Fam20C-mediated phosphorylation of osteopontin is critical for its secretion but dispensable for its action as a cytokine in the activation of hepatic stellate cells in liver fibrogenesis.

Osteopontin (OPN) is a phosphoglycoprotein secreted into the extracellular matrix upon liver injury, acting as a cytokine stimulates the deposition of fibrillary collagen in liver fibrogenesis. In livers of mice subjected to bile duct ligation (BDL) and in cultured activated hepatic stellate cells (HSCs), we show that OPN, besides being overexpressed, is substantially phosphorylated by family with sequence similarity 20, member C (Fam20C), formerly known as Golgi casein kinase (G-CK), which is exclusively resident in the Golgi apparatus. In both experimental models, Fam20C becomes overactive when associated with a 500-kDa multiprotein complex, as compared with the negligible activity in livers of sham-operated rats and in quiescent HSCs. Fam20C knockdown not only confirmed the role of Fam20C itself in OPN phosphorylation, but also revealed that phosphorylation was essential for OPN secretion. However, OPN acts as a fibrogenic factor independently of its phosphorylation state, as demonstrated by the increased expression of Collagen-I by HSCs incubated with either a phosphorylated or nonphosphorylated form of recombinant OPN. Collectively, our results confirm that OPN promotes liver fibrosis and highlight Fam20C as a novel factor driving this process by favoring OPN secretion from HSCs, opening new avenues for deciphering yet unidentified mechanisms underlying liver fibrogenesis.

Human Sperm Capacitation Involves the Regulation of the Tyr-Phosphorylation Level of the Anion Exchanger 1 (AE1).

Bicarbonate uptake is one of the early steps of capacitation, but the identification of proteins regulating anion fluxes remains elusive. The aim of this study is to investigate the role of sperm solute carrier 4 (SLC4) A1 (spAE1) in the capacitation process. The expression, location, and tyrosine-phosphorylation (Tyr-P) level of spAE1 were assessed. Thereby, it was found that 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), an SLC4 family channel blocker, inhibited capacitation in a dose-dependent manner by decreasing acrosome reaction (ARC% 24.5 ± 3.3 vs 64.9 ± 4.3, p < 0.05) and increasing the percentage of not viable cells (NVC%), comparable to the inhibition by I-172, a cystic fibrosis transmembrane conductance regulator (CFTR) blocker (AR% 30.5 ± 4.4 and NVC% 18.6 ± 2.2). When used in combination, a synergistic inhibitory effect was observed with a remarkable increase of the percentage of NVC (45.3 ± 4.1, p < 0.001). spAE1 was identified in sperm membrane as a substrate for Tyr-protein kinases Lyn and Syk, which were identified as both soluble and membrane-bound pools. spAE1-Tyr-P level increased in the apical region of sperm under capacitating conditions and was negatively affected by I-172 or DIDS, and, to a far greater extent, by a combination of both. In conclusion, we demonstrated that spAE1 is expressed in sperm membranes and it is phosphorylated by Syk, but above all by Lyn on Tyr359, which are involved in sperm viability and capacitation.

THz Water Transmittance and Leaf Surface Area: An Effective Nondestructive Method for Determining Leaf Water Content.

Water availability is a major limiting factor in plant productivity and plays a key role in plant species distribution over a given area. New technologies, such as terahertz quantum cascade lasers (THz-QCLs) have proven to be non-invasive, effective, and accurate tools for measuring and monitoring leaf water content. This study explores the feasibility of using an advanced THz-QCL device for measuring the absolute leaf water content in Corylus avellana L., Laurus nobilis L., Ostrya carpinifolia Scop., Quercus ilex L., Quercus suber L., and Vitis vinifera L. (cv. Sangiovese). A recently proposed, simple spectroscopic technique was used, consisting in determining the transmission of the THz light beam through the leaf combined with a photographic measurement of the leaf area. A significant correlation was found between the product of the leaf optical depth (τ) and the leaf surface area (LA) with the leaf water mass (Mw) for all the studied species (Pearson's r test, p ≤ 0.05). In all cases, the best fit regression line, in the graphs of τLA as a function of Mw, displayed R2 values always greater than 0.85. The method proposed can be combined with water stress indices of plants in order to gain a better understanding of the leaf water management processes or to indirectly monitor the kinetics of leaf invasion by pathogenic bacteria, possibly leading to the development of specific models to study and fight them.

