Mammalian sperm cells must undergo several processes collectively called capacitation before carrying out the acrosome reaction (AR), which is required for sperm penetration into the oocyte. The spontaneous acrosomal reaction (SAR), which can occur before the sperm cell reaches the vicinity of the oocyte, impairs the fertilizing ability of the sperm. This study examined the role of the Fer tyrosine kinase in sperm fertilizing activity. Inhibition of the Fer activity led to a 75% reduction in IVF rates in mice, indicating a critical role for Fer in fertilization. Further investigation of Fer’s role during sperm capacitation focused on its potential interaction with the epidermal growth factor receptor (EGFR). Inhibition of Fer during capacitation significantly decreased the EGFR activation state and increased the incidence of SAR, whereas inhibition of Fer during the acrosome reaction step had no effect on the EGF-induced AR. The effects of Fer inhibition on EGFR activation and SAR enhancement are mediated by the Ca2+ channel, CatSper. Notably, reduction in Ca2+ influx by CatSper inhibition revealed a significant increase in Fer phosphorylation/activation, while increasing intracellular Ca2+ concentrations completely inhibited this effect. Additionally, we show that Fer activation depends on a signaling cascade involving protein kinase A (PKA) that leads to EGFR activation through the following pathway: HCO3− → SAC → cAMP → PKA → Src → Fer → EGFR. Collectively, we decipher in this work a new regulatory cascade that leads to the Fer-directed activation of EGFR in sperm capacitation.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a high percentage of morbidity. The deciphering and identification of novel targets and tools for intervening with its adverse progression are therefore of immense importance. To address this goal we adopted a specific inhibitor of the intracellular tyrosine kinase Fer, whose expression level is upregulated in PDAC tumors, and is associated with poor prognosis of patients. Subjecting PDAC cells to the E260-Fer inhibitor, unraveled its simultaneous effects on the mitochondria, and on a non-mitochondrial ERK1/2 regulatory cascade. E260 caused severe mitochondrial deformation, resulting in cellular- aspartate and ATP depletion, and followed by the activation of the metabolic sensor AMPK. This led to the phosphorylation and deactivation of the bona fide AMPK substrate, RAPTOR, which serves as a positive regulator of the mTORC1 metabolic hub. Accordingly, this resulted in the inhibition of the mTORC1 activity. In parallel, E260 downregulated the activation state of the ERK1/2 kinases, and their ability to neutralize the mTORC1 suppressor TSC2, thereby accentuating the inhibition of mTORC1. Importantly, both activation of AMPK and downregulation of ERK1/2 and mTORC1 were also achieved upon the knockdown of Fer, corroborating the regulatory role of Fer in these processes. Concomitantly, in PDAC tumors and not in healthy pancreatic tissues, the expression levels of Fer demonstrate moderate but statistically significant positive correlation with the expression levels of mTOR and its downstream effector LARP1. Finally, targeting the Fer driven activation of mTORC1, culminated in necrotic death of the treated PDAC cells, envisaging a new intervention tool for the challenging PDAC disease.

Background:Spontaneous Bacterial Peritonitis (SBP) poses a significant risk to cirrhosis patients with ascites, emphasizing the critical need for early detection and intervention. This retrospective observational study spanning a decade aimed to devise predictive models for SBP using routine laboratory tests. Additionally, it aimed to propose a novel scoring system to aid SBP diagnosis. Methods:Data analysis encompassed 229 adult cirrhotic patients hospitalized for ascites between 2012 and 2021. Exclusions eliminated cases of secondary ascites unrelated to liver cirrhosis. Patients were categorized into SBP-positive (n=110) and SBP-negative (n=119) groups. Comparative analysis of demographic details and various laboratory indicators (Neutrophil-to-Lymphocyte Ratio (NLR), Mean Platelet Volume (MPV), C-Reactive Protein (CRP), Platelet (PLT), Alanine Transaminase (ALT), Aspartate Amino Transferase (AST), Potassium (K), Sodium (Na), Total Bilirubin (TB) and International Normalized Ratio (INR) was performed between the groups. The study presented effective SBP prediction models for prompt diagnosis and treatment: a multivariate logistic regression model and a simple scoring system. Findings:The study advocates early diagnosis and rapid treatment for all cirrhotic patients with ascites, regardless of cirrhosis stage. Furthermore, it recommends initiating SBP treatment for patients scoring 2-3 in the proposed scoring system while excluding SBP findings for those scoring zero. Conclusion:Combining age, sex, and specific laboratory tests (MPV, NLR, CRP, TB, and INR) within random forest models and a simple scoring system enables swift and accurate SBP diagnosis.

