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Background: It is generally believed that hepatitis B virus (HBV) core protein (HBc) dephosphorylation (de-P) is important for viral DNA synthesis and virion secretion. HBV polymerase contains four domains for terminal protein, spacer, reverse transcriptase, and RNase H activities. Methods: HBV Polymerase mutants were transfected into HuH-7 cells and assayed for replication and HBc de-P by the Phos-tag gel analysis. Infection assay was performed by using a HepG2-NTCP-AS2 cell line. Results: Here, we show that a novel phosphatase activity responsible for HBc de-P can be mapped to the C-terminal domain of the polymerase overlapping with the RNase H domain. Surprisingly, while HBc de-P is crucial for viral infectivity, it is essential for neither viral DNA synthesis nor virion secretion. The potential origin, significance, and mechanism of this polymerase-associated phosphatase activity are discussed in the context of an electrostatic homeostasis model. The Phos-tag gel analysis revealed an intriguing pattern of "bipolar distribution" of phosphorylated HBc and a de-P HBc doublet. Conclusions: It remains unknown if such a polymerase-associated phosphatase activity can be found in other related biosystems. This polymerase-associated phosphatase activity could be a druggable target in clinical therapy for hepatitis B. [ABSTRACT FROM AUTHOR]

Metaplastic breast carcinoma (MBC), a category of breast cancer, includes different histological types, which are occasionally mixed and heterogeneous. Considering the heterogeneity of cancer cells in a tumour mass has become highly significant, not only from a biological aspect but also for clinical management of recurrence. This study aimed to analyse the immunohistochemical and molecular profiles of each MBC component of a tumour mass. Twenty-five MBC tumours were histologically evaluated, and the most frequent MBC component (c) was squamous cell carcinoma (SCC), followed by spindle cell carcinoma (SpCC). A total of 69 components of MBC and non-MBC in formalin-fixed paraffin-embedded sections were examined for 7 markers by immunohistochemistry. SCC(c) were significantly PTEN negative and CK14 positive, and SpCC(c) were significantly E-cadherin negative and vimentin positive. Multivariate analyses revealed that immunohistochemical profiles of normal/intraductal (IC)(c), no special type (NST)(c), and MBC(c) differed; moreover, SCC(c) and SpCC(c) were distinctly grouped. PTEN gene mutation was detected only in SCC(c) (2/7), but not in SpCC(c). Next-generation sequence analyses for 2 cases with tumours containing SCC(c) demonstrated that PTEN gene mutation increased progressively from IC(c) to NST(c) to SCC(c). In conclusion, the immunohistochemical and molecular profiles of the SCC(c) of MBC are distinct from those of the SpCC(c). [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and trehalose, also play crucial role in bacterial physiology and virulence. However, some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known. Here we structurally and functionally characterized a conserved hypothetical gene Rv3400. We determined the crystal structure of Rv3400 at 1.7 Å resolution. The crystal structure revealed that Rv3400 adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins. Our comparative structural analysis suggested that Rv3400 could perform either phosphatase or pyrophosphatase or β‐phosphoglucomutase (β‐PGM) activity. Using biochemical studies, we further confirmed that Rv3400 performs β‐PGM activity and hence, Rv3400 encodes for β‐PGM in Mtb. Our data also confirm that Mtb β‐PGM is a metal dependent enzyme having broad specificity for divalent metal ions. β‐PGM converts β‐D‐glucose‐1‐phosphate to β‐D‐glucose‐6‐phosphate which is required for the generation of ATP and NADPH through glycolysis and pentose phosphate pathway, respectively. Using site directed mutagenesis followed by biochemical studies, we show that two Asp residues in the highly conserved DxD motif, D29 and D31, are crucial for enzyme activity. While D29A, D31A, D29E, D31E and D29N mutants lost complete activity, D31N mutant retained about 30% activity. This study further helps in understanding the role of β‐PGM in the physiology of Mtb. [ABSTRACT FROM AUTHOR]

Background: T cell receptor (TCR) signaling and T cell activation are tightly regulated by gatekeepers to maintain immune tolerance and avoid autoimmunity. The TRAIL receptor (TRAIL-R) is a TNF-family death receptor that transduces apoptotic signals to induce cell death. Recent studies have indicated that TRAIL-R regulates T cell-mediated immune responses by directly inhibiting T cell activation without inducing apoptosis; however, the distinct signaling pathway that regulates T cell activation remains unclear. In this study, we screened for intracellular TRAIL-R-binding proteins within T cells to explore the novel signaling pathway transduced by TRAIL-R that directly inhibits T cell activation. Methods: Whole-transcriptome RNA sequencing was used to identify gene expression signatures associated with TRAIL-R signaling during T cell activation. High-throughput screening with mass spectrometry was used to identify the novel TRAIL-R binding proteins within T cells. Co-immunoprecipitation, lipid raft isolation, and confocal microscopic analyses were conducted to verify the association between TRAIL-R and the identified binding proteins within T cells. Results: TRAIL engagement downregulated gene signatures in TCR signaling pathways and profoundly suppressed phosphorylation of TCR proximal tyrosine kinases without inducing cell death. The tyrosine phosphatase SHP-1 was identified as the major TRAIL-R binding protein within T cells, using high throughput mass spectrometry-based proteomics analysis. Furthermore, Lck was co-immunoprecipitated with the TRAIL-R/SHP-1 complex in the activated T cells. TRAIL engagement profoundly inhibited phosphorylation of Lck (Y394) and suppressed the recruitment of Lck into lipid rafts in the activated T cells, leading to the interruption of proximal TCR signaling and subsequent T cell activation. Conclusions: TRAIL-R associates with phosphatase SHP-1 and transduces a unique and distinct immune gatekeeper signal to repress TCR signaling and T cell activation via inactivating Lck. Thus, our results define TRAIL-R as a new class of immune checkpoint receptors for restraining T cell activation, and TRAIL-R/SHP-1 axis can serve as a potential therapeutic target for immune-mediated diseases. [ABSTRACT FROM AUTHOR]

Phosphatases are important regulators of protein phosphorylation and various cellular processes, and they serve as counterparts to kinases. In this study, our comprehensive analysis of oomycete complete proteomes unveiled the presence of approximately 3833 phosphatases, with most species estimated to have between 100 and 300 putative phosphatases. Further investigation of these phosphatases revealed a significant increase in protein serine/threonine phosphatases (PSP) within oomycetes. In particular, we extensively studied the metallo‐dependent protein phosphatase (PPM) within the PSP family in the model oomycete Phytophthora sojae. Our results showed notable differences in the expression patterns of PPMs throughout 10 life stages of P. sojae, indicating their vital roles in various stages of oomycete pathogens. Moreover, we identified 29 PPMs in P. sojae, and eight of them possessed accessory domains in addition to phosphate domains. We investigated the biological function of one PPM protein with an extra PH domain (PPM1); this protein exhibited high expression levels in both asexual developmental and infectious stages. Our analysis confirmed that PPM1 is indeed an active protein phosphatase, and its accessory domain does not affect its phosphatase activity. To delve further into its function, we generated knockout mutants of PPM1 and validated its essential roles in mycelial growth, sporangia and oospore production, as well as infectious stages. To the best of our knowledge, this study provides the first comprehensive inventory of phosphatases in oomycetes and identifies an important phosphatase within the expanded serine/threonine phosphatase group in oomycetes. [ABSTRACT FROM AUTHOR]

The availability of phosphorus (P) in ultisol acid soils presents a significant challenge due to its attachment to aluminum (Al) or iron (Fe) compounds. A potential solution to address this issue is the utilization of phosphate solubilizing bacteria (PSB). Therefore, this study aimed to analyze the potential of PSB originating from upland rice cultivation on ultisol soils. The bacterial isolates were obtained from soil samples taken from the rhizosphere area and root tissue of upland rice plants cultivated in Payabenua and Saing Villages, Bangka Regency. The pathogenicity testing encompassed hypersensitivity and hemolysis tests, while the P solubilization included the evaluation of the phosphate solubilizing index (PSI) and P dissolution. Subsequently, the selected isolates were subjected to phosphatase enzyme and organic acid content assessment. The results showed a total of 120 isolates, predominantly distributed in the Payabenua area and primarily consisting of endophytic bacteria. Among the six selected isolates, genus Burkholderia dominated four isolates, while the remaining isolates belonged to genus Serratia. Furthermore, in Burkholderia vietnamiensis, the solubility value of P in AlPO4 and Ca3(PO4)2 liquid media exhibited a range of 0.0013 to 0.0344% and 0.0008 to 0.1842%, respectively. [ABSTRACT FROM AUTHOR]