Lyn sustains oncogenic signaling in chronic lymphocytic leukemia by strengthening SET-mediated inhibition of PP2A.

Aberrant protein kinase activities, and the consequent dramatic increase of Ser/Thr and -Tyr phosphorylation, promote the deregulation of the survival pathways in chronic lymphocytic leukemia (CLL), which is crucial to the pathogenesis and progression of the disease. In this study, we show that the tumor suppressor protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, is in an inhibited form because of the synergistic contribution of 2 events, the interaction with its physiologic inhibitor SET and the phosphorylation of Y307 of the catalytic subunit of PP2A. The latter event is mediated by Lyn, a Src family kinase previously found to be overexpressed, delocalized, and constitutively active in CLL cells. This Lyn/PP2A axis accounts for the persistent high level of phosphorylation of the phosphatase's targets and represents a key connection linking phosphotyrosine- and phosphoserine/threonine-mediated oncogenic signals. The data herein presented show that the disruption of the SET/PP2A complex by a novel FTY720-analog (MP07-66) devoid of immunosuppressive effects leads to the reactivation of PP2A, which in turn triggers apoptosis of CLL cells. When used in combination with SFK inhibitors, the action of MP07-66 is synergistically amplified, providing a new option in the therapeutic strategy for CLL patients.

Targeted activation of the SHP-1/PP2A signaling axis elicits apoptosis of chronic lymphocytic leukemia cells.

Lyn, a member of the Src family of kinases, is a key factor in the dysregulation of survival and apoptotic pathways of malignant B cells in chronic lymphocytic leukemia. One of the effects of Lyn's action is spatial and functional segregation of the tyrosine phosphatase SHP-1 into two pools, one beneath the plasma membrane in an active state promoting pro-survival signals, the other in the cytosol in an inhibited conformation and unable to counter the elevated level of cytosolic tyrosine phosphorylation. We herein show that SHP-1 activity can be elicited directly by nintedanib, an agent also known as a triple angiokinase inhibitor, circumventing the phospho-S591-dependent inhibition of the phosphatase, leading to the dephosphorylation of pro-apoptotic players such as procaspase-8 and serine/threonine phosphatase 2A, eventually triggering apoptosis. Furthermore, the activation of PP2A by using MP07-66, a novel FTY720 analog, stimulated SHP-1 activity via dephosphorylation of phospho-S591, which unveiled the existence of a positive feedback signaling loop involving the two phosphatases. In addition to providing further insights into the molecular basis of this disease, our findings indicate that the PP2A/SHP-1 axis may emerge as an attractive, novel target for the development of alternative strategies in the treatment of chronic lymphocytic leukemia.

Lyn-mediated procaspase 8 dimerization blocks apoptotic signaling in B-cell chronic lymphocytic leukemia.

Lyn, a member of the group of tyrosine kinases named the Src family kinases (SFKs), is overexpressed, associated with an aberrant multiprotein complex and constitutively active in B-cell chronic lymphocytic leukemia (B-CLL) cells, resulting in a high level of tyrosine phosphorylation and contributing to their resistance to apoptosis. By using biochemical and bioinformatics tools, we identified procaspase-8 (procasp8), the caspase-8 zymogen, as a cytosolic target for Lyn in B-CLL cells, the phosphorylation of which at Tyr380 promotes the formation of an inactive procasp8 homodimer. This complex remains segregated in the cytosol and appears to be crucial in mediating the antiapoptotic function of Lyn in this disease. The significance of the Lyn-procasp8 axis in impairing apoptosis in B-CLL cells was further confirmed by pharmacological and genetic inhibition of procasp8, which drastically reduced the apoptosis induced by the SFK inhibitors PP2 and dasatinib. Our data highlight that Lyn's dysregulated expression, activity, and localization in B-CLLs support resistance to cell demise by inhibiting an early player of apoptotic signaling, and potentially broaden the perspectives of developing new strategies for the treatment of this disease.