Fer and its sperm and cancer specific variant, FerT, are non-receptor tyrosine kinases which play roles in cancer progression and metastasis. Recent studies have shed light on the regulatory role of these kinases in ensuring proper sperm function. Comparison of the regulatory cascades in which Fer and FerT are engaged in sperm and cancer cells presents an interesting picture, in which similar regulatory interactions of these enzymes are integrated in a similar or different regulatory context in the two cell types. These diverse compositions extend from the involvement of Fer in modulation of actin cytoskeleton integrity and function, to the unique regulatory interactions of Fer with PARP-1 and the PP1 phosphatase. Furthermore, recent findings link the metabolic regulatory roles of Fer and FerT in sperm and cancer cells. In the current review, we discuss the above detailed aspects, which portray Fer and FerT as new regulatory links between sperm and malignant cells. This perspective view can endow us with new analytical and research tools that will deepen our understanding of the regulatory trajectories and networks that govern these two multi-layered systems.

Supplementary Table S2: summarizing tumor progression in immuno-compromised mice, up to 6 weeks after being subcutaneously injected with 4X106 NIH3T3-MTS- MycFerT, NIH3T3-MycFerT or NIH3T3-pIRES cells.

Supplementary Figure S1: shows the induction of cellular ROS levels upon the down-regulation of Fer or FerT using different siRNA's.

Supplementary Figure S2: demonstrates the different tumor formation promoting activities of mitochondrial directed and undirected FerT in immuno-compromised mice

Supplementary Table S1: lists the different siRNA's used in the currently described work

Supplementary Data legends. This File contains the legends for the supplementary Figures and tables

The physiological acrosome reaction occurs after mammalian spermatozoa undergo a process called capacitation in the female reproductive tract. Only acrosome reacted spermatozoon can penetrate the egg zona-pellucida and fertilize the egg. Sperm also contain several mechanisms that protect it from undergoing spontaneous acrosome reaction (sAR), a process that can occur in sperm before reaching proximity to the egg and that abrogates fertilization. We previously showed that calmodulin-kinase II (CaMKII) and phospholipase D (PLD) are involved in preventing sAR through two distinct pathways that enhance F-actin formation during capacitation. Here, we describe a novel additional pathway involving the tyrosine kinase Fer in a mechanism that also prevents sAR by enhancing actin polymerization during sperm capacitation. We further show that protein-kinase A (PKA) and the tyrosine-kinase Src, as well as PLD, direct Fer phosphorylation/activation. Activated Fer inhibits the Ser/Thr phosphatase PP1, thereby leading to CaMKII activation, actin polymerization, and sAR inhibition.