Background and Aims Soils in south-western Australia are severely phosphorus (P) impoverished, and plants in this region have evolved a variety of P-acquisition strategies. Phosphorus acquisition by Adenanthos cygnorum (Proteaceae) is facilitated by P-mobilizing neighbours which allows it to extend its range of habitats. However, we do not know if other Adenanthos species also exhibit a strategy based on facilitation for P acquisition in P-impoverished environments. Methods We collected leaf and soil samples of Adenanthos barbiger , A. cuneatus , A. meisneri, A. obovatus , A. sericeus and Adenanthos sp. Whicher Range (G.J. Keighery 9736) growing in their natural habitats at different locations within the severely P-limited megadiverse environment of south-western Australia. Hydroponic experiments were conducted to collect the carboxylates exuded by cluster roots. Pot experiments in soil were carried out to measure rhizosheath phosphatase activity. Key Results We found no evidence for facilitation of P uptake in any of the studied Adenanthos species. Like most Proteaceae, A. cuneatus , A. meisneri , A. obovatus , A. sericeus and Adenanthos sp. Whicher Range (G.J. Keighery 9736) expressed P-mining strategies, including the formation of cluster roots. Cluster roots of A. obovatus were less effective than those of the other four Adenanthos species. In contrast to what is known for most Proteaceae, we found no cluster roots for A. barbiger. This species probably expressed a post-fire P-acquisition strategy. All Adenanthos species used P highly efficiently for photosynthesis, like other Proteaceae in similar natural habitats. Conclusions Adenanthos is the first genus of Proteaceae found to express multiple P-acquisition strategies. The diversity of P-acquisition strategies in these Proteaceae, coupled with similarly diverse strategies in Fabaceae and Myrtaceae, demonstrates that caution is needed in making family- or genus-wide extrapolations about the strategies exhibited in severely P-impoverished megadiverse ecosystems. [ABSTRACT FROM AUTHOR]

Two Ni(II) complexes [(Ni L1)(CH3COO) (H2O)2](1) and [{(NiL2) (CH3COO)}2 (µ-H2O)] (2) having different coordination environments around metal centres have been selected to investigate structure-activity correlation for DNA binding and homogenous phosphatase-like activities. Complex 1 is a monomer in which the octahedral Ni(II) center is coordinated to phenolate oxygen along with two nitrogen atoms from imine and amine groups of the deprotonated ligand HL1. An oxygen atom of the acetate group is coordinated in monodentate mode, and two coordinated water molecules occupy the other two axial sites. Complex 2 is a dimer where each Ni(II) centre is coordinated in an octahedral environment through one imine nitrogen, one amine nitrogen, two bridging phenolate oxygens from two deprotonated ligands (HL2) molecules, a mono-dentate acetate group, and a bridging water molecule. The role of structural features of both the complexes has been studied in DNA binding activity and phosphate ester bond cleavage of bis(4-nitrophenyl) phosphate (BNPP) as a model substrate. Complex 2 showed higher DNA binding ability than complex 1 due to its stable bridging structure and high nuclearity. The catalytic phosphate ester bond hydrolysis of BNPP was explored with both complexes spectrophotometrically. The dinuclear complex 2 also exhibited a higher rate of acceleration in the BNPP hydrolysis than mononuclear complex 1. The active nucleophile from the coordinated water and cooperativity in two metal centres of complex 2 are the key features to cleave the phosphate ester bond of the substrate. Further, the first-order rate constants and various kinetic parameters based on the Michaelis-Menten equation were calculated for each complex. A significant phosphatase-like activity for complex 2 has been observed with a turnover number of 1.23 × 10−2 s−1. A structure-activity relationship for phosphatase-like catalytic activities of two mono and di nuclear Ni(II) complexes has been studied. Dinuclear complex 2 exhibits higher phosphatase-like activity due to coordinated water molecules and cooperativity between two metal centres. [ABSTRACT FROM AUTHOR]

Post‐translational modifications are key in the regulation of activity, structure, localization, and stability of most proteins in eukaryotes. Phosphorylation is potentially the most studied post‐translational modification, also due to its reversibility and thereby the regulatory role this modification often plays. While most research attention was focused on kinases in the past, phosphatases remain understudied, most probably because the addition and presence of the modification is more easily studied than its removal and absence. Here, we report the identification of an uncharacterized protein tyrosine phosphatase PPH‐7 in C. elegans, a member of the evolutionary conserved PTPN family of phosphatases. Lack of PPH‐7 function led to reduction of fertility and embryonic lethality at elevated temperatures. Proteomics revealed changes in the regulation of targets of the von Hippel–Lindau (VHL) E3 ligase, suggesting a potential role for PPH‐7 in the regulation of VHL. [ABSTRACT FROM AUTHOR]

Purpose: Soil available phosphate (AP) is largely dependent on phoD-harboring bacteria, which can release alkaline phosphatase (ALP) to transform insoluble P for plant absorption. However, the way of phoD-harboring bacterial communities responding to restoration measures in alpine ecosystems, which are among the least studied and most vulnerable ecosystems, remains largely unknown. This knowledge is fundamental for formulating effective ecosystem management and soil conservation policies. Materials and methods: By combining quantitative PCR and amplicon sequencing, we examined the alterations in phoD-harboring bacterial communities across four distinct meadow types, and explored the potential environmental drivers of alpine soil P availability. Results and discussion: The results indicated that the fenced and fenced + reseeded meadows exhibited higher ALP activity and soil AP content compared to the grazed meadow, but lower than that of the undegraded meadow. The fenced meadow had the highest phoD-harboring bacterial community diversity. Rare genera such as Rhizobium, Breoghania, and Actinomadura, played a critical role in regulating ALP activity. A structural equation model demonstrated that soil pH, nutrient supply (e.g., soil organic carbon, NO3−-N), and vegetation together drove the improvement of soil P availability by enhancing ALP activity, which was closely related to phoD-harboring bacterial communities. These findings suggest that fencing could promote alpine soil P availability by adjusting phoD-harboring bacteria and ALP activity. Conclusions: Our results demonstrate the beneficial impacts of grazing-to-fencing conversion on meadows in alpine ecosystems and can contribute to the development of sustainable management strategies for degraded alpine ecosystems. [ABSTRACT FROM AUTHOR]

Circular economy recycling-derived fertilizers (RDF) have the potential to replace linear economy fertilizers such as unsustainable superphosphates. Here, effects of ash RDF treatments in Irish grassland cultivation were investigated in a simulated second growing season. Soil fertilized in a preceding pot trial with superphosphate (SP), poultry-litter ash (PLA) and sewage-sludge ash (SSA) at P concentration of 60 kg P ha−1 and a P-free control (SP0) was reused in a microcosm trial. Lolium perenne was cultivated for 54 days in six replicates with a full complement of micro- and macro-nutrients other than P. PLA treatments provided higher dry weight shoot yields than SP0, while SSA and SP overlapped with SP0 and PLA. Most probable number (MPN) analysis showed that phosphonate- and phytate-utilizing bacterial abundance was significantly increased in PLA. Alkaline (phoD) phosphomonoesterase gene fragments were significantly more abundant (qPCR) in the ashes than the superphosphate or P-free control. Bacterial communities were significantly affected by the P application. Similarly, a significant separation of treatments was confirmed in a canonical correspondence analysis of the phoD-harboring community. The genera Streptomyces and Xanthomonas were significantly higher in abundance in the ash RDFs. These results demonstrated the potential benefits of ash RDF treatments as an alternative P source. [ABSTRACT FROM AUTHOR]

IntroductionReversible protein phosphorylation is an abundant post-translational modification dynamically regulated by opposing kinases and phosphatases. Protein phosphorylation has been extensively studied in cell division, where waves of cyclin-dependent kinase activity, peaking in mitosis, drive the sequential stages of the cell cycle. Here we developed and employed a strategy to specifically probe kinase or phosphatase substrates at desired times or experimental conditions in the model organism Saccharomyces cerevisiae.MethodsWe combined auxin-inducible degradation (AID) with mass spectrometry-based phosphoproteomics, which allowed us to arrest physiologically normal cultures in mitosis prior to rapid phosphatase degradation and phosphoproteome analysis.Results and discussionOur results revealed that protein phosphatase 2A coupled with its B56 regulatory subunit, Rts1 (PP2ARts1), is involved in dephosphorylation of numerous proteins in mitosis, highlighting the need for phosphatases to selectively maintain certain proteins in a hypophosphorylated state in the face of high mitotic kinase activity. Unexpectedly, we observed elevated phosphorylation at many sites on several subunits of the fungal eisosome complex following rapid Rts1 degradation. Eisosomes are dynamic polymeric assemblies that create furrows in the plasma membrane important in regulating nutrient import, lipid metabolism, and stress responses, among other things. We found that PP2ARts1-mediated dephosphorylation of eisosomes promotes their plasma membrane association and we provide evidence that this regulation impacts eisosome roles in metabolic homeostasis. The combination of rapid, inducible protein degradation with proteomic profiling offers several advantages over common protein disruption methods for characterizing substrates of regulatory enzymes involved in dynamic biological processes.