Fatty Acid-Derived Pro-Toxicants of the Rat Selective Toxicant Norbormide.

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. However, as an acute vasoactive, NRB has a rapid onset of action which makes it relatively unpalatable to rats, often leading to sublethal uptake and accompanying bait shyness. A series of NRB-derived pro-toxicants (3a - i, 4a - i, and 5a - i) were prepared in an effort to 'mask' this acute response and improve both palatability and efficacy. Their synthesis, in vitro biological evaluation (vasocontractile response in rat vasculature, stability in selected rat media) and palatability/efficacy in Sprague-Dawley, wild Norway, and wild ship rats is described. Most notably, pro-toxicant 3d was revealed to be free of all pre-cleavage vasoconstrictory activity in rat caudal artery and was subsequently demonstrated to release NRB in the presence of rat blood, liver, and pancreatic enzymes. Moreover, it consistently displayed a high level of acceptance by rats in a two-choice bait-palatability and efficacy trial, with accompanying high mortality. On this evidence, fatty acid ester prodrugs would appear to offer a promising platform for the further development of NRB-derived toxicants with enhanced palatability and efficacy profiles.

The tyrosine phosphatase SHP-1 inhibits proliferation of activated hepatic stellate cells by impairing PDGF receptor signaling.

The dimerization and auto-transphosphorylation of platelet-derived growth factor receptor (PDGFR) upon engagement by platelet-derived growth factor (PDGF) activates signals promoting the mitogenic response of hepatic stellate cells (HSCs) due to liver injury, thus contributing to the development of hepatic fibrosis. We demonstrate that the tyrosine phosphatases Src homology 2 domain-containing phosphatase 1 and 2 (SHP-1 and SHP-2) act as crucial regulators of a complex signaling network orchestrated by PDGFR activation in a spatio-temporal manner with diverse and opposing functions in HSCs. In fact, silencing of either phosphatase shows that SHP-2 is committed to PDGFR-mediated cell proliferation, whereas SHP-1 dephosphorylates PDGFR hence abrogating the downstream signaling pathways that result in HSC activation. In this regard, SHP-1 as an off-switch of PDGFR signaling appears to emerge as a valuable molecular target to trigger as to prevent HSC proliferation and the fibrogenic effects of HSC activation. We show that boswellic acid, a multitarget compound with potent anti-inflammatory action, exerts an anti-proliferative effect on HSCs, as in other cell models, by upregulating SHP-1 with subsequent dephosphorylation of PDGFR-ß and downregulation of PDGF-dependent signaling after PDGF stimulation. Moreover, the synergism resulting from the combined use of boswellic acid and imatinib, which directly inhibits PDGFR-ß activity, on activated HSCs offers new perspectives for the development of therapeutic strategies that could implement molecules affecting diverse players of this molecular circuit, thus paving the way to multi-drug low-dose regimens for liver fibrosis.

Label-free fluorescence detection of kinase activity using a gold nanoparticle based indicator displacement assay.