Metabolic plasticity is a hallmark of the ability of metastatic cancer cells to survive under stressful conditions. The intracellular Fer kinase is a selective constituent of the reprogramed mitochondria and metabolic system of cancer cells. In the current work, we deciphered the modulatory roles of Fer in the reprogrammed metabolic systems of metastatic, lung (H358), non-small cell lung cancer (NSCLC), and breast (MDA-MB-231), triple-negative breast cancer (TNBC), carcinoma cells. We show that H358 cells devoid of Fer (H358ΔFer), strictly depend on glucose for their proliferation and growth, and fail to compensate for glucose withdrawal by oxidizing and metabolizing glutamine. Furthermore, glucose deficiency caused increased reactive oxygen species (ROS) production and induction of a DNA damage response (DDR), accompanied by the onset of apoptosis and attenuated cell-cycle progression. Analysis of mitochondrial function revealed impaired respiratory and electron transport chain (ETC) complex 1 (comp. I) activity in the Fer-deficient H358ΔFer cells. This was manifested by decreased levels of NAD+ and ATP and relatively low abundance of tricarboxylic acid (TCA) cycle metabolites. Impaired electron transport chain comp. I activity and dependence on glucose were also confirmed in Fer-deficient, MDA-MB-231ΔFer cells. Although both H358ΔFer and MDA-MB-231ΔFer cells showed a decreased aspartate level, this seemed to be compensated by the predominance of pyrimidines synthesis over the urea cycle progression. Notably, absence of Fer significantly impeded the growth of H358ΔFer and MDA-MB-231ΔFer xenografts in mice provided with a carb-deficient, ketogenic diet. Thus, Fer plays a key role in the sustention of metabolic plasticity of malignant cells. In compliance with this notion, targeting Fer attenuates the progression of H358 and MDA-MB-231 tumors, an effect that is potentiated by a glucose-restrictive diet.

Insulin-regulated glucose homeostasis is a critical and intricate physiological process, of which not all regulatory components have been deciphered. One of the key players in modulating glucose uptake by cells is the glucose transporter-GLUT4. In this study, we aimed to explore the regulatory role of the trans-Golgi-associated protein-TATA Element Modulatory Factor (TMF1) in the GLUT4 mediated, insulin-directed glucose uptake. By establishing and using TMF1-/- myoblasts and mice, we examined the effect of TMF1 absence on the insulin driven functioning of GLUT4. We show that TMF1 is upregulated by insulin in myoblasts, and is essential for the formation of insulin responsive, glucose transporter GLUT4-containing vesicles. Absence of TMF1 leads to the retention of GLUT4 in perinuclear compartments, and to severe impairment of insulin-stimulated GLUT4 trafficking throughout the cytoplasm and to the cell plasma membrane. Accordingly, glucose uptake is impaired in TMF1-/- cells, and TMF1-/- mice are hyperglycemic. This is reflected by the mice impaired blood glucose clearance and increased blood glucose level. Correspondingly, TMF1-/- animals are leaner than their normal littermates. Thus, TMF1 is a novel effector of insulin-regulated glucose homeostasis, and dys-functioning of this protein may contribute to the onset of a diabetes-like disorder.

Aerobic glycolysis is an important metabolic adaptation of cancer cells. However, there is growing evidence that reprogrammed mitochondria also play an important metabolic role in metastatic dissemination. Two constituents of the reprogrammed mitochondria of cancer cells are the intracellular tyrosine kinase Fer and its cancer- and sperm-specific variant, FerT. Here, we show that Fer and FerT control mitochondrial susceptibility to therapeutic and hypoxic stress in metastatic colon (SW620) and non-small cell lung cancer (NSCLC-H1299) cells. Fer- and FerT-deficient SW620 and H1299 cells (SW∆Fer/FerT and H∆Fer/FerT cells, respectively) become highly sensitive to metformin treatment and to hypoxia under glucose-restrictive conditions. Metformin impaired mitochondrial functioning that was accompanied by ATP deficiency and robust death in SW∆Fer/FerT and H∆Fer/FerT cells compared to the parental SW620 and H1299 cells. Notably, selective knockout of the fer gene without affecting FerT expression reduced sensitivity to metformin and hypoxia seen in SW∆Fer/FerT cells. Thus, Fer and FerT modulate the mitochondrial susceptibility of metastatic cancer cells to hypoxia and metformin. Targeting Fer/FerT may therefore provide a novel anticancer treatment by efficient, selective, and more versatile disruption of mitochondrial function in malignant cells.