ABSTRACT Cryptococcus neoformans is an environmentally acquired fungal pathogen that causes over 140,000 deaths per year. Cryptococcal infection occurs when infectious particles are deposited into the lung, where they encounter host phagocytic cells. C. neoformans may be engulfed by these phagocytes, an important step of infection that leads to outcomes ranging from termination of infection to cryptococcal dissemination. To study this critical process, we screened approximately 4,700 cryptococcal gene deletion mutants for altered uptake, using primary mouse and human phagocytic cells. Among the hits of these two screens, we identified 93 mutants with perturbed uptake in both systems, as well as others with differences in uptake by only one cell type. We further screened the hits for changes in thickness of the capsule, a protective polysaccharide layer around the cell which is an important cryptococcal virulence factor. The combination of our three screens yielded 45 mutants, including one lacking the phosphatidylinositol-4-phosphate phosphatase Sac1. In this work, we implicate Sac1 in both host cell uptake and capsule production. We found that sac1 mutants exhibit lipid trafficking defects, reductions in secretory system function, and changes in capsule size and composition. Many of these changes occur specifically in tissue culture media, highlighting the role of Sac1 phosphatase activity in responding to the stress of host-like conditions. Overall, these findings show how genome-scale screening can identify cellular factors that contribute to our understanding of cryptococcal biology and demonstrate the role of Sac1 in determining fungal virulence.IMPORTANCECryptococcus neoformans is a fungal pathogen with significant impact on global health. Cryptococcal cells inhaled from the environment are deposited into the lungs, where they first contact the human immune system. The interaction between C. neoformans and host cells is critical because this step of infection can determine whether the fungal cells die or proliferate within the human host. Despite the importance of this stage of infection, we have limited knowledge of cryptococcal factors that influence its outcome. In this study, we identify cryptococcal genes that affect uptake by both human and mouse cells. We also identify mutants with altered capsule, a protective coating that surrounds the cells to shield them from the host immune system. Finally, we characterize the role of one gene, SAC1, in these processes. Overall, this study contributes to our understanding of how C. neoformans interacts with and protects itself from host cells.

Introduction: Dual specific phosphatases (DUSPs) are mitogen-activated protein kinase (MAPK) regulators, which also serve as drug targets for treating various vascular diseases. Previously, we have presented mechanistic characterizations of DUSP5 and its interaction with pERK, proposing a dual active site.Methods: Herein, we characterize the interactions between the DUSP5 phosphatase domain and the pT-E-pY activation loop of ERK2, with specific active site assignments. We also report the full NMR chemical shift assignments of DUSP5 that now enable chemical shift perturbation and dynamics studies.Results and Discussion: Both phosphates of the pT-E-pY tripeptide are dephosphorylated, based on 31P NMR; but, steady state kinetic studies of the tripeptide both as a substrate and as an inhibitor indicate a preference for binding and dephosphorylation of the phospho-tyrosine before the phospho-threonine. Catalytic efficiency (kcat/Km) is 3.7 M−1S−1 for T-E-pY vs 1.3 M−1S−1 for pT-E-Y, although the diphosphorylated peptide (pT-E-pY) is a better substrate than both, with kcat/Km = 18.2 M−1S−1 . Steady state inhibition studies with the pNPP substrate yields Kis values for the peptide inhibitors of: 15.82 mM (pT-E-Y), 4.932 mM (T-E-pY), 1.672 mM (pT-E-pY). Steady state inhibition studies with pNPP substrate and with vanadate or phosphate inhibitors indicated competitive inhibition with Kis values of 0.0006122 mM (sodium vanadate) and 17.32 mM (sodium phosphate), similar to other Protein Tyrosine Phosphatases with an active site cysteine nucleophile that go through a five-coordinate high energy transition state or intermediate. Molecular dynamics (MD) studies confirm preferential binding of the diphosphorylated peptide, but with preference for binding the pY over the pT reside in the catalytic site proximal to the Cys263 nucleophile. Based on MD, the monophosphorylated peptide binds tighter if phosphorylated on the Tyr vs the Thr. And, if the starting pose of the docked diphosphorylated peptide has pT in the catalytic site, it will adjust to have the pY in the catalytic site, suggesting a dynamic shifting of the peptide orientation. 2D 1H-15N HSQC chemical shift perturbation studies confirm that DUSP5 with tripeptide bound is in a dynamic state, with extensive exchange broadening observed—especially of catalytic site residues. The availability of NMR chemical shift assignments enables additional future studies of DUSP5 binding to the ERK2 diphosphorylated activation loop.Summary: These studies indicate a preference for pY before pT binding, but with ability to bind and dephosphorylate both residues, and with a dynamic active site pocket that accommodates multiple tripeptide orientations.

Abstract Biosynthesis is the application of enzymes in microbial cell factories and has emerged as a promising alternative to chemical synthesis. However, natural enzymes with limited catalytic performance often need to be engineered to meet specific needs through a time‐consuming trial‐and‐error process. This study presents a quantum mechanics (QM)‐incorporated design–build–test–learn (DBTL) framework to rationally design phosphatase BT4131, an enzyme with an ambiguous substrate spectrum involved in N‐acetylglucosamine (GlcNAc) biosynthesis. First, mutant M1 (L129Q) is designed using force field‐based methods, resulting in a 1.4‐fold increase in substrate preference (kcat/Km) toward GlcNAc‐6‐phosphate (GlcNAc6P). QM calculations indicate that the shift in substrate preference is caused by a 13.59 kcal mol−1 reduction in activation energy. Furthermore, an iterative computer‐aided design is conducted to stabilize the transition state. As a result, mutant M4 (I49Q/L129Q/G172L) with a 9.5‐fold increase in kcat‐GlcNAc6P/Km‐GlcNAc6P and a 59% decrease in kcat‐Glc6P/Km‐Glc6P is highly desirable compared to the wild type in the GlcNAc‐producing chassis. The GlcNAc titer increases to 217.3 g L−1 with a yield of 0.597 g (g glucose)−1 in a 50‐L bioreactor, representing the highest reported level. Collectively, this DBTL framework provides an easy yet fascinating approach to the rational design of enzymes for industrially viable biocatalysts.

Aims: To investigate the effects of senescent osteocytes on bone homeostasis in the progress of age-related osteoporosis and explore the underlying mechanism. Methods: In a series of in vitro experiments, we used tert-Butyl hydroperoxide (TBHP) to induce senescence of MLO-Y4 cells successfully, and collected conditioned medium (CM) and senescent MLO-Y4 cell-derived exosomes, which were then applied to MC3T3-E1 cells, separately, to evaluate their effects on osteogenic differentiation. Furthermore, we identified differentially expressed microRNAs (miRNAs) between exosomes from senescent and normal MLO-Y4 cells by high-throughput RNA sequencing. Based on the key miRNAs that were discovered, the underlying mechanism by which senescent osteocytes regulate osteogenic differentiation was explored. Lastly, in the in vivo experiments, the effects of senescent MLO-Y4 cell-derived exosomes on age-related bone loss were evaluated in male SAMP6 mice, which excluded the effects of oestrogen, and the underlying mechanism was confirmed. Results: The CM and exosomes collected from senescent MLO-Y4 cells inhibited osteogenic differentiation of MC3T3-E1 cells. RNA sequencing detected significantly lower expression of miR-494-3p in senescent MLO-Y4 cell-derived exosomes compared with normal exosomes. The upregulation of exosomal miR-494-3p by miRNA mimics attenuated the effects of senescent MLO-Y4 cell-derived exosomes on osteogenic differentiation. Luciferase reporter assay demonstrated that miR-494-3p targeted phosphatase and tensin homolog (PTEN), which is a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overexpression of PTEN or inhibition of the PI3K/AKT pathway blocked the functions of exosomal miR-494-3p. In SAMP6 mice, senescent MLO-Y4 cell-derived exosomes accelerated bone loss, which was rescued by upregulation of exosomal miR-494-3p. Conclusion: Reduced expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenic differentiation and accelerates age-related bone loss via PTEN/PI3K/AKT pathway. Cite this article: Bone Joint Res 2024;13(2):52–65.