A straightforward indicator-displacement assay (IDA) has been developed for the quantitative analysis of ATP→ADP conversion. The IDA relies on the use of gold nanoparticles passivated with a monolayer of thiols terminating with a 1,4,7-triazacyclononane (TACN)·Zn(2+) head group. The analytes ATP and ADP compete to a different extent with a fluorescent probe for binding to the monolayer surface. In the presence of ATP the fluorescent probe is free in solution, whereas in the presence of ADP the fluorescent probe is captured by the nanoparticles and its fluorescence is quenched. The linear response of the fluorescence signal towards different ratios of ATP : ADP permitted the detection of protein kinase activity simply by adding aliquots of the enzyme solution to the assay solution followed by measurement of the fluorescent intensity. The assay poses no restrictions on the target kinase nor does it require labeling of the kinase substrate. The assay was tested on the protein kinases PIM-1 and Src and validated through a direct comparison with the classical radiometric assay using the [γ-(32)P]-labeled ATP.

CFTR mutations altering CFTR fragmentation.

Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.

Symphonies of Growth: Unveiling the Impact of Sound Waves on Plant Physiology and Productivity.

The application of sound wave technology to different plant species has revealed that variations in the Hz, sound pressure intensity, treatment duration, and type of setup of the sound source significantly impact the plant performance. A study conducted on cotton plants treated with Plant Acoustic Frequency Technology (PAFT) highlighted improvements across various growth metrics. In particular, the treated samples showed increases in the height, size of the fourth expanded leaf from the final one, count of branches carrying bolls, quantity of bolls, and weight of individual bolls. Another study showed how the impact of a 4 kHz sound stimulus positively promoted plant drought tolerance. In other cases, such as in transgenic rice plants, GUS expression was upregulated at 250 Hz but downregulated at 50 Hz. In the same way, sound frequencies have been found to enhance the osmotic potential, with the highest observed in samples treated with frequencies of 0.5 and 0.8 kHz compared to the control. Furthermore, a sound treatment with a frequency of 0.4 kHz and a sound pressure level (SPL) of 106 dB significantly increased the paddy rice germination index, as evidenced by an increase in the stem height and relative fresh weight. This paper presents a complete, rationalized and updated review of the literature on the effects of sound waves on the physiology and growth parameters of sound-treated plants.

Allocation of Nutrients and Leaf Turnover Rate in Poplar under Ambient and Enriched Ozone Exposure and Soil Nutrient Manipulation.

An excess of ozone (O3) is currently stressing plant ecosystems and may negatively affect the nutrient use of plants. Plants may modify leaf turnover rates and nutrient allocation at the organ level to counteract O3 damage. We investigated leaf turnover rate and allocation of primary (C, N, P, K) and secondary macronutrients (Ca, S, Mg) under various O3 treatments (ambient concentration, AA, with a daily hourly average of 35 ppb; 1.5 × AA; 2.0 × AA) and fertilization levels (N: 0 and 80 kg N ha-1 y-1; P: 0 and 80 kg N ha-1 y-1) in an O3-sensitive poplar clone (Oxford: Populus maximowiczii Henry × P. berolinensis Dippel) in a Free-Air Controlled Exposure (FACE) experiment. The results indicated that both fertilization and O3 had a significant impact on the nutrient content. Specifically, fertilization and O3 increased foliar C and N contents (+5.8% and +34.2%, respectively) and root Ca and Mg contents (+46.3% and +70.2%, respectively). Plants are known to increase the content of certain elements to mitigate the damage caused by high levels of O3. The leaf turnover rate was accelerated as a result of increased O3 exposure, indicating that O3 plays a main role in influencing this physiological parameter. A PCA result showed that O3 fumigation affected the overall allocation of primary and secondary elements depending on the organ (leaves, stems, roots). As a conclusion, such different patterns of element allocation in plant leaves in response to elevated O3 levels can have significant ecological implications.

Neural stem/progenitor cells from olfactory neuroepithelium collected by nasal brushing as a cell model reflecting molecular and cellular dysfunctions in schizophrenia.

