known to impact the fate of cells and increase or decrease the risk of a variety of complex diseases including cancer forms. Therefore, the identification of variants associated with cancer susceptibility in Saudi population may protect individuals from cancer or aid in patient-tailored therapies. The endo-lysosomal ion transport genes responsible for cationic ion homeostasis within the cell. We screened 703 single-nucleotide polymorphisms (SNPs) of the endo-lysosomal ion transporter genes in the Saudi population and identified cancer-associated variants that have been reported in other populations. Methods: Utilizing previously derived local data of Whole-Exome Sequencing (WES), we examined SNPs of TPCN1, TPCN2, P2RX4, TRPM7, TRPV4, TRPV4, and TRPV6 genes. The SNPs were identified for those genes by our in-house database. We predicted the pathogenicity of these variants using in silico tools CADD, Polyphen-2, SIFT, PrimateAI, and FATHMM-XF. Then, we validated our findings by exploring the genetics database (VarSome, dbSNP NCB, OMIM, ClinVar, Ensembl, and GWAS Catalog) to further link cancer risk. Results: The WES database yielded 703 SNPs found in TPCN2, P2RX4, TRPM7, TRPV4, and TRPV6 genes in 1,144 subjects. The number of variants that were found to be common in our population was 150 SNPs. We identified 13 coding-region non-synonymous variants of the endo-lysosomal genes that were most common with a minor allele frequency (MAF) of ≥ 1 %. Twelve of these variants are rs2376558, rs3750965, rs61746574, rs35264875, rs3829241, rs72928978, rs25644, rs8042919, rs17881456, rs4987682, rs4987667, and rs4987657 that were classified as cancer-associated genes. Conclusion: Our study highlighted cancer-associated SNPs in the endo-lysosomal genes among Saudi individuals. The allelic frequencies on polymorphic variants confer susceptibility to complex diseases that are comparable to other populations. There is currently insufficient clinical data supporting the link between these SNPs and cancer risk in the Saudi population. Our data argues for initiating future cohort studies in which individuals with the identified SNPs are monitored and assessed for their likelihood of developing malignancies and therapy outcomes.

Endolysosomal two-pore channel 2 plays opposing roles in primary and metastatic malignant melanoma cells.

The ion channel two-pore channel 2 (TPC2), localised on the membranes of acidic organelles such as endo-lysosomes and melanosomes, has been shown to play a role in pathologies including cancer, and it is differently expressed in primary versus metastatic melanoma cells. Whether TPC2 plays a pro- or anti-oncogenic role in different tumour conditions is a relevant open question which we have explored in melanoma at different stages of tumour progression. The behaviour of primary melanoma cell line B16F0 and its metastatic subline B16F10 were compared in response to TPC2 modulation by silencing (by small interfering RNA), knock-out (by CRISPR/Cas9) and overexpression (by mCherry-TPC2 transfected plasmid). TPC2 silencing increased cell migration, epithelial-to-mesenchymal transition and autophagy in the metastatic samples, but abated them in the silenced primary ones. Interestingly, while TPC2 inactivation failed to affect markers of proliferation in both samples, it strongly enhanced the migratory behaviour of the metastatic cells, again suggesting that in the more aggressive phenotype TPC2 plays a specific antimetastatic role. In line with this, overexpression of TPC2 in B16F10 cells resulted in phenotype rescue, that is, a decrease in migratory ability, thus collectively resuming traits of the B16F0 primary cell line. Our research shows a novel role of TPC2 in melanoma cells that is intriguingly different in initial versus late stages of cancer progression.

Loss of two-pore channel 2 function in melanoma-derived tumours reduces tumour growth in vivo but greatly increases tumour-related toxicity in the organism.