Abstract Background The most aggressive form of breast cancer is triple-negative breast cancer (TNBC), which lacks expression of the estrogen receptor (ER) and progesterone receptor (PR), and does not have overexpression of the human epidermal growth factor receptor 2 (HER2). Treatment options for women with TNBC tumors are limited, unlike those with ER-positive tumors that can be treated with hormone therapy, or those with HER2-positive tumors that can be treated with anti-HER2 therapy. Therefore, we have sought to identify novel targeted therapies for TNBC. In this study, we investigated the potential of a novel phosphatase, NUDT5, as a potential therapeutic target for TNBC. Methods The mRNA expression levels of NUDT5 in breast cancers were investigated using TCGA and METABRIC (Curtis) datasets. NUDT5 ablation was achieved through siRNA targeting and NUDT5 inhibition with the small molecule inhibitor TH5427. Xenograft TNBC animal models were employed to assess the effect of NUDT5 inhibition on in vivo tumor growth. Proliferation, death, and DNA replication assays were conducted to investigate the cellular biological effects of NUDT5 loss or inhibition. The accumulation of 8-oxo-guanine (8-oxoG) and the induction of γH2AX after NUDT5 loss was determined by immunofluorescence staining. The impact of NUDT5 loss on replication fork was assessed by measuring DNA fiber length. Results In this study, we demonstrated the significant role of an overexpressed phosphatase, NUDT5, in regulating oxidative DNA damage in TNBCs. Our findings indicate that loss of NUDT5 results in suppressed growth of TNBC both in vitro and in vivo. This growth inhibition is not attributed to cell death, but rather to the suppression of proliferation. The loss or inhibition of NUDT5 led to an increase in the oxidative DNA lesion 8-oxoG, and triggered the DNA damage response in the nucleus. The interference with DNA replication ultimately inhibited proliferation. Conclusions NUDT5 plays a crucial role in preventing oxidative DNA damage in TNBC cells. The loss or inhibition of NUDT5 significantly suppresses the growth of TNBCs. These biological and mechanistic studies provide the groundwork for future research and the potential development of NUDT5 inhibitors as a promising therapeutic approach for TNBC patients.

YqeK is a bacterial HD-domain metalloprotein that hydrolyzes the putative second messenger diadenosine tetraphosphate (Ap4A). Elevated Ap4A levels are primarily observed upon exposure of bacteria to factors such as heat or oxidative stress and cause pleiotropic effects, including antibiotic sensitivity and disrupted biofilm formation. Ap4A thus plays a central role in bacterial physiology and metabolism, and its hydrolysis by YqeK is intimately linked to the ability of these microbes to cope with stress. Although YqeK is reported to hydrolyze Ap4A under aerobic conditions, all four existing crystal structures reveal an active site that consists of a diiron center, portraying a cryptic chemical nature for the active metallocofactor. This study examines two YqeK proteins from two ecologically diverse parent organisms: the obligate anaerobe Clostridium acetobutylicum and the facultative aerobe Bacillus halodurans. Both enzymes utilize Fe-based cofactors for catalysis, while under ambient or oxidative conditions, Bh YqeK hydrolyzes Ap4A more efficiently compared to Ca YqeK. This redox-dependent activity difference stems from the following two molecular mechanisms: the incorporation of mixed-metal, Fe-based bimetallic cofactors, in which the second metal is redox inert (i.e., Fe–Zn) and the upshift of the Fe–Fe cofactor reduction potentials. In addition, three strictly conserved, positively charged residues vicinal to the active site are critical for tuning Ap4A hydrolysis. In conclusion, YqeK is an Fe-dependent phosphohydrolase that appears to have evolved to permit Ap4A hydrolysis under different environmental niches (aerobic vs. anaerobic) by expanding its cofactor configuration and O2 tolerance.

Bioconversion of cellulosic biomass, such as sugarcane bagasse (SB), into valuable products by fermentation and saccharification processes can reduce environmental pollution. Therefore, this study was designed to saccharify the SB by thermostable cellulase enzyme and to use the hydrolysate for the release of phosphorus and phosphatases production. Initially, a thermostable cellulase was obtained from a novel bacterium, Neobacillus sedimentimangrovi UE25, using alkali and ionic liquid pretreated SB. The strain produced 110 IU mL−1 of endoglucanase. The enzyme was utilized to saccharify SB and the factors affecting this process were optimized by employing central composite design (CCD). A yield of 45 mg g−1 of reducing sugars was obtained under the optimized conditions, i.e., temperature 58 °C, pH 5.1, endoglucanase units 17.849, and substrate combination 0.48 (i.e., a mix of pretreated and untreated SB in the ratio of 48% (w/v) to 52% (w/v)) in a relatively shorter reaction time of 18 h 24 min. The hydrolysate from the process was utilized to cultivate Pseudomonas species that produced 54 and 57 IU mL−1 alkaline and acid phosphatases, respectively. Scanning electron microscopy with EDX-, FTIR-, and NMR-based elucidation of the substrate showed removal of lignin by the pretreatment process and utilization of cellulose in fermented and saccharified substrate. This study supports the idea of utilization of industrial waste by-product for the production of valuable products. [ABSTRACT FROM AUTHOR]

Soil phosphorus (P) partitioning could contribute to species diversity and structure in plant communities, but field-scale evidence for P partitioning remains scarce. We hypothesized that the presence of P partitioning could be inferred from statistical associations between the spatial distributions of plants and chemical forms of bioavailable soil P. We investigated this in a diverse tropical tree community on Barro Colorado Island, Panama. We quantified potentially bioavailable forms of soil P by extraction in 2 mM citric acid followed by treatment with phosphatase enzymes. We then linked these P forms to the distribution of 189 tree species in a 50 ha forest dynamics plot by testing species–P associations against null models of random dispersal. We found that 20% of tree species were significantly (α = 0.05) associated with the depletion of at least one soil organic P fraction, although around half of these associations might be false rejections of the null hypothesis due to type I error. Species in the Fabaceae (legumes), which are known to express high rates of phosphatase in their roots, were most frequently associated with soil P fractions. We interpret our findings as evidence of widespread P partitioning at the community scale, affecting a relatively small proportion of tree species in this moderately fertile forest. We predict that stronger evidence of partitioning will be found at sites with lower P availability. [ABSTRACT FROM AUTHOR]

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Å and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant. [ABSTRACT FROM AUTHOR]

Background: Phosphorus (P) is an essential nutrient for plant growth, taking part in primary cellular metabolic processes as a structural component of key biomolecules. Soil processes as adsorption, precipitation, and coprecipitation can affect P bioavailability, leading to limited plant growth and excessive use of P fertilizers, with adverse impacts on the environment and progressive depletion of P reserves. To cope with P stress, plants undergo several growth, development, and metabolic adjustments, aimed at increasing P-acquisition and -utilization efficiency. Recently, strigolactones (SLs) have emerged as newly defined hormones that mediate multiple levels of morphological, physiological and biochemical changes in plants as part of the P acclimation strategies to optimize growth. Therefore, understanding the soil processes affecting P availability and P acquisition strategies by plants can contribute to improved agronomical practices, resources optimization and environmental protection, and the development of plants with high P use efficiency for enhanced agricultural productivity. Scope: In this review, we discuss the range of abiotic processes that control P retention in soil and how different concentrations or degrees of P bioavailability can trigger various responses in plants, while critically highlighting the inconsistent conditions under which experiments evaluating aspects of P nutrition in plants have been conducted. We also present recent advances in elucidating the role of SLs in the complex P signalling pathway, with a special focus on what has been discovered so far in the model plant tomato (Solanum lycopersicum L.). [ABSTRACT FROM AUTHOR]