OBJECTIVES: Neural stem/progenitor cells derived from olfactory neuroepithelium (hereafter olfactory neural stem/progenitor cells, ONSPCs) are emerging as a potential tool in the exploration of psychiatric disorders. The present study intended to assess whether ONSPCs could help discern individuals with schizophrenia (SZ) from non-schizophrenic (NS) subjects by exploring specific cellular and molecular features. METHODS: ONSPCs were collected from 19 in-patients diagnosed with SZ and 31 NS individuals and propagated in basal medium. Mitochondrial ATP production, expression of ß-catenin and cell proliferation, which are described to be altered in SZ, were examined in freshly isolated or newly thawed ONSPCs after a few culture passages. RESULTS: SZ-ONSPCs exhibited a lower mitochondrial ATP production and insensitivity to agents capable of positively or negatively affecting ß-catenin expression with respect to NS-ONSPCs. As to proliferation, it declined in SZ-ONSPCs as the number of culture passages increased compared to a steady level of growth shown by NS-ONSPCs. CONCLUSIONS: The ease and safety of sample collection as well as the differences observed between NS- and SZ-ONSPCs, may lay the groundwork for a new approach to obtain biological material from a large number of living individuals and gain a better understanding of the mechanisms underlying SZ pathophysiology.

Structural features underlying the selectivity of the kinase inhibitors NBC and dNBC: role of a nitro group that discriminates between CK2 and DYRK1A.

8-hydroxy-4-methyl-9-nitrobenzo(g)chromen-2-one (NBC) has been found to be a fairly potent ATP site-directed inhibitor of protein kinase CK2 (Ki = 0.22 µM). Here, we show that NBC also inhibits PIM kinases, especially PIM1 and PIM3, the latter as potently as CK2. Upon removal of the nitro group, to give 8-hydroxy-4-methyl-benzo(g)chromen-2-one (here referred to as "denitro NBC", dNBC), the inhibitory power toward CK2 is almost entirely lost (IC(50) > 30 µM) whereas that toward PIM1 and PIM3 is maintained; in addition, dNBC is a potent inhibitor of a number of other kinases that are weakly inhibited or unaffected by NBC, with special reference to DYRK1A whose IC(50) values with NBC and dNBC are 15 and 0.60 µM, respectively. Therefore, the observation that NBC, unlike dNBC, is a potent inducer of apoptosis is consistent with the notion that this effect is mediated by inhibition of endogenous CK2. The structural features underlying NBC selectivity have been revealed by inspecting its 3D structure in complex with the catalytic subunit of Z. mays CK2. The crucial role of the nitro group is exerted both through a direct electrostatic interaction with the side chain of Lys68 and, indirectly, by enhancing the acidic dissociation constant of the adjacent hydroxyl group which interacts with a conserved water molecule in the deepest part of the cavity. By contrast, the very same nitro group is deleterious for the binding to the active site of DYRK1A, as disclosed by molecular docking. This provides the rationale for preferential inhibition of DYRK1A by dNBC.

Post-COVID-19 and Post-COVID-19 Vaccine Arthritis, Polymyalgia Rheumatica and Horton's Arteritis: A Single-Center Assessment of Clinical, Serological, Genetic, and Ultrasonographic Biomarkers.

The potential role of the COVID-19 vaccine and infection to induce autoimmunity is currently underestimated despite the literature emphasizing arthralgia as a common adverse event. We aimed to study the impact of rheumatological complications post-COVID-19 (PC) and post-COVID-19 vaccine (PCV), comparing undifferentiated arthritis (UA) to Polymyalgia Rheumatica, Horton's Arteritis (PMR-HA) and isolated arthritis to UA with "connective-like" accompanying symptoms. We retrospectively included 109 patients with at least 6 months of follow-up, analyzing serum biomarkers, joint ultrasound (US), lung HRCT, DLCO, and HLA haplotypes. There were 87 UA patients showing increased gastrointestinal and lung involvement (p = 0.021 and p = 0.012), higher anti-spike protein IgG levels (p = 0.003), and anti-SARS-CoV-2 IgG positivity (p = 0.003). Among them, 66 cases progressed to ACR-EULAR 2010 early arthritis after 3 months, whereas PMR-HA patients were more commonly PCV (81.8%, p = 0.008), demonstrating higher CRP (p = 0.007) and ESR (p = 0.006) levels, a lower rate of ANA positivity (p = 0.005), and a higher remission rate after six months (p = 0.050). In UA patients, the prevalent HLA was DRB1*11 and C*07 (36.8% and 42.1%). Serum calprotectin, interleukin-6, and C*07 (p = 0.021, 0.041, 0.018) seemed more specific for isolated UA. Conversely, "connective-like" arthritis showed poorer DLCO (p = 0.041) and more frequent US synovitis (p = 0.041). In conclusion, UA is a frequent common PC and PCV complication and may persist over time when compared to PMR-HA.