BACKGROUND: Melanoma, a severe form of skin cancer, poses significant health risks due to its aggressive nature and potential for metastasis. The role of two-pore channel 2 (TPC2) in the development and progression of melanoma remains poorly understood. This study aims to investigate the impact of TPC2 knockout (KO) on melanoma-derived tumors, focusing on tumour growth and related toxicity in the organism. METHODS: The study utilized CHL-1 and B16 melanoma cell lines with TPC2 KO to assess the changes in proliferation dynamics. Methods included real-time monitoring of cell proliferation using the xCELLigence system, in vivo tumour growth assays in mice, histopathological analyses, inflammation marker assessment, and quantitative PCR (qPCR) for gene expression analysis RESULTS: TPC2 KO was found to significantly alter the proliferation dynamics of CHL-1 and B16 melanoma cells. The in vivo studies demonstrated reduced tumor growth in TPC2 KO cell-derived tumors. However, a notable increase in tumor-related toxicity in affected organs, such as the liver and spleen, was observed, indicating a complex role of TPC2 in melanoma pathology. CONCLUSIONS: The loss of TPC2 function in melanoma cells leads to reduced tumour growth but exacerbates tumour-related toxicity in the organism. These findings highlight the dual role of TPC2 in melanoma progression and its potential as a therapeutic target. Further research is needed to fully understand the mechanisms underlying these effects and to explore TPC2 as a treatment target in melanoma.

Clinicopathological features and survival of rare primary pulmonary lymphoepithelial carcinoma: A cohort from a single center.

BACKGROUND: Primary pulmonary lymphoepithelial carcinoma (PLEC) is a rare subtype of nonsmall cell lung cancer. This study aimed to investigate the clinicopathological and prognostic characteristics of resected primary PLEC. MATERIALS AND METHODS: In this retrospective study, 95 consecutive patients with primary PLEC, who received radical surgical resection treatment, were examined from October 2009 to January 2022. The clinicopathological features and their association with survival outcomes were analyzed. RESULTS: Primary PLEC predominated in relatively younger patients and nonsmokers, who lacked driver mutations and were always positive for immunohistochemical markers of the squamous cell lineage. Further, 21.1% of patients had abnormally elevated preoperative serum marker fragments of cytokeratin 19 (Cyfra21-1). The median follow-up time was 43.5 months. The 1-, 3-, and 5-year recurrence-free survival (RFS) rates were 96.5%, 81.8%, and 64.3%, respectively. The median RFS time was not reached. Cox univariate survival analysis showed that patients with positive lymph nodes had significantly worse RFS than those with negative ones (p = 0.017). The patients with open surgery experienced significantly worse RFS than those with video-assisted thoracoscopic surgery (p = 0.038). The multivariate survival analysis confirmed that only lymph node involvement (hazard ratio: 2.769; 95% confidence interval: 1.171-6.548, p = 0.020) was an independent prognostic factor. CONCLUSIONS: Primary PLEC is a rare type of lung cancer with a favorable outcome, more common in young and nonsmoking Asian populations. Driver gene mutations are rare. Regional lymph node metastasis is an independent prognostic factor for RFS after radical surgical resection.

Relevance of lysosomal Ca2+ signalling machinery in cancer.

In comparison with normal cells, cancer cells are equipped with a higher number of lysosomes, involved in degradative and non-degradative roles. In particular, the lysosome is a Ca2+signalling hub, and the enhancement of this interconnected machinery in cancer cells has recently prompted investigations into the role that lysosomal ion channels play in oncology. The present review reports findings about the emerging role of lysosomal Ca2+channels: Two-Pore Channels (TPCs), Transient Receptor Potential Cation Channels (TRPMLs; mucolipins), and Purinergic X Receptor 4 (P2×4R), in a variety of cancer models, highlighting their impact on crucial functions such as the regulation of autophagy and the composition of the tumour microenvironment, including the secretion-mediated interplay with immune and endothelial cells. Notably, recent evidence indicates that, by regulating tumour secretome, lysosomal Ca2+ signalling can affect the composition of the tumour-infiltrating immune cell repertoire. Intriguingly, the data so far available show that the protumoral/antitumoral role of lysosomal Ca2+ channels can differ according to the specific genetic context, types of cancer and the malignancy stage, and signals from the microenvironment.

TPC2 targeting evolution: Leveraging therapeutic opportunities for cancer.

Growing evidence implicates a vital role for TPC2/Ca2+ signaling in pathophysiological processes attributed to cancer, raising questions regarding the utility of TPC2 as a cancer therapeutic target. In this issue of Cell Chemical Biology, Müller et al. (2021) develop TPC2 inhibitors, SG005 and SG-094, exhibiting anti-tumor effects with potential translational relevance.