SHP2 phosphatase promotes full activation of the RTK-dependent Ras/MAPK pathway. Its mutations can drive cancer and RASopathies, a group of neurodevelopmental disorders (NDDs). Here we ask how same residue mutations in SHP2 can lead to both cancer and NDD phenotypes, and whether we can predict what the outcome will be. We collected and analyzed mutation data from the literature and cancer databases and performed molecular dynamics simulations of SHP2 mutants. We show that both cancer and Noonan syndrome (NS, a RASopathy) mutations favor catalysis-prone conformations. As to cancer versus RASopathies, we demonstrate that cancer mutations are more likely to accelerate SHP2 activation than the NS mutations at the same genomic loci, in line with NMR data for K-Ras4B more aggressive mutations. The compiled experimental data and dynamic features of SHP2 mutants lead us to propose that different from strong oncogenic mutations, SHP2 activation by NS mutations is less likely to induce a transition of the ensemble from the SHP2 inactive state to the active state. Strong signaling promotes cell proliferation, a hallmark of cancer. Weak, or moderate signals are associated with differentiation. In embryonic neural cells, dysregulated differentiation is connected to NDDs. Our innovative work offers structural guidelines for identifying and correlating mutations with clinical outcomes, and an explanation for why bearers of RASopathy mutations may have a higher probability of cancer. Finally, we propose a drug strategy against SHP2 variants-promoting cancer and RASopathies. [ABSTRACT FROM AUTHOR]

Insulin signaling cascade in peripheral insulin-sensitive tissues regulates whole-body glucose metabolism. Any deregulation in this pathway leads to insulin resistance, ultimately leading to metabolic diseases like type 1 diabetes, type 2 diabetes, and obesity. Insulin signaling in the brain has also been studied for many decades and associated with many primary functions like maintenance of synaptic plasticity, regulation of cognition, and circadian rhythm. Importantly, neuronal insulin signaling has also been associated with the regulation of neuronal glucose uptake. Any impairment in neuronal insulin signaling affecting neuronal glucose uptake has been associated with neurodegenerative disorders like Alzheimer’s disease, the process now being termed as type 3 diabetes. Since the criticality lies in proper signaling cascade, determining important points of deregulation is important. In this review, we have discussed some critical points of such deregulation, dividing them into two classes of enzymes: kinases and phosphatases. We have highlighted their individual roles in neuronal insulin signaling, along with their possible implications in neuronal insulin resistance. Future strategies targeting these nodes in neuronal insulin signaling might be helpful in exploring potential therapeutic opportunities to overcome neuronal insulin resistance and related neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The SH2-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) enzyme opposes the activity of PI3K and therefore is of interest in the treatment of inflammatory disorders. Recent results also indicate that SHIP1 promotes phagolysosomal degradation of lipids by microglia, suggesting that the enzyme may be a target for the treatment of Alzheimer's disease. Therefore, small molecules that increase SHIP1 activity may have benefits in these areas. Recently we discovered a bis-sulfonamide that increases the enzymatic activity of SHIP1. A series of similar SHIP1 activators have been synthesized and evaluated to determine structure–activity relationships and improve in vivo stability. Some new analogs have now been found with improved potency. In addition, both the thiophene and the thiomorpholine in the parent structure can be replaced by groups without a low valent sulfur atom, which provides a way to access activators that are less prone to oxidative degradation. [ABSTRACT FROM AUTHOR]

Studies were carried out to determine the impact of the humic preparation on the phosphate regime of ordinary chernozem. The work was carried out in 2017–2022 under the conditions of the Rostov oblast on winter wheat crops. During the field experiment, the following variants were studied: without treatments (control); and treatment with BIO-Don at a dose of 2 L/ha. Mobile phosphates (according to the Machigin method) and phosphatase activity (according to Galstyan and Arutyunyan) were determined in 4 stages: sampling 1—resumption of spring growing season before the introduction of mineral supplements; sampling 2—2 weeks after treatment with humic preparation (tillering); sampling 3—2 weeks after treatment (heading); sampling 4—after harvesting. Spraying with a solution of a humic preparation of vegetative plants was carried out in the phase of tillering and heading of winter wheat. This resulted in dry conditions in 2017–2018 to a slight increase in the content of mobile phosphates from 19.1 to 23.8 mg/kg of soil (between the first and fourth samplings), the difference in comparison with the control was 0.4–3.2 mg/kg; in 2018–2019 and 2019–2020, to a significant increase in the value of this indicator from 21.1 to 34.7 mg/kg and from 19.7 to 58.0 mg/kg, according to periods. In 2020–2021, a sharp increase in the content of mobile forms of phosphorus in the tillering phase by 20–25% was recorded when treated with BIO-Don, compared to the control, to 59.2 mg/kg and a decrease by harvesting to 26.0 mg/kg. In 2021–2022, the amount of phosphates varied in the range from 35.0 to 21.6 mg/kg. At elevated and high concentrations of mobile phosphates (in 2019–2021 in the range of 45–65 mg/kg P2O5), no increase in enzymatic activity was observed. Under conditions of lack of moisture (2017–2018), in the variant with BIO-Don treatment, a significant decrease in soil phosphatase activity during the first and fourth sampling by 0.68 and 0.61 mg P2O5/100 g/L/h, respectively, was revealed. The dynamics of enzyme activity varied from high to moderate. Under favorable humid conditions in 2018–2019 (HTC = 0.7) an increase in the mobility of phosphates due to their dissolution in soil moisture suspended the activation of phosphatase. [ABSTRACT FROM AUTHOR]

A factorial experiment was carried out in pots in the agricultural canopy of the College of Agriculture, University of Al-Muthanna during the winter agricultural season 2020-2021 and by Completely random design (CRD) to study the effect of the Mycorrhizal fungus Glomus mosseae and phosphate rock on the availability of phosphorous in the soil and the growth of wheat plant, as three levels (0, 60, 120) kg P.hectare-1 of phosphate rock were used. It is was calculated on the basis of the phosphate rock content of phosphorous, which amounted to 10.2% P and the soil was inoculated with mycorrhiza, without pollination, sterile and non-sterile soil, and with three replications. The results showed significant increase in plant height and dry matter weight for the vegetative and root groups and ready phosphorus in the soil and for the two growth stages as a result of inoculation with the fungus G. mosseae, as the percentage of increase in plant height was 22.16%, 16.94% and in the vegetative dry weight of 28.68% and 58.46% and in phosphorus. The addition of phosphate rock individually also achieved a significant increase on the rates of the studied traits, as the plant height was 23.5 cm and 43.37 cm for the first and second stages in succession with the third level of phosphate rock, while the dry weight was recorded. [ABSTRACT FROM AUTHOR]

In response to different immune challenges, immune cells migrate to specific sites in the body, where they perform their functions such as defense against infection, inflammation regulation, antigen recognition, and immune surveillance. Therefore, the migration ability is a fundamental aspect of immune cell function. Phosphoinositide signaling plays critical roles in modulating immune cell migration by controlling cell polarization, cytoskeletal rearrangement, protrusion formation, and uropod contraction. Upon chemoattractant stimulation, specific phosphoinositide kinases and phosphatases control the local phosphoinositide levels to establish polarized phosphoinositide distribution, which recruits phosphoinositide effectors to distinct subcellular locations to facilitate cell migration. In this Special Issue of "Molecular Mechanisms Underlying Cell Adhesion and Migration", we discuss the significance of phosphoinositide production and conversion by phosphoinositide kinases and phosphatases in the migration of different types of immune cells. [ABSTRACT FROM AUTHOR]

An incubation experiment was conducted to study the effect of clay content and composition on organic nitrogen mineralization. The experiment measured the mineralization of organic nitrogen from alfalfa residues, enzyme activities, and microbial biomass nitrogen in mixtures of sand, kaolinite, and non-layered colloids (NLCs) with sand as a control. The study found that as the contents of kaolinite and NLCs increased, the mineralization of organic nitrogen and enzyme activity decreased, but microbial biomass nitrogen increased. The maximum decrease in organic nitrogen mineralization was 88.6%, and microbial biomass nitrogen increased from 4.7 to 15.5%. The acid and alkaline phosphatase activities also decreased by 86.0 and 93.6%, respectively, with an increase in clay content. The specific surface area (SSA) of the mixtures showed an inverse relationship with enzyme activity and mineralization of organic nitrogen. Inactivation of extracellular enzymes by adsorption on the surfaces of kaolinite and NLCs, and decreased accessibility of organic nitrogen substrate molecules due to adsorption, reduced the mineralization of organic nitrogen. Microbial biomass nitrogen increased as the water holding capacity of the mixtures increased, indicating the importance of water-filled pores in accommodating active microbial biomass and protecting it from desiccation and predators. [ABSTRACT FROM AUTHOR]