MeltSerts technology (brinzolamide ocular inserts via hot-melt extrusion): QbD-steered development, molecular dynamics, in vitro, ex vivo and in vivo studies.

The research work aimed to develop a robust sustained release biocompatible brinzolamide (BRZ)-loaded ocular inserts (MeltSerts) using hot-melt extrusion technology with enhanced solubility for glaucoma management. A 32 rotatable central composite design was employed for the optimization of the MeltSerts to achieve sustained release. The effect of two independent factors was examined: Metolose® SR 90SH-100000SR (HPMC, hydroxypropyl methyl cellulose) and Kolliphor® P 407 (Poloxamer 407, P407). The drug release (DR) of BRZ at 0.5 h and 8 h were adopted as dependent responses. The factorial analysis resulted in an optimum composition of 50.00 % w/w of HPMC and 15.00 % w/w of P407 which gave % DR of 9.11 at 0.5 h and 69.10 at 8 h. Furthermore, molecular dynamic simulations were performed to elucidate various interactions between BRZ, and other formulation components and it was observed that BRZ showed maximum interactions with HPC and HPMC with an occupancy of 92.82 and 52.87 %, respectively. Additionally, molecular docking studies were performed to understand the interactions between BRZ and mucoadhesive polymers with ocular mucin (MUC-1). The results indicated a docking score of only -5.368 for BRZ alone, whereas a significantly higher docking score was observed for the optimized Meltserts -6.977, suggesting enhanced retention time of the optimized MeltSerts. SEM images displayed irregular surfaces, while EDS analysis validated uniform BRZ distribution in the optimized formulation. The results of the ocular irritancy studies both ex vivo and in vivo demonstrated that MeltSerts are safe for ocular use. The results indicate that the developed MeltSerts Technology has the potential to manufacture ocular inserts with cost-effectiveness, one-step processability, and enhanced product quality. Nonetheless, it also offers a once-daily regimen, consequently decreasing the dosing frequency, preservative exposure, and ultimately better glaucoma management.

Terahertz imaging for non-invasive classification of healthy and cimiciato-infected hazelnuts.

The development of new non-invasive approaches able to recognize defective food is currently a lively field of research. In particular, a simple and non-destructive method able to recognize defective hazelnuts, such as cimiciato-infected ones, in real-time is still missing. This study has been designed to detect the presence of such damaged hazelnuts. To this aim, a measurement setup based on terahertz (THz) radiation has been developed. Images of a sample of 150 hazelnuts have been acquired in the low THz range by a compact and portable active imaging system equipped with a 0.14 THz source and identified as Healthy Hazelnuts (HH) or Cimiciato Hazelnut (CH) after visual inspection. All images have been analyzed to find the average transmission of the THz radiation within the sample area. The differences in the distribution of the two populations have been used to set up a classification scheme aimed at the discrimination between healthy and injured samples. The performance of the classification scheme has been assessed through the use of the confusion matrix on 50 samples. The False Positive Rate (FPR) and True Negative Rate (TNR) are 0% and 100%, respectively. On the other hand, the True Positive Rate (TPR) and False Negative Rate (FNR) are 75% and 25%, respectively. These results are relevant from the perspective of the development of a simple, automatic, real-time method for the discrimination of cimiciato-infected hazelnuts in the processing industry.