A switch from catabolic to anabolic metabolism, a major hallmark of cancer, enables rapid cell duplication, and is driven by multiple oncogenic alterations, including PIK3CA mutation, MYC amplification, and TP53 loss. However, tumor growth requires active mitochondrial function and oxidative phosphorylation (OXPHOS). Recently, loss of the retinoblastoma (RB1) tumor suppressor in breast cancer was shown to induce mitochondrial protein translation (MPT) and OXPHOS. Here, we discuss how increased OXPHOS can enhance anabolic metabolism and cell proliferation, as well as cancer stemness and metastasis. Mitochondrial STAT3, FER/FER-T, and CHCHD2 are also implicated in OXPHOS. We propose that RB1 loss represents a prototypic oncogenic alteration that promotes OXPHOS, that aggressive tumors acquire lethal combinations of oncogenes and tumor suppressors that stimulate anabolism versus OXPHOS, and that targeting both metabolic pathways would be therapeutic.

Disruption of the reprogrammed energy management system of malignant cells is a prioritized goal of targeted cancer therapy. Two regulators of this system are the Fer kinase, and its cancer cell specific variant, FerT, both residing in subcellular compartments including the mitochondrial electron transport chain. Here, we show that a newly developed inhibitor of Fer and FerT, E260, selectively evokes metabolic stress in cancer cells by imposing mitochondrial dysfunction and deformation, and onset of energy-consuming autophagy which decreases the cellular ATP level. Notably, Fer was also found to associate with PARP-1 and E260 disrupted this association thereby leading to PARP-1 activation. The cooperative intervention with these metabolic pathways leads to energy crisis and necrotic death in malignant, but not in normal human cells, and to the suppression of tumors growth in vivo. Thus, E260 is a new anti-cancer agent which imposes metabolic stress and cellular death in cancer cells.The tyrosine-kinases Fer/FerT associate with the mitochondrial electron transport chain in cancer cells supporting their metabolic reprogramming. Here the authors discover a compound that disrupts Fer /FerT activity and selectively induces cell death of cancer cell lines displaying anti-tumor activity in vivo.

High level of discordance between aPTT and anti-factor Xa (anti-F10a) has been reported in patients with left ventricular assist devices (LVADs). In this study we sought to determine the biochemical basis for discordance by measuring coagulation factors in patients with LVADs. A prospective study of consecutive CF-LVAD patients admitted for IV unfractionated heparin (UFH) therapy as bridge to therapeutic INR or for suspected device thrombosis. Simultaneous anti-F10a, activated partial thromboplastin time (aPTT), international normalization ratio (INR), LDH, vitamin K dependent factors (2,7,9,10) and coagulation factors 8,11 and 12 were measured. The incidence of discordance was compared between coagulation factors. 62 patients were included, the overall concordance between anti-F10a and aPTT was only 29.9%, (205/686 paired samples). The frequency of discordance between samples with low vitamin K factors (n=57) vs normal vitamin K factors (n=17) were similar 35.1% vs 35.3% (p=0.99), respectively. Factor 8 was elevated in 50 out of 52 patients (96%) with an average level of 295% ± 120% (reference range 60-140%). Factor 12 was low in 12 out of 75 samples (16%) (9/52 patients) with an average of 88% ± 31 (reference range 50-186%), samples drawn with a low factor 12 were discordant 90.1%. 6/74 samples had a low factor V, samples with a low factor 5 had a 100% discordance rate. CF LVAD patients had a supra-therapeutic aPTT despite a therapeutic anti-FXa. Warfarin administration results in parked prolongation of the aPTT relative to anti-factor Xa, however the discordance cannot be explained based on warfarin administration alone. Elevated factor 8 was common, and not associated with discordance; Low factor 12 and 5 levels were observed in 24 % of samples and associated with a high incidence of discordance. Further investigation is required to determine the significance of factor 12 deficiency and elevated factor 8 levels in CF-LVADs.