In this study, agronomic and functional characteristics of potato (Solanum tuberosum) plants constitutively overexpressing the protein phosphatase 2A (PP2A) catalytic subunit StPP2Ac2b (StPP2Ac2b-OE) were evaluated. StPP2Ac2b-OE plants display reduced vegetative growth, tuber yield and tuber weight under well-watered and drought conditions. Leaves of StPP2Ac2b-OE plants show an increased rate of water loss, associated with an impaired ability to close stomata in response to abscisic acid. StPP2Ac2b-OE lines exhibit larger stomatal size and reduced stomatal density. These altered stomatal characteristics might be responsible for the impaired stomatal closure and the elevated transpiration rates, ultimately leading to increased sensitivity to water-deficit stress and greater yield loss under drought conditions. Overexpression of StPP2Ac2b accelerates senescence in response to water-deficit stress, which could also contribute to the increased sensitivity to drought. Actively photosynthesising leaves of StPP2Ac2b-OE plants exhibit elevated levels of carbohydrates and a down-regulation of the sucrose transporter StSWEET11 , suggesting a reduced sucrose export from leaves to developing tubers. This effect, combined with the hindered vegetative development, may contribute to the reduced tuber weight and yield in StPP2Ac2b-OE plants. These findings offer novel insights into the physiological functions of PP2A in potato plants and provide valuable information for enhancing potato productivity by modulating the expression of StPP2Ac2b. This study provides novel insights into the physiological functions of protein phosphatase 2A in potato (Solanum tuberosum) plants. The results obtained reveal that the catalytic subunit StPP2Ac2b is involved in stomatal development, senescence induced by water-deficit stress and sucrose export from leaves. Manipulating the expression of StPP2Ac2b has significant effects on these physiological processes, ultimately impacting on important agronomic traits. This work presents valuable information for improving potato productivity by modulating the expression of StPP2Ac2b. [ABSTRACT FROM AUTHOR]

ABSTRACTThe gram-positive bacterium Staphylococcus aureus can invade non-professional phagocytic cells by associating with the plasma protein fibronectin to exploit host cell integrins. Integrin-mediated internalization of these pathogens is facilitated by the local production of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) via an integrin-associated isoform of phosphatidylinositol-5′ kinase. In this study, we addressed the role of PI-4,5-P2-directed phosphatases on internalization of S. aureus. ShRNA-mediated knockdown of individual phosphoinositide 5-phosphatases revealed that synaptojanin1 (SYNJ1) is counteracting invasion of S. aureus into mammalian cells. Indeed, shRNA-mediated depletion as well as genetic deletion of synaptojanin1 via CRISPR/Cas9 resulted in a gain-of-function phenotype with regard to integrin-mediated uptake. Surprisingly, the surface level of integrins was slightly downregulated in Synj1-KO cells. Nevertheless, these cells showed enhanced local accumulation of PI-4,5-P2 and exhibited increased internalization of S. aureus. While the phosphorylation level of the integrin-associated protein tyrosine kinase FAK was unaltered, the integrin-binding and -activating protein talin was enriched in the vicinity of S. aureus in synaptojanin1 knockout cells. Scanning electron microscopy revealed enlarged membrane invaginations in the absence of synaptojanin1 explaining the increased capability of these cells to internalize integrin-bound microorganisms. Importantly, the enhanced uptake by Synj1-KO cells and the exaggerated morphological features were rescued by the re-expression of the wild-type enzyme but not phosphatase inactive mutants. Accordingly, synaptojanin1 activity limits integrin-mediated invasion of S. aureus, corroborating the important role of PI-4,5-P2 during this process.IMPORTANCEStaphylococcus aureus, an important bacterial pathogen, can invade non-professional phagocytes by capturing host fibronectin and engaging integrin α5β1. Understanding how S. aureus exploits this cell adhesion receptor for efficient cell entry can also shed light on the physiological regulation of integrins by endocytosis. Previous studies have found that a specific membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), supports the internalization process. Here, we extend these findings and report that the local levels of PIP2 are controlled by the activity of the PIP2-directed lipid phosphatase Synaptojanin1. By dephosphorylating PIP2 at bacteria-host cell attachment sites, Synaptojanin1 counteracts the integrin-mediated uptake of the microorganisms. Therefore, our study not only generates new insight into subversion of cellular receptors by pathogenic bacteria but also highlights the role of host cell proteins acting as restriction factors for bacterial invasion at the plasma membrane.

Plant development, kinds, and benefits of bio-fertilizer inoculation will be discussed in this paper, Studies have shown that using bio-fertilizers increases the growth of the host plant, as well as improving the root growth and increasing the root system area, which leads to an increase in water and nutrient absorption. Also, the use of biological fertilizers helps reduce the toxicity in plant products as a result of a decrease in chemical fertilizers, In addition to increasing plant resistance to stress conditions like salt, drought, and temperature, bio-fertilizers can also increase soil fertility by contributing to soil aggregation and increasing the activity of soil enzymes like dehydrogenase, phosphatase, and urease, for example. These enzymes help plants be stronger against stress conditions like salt, drought, and temperature

Soil health depletion due to intensive tillage operations is a global issue in the agricultural sector. Conservation tillage (CT) which involves non-inversion tillage and leaving ∼30% of the soil surface covered with crop residues, is a strategy designed to enhance soil health. However, no comprehensive study to investigate the long-term effect of CT on soil biological activity and the soil nutrient supply has yet been widely carried out. Biological and chemical soil properties were assessed at depths 0–5, 10–15, and 20–25 cm depths after 18 years of CT and conventional tillage practice (PT). Various stages in the vegetative growth of maize were investigated in 2021 in Hungary. The findings indicated that tillage intensity, soil depth, and growth stages all significantly influenced soil enzyme activities and the concentration of soil nutrients. Less soil disturbance resulted in a significantly larger concentration of soil carbon parameters (total organic carbon and labile carbon) in CT plots, where the activity of β-glucosidase and dehydrogenase (DHA) in the upper soil layer increased significantly (0.7–2.6 and 2.6–4.7 times, respectively) compared to PT. The high amount of organic matter and the greater resistance to erosion observed in CT also contributed to the higher concentration of available nutrients (NH4, NO3, Ca, K) and total P in the surface soil layer. Phosphatase activity was highest in the mid-stage of vegetative growth and was positively correlated to the total P concentration. The alterations in soil water content were clearly negatively correlated with the change in DHA and phosphatase activity. Overall, due to the more balanced environmental conditions, the decomposition of organic substances was more balanced and slower in CT than in PT. This implied that the mobilization of nutrients in the soil was more balanced as well, and that the nutrients were released gradually. The enhancement of the soil nutrient-supplying capacity achieved by means of long-term conservation tillage provides a promising strategy for sustainable nutrient management.

Mycorrhizal fungi, a category of fungi that form symbiotic relationships with plant roots, can participate in the induction of plant disease resistance by secreting phosphatase enzymes. While extensive research exists on the mechanisms by which mycorrhizal fungi induce resistance, the specific contributions of phosphatases to these processes require further elucidation. This article reviews the spectrum of mycorrhizal fungi-induced resistance mechanisms and synthesizes a current understanding of how phosphatases mediate these effects, such as the induction of defense structures in plants, the negative regulation of plant immune responses, and the limitation of pathogen invasion and spread. It explores the role of phosphatases in the resistance induced by mycorrhizal fungi and provides prospective future research directions in this field.

The use of reclaimed wastewater for irrigation could result in the release of pharmaceutically active compounds (PhACs) and their metabolites into the agroecosystem. In this study, we investigated the fate of carbamazepine (CBZ) and its metabolites, with the aim of clarifying their behavior in a soil–plant system in a greenhouse experiment. The research was carried out using irrigation water especially fortified with high doses of CBZ (200 or 600 ppb) in order to evaluate the dynamics of CBZ and its metabolites in the soil and basil organs. The results of the study showed that CBZ is easily absorbed by the aerial part of the basil plant. The soil contained two metabolites of CBZ, namely acridine and carbamazepine-10,11-epoxide, as revealed by high-resolution mass spectrometry analyses. In addition, acridine was found in the aerial parts of basil plants. Furthermore, the greater presence of CBZ and its metabolites in bulk soil indicated a positive role of the basil rhizosphere in the degradation of such compounds or a positive role of the plant in the removal of the contaminant by uptake. Considering the observed morphological parameters and the mean CBZ content in wastewater, significantly lower than that used in the experiment, basil can be considered resistant to the application of irrigation water contaminated with CBZ.