Mitapivat reprograms the RBC metabolome and improves anemia in a mouse model of hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common, nonimmune, hereditary, chronic hemolytic anemia after hemoglobinopathies. The genetic defects in membrane function causing HS lead to perturbation of the RBC metabolome, with altered glycolysis. In mice genetically lacking protein 4.2 (4.2-/-; Epb42), a murine model of HS, we showed increased expression of pyruvate kinase (PK) isoforms in whole and fractioned RBCs in conjunction with abnormalities in the glycolytic pathway and in the glutathione (GSH) system. Mitapivat, a PK activator, metabolically reprogrammed 4.2-/- mouse RBCs with amelioration of glycolysis and the GSH cycle. This resulted in improved osmotic fragility, reduced phosphatidylserine positivity, amelioration of RBC cation content, reduction of Na/K/Cl cotransport and Na/H-exchange overactivation, and decrease in erythroid vesicles release in vitro. Mitapivat treatment significantly decreased erythrophagocytosis and beneficially affected iron homeostasis. In mild-to-moderate HS, the beneficial effect of splenectomy is still controversial. Here, we showed that splenectomy improves anemia in 4.2-/- mice and that mitapivat is noninferior to splenectomy. An additional benefit of mitapivat treatment was lower expression of markers of inflammatory vasculopathy in 4.2-/- mice with or without splenectomy, indicating a multisystemic action of mitapivat. These findings support the notion that mitapivat treatment should be considered for symptomatic HS.

Tyrosine phosphorylation and liver regeneration: A glance at intracellular transducers.

Liver regeneration (LR) is a compensatory growth that occurs in response to resection or injury of the liver aimed at restoring the liver mass and maintaining body homeostasis. The activation of intracellular signaling pathways due to extracellular stimuli mainly reflects a highly coordinated spatial and temporal organization of phosphotyrosine-based signals generated by the concerted action of three basic functional modules, namely protein tyrosine kinases, protein tyrosine phosphatases, and the Src homology 2 (SH2) domain. In this review, we have selected a set of signaling proteins downstream of activated cytokine and growth factor receptors that highlight the multifaceted aspects of tyrosine phosphorylation with their impact on the course of LR. Besides being a process of remarkable biological interest, LR has recently emerged as a model for dissecting molecular mechanisms underlying diverse pathophysiological states, offering new perspectives in primarily, but not only, managing life-threatening liver diseases.

Interaction between the SH3 domain of Src family kinases and the proline-rich motif of HTLV-1 p13: a novel mechanism underlying delivery of Src family kinases to mitochondria.

The association of the SH3 (Src homology 3) domain of SFKs (Src family kinases) with protein partners bearing proline-rich motifs has been implicated in the regulation of SFK activity, and has been described as a possible mechanism of relocalization of SFKs to subcellular compartments. We demonstrate in the present study for the first time that p13, an accessory protein encoded by the HTLV-1 (human T-cell leukaemia virus type 1), binds the SH3 domain of SFKs via its C-terminal proline-rich motif, forming a stable heterodimer that translocates to mitochondria by virtue of its N-terminal mitochondrial localization signal. As a result, the activity of SFKs is dramatically enhanced, with a subsequent increase in mitochondrial tyrosine phosphorylation, and the recognized ability of p13 to insert itself into the inner mitochondrial membrane and to perturb the mitochondrial membrane potential is abolished. Overall, the present study, in addition to confirming that the catalytic activity of SFKs is modulated by interactors of their SH3 domain, leads us to hypothesize a general mechanism by which proteins bearing a proline-rich motif and a mitochondrial localization signal at the same time may act as carriers of SFKs into mitochondria, thus contributing to the regulation of mitochondrial functions under various pathophysiological conditions.