TMF/ARA160 is known to be a TATA element Modulatory Factor (TMF). It was initially identified as a DNA-binding factor and a coactivator of the Androgen receptor. It was also characterized as a Golgi-associated protein, which is essential for acrosome formation during functional sperm development. However, the molecular roles of TMF in this intricate process have not been revealed. Here, we show that during spermiogenesis, TMF undergoes a dynamic change of localization throughout the Golgi apparatus. Specifically, TMF translocates from the cis-Golgi to the trans-Golgi network and to the emerging vesicles surface, as the round spermatids develop. Notably, lack of TMF led to an abnormal spatial orientation of the Golgi and to the deviation of the trans-Golgi surface away from the nucleus of the developing round spermatids. Concomitantly, pro-acrosomal vesicles derived from the TMF-/- Golgi lacked targeting properties and did not tether to the spermatid nuclear membrane thereby failing to form the acrosome anchoring scaffold, the acroplaxome, around the cell-nucleus. Absence of TMF also perturbed the positioning of microtubules, which normally lie in proximity to the Golgi and are important for maintaining Golgi spatial orientation and dynamics and for chromatoid body formation, which is impaired in TMF-/- spermatids. In-silico evaluation combined with molecular and electron microscopic analyses revealed the presence of a microtubule interacting domain (MIT) in TMF, and confirmed the association of TMF with microtubules in spermatogenic cells. Furthermore, the MIT domain in TMF, along with microtubules integrity, are required for stable association of TMF with the Golgi apparatus. Collectively, we show here for the first time that a Golgi and microtubules associated protein is crucial for maintaining proper Golgi orientation during a cell developmental process.

Background: Several epidemiological studies have shown that diets rich in carotenoids are associated with a reduced risk of cardiovascular disease. However, administration of synthetic all-trans b-carotene was reported to have no effect on cardiovascular disease. We previously showed that the 9-cis b-carotene-rich powder of the alga Dunaliella bardawil inhibits atherogenesis and reduces plasma non-HDL cholesterol levels in mice.Context and purpose of this study: We sought to study whether isolated 9-cis b-carotene inhibits atherogenesis in a murine model of atherosclerosis.Results: Twelve-week-old female LDL receptor knockout mice (LDLR-/-) were pretreated for 2 weeks with regular chow diet fortified with the alga Dunaliella powder, 9-cis β-carotene isomer, all-trans β-carotene isomer, or 9-cis retinoic acid, followed by 10 weeks of a high-fat diet with the same fortifications. In contrast to Dunaliella, 9-cis β-carotene did not inhibit the high fat diet-induced elevation of plasma cholesterol. In addition, diet fortification with Dunaliella powder, β-carotene isomers, or 9-cis retinoic acid did not change the plasma retinol or retinoic acid levels. Nevertheless, 9-cis β-carotene significantly inhibited atherogenesis compared to the control mice (39% reduction).Conclusions: The results suggest that 9-cis β-carotene should be considered as an anti-atherogenic agent in the human diet.Key words: Atherosclerosis, Dunaliella, 9CBC, LDLR-/- mice

Tata Element Modulatory Factor (TMF/ARA160) is a multifunctional Golgi-associated protein, which accumulates in colonic enterocytes and goblet cells. Mice lacking TMF/ARA160 (TMF-/-) produce thick and uniform colonic mucus that resists adherent bacterial colonization and diminishes susceptibility of these mice to induced acute colitis, through a mechanism that is not fully understood. Here, we show that mucus secretion by goblet cells is altered in the colon of TMF-/- mice, resulting in the formation of a highly oligomerized colonic gel-forming mucin, MUC2. Microbiome analysis revealed a shift in the microbiota of TMF-/- mice leading to predominance of the Firmicutes phylum and a significantly higher abundance of probiotic beneficial bacterial species. Notably, this trait was transmissible, and when cohoused with wild-type animals, TMF-/- mice influenced the microbiota and diminished the susceptibility of wildtype mice to chemically induced dextran sulfate sodium colitis. Thus, altered mucus secretion in TMF-/- mouse colons is accompanied by a reprogrammed intestinal microbiota, leading to a transmissible reduced sensitivity to induced colitis.