The role of genetic polymorphisms in endolysosomal ion channels TPC2 and P2RX4 in cancer pathogenesis, prognosis, and diagnosis: a genetic association in the UK Biobank.

Recent studies have implicated important roles for endolysosomal ion channels in cancer biology. We used UK Biobank data to characterise the relationships between genetic variants in two genes coding for endolysosomal ion channels-i.e. TPCN2 and P2RX4-and cancer in terms of the definition of tumour types, susceptibility, and prognosis. We investigated these relationships at both global and local levels with regard to specific types of cancer, including malignant neoplasms of the brain, breast, bronchus, lung, colon, lymphoid and haematopoietic systems, skin, ovary, prostate, rectum, thyroid gland, lip, oral cavity, pharynx, and urinary tract. Apart from rs3829241 (p value < 0.05), all the genetic variants were in Hardy-Weinberg equilibrium. We included 468,436 subjects in the analysis and stratified them into two major cohorts: cancer-free controls (385,253) and cancer cases (83,183). For the first time, we report novel associations between genetic variants of TPCN2 and P2RX4 and cancer/cancer subtypes in the UK Biobank's population. Genotype GG in TPCN2 rs3750965 was significantly associated with a decreased risk of cancer and an increased risk of lip, oral cavity, and pharynx cancer and cancer recurrence in patients with prostate cancer, and genotypes GA/GG were associated with a significantly lower risk of developing various malignant neoplasms (involving melanoma, prostate, mesothelial, and soft tissues). rs35264875:TA was associated with a high risk of cancer at the global level, with subtypes of cancer at the local level (including breast, colon, prostate, and stated or presumed primary cancer of lymphoid, haematopoietic, and related tissue), and with a significantly low risk of cancer metastasis. rs72932540:GA was associated with a higher incidence of cancer/cancer subtypes (including breast, melanoma, and rectal cancer), and genotypes GA/GG were associated with an increased risk of prostate cancer. The P2RX4 rs25644 allele GG was associated with a high risk of prostate cancer, whereas it was associated with a low risk of cancer recurrence in patients with prostate cancer. Genotypes GA/GG in rs28360472 were associated with an increased risk of breast, mesothelial, and soft tissue cancers but with a decreased risk of colon cancer. We also provide insights into the pathophysiological contributions made by these significant polymorphisms to cancer/cancer subtypes and their effects on expression or channel activity. Further investigations of these genetic variants could help identify novel cancer biomarkers and facilitate the development of new diagnostic and therapeutic strategies. This would constitute a further step towards personalised cancer care.

Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination.

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.

Deciphering the Role of Ca2+ Signalling in Cancer Metastasis: From the Bench to the Bedside.

Metastatic cancer is one of the major causes of cancer-related mortalities. Metastasis is a complex, multi-process phenomenon, and a hallmark of cancer. Calcium (Ca2+) is a ubiquitous secondary messenger, and it has become evident that Ca2+ signalling plays a vital role in cancer. Ca2+ homeostasis is dysregulated in physiological processes related to tumour metastasis and progression-including cellular adhesion, epithelial-mesenchymal transition, cell migration, motility, and invasion. In this review, we looked at the role of intracellular and extracellular Ca2+ signalling pathways in processes that contribute to metastasis at the local level and also their effects on cancer metastasis globally, as well as at underlying molecular mechanisms and clinical applications. Spatiotemporal Ca2+ homeostasis, in terms of oscillations or waves, is crucial for hindering tumour progression and metastasis. They are a limited number of clinical trials investigating treating patients with advanced stages of various cancer types. Ca2+ signalling may serve as a novel hallmark of cancer due to the versatility of Ca2+ signals in cells, which suggests that the modulation of specific upstream/downstream targets may be a therapeutic approach to treat cancer, particularly in patients with metastatic cancers.

Loss of Two-Pore Channel 2 (TPC2) Expression Increases the Metastatic Traits of Melanoma Cells by a Mechanism Involving the Hippo Signalling Pathway and Store-Operated Calcium Entry.