Enzymes play a crucial role in various industrial production and pharmaceutical developments, serving as catalysts for numerous biochemical reactions. Determining the optimal catalytic temperature (Topt) of enzymes is crucial for optimizing reaction conditions, enhancing catalytic efficiency, and accelerating the industrial processes. However, due to the limited availability of experimentally determined Topt data and the insufficient accuracy of existing computational methods in predicting Topt, there is an urgent need for a computational approach to predict the Topt values of enzymes accurately. In this study, using phosphatase (EC 3.1.3.X) as an example, we constructed a machine learning model utilizing amino acid frequency and protein molecular weight information as features and employing the K-nearest neighbors regression algorithm to predict the Topt of enzymes. Usually, when conducting engineering for enzyme thermostability, researchers tend not to modify conserved amino acids. Therefore, we utilized this machine learning model to predict the Topt of phosphatase sequences after removing conserved amino acids. We found that the predictive model’s mean coefficient of determination (R2) value increased from 0.599 to 0.755 compared to the model based on the complete sequences. Subsequently, experimental validation on 10 phosphatase enzymes with undetermined optimal catalytic temperatures shows that the predicted values of most phosphatase enzymes based on the sequence without conservative amino acids are closer to the experimental optimal catalytic temperature values. This study lays the foundation for the rapid selection of enzymes suitable for industrial conditions.

Purpose: Application of organic manures, along with fertilizers, is important to maintain soil fertility to achieve higher productivity. In reality, the farmers apply organic manures and fertilizers in different proportions and quantities depending on their availability and purchasing power of farmers. Organic manures also help to enhance soil carbon stocks and soil biological activities. Hence, this study aimed to assess the role of organic manures on soil carbon stocks and soil enzymes in intensively managed ginger production systems.Method: Farmers’ fields based study was conducted in Shivamogga district coming under Hilly Zone of Karnataka, India. The information on use of organic manures and fertilizers for ginger production were collected from 60 growers and they were grouped into three categories using the K-factorization statistical tool. Soil samples were collected after crop harvest and analysed for soil carbon stocks and soil enzyme activities. The mean values of 3 categories of farmers were compared using suitable statistical tools.Results: Added organic manure and the soil organic carbon (SOC) varied significantly among high and low organic manure applications. Incremental variations were observed in the order of Category 3 > Category 2 > Category 1 ginger fields. Enzyme activities were found higher in fields applied with high organic manures. Both soil carbon stocks and soil enzyme activities exhibited strong and positive correlations with organic manures.Conclusion: The study revealed that application of organic manures in intensively managed ginger production systems helps to enhance soil carbon stocks and maintain soil biological activities.

Cyanobacteria that grow above seawater salinity at temperatures above 45°C have rarely been studied.The present study is an attempt to decipher these unknown facts where the unique properties of phosphatase enzymes in a thermo-halotolerant Iceland clone 2 Leptolyngbyahas been studied under some important environmental conditions that play a distinctive rolein the growth of these extremophiles in those adverse ecological niches. Leptolyngbyaused in this present study is a unique species having 2 extreme characteristics of tolerance to high salt concentrations and temperature, so it is of intrinsic and scientific interest to study the phosphate dynamics and its variability under different ecological factors.The results of these experiments clearly show that under very adverse conditions of low light or high temperature and very high salt concentrations (almost 3 times of salt present in seawater 90g/L) this extremophile has the capacity to maintain its growth and metabolism which is the key to its survival in these extreme habitats.Observations from growth experiments under different environmental conditions(Temperatures, pH, salt concentrations, different light intensities) under laboratory conditions were found to be like its diverse patterns and adaptive ability in the extreme environment this organism has been isolated from. Phosphatase activity as a wayof understanding how P is metabolized under extreme conditions revealed that the highest phosphatase activity was observed in high salt concentrations (3 times that of seawater) and high temperatures of 45°C and low light intensities that is a very significant observation and scientifically important.

ABSTRACTCell differentiation is an essential biological process that is subject to strict temporal regulation. Caulobacter crescentus undergoes obligate differentiation from a swarmer cell to a stationary, replication-competent stalked cell with each cell cycle. We report that the switch from phosphatase to kinase activity of the histidine kinase PleC contributes to timing this differentiation event. PleC Per-Arnt-Sim (PAS) domain interaction with the polar scaffold protein PodJ localizes PleC to the cell pole and inhibits in vivo kinase activity. Upon PodJ degradation, released PleC switches to its kinase form and phosphorylates the PleD diguanylate cyclase, initiating the signaling pathway responsible for differentiation. While PodJ inhibits PleC kinase activity, it does not impact PleC phosphatase activity on DivK, which is required for pili biogenesis and flagellar rotation. Thus, PleC PAS domains affect enzymatic function on diverse substrates by relying on context-dependent binding partners, thereby controlling the timing of Caulobacter cell differentiation.IMPORTANCEThe process of cell differentiation is highly regulated in both prokaryotic and eukaryotic organisms. The aquatic bacterium, Caulobacter crescentus, undergoes programmed cell differentiation from a motile swarmer cell to a stationary stalked cell with each cell cycle. This critical event is regulated at multiple levels. Kinase activity of the bifunctional enzyme, PleC, is limited to a brief period when it initiates the molecular signaling cascade that results in cell differentiation. Conversely, PleC phosphatase activity is required for pili formation and flagellar rotation. We show that PleC is localized to the flagellar pole by the scaffold protein, PodJ, which is known to suppress PleC kinase activity in vitro. PleC mutants that are unable to bind PodJ have increased kinase activity in vivo, resulting in premature differentiation. We propose a model in which PodJ regulation of PleC’s enzymatic activity contributes to the robust timing of cell differentiation during the Caulobacter cell cycle.

Soil organic phosphorus (P) mineralization is catalyzed by phosphatases synthesized by plants or microorganisms, particularly when organic P is present and inorganic P is limited. We investigated how P status affects the substrate induction response of phosphatase activities and changes in labile inorganic P (ΔNaHCO3-Pi) by adding phosphodiester to long-term low P and high P-fertilized soils. Phosphodiester addition significantly increased acid phosphomonoesterase activity and ΔNaHCO3-Pi, while exhibiting non-significant effect on phosphodiesterase activity. No significant differences were observed in phosphodiester-induced phosphatase activities and ΔNaHCO3-Pi between the low P and high P-fertilized soils. Our results indicate that phosphodiester may incentivize microbially mediated enzymatic P mineralization not necessarily for P but possibly for energy. This emphasizes the importance of phosphodiester in the development of microbial strategies for mobilizing soil P.

ABSTRACTApicomplexan parasites, including Toxoplasma gondii, encode many plant-like proteins, which play significant roles and present attractive targets for drug development. In this study, we have characterized the plant-like protein phosphatase PPKL, which is unique to the parasite and absent in its mammalian host. We have shown that its localization changes as the parasite divides. In non-dividing parasites, it is present in the cytoplasm, nucleus, and preconoidal region. As the parasite begins division, PPKL is enriched in the preconoidal region and the cortical cytoskeleton of nascent parasites. Later in the division, PPKL is present in the basal complex ring. Conditional knockdown of PPKL showed that it is essential for parasite propagation. Moreover, parasites lacking PPKL exhibit uncoupling of division, with normal DNA duplication but severe defects in forming daughter parasites. While PPKL depletion does not impair the duplication of centrosomes, it affects the stability of cortical microtubules. Both co-immunoprecipitation and proximity labeling identified the kinase DYRK1 as a potential functional partner of PPKL. Complete knockout of DYRK1 causes parasites to exhibit division defects with predominantly asynchronous divisions. Global phosphoproteomics analysis revealed a significant increase in phosphorylation of the microtubule-associated protein SPM1 in PPKL-depleted parasites, suggesting that PPKL regulates cortical microtubules by mediating the phosphorylation state of SPM1. More importantly, the phosphorylation of cell cycle-associated kinase Crk1, a known regulator of daughter cell assembly, is altered in PPKL-depleted parasites. Thus, we propose that PPKL regulates daughter parasite development by influencing the Crk1-dependent signaling pathway.IMPORTANCEToxoplasma gondii can cause severe disease in immunocompromised or immunosuppressed patients and during congenital infections. Treating toxoplasmosis presents enormous challenges since the parasite shares many biological processes with its mammalian hosts, which results in significant side effects with current therapies. Consequently, proteins that are essential and unique to the parasite represent favorable targets for drug development. Interestingly, Toxoplasma, like other members of the phylum Apicomplexa, has numerous plant-like proteins, many of which play crucial roles and do not have equivalents in the mammalian host. In this study, we found that the plant-like protein phosphatase PPKL appears to be a key regulator of daughter parasite development. With the depletion of PPKL, the parasite shows severe defects in forming daughter parasites. This study provides novel insights into the understanding of parasite division and offers a new potential target for the development of antiparasitic drugs.

AbstractPhosphorus (P) plays a tremendous and determinant role in plant growth. However, its availability is hampered by the presence of metallic ions in soils. Therefore, the present work has been initiated to analyze the P-solubilization, organic acid, and phosphatase production potency of Bacillus velezensis AR1. Quantification of P-solubilization ranged from 150.6 to 491.3 and from 2.8 to 54.4 mg L−1 in the tricalcium phosphate (Ca-P) and aluminum phosphate (Al-P) medium, respectively. High-performance liquid chromatography analysis of organic acid detection showed variations, whereby 446.88 and 173.73 mg L−1 of succinic and 2-keto gluconic acids in the medium supplemented by Ca-P, and 12.59 and 11.83 mg L−1 of 2-keto gluconic and fumaric acids in the medium supplemented by Al-P were produced, respectively. There were also remarkable extracellular acid and alkaline phosphatase activities. Furthermore, inoculated and uninoculated 5- and 12-day-old culture broths of Ca-P and Al-P were applied to investigate their effect on pea plant growth. The result indicated that the highest plant height and tissue dry weight from the 5-day-old Al-P-modified culture (Al-5), and the highest root length from the 12-day-old Al-P-modified culture (AL-12) were recorded due to the solubilized phosphorus in the culture broth, even compared to the standard fertilizer. Similarly, the highest total nitrogen and total phosphorus were recorded due to Ca-P12 and Ck, respectively. These results concluded that the bacteria isolate B. velezensis AR1 could be developed into a bioinoculant for phosphate solubilization.

Two Zn(II)‐based compounds, [Zn2L1(OAc)3(MeOH)] (1) and [Zn2L2(OAc)3]n (2), have been reported where HL1 is (E)‐4‐bromo‐2‐methoxy‐6‐(((2‐morpholino ethyl)imino) methyl)phenol and HL2 is (E)‐4‐bromo‐2‐methoxy‐6‐(((2‐(piperazine‐1‐yle)ethyl)imino)methyl) phenol. Single‐crystal X‐ray diffraction (SCXRD) analysis unveils vivid change in structural arrangements and dimensionality from 1 to 2 due to change in coordinated atom from oxygen to nitrogen of the ligands. SCXRD study shows that compound 1 is dinuclear but compound 2 has a 1‐dimensional polymeric structure having helical chain. Structural diversity greatly influences the catalytic activity. Compound 1 acts as excellent catalyst for conversion of 3, 5‐di‐tert‐butyl catechol (3, 5‐DTBC) to 3, 5‐di‐tert‐butylbenzoquinone (3, 5‐DTBQ) with the turnover number (kcat) value of 34.94 sec−1. Further, compound 1 reveals phosphatase like activity for conversion of disodium salt of (4‐nitrophenyl)‐phosphate hexahydrate to p‐nitrophenolate with the kcat value of 24.64 sec−1. Interestingly, compound 2 does not show any catalytic activity. To correlate this distinctly different catalytic behavior of two compounds, DFT calculation was carried out. The calculation reveals that detachment of coordinated methanol from coordination sphere of zinc in compound 1 is energetically favourable which creates room for substrate binding, resulting in high catalytic activity. By contrast, in compound 2, detachment of piperazine or Zn−O of –COOH group is energetically unfavourable, resulting in no catalytic activity. [ABSTRACT FROM AUTHOR]

NLRP3 is a prototypical sensor protein connecting cellular stress to proinflammatory signaling. A complex array of regulatory steps is required to switch NLRP3 from an inactive state into a primed entity that is poised to assemble an inflammasome. Accumulating evidence suggests that posttranslational mechanisms are critical. In particular, phosphorylation/dephosphorylation and ubiquitylation/deubiquitylation reactions have been reported to regulate NLRP3. Taken individually, several post-translational modifications appear to be essential. However, it remains difficult to understand how they may be coordinated, whether there is a unique sequence of regulatory steps accounting for the functional maturation of NLRP3, or whether the sequence is subject to variations depending on cell type, the stimulus, and other parameters such as the cellular context. This review will focus on the regulation of the NLRP3 inflammasome by phosphorylation and dephosphorylation, and on kinases and phosphatases that have been reported to modulate NLRP3 activity. The aim is to try to integrate the current understanding and highlight potential gaps for further studies. [ABSTRACT FROM AUTHOR]

Endothelin (ET)-1 is an endothelial-derived peptide that exerts biphasic effects on nitric oxide (NO) levels in endothelial cells such that acute exposure stimulates-while sustained exposure attenuates-NO production. Although the mechanism involved in the decrease in NO generation has been identified but the signaling involved in the acute increase in NO is still unresolved. This was the focus of this study. Our data indicate that exposing pulmonary arterial endothelial cells (PAEC) to ET-1 led to an increase in NO for up to 30min after which levels declined. These effects were attenuated by ET receptor antagonists. The increase in NO correlated with significant increases in pp60Src activity and increases in eNOS phosphorylation at Tyr83 and Ser1177. The ET-1 mediated increase in phosphorylation and NO generation were attenuated by the over-expression of a pp60Src dominant negative mutant. The increase in pp60Src activity correlated with a reduction in the interaction of Caveolin-1 with pp60Src and the calcineurin-mediated dephosphorylation of caveolin-1 at three previously unidentified sites: Thr91, Thr93, and Thr95. The calcineurin inhibitor, Tacrolimus, attenuated the acute increase in pp60Src activity induced by ET-1 and a calcineurin siRNA attenuated the ET-1 mediated increase in eNOS phosphorylation at Tyr83 and Ser1177 as well as the increase in NO. By using a Caveolin-1 celluSpot peptide array, we identified a peptide targeting a sequence located between aa 41–56 as the pp60Src binding region. This peptide fused to the TAT sequence was found to decrease caveolin-pp60Src interaction, increased pp60Src activity, increased eNOS pSer1177 and NO levels in PAEC and induce vasodilation in isolated aortic rings in wildtype but not eNOS knockout mice. Together, our data identify a novel mechanism by which ET-1 acutely increases NO via a calcineurin-mediated dephosphorylation of caveolin-1 and the subsequent stimulation of pp60Src activity, leading to increases in phosphorylation of eNOS at Tyr83 and Ser1177. [Display omitted] • ET-1 acutely increases NO in PAEC via dephosphorylation of caveolin-1. • Dephosphorylation of caveolin-1 activates pp60Src and increases eNOS phosphorylation. • pp60Src activity correlates with a reduction in its interaction with Caveolin-1. • The pp60Src interaction region was localized to aa41-56 in caveolin-1. • A synthetic caveolin-1 peptide (CavSrc) based on the Caveolin-1 interaction region stimulated pp60Src activity and NO levels in PAEC. • CavSrc induced vasodilation in isolated aortic rings from wildtype but not eNOS-/- mice. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 emerged in the human population in late 2019, and human-to-human transmission has dominated the evolutionary landscape and driven the selection of different lineages. The first major change that resulted in increased transmission was the D614G substitution in the spike protein. This was accompanied by the P323L substitution in the viral RNA-dependent RNA polymerase (NSP12). Together with D614G, these changes are the root of the predominant global SARS-CoV-2 landscape. Here, we found that NSP12 formed an interactome with cellular proteins. The functioning of NSP12 was dependent on the T-complex protein ring complex, a molecular chaperone. In contrast, there was a differential association between NSP12 variants and components of a phosphatase complex (PP2/PP2A and STRN3). The virus expressing NSP12L323 was less sensitive to perturbations in PP2A and supports the paradigm that ongoing genotype to phenotype adaptation of SARS-CoV-2 in humans is not exclusively restricted to the spike protein. [ABSTRACT FROM AUTHOR]