Melanoma is one of the most aggressive and treatment-resistant human cancers. The two-pore channel 2 (TPC2) is located on late endosomes, lysosomes and melanosomes. Here, we characterized how TPC2 knockout (KO) affected human melanoma cells derived from a metastatic site. TPC2 KO increased these cells' ability to invade the extracelullar matrix and was associated with the increased expression of mesenchymal markers ZEB-1, Vimentin and N-Cadherin, and the enhanced secretion of MMP9. TPC2 KO also activated genes regulated by YAP/TAZ, which are key regulators of tumourigenesis and metastasis. Expression levels of ORAI1, a component of store-operated Ca2+ entry (SOCE), and PKC-ßII, part of the HIPPO pathway that negatively regulates YAP/TAZ activity, were reduced by TPC2 KO and RNA interference knockdown. We propose a cellular mechanism mediated by ORAI1/Ca2+/PKC-ßII to explain these findings. Highlighting their potential clinical significance, patients with metastatic tumours showed a reduction in TPC2 expression. Our research indicates a novel role of TPC2 in melanoma. While TPC2 loss may not activate YAP/TAZ target genes in primary melanoma, in metastatic melanoma it could activate such genes and increase cancer aggressiveness. These findings aid the understanding of tumourigenesis mechanisms and could provide new diagnostic and treatment strategies for skin cancer and other metastatic cancers.

Targeting Two-Pore Channels: Current Progress and Future Challenges.

Two-pore channels (TPCs) are cation-permeable channels located on endolysosomal membranes and important mediators of intracellular Ca2+ signalling. TPCs are involved in various pathophysiological processes, including cell growth and development, metabolism, and cancer progression. Most studies of TPCs have used TPC-/- cell or whole-animal models, or Ned-19, an indirect inhibitor. The TPC activation mechanism remains controversial, which has made it difficult to develop selective modulators. Recent studies of TPC structure and their interactomes are aiding the development of direct pharmacological modulators. This process is still in its infancy, but will facilitate future research and TPC targeting for therapeutical purposes. Here, we review the progress of current research into TPCs, including recent insights into their structures, functional roles, mechanisms of activation, and pharmacological modulators.

Endolysosomal Ca2+ Signaling in Cancer: The Role of TPC2, From Tumorigenesis to Metastasis.

Ca2+ homeostasis is dysregulated in cancer cells and affects processes such as tumorigenesis, angiogenesis, autophagy, progression, and metastasis. Emerging evidence has suggested that endolysosomal cation channels sustain several cancer hallmarks involving proliferation, metastasis, and angiogenesis. Here, we investigate the role of TPC1-2, TRPML1-3, and P2×4 in cancer, with a particular focus on the role of TPC2 in cancer development, melanoma, and other cancer types as well as its endogenous and exogenous modulators. It has become evident that TPC2 plays a role in cancer; however, the precise mechanisms underlying its exact role remain elusive. TPC2 is a potential candidate for cancer biomarkers and a druggable target for future cancer therapy.

The eggstraordinary story of how life begins.

More than 15 years have elapsed since the identification of phospholipase C ζ1 (PLCζ) from a genomic search for mouse testis/sperm-specific PLCs. This molecule was proposed to represent the sperm factor responsible for the initiation of calcium (Ca2+ ) oscillations required for egg activation and embryo development in mammals. Supporting evidence for this role emerged from studies documenting its expression in all mammals and other vertebrate species, the physiological Ca2+ rises induced by injection of its messenger RNA into mammalian and nonmammalian eggs, and the lack of expression in infertile males that fail intracytoplasmic sperm injection. In the last year, genetic animal models have added support to its role as the long sought-after sperm factor. In this review, we highlight the findings that demonstrated the role of Ca2+ as the universal signal of egg activation and the experimental buildup that culminated with the identification of PLCζ as the soluble sperm factor. We also discuss the structural-functional properties that make PLCζ especially suited to evoke oscillations in eggs. Lastly, we examine unresolved aspects of the function and regulation of PLCζ and whether or not it is the only sperm factor in mammalian sperm.

PLCζ is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but conception can occur in its absence.

Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca2+ oscillations that normally initiate embryogenesis. Consistent with this, recombinant PLCζ induces Ca2+ oscillations in eggs and debilitating mutations in the PLCZ1 gene are associated with infertility in men. However, there has been no evidence that knockout of the gene encoding PLCζ abolishes the ability of sperm to induce Ca2+ oscillations in eggs. Here, we show that sperm derived from Plcz1-/- male mice fail to trigger Ca2+ oscillations in eggs, cause polyspermy and thus demonstrate that PLCζ is the physiological trigger of these Ca2+ oscillations. Remarkably, some eggs fertilized by PLCζ-null sperm can develop, albeit at greatly reduced efficiency, and after a significant time-delay. In addition, Plcz1-/- males are subfertile but not sterile, suggesting that in the absence of PLCζ, spontaneous egg activation can eventually occur via an alternative route. This is the first demonstration that in vivo fertilization without the normal physiological trigger of egg activation can result in offspring. PLCζ-null sperm now make it possible to resolve long-standing questions in fertilization biology, and to test the efficacy and safety of procedures used to treat human infertility.

Synthesis of the Ca2+-mobilizing messengers NAADP and cADPR by intracellular CD38 enzyme in the mouse heart: Role in ß-adrenoceptor signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation-contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38-/- mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38-/- myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of ß-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38-/- myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38-/- mice. Whole hearts isolated from CD38-/- mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of ß-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.

Calcium signals regulated by NAADP and two-pore channels--their role in development, differentiation and cancer.

Ca(2+) signals regulate a wide range of physiological processes. Intracellular Ca(2+) stores can be mobilized in response to extracellular stimuli via a range of signal transduction mechanisms, often involving recruitment of diffusible second messenger molecules. The Ca(2+) mobilizing messengers InsP 3 and cADPR release Ca(2+) from the endoplasmic reticulum via InsP 3 and ryanodine receptors, respectively, while a third messenger, NAADP, releases Ca(2+) from acidic endosomes and lysosomes. Bidirectional communication between the ER and acidic organelles has functional relevance for endolysosomal function as well as for the generation of Ca(2+) signals. The two-pore channels (TPCs) are currently strong candidates for being key components of NAADP-regulated Ca(2+) channels. Ca(2+) signals have been shown to play important roles in embryonic development and cell differentiation; however, much remains to be established about the exact signalling mechanisms involved. Investigation of the role of NAADP and TPCs in development and differentiation is still at an early stage, but recent studies have suggested that they play important roles at key developmental stages in vivo and are important mediators of differentiation of neurons, skeletal muscle cells and osteoclasts in vitro. NAADP signals and TPCs have also been implicated in autophagy, an important process in differentiation. Moreover, potential links between TPC2 and cancer have been recently identified. Further studies will be required to identify the precise mechanisms of action of TPCs and their link with NAADP signalling, and to relate these to their roles in differentiation and other key developmental processes in the cell and organism.

VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2-dependent Ca2+ signaling.

Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca(2+) signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca(2+) mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca(2+) stores, resulting in Ca(2+) release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2(-/-) mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca(2+) release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca(2+) release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2(-/-) mice, but was unaffected in Tpcn1(-/-) animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca(2+) signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.

Preparation and use of sea urchin egg homogenates for studying NAADP-mediated Ca²âº release.

NAADP and other Ca(2+)-mobilizing messengers are membrane impermeant and thus must be added directly to cell-free or broken-cell preparations to effect Ca(2+) release. The sea urchin egg homogenate, where the biological activity of NAADP was first reported, remains the gold standard cell-free system for studying NAADP-mediated Ca(2+) release. Here we describe how to prepare sea urchin egg homogenate and use it to measure NAADP-mediated Ca(2+) release.

Variance in total levels of phospholipase C zeta (PLC-ζ) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte activation capability.

OBJECTIVE: To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability. DESIGN: Laboratory study. SETTING: University laboratory. PATIENT(S): Control subjects (men with proven oocyte activation capacity; n = 16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n = 5). INTERVENTION(S): Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm. MAIN OUTCOME MEASURE(S): Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men. RESULT(S): Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern. CONCLUSION(S): The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis.