HMGA1 chromatin regulators induce transcriptional networks involved in GATA2 and proliferation during MPN progression.

Myeloproliferative neoplasms (MPNs) transform to myelofibrosis (MF) and highly lethal acute myeloid leukemia (AML), although the actionable mechanisms driving progression remain elusive. Here, we elucidate the role of the high mobility group A1 (HMGA1) chromatin regulator as a novel driver of MPN progression. HMGA1 is upregulated in MPN, with highest levels after transformation to MF or AML. To define HMGA1 function, we disrupted gene expression via CRISPR/Cas9, short hairpin RNA, or genetic deletion in MPN models. HMGA1 depletion in JAK2V617F AML cell lines disrupts proliferation, clonogenicity, and leukemic engraftment. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2V617F mice, decreasing erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, and expansion of stem and progenitors, while preventing splenomegaly and fibrosis within the spleen and BM. RNA-sequencing and chromatin immunoprecipitation sequencing revealed HMGA1 transcriptional networks and chromatin occupancy at genes that govern proliferation (E2F, G2M, mitotic spindle) and cell fate, including the GATA2 master regulatory gene. Silencing GATA2 recapitulates most phenotypes observed with HMGA1 depletion, whereas GATA2 re-expression partially rescues leukemogenesis. HMGA1 transactivates GATA2 through sequences near the developmental enhancer (+9.5), increasing chromatin accessibility and recruiting active histone marks. Further, HMGA1 transcriptional networks, including proliferation pathways and GATA2, are activated in human MF and MPN leukemic transformation. Importantly, HMGA1 depletion enhances responses to the JAK2 inhibitor, ruxolitinib, preventing MF and prolonging survival in murine models of JAK2V617F AML. These findings illuminate HMGA1 as a key epigenetic switch involved in MPN transformation and a promising therapeutic target to treat or prevent disease progression.

Knockdown of phosphatases of regenerating liver-1 prolongs the lifespan of Caenorhabditis elegans via activating DAF-16/FOXO.

Phosphatases of regenerating liver (PRLs) are dual-specificity protein phosphatases. The aberrant expression of PRLs threatens human health, but their biological functions and pathogenic mechanisms are unclear yet. Herein, the structure and biological functions of PRLs were investigated using the Caenorhabditis elegans (C. elegans). Structurally, this phosphatase in C. elegans, named PRL-1, consisted of a conserved signature sequence WPD loop and a single C(X)5 R domain. Besides, by Western blot, immunohistochemistry and immunofluorescence staining, PRL-1 was proved to mainly express in larval stages and express in intestinal tissues. Afterward, by feeding-based RNA-interference method, knockdown of prl-1 prolonged the lifespan of C. elegans but also improved their healthspan, such as locomotion, pharyngeal pumping frequency, and defecation interval time. Furthermore, the above effects of prl-1 appeared to be taken without acting on germline signaling, diet restriction pathway, insulin/insulin-like growth factor 1 signaling pathway, and SIR-2.1 but through a DAF-16-dependent pathway. Moreover, knockdown of prl-1 induced the nuclear translocation of DAF-16, and upregulated the expression of daf-16, sod-3, mtl-1, and ctl-2. Finally, suppression of prl-1 also reduced the ROS. In conclusion, suppression of prl-1 enhanced the lifespan and survival quality of C. elegans, which provides a theoretical basis for the pathogenesis of PRLs in related human diseases.

Bioinformatics analyses of combined databases identify shared differentially expressed genes in cancer and autoimmune disease.

BACKGROUND: Inadequate immunity caused by poor immune surveillance leads to tumorigenesis, while excessive immunity due to breakdown of immune tolerance causes autoimmune genesis. Although the function of immunity during the onset of these two processes appears to be distinct, the underlying mechanism is shared. To date, gene expression data for large bodies of clinical samples are available, but the resemblances of tumorigenesis and autoimmune genesis in terms of immune responses remains to be summed up. METHODS: Considering the high disease prevalence, we chose invasive ductal carcinoma (IDC) and systemic lupus erythematosus (SLE) to study the potential commonalities of immune responses. We obtained gene expression data of IDC/SLE patients and normal controls from five IDC databases (GSE29044, GSE21422, GSE22840, GSE15852, and GSE9309) and five SLE databases (GSE154851, GSE99967, GSE61635, GSE50635, and GSE17755). We intended to identify genes differentially expressed in both IDC and SLE by using three bioinformatics tools including GEO2R, the limma R package, and Weighted Gene Co-expression Network Analysis (WGCNA) to perform function enrichment, protein-protein network, and signaling pathway analyses. RESULTS: The mRNA levels of signal transducer and activator of transcription 1 (STAT1), 2'-5'-oligoadenylate synthetase 1 (OAS1), 2'-5'-oligoadenylate synthetase like (OASL), and PML nuclear body scaffold (PML) were found to be differentially expressed in both IDC and SLE by using three different bioinformatics tools of GEO2R, the limma R package and WGCNA. From the combined databases in this study, the mRNA levels of STAT1 and OAS1 were increased in IDC while reduced in SLE. And the mRNA levels of OASL and PML were elevated in both IDC and SLE. Based on Kyoto Encyclopedia of Genes and Genomes pathway analysis and QIAGEN Ingenuity Pathway Analysis, both IDC and SLE were correlated with the changes of multiple components involved in the Interferon (IFN)-Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. CONCLUSION: The expression levels of STAT1 and OAS1 manifest the opposite expression tendency across cancer and autoimmune disease. They are components in the IFN-JAK-STAT signaling pathway related to both tumorigenesis and autoimmune genesis. STAT1 and OAS1-associated IFN-JAK-STAT signaling could explain the commonalities during tumorigenesis and autoimmune genesis and render significant information for more precise treatment from the point of immune homeostasis.

Efficacy and safety of Oral LL-37 against the Omicron BA.5.1.3 variant of SARS-COV-2: A randomized trial.

Recombinant LL-37 Lactococcus lactis (Oral LL-37) was designed to prevent progression of COVID-19 by targeting virus envelope, however, effectiveness and safety of Oral LL-37 in clinical application was unclear. A total of 238 adult inpatients, open-labelled, randomized, placebo-controlled, single-center study was conducted to investigate the primary end points, including negative conversion time (NCT) of SARS-CoV-2 RNA and adverse events (AEs). As early as intervened on 6th day of case confirmed, Oral LL-37 could significantly shorten NCT (LL-37 9.80 ± 2.67 vs. placebo 14.04 ± 5.89, p < 0.01). For Oral LL-37, as early as treated in 6 days, the adjusted hazard ratio (HR) for a primary event of nucleic acid negative outcome was 6.27-fold higher than 7-day-later (HR: 6.276, 95% confidence interval [CI]: 3.631-10.848, p < 0.0001), and the adjusted HR of Oral LL-37 within 6 days is higher than placebo (HR: 2.427 95% CI: 1.239-4.751, p = 0.0097). No severe AEs were observed during hospitalization and follow-up investigation. This study shows that early intervention of Oral LL-37 incredibly reduces NCT implying a potential for clearance of Omicron BA.5.1.3 without evident safety concerns.

SHP2 participates in decidualization by activating ERK to maintain normal nuclear localization of progesterone receptor.

In brief: The establishment and maintenance of embryo implantation and pregnancy require decidualization of endometrial stromal cells. This paper reveals that SHP2 ensures the correct subcellular localization of progesterone receptor, thereby safeguarding the process of decidualization. Abstract: Decidualization is the process of conversion of endometrial stromal cells into decidual stromal cells, which is caused by progesterone production that begins during the luteal phase of the menstrual cycle and then increases throughout pregnancy dedicated to support embryonic development. Decidualization deficiency is closely associated with various pregnancy complications, such as recurrent miscarriage (RM). Here, we reported that Src-homology-2-containing phospho-tyrosine phosphatase (SHP2), a key regulator in the signal transduction process downstream of various receptors, plays an indispensable role in decidualization. SHP2 expression was upregulated during decidualization. SHP2 inhibitor RMC-4550 and shRNA-mediated SHP2 reduction resulted in a decreased level of phosphorylation of ERK and aberrant cytoplasmic localization of progesterone receptor (PR), coinciding with reduced expression of IGFBP1 and various other target genes of decidualization. Solely inhibiting ERK activity recapitulated these observations. Administration of RMC-4550 led to decidualization deficiency and embryo absorption in mice. Moreover, reduced expression of SHP2 was detected in the decidua of RM patients. Our results revealed that SHP2 is key to PR's nuclear localization, thereby indispensable for decidualization and that reduced expression of SHP2 might be engaged in the pathogenesis of RM.

Clinical characteristics of patients with early-onset diabetes mellitus: a single-center retrospective study.

BACKGROUND: The prevalence of diabetes mellitus (DM) is dramatically increasing around the world, and patients are getting younger with changes in living standards and lifestyle. This study summarized and analyzed the clinical characteristics of different types of newly diagnosed diabetes mellitus patients with an onset age between 18 and 40 years to provide clinical evidence for the early diagnosis and treatment of diabetes, reduce short-term and long-term complications and offer scientific and personalized management strategies. METHODS: A total of 655 patients newly diagnosed with early-onset diabetes mellitus in the Department of Endocrinology, the First Medical Center of PLA General Hospital from January 2012 to December 2022 were retrospectively enrolled in this study, with an onset age of 18-40 years. Their clinical data were collected and investigated. All patients were divided into two groups according to whether they presented with diabetic microangiopathy. Similarly, patients with early-onset type-2 diabetes were grouped in accordance with whether they had ketosis at the time of diagnosis. Binary logistic regression analysis was performed to analyze risk factors, and receiver-operating characteristic (ROC) analysis was used to explore the predictive value of significant risk factors. RESULTS: The findings were as follows: (1) Of 655 enrolled patients, 477 (72.8%) were male and 178 (27.1%) were female, with a mean age of onset of was 29.73 years ± 0.24 SD. (2) The prevalence of early-onset diabetes was gradually increasing. Type-2 diabetes was the most common type of early-onset diabetes (491, 75.0%). The ages of onset of early-onset type-1 diabetes, type-2 diabetes and LADA were mainly 18-24 years, 25-40 years and 33-40 years, respectively. (3) Initial clinical manifestations of early-onset diabetes were classic diabetes symptoms (361, 55.1%), followed by elevated blood glucose detected through medical examination (207, 31.6%). (4) Binary logistic regression analysis suggested that high serum uric acid (UA), a high urinary albumin-to-creatinine ratio (UACR) and diabetic peripheral neuropathy (DPN) were risk factors for microangiopathy in early-onset diabetes patients (P < 0.05). The area under the curve (AUC) on ROC analysis of the combination of UA, UACR and DPN was 0.848, 95% CI was 0.818 ~ 0.875, sensitivity was 73.8% and specificity was 85.9%, which had higher predictive value than those of UA, UACR and DPN separately. (5) Weight loss, high glycosylated hemoglobin (HbA1c) and young onset age were risk factors for ketosis in patients with early-onset type-2 diabetes (P < 0.05). CONCLUSION: (1) Men were more likely to have early-onset diabetes than women. (2) Early-onset diabetes patients with high serum uric acid levels, high UACRs and peripheral neuropathy were prone to microangiopathy. Comprehensive evaluation of these risk factors could have higher predictive value in the prediction, diagnosis and treatment of microvascular lesions. (3) Patients with weight loss at onset, high HbA1c and young onset age were more likely to develop ketosis. Attention should be given to the metabolic disorders of these patients.

Efficacy of SCF drug conjugate targeting c-KIT in gastrointestinal stromal tumor.

BACKGROUND: Gastrointestinal stromal tumor (GIST) is a rare type of cancer that occurs in the gastrointestinal tract. The majority of GIST cases carry oncogenic forms of KIT, the receptor for stem cell factor (SCF). Small molecule kinase inhibitor imatinib is effective in prolonging the survival of GIST patients by targeting KIT. However, drug resistance often develops during the therapeutic treatment. Here, we produced a SCF-emtansine drug conjugate (SCF-DM1) with favorable drug efficacy towards GIST cells. METHODS: Recombinant human SCF (rhSCF) was expressed in E. coli cells and further purified with Ni-NTA Sepharose and Phenyl Sepharose. It was then conjugated with DM1, and the conjugated product SCF-DM1 was evaluated using in vitro cell-based assays and in vivo xenograft mouse model. RESULTS: SCF-DM1 was effective in inhibiting imatinib-sensitive and -resistant GIST cell lines and primary tumor cells, with IC50 values of < 30 nM. It induced apoptosis and cell cycle arrest in GIST cells. In xenograft mouse model, SCF-DM1 showed favorable efficacy and safety profiles. CONCLUSIONS: rhSCF is a convenient and effective vector for drug delivery to KIT positive GIST cells. SCF-DM1 is an effective drug candidate to treat imatinib-sensitive and -resistant GIST.

Expression of a recombinant FLT3 ligand and its emtansine conjugate as a therapeutic candidate against acute myeloid leukemia cells with FLT3 expression.

BACKGROUND: Most patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods. Gain-of-function FMS-like tyrosine kinase 3 (FLT3) mutations are present in 30-40% of AML patients and serve as an attractive therapeutic target. In addition, FLT3 is aberrantly expressed on blasts in > 90% of patients with AML, making the FLT3 ligand-based drug conjugate a promising therapeutic strategy for the treatment of patients with AML. Here, E. coli was used as a host to express recombinant human FLT3 ligand (rhFL), which was used as a specific vehicle to deliver cytotoxic drugs to FLT3 + AML cells. METHODS: Recombinant hFL was expressed and purified from induced recombinant BL21 (DE3) E. coli. Purified rhFL and emtansine (DM1) were conjugated by an N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) linker. We evaluated the potency of the conjugation product FL-DM1 against FLT3-expressing AML cells by examining viability, apoptosis and the cell cycle. The activation of proteins related to the activation of FLT3 signaling and apoptosis pathways was detected by immunoblotting. The selectivity of FL-DM1 was assessed in our unique HCD-57 cell line, which was transformed with the FLT3 internal tandem duplication mutant (FLT3-ITD). RESULTS: Soluble rhFL was successfully expressed in the periplasm of recombinant E. coli. The purified rhFL was bioactive in stimulating FLT3 signaling in AML cells, and the drug conjugate FL-DM1 showed activity in cell signaling and internalization. FL-DM1 was effective in inhibiting the survival of FLT3-expressing THP-1 and MV-4-11 AML cells, with half maximal inhibitory concentration (IC50) of 12.9 nM and 1.1 nM. Additionally, FL-DM1 induced caspase-3-dependent apoptosis and arrested the cell cycle at the G2/M phase. Moreover, FL-DM1 selectively targeted HCD-57 cells transformed by FLT3-ITD but not parental HCD-57 cells without FLT3 expression. FL-DM1 can also induce obvious apoptosis in primary FLT3-positive AML cells ex vivo. CONCLUSIONS: Our data demonstrated that soluble rhFL can be produced in a bioactive form in the periplasm of recombinant E. coli. FL can be used as a specific vehicle to deliver DM1 into FLT3-expressing AML cells. FL-DM1 exhibited cytotoxicity in FLT3-expressing AML cell lines and primary AML cells. FL-DM1 may have potential clinical applications in treating patients with FLT3-positive AML.

Structure analysis of the receptor binding of 2019-nCoV.

2019-nCoV is a newly identified coronavirus with high similarity to SARS-CoV. We performed a structural analysis of the receptor binding domain (RBD) of spike glycoprotein responsible for entry of coronaviruses into host cells. The RBDs from the two viruses share 72% identity in amino acid sequences, and molecular simulation reveals highly similar ternary structures. However, 2019-nCoV has a distinct loop with flexible glycyl residues replacing rigid prolyl residues in SARS-CoV. Molecular modeling revealed that 2019-nCoV RBD has a stronger interaction with angiotensin converting enzyme 2 (ACE2). A unique phenylalanine F486 in the flexible loop likely plays a major role because its penetration into a deep hydrophobic pocket in ACE2. ACE2 is widely expressed with conserved primary structures throughout the animal kingdom from fish, amphibians, reptiles, birds, to mammals. Structural analysis suggests that ACE2 from these animals can potentially bind RBD of 2019-nCoV, making them all possible natural hosts for the virus. 2019-nCoV is thought to be transmitted through respiratory droplets. However, since ACE2 is predominantly expressed in intestines, testis, and kidney, fecal-oral and other routes of transmission are also possible. Finally, antibodies and small molecular inhibitors that can block the interaction of ACE2 with RBD should be developed to combat the virus.

The Protein Tyrosine Phosphatase MEG2 Regulates the Transport and Signal Transduction of Tropomyosin Receptor Kinase A.

Protein tyrosine phosphatase MEG2 (PTP-MEG2) is a unique nonreceptor tyrosine phosphatase associated with transport vesicles, where it facilitates membrane trafficking by dephosphorylation of the N-ethylmaleimide-sensitive fusion factor. In this study, we identify the neurotrophin receptor TrkA as a novel cargo whose transport to the cell surface requires PTP-MEG2 activity. In addition, TrkA is also a novel substrate of PTP-MEG2, which dephosphorylates both Tyr-490 and Tyr-674/Tyr-675 of TrkA. As a result, overexpression of PTP-MEG2 down-regulates NGF/TrkA signaling and blocks neurite outgrowth and differentiation in PC12 cells and cortical neurons.

Plasma exosome microRNAs are indicative of breast cancer.

BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. METHODS: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. RESULTS: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. CONCLUSIONS: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.

Elevated levels of mast cells are involved in pruritus associated with polycythemia vera in JAK2V617F transgenic mice.

Pruritus occurs frequently in patients with polycythemia vera (PV), and the pathophysiology of PV-associated pruritus is unclear. We have previously demonstrated that transgenic mice expressing JAK2V617F displayed clear PV-like phenotypes. In the current study, we found frequent occurrence of pruritus with aged JAK2V617F transgenic mice and further investigated the underlying mechanisms by studying mast cells, key players in allergic reactions and anaphylaxis. Massive accumulations of mast cells were observed in the skin of pruritic JAK2V617F transgenic mice. In vitro culture yielded much higher mast cell counts from the bone marrow, spleen, peripheral blood, and peritoneal cavity of JAK2V617F transgenic mice than from controls. Cultured mast cells from JAK2V617F transgenic mice exhibited enhanced proliferative signals, relative resistance to cell death upon growth factor deprivation, and a growth advantage over control cells under suboptimal growth conditions. However, these mast cells displayed normal morphology and contained normal levels of mast cell proteases before and after degranulation. Finally, the JAK2 inhibitor G6 effectively reduced mast cell numbers and alleviated pruritus in JAK2V617F transgenic mice. Collectively, these data demonstrate that mast cells are involved in PV-associated pruritogenesis and that JAK2 inhibitors are potential antipruritus drugs.

Transposon mutagenesis reveals cooperation of ETS family transcription factors with signaling pathways in erythro-megakaryocytic leukemia.

To define genetic lesions driving leukemia, we targeted cre-dependent Sleeping Beauty (SB) transposon mutagenesis to the blood-forming system using a hematopoietic-selective vav 1 oncogene (vav1) promoter. Leukemias of diverse lineages ensued, most commonly lymphoid leukemia and erythroleukemia. The inclusion of a transgenic allele of Janus kinase 2 (JAK2)V617F resulted in acceleration of transposon-driven disease and strong selection for erythroleukemic pathology with transformation of bipotential erythro-megakaryocytic cells. The genes encoding the E-twenty-six (ETS) transcription factors Ets related gene (Erg) and Ets1 were the most common sites for transposon insertion in SB-induced JAK2V617F-positive erythroleukemias, present in 87.5% and 65%, respectively, of independent leukemias examined. The role of activated Erg was validated by reproducing erythroleukemic pathology in mice transplanted with fetal liver cells expressing translocated in liposarcoma (TLS)-ERG, an activated form of ERG found in human leukemia. Via application of SB mutagenesis to TLS-ERG-induced erythroid transformation, we identified multiple loci as likely collaborators with activation of Erg. Jak2 was identified as a common transposon insertion site in TLS-ERG-induced disease, strongly validating the cooperation between JAK2V617F and transposon insertion at the Erg locus in the JAK2V617F-positive leukemias. Moreover, loci expressing other regulators of signal transduction pathways were conspicuous among the common transposon insertion sites in TLS-ERG-driven leukemia, suggesting that a key mechanism in erythroleukemia may be the collaboration of lesions disturbing erythroid maturation, most notably in genes of the ETS family, with mutations that reduce dependence on exogenous signals.

Specific dephosphorylation of Janus Kinase 2 by protein tyrosine phosphatases.

Many protein kinases are activated through phosphorylation of an activation loop thereby turning on downstream signaling pathways. Activation of JAK2, a nonreceptor tyrosine kinase with an important role in growth factor and cytokine signaling, requires phosphorylation of the 1007 and 1008 tyrosyl residues. Dephosphorylation of these two sites by phosphatases presumably inactivates the enzyme, but the underlying mechanism is not known. In this study, we employed MALDI-TOF/TOF and triple quadrupole mass spectrometers to analyze qualitatively and quantitatively the dephosphorylation process by using synthetic peptides derived from the tandem autophosphorylation sites (Y1007 and Y1008) of human JAK2. We found that tyrosine phosphatases catalyzed the dephosphorylation reaction sequentially, but different enzymes exhibited different selectivity. Protein tyrosine phosphatase 1B caused rapid dephosphorylation of Y1008 followed by Y1007, while SHP1 and SHP2 selectively dephosphorylated Y1008 only, and yet HePTP randomly removed a single phosphate from either Y1007 or Y1008, leaving behind mono-phosphorylated peptides. The specificity of dephosphorylation was further confirmed by molecular modeling. The data reveal multiple modes of JAK2 regulation by tyrosine phosphatases, reflecting a complex, and intricate interplay between protein phosphorylation and dephosphorylation.

RBPJ mutations identified in two families affected by Adams-Oliver syndrome.

Through exome resequencing, we identified two unique mutations in recombination signal binding protein for immunoglobulin kappa J (RBPJ) in two independent families affected by Adams-Oliver syndrome (AOS), a rare multiple-malformation disorder consisting primarily of aplasia cutis congenita of the vertex scalp and transverse terminal limb defects. These identified mutations link RBPJ, the primary transcriptional regulator for the Notch pathway, with AOS, a human genetic disorder. Functional assays confirmed impaired DNA binding of mutated RBPJ, placing it among other notch-pathway proteins altered in human genetic syndromes.

Molecular cloning and characterization of a tyrosine phosphatase from Monosiga brevicollis.

Protein tyrosine phosphorylation is thought to be a unique feature of multicellular animals. Interestingly, the genome of the unicellular protist Monosiga brevicollis reveals a surprisingly high number and diversity of protein tyrosine kinases, protein tyrosine phosphatases (PTPs), and phosphotyrosine-binding domains. Our study focuses on a hypothetical SH2 domain-containing PTP (SHP), which interestingly has a predicted structure that is distinct from SHPs found in animals. In this study, we isolated cDNA of the enzyme and discovered that its actual sequence was different from the predicted sequence as a result of non-consensus RNA splicing. Contrary to the predicted structure with one SH2 domain and a disrupted phosphatase domain, Monosiga brevicollis SHP (MbSHP) contains two SH2 domains and an intact PTP domain, closely resembling SHP enzymes found in animals. We further expressed the full-length and SH2 domain-truncated forms of the enzyme in Escherichiacoli cells and characterized their enzymatic activities. The double-SH2 domain-truncated form of the enzyme effectively dephosphorylated a common PTP substrate with a specific activity among the highest in characterized PTPs, while the full-length and the N-terminal SH2 domain-truncated forms of the enzyme showed much lower activity with altered pH dependency and responses to ionic strength and common PTP inhibitors. This indicates that SH2 domains suppress the catalytic activity. SHP represents a highly conserved ancient PTP, and studying MbSHP should provide a better understanding about the evolution of tyrosine phosphorylation.

Caenorhabditis elegans eyes absent ortholog EYA-1 is required for stress resistance.

Eyes absent (Eya) is a highly conserved transcription cofactor and protein phosphatase that regulates multiple developmental processes throughout the metazoans. It is a dual function protein, working as a transcription factor in the nucleus and as a tyrosine phosphatase in the cytoplasm. In this study, we isolated EYA-1 of Caenorhabditis elegans, the only homolog of Eyes absent, and set up an effective feeding-based RNAi (RNA interference) against the gene. We found that knockdown of EYA-1 decreased heat and oxidative stress tolerance and accelerated the onset of paralysis mediated by Aß1-42 proteotoxicity and polyQ. Under heat stress (35°C), EYA-1 knockdown shortened the mean lifespan by 16.8%, which could be attributed to decrease in heat shock protein-16.2 (hsp-16.2) expression. Under oxidative stress, EYA-1 knockdown could shorten the mean lifespan by 18.7%, which could be attributed to intracellular ROS accumulation and the decrease of superoxide dismutase-3 (sod-3) protein expression. Moreover, EYA-1 knockdown animals also showed increased lipofuscin accumulation under oxidative stress. Further studies demonstrated that EYA-1 knockdown could not inhibit daf-16 nuclear accumulation in wild-type worms in response to stress. On the other hand, EYA-1 deficiency did not further reduce stress resistance of daf-16 mutants, which are stress sensitive. Quantitative real-time PCR results also showed that the expression of two daf-16 target genes, hsp-12.3 and sod-3, was downregulated in EYA-1 RNAi-treated worms under stress. All this evidence indicates EYA-1 is required for stress resistance of worms, and it might act downstream of daf-16 to regulate expression of stress resistance-associated genes.

A metagenomics study reveals the gut microbiome as a sex-specific modulator of healthy aging in Hainan centenarians.

BACKGROUND: Sex differences in health status and life expectancy are widely accepted to exist. The mechanisms underlying it are still poorly understood. In this study, we aimed to clarify the influences and contributions of sex on the gut microbiome in healthy centenarians and to explore the different roles played by the gut microbiome in healthy aging between the sexes. RESULTS: Taking covariates of different dimensions into account (social demographics, anthropometry, the activities of daily living, dietary structure, mental state, blood tests, lifestyle and disease history), our data showed that sex was one of the most significant covariates affecting the gut microbiome of healthy centenarians at both the species and Kyoto Encyclopedia of Genes and Genomes Orthology (KO) levels. The beta diversity between the sexes were significantly different (Adonis test: p = 0.011, R2 = 0.031), and the male centenarians had a greater alpha diversity than the females (Simpson and Shannon test: P<0.05). At the species level, we identified 31 species enriched in males and 7 species enriched in females. The composition and function patterns of the microbiome varied between the sexes. Further functional analysis showed that males' gut microbiome exhibited greater resistance to oxidative stress compared to females. In contrast to men, the species associated with healthy aging dominated among healthy female centenarians, while the species associated with unhealthy aging were relatively rare. CONCLUSIONS: The present study reveals that the gut microbiome structure and resistance to oxidative stress in healthy centenarians differ between the sexes and provides new insights into the possible sex-specific role of the gut microbiome in healthy aging.

The small molecule inhibitor G6 significantly reduces bone marrow fibrosis and the mutant burden in a mouse model of Jak2-mediated myelofibrosis.

Philadelphia chromosome-negative myeloproliferative neoplasms, including polycythemia vera, essential thrombocytosis, and myelofibrosis, are disorders characterized by abnormal hematopoiesis. Among these myeloproliferative neoplasms, myelofibrosis has the most unfavorable prognosis. Furthermore, currently available therapies for myelofibrosis have little to no efficacy in the bone marrow and hence, are palliative. We recently developed a Janus kinase 2 (Jak2) small molecule inhibitor called G6 and found that it exhibits marked efficacy in a xenograft model of Jak2-V617F-mediated hyperplasia and a transgenic mouse model of Jak2-V617F-mediated polycythemia vera/essential thrombocytosis. However, its efficacy in Jak2-mediated myelofibrosis has not previously been examined. Here, we hypothesized that G6 would be efficacious in Jak2-V617F-mediated myelofibrosis. To test this, mice expressing the human Jak2-V617F cDNA under the control of the vav promoter were administered G6 or vehicle control solution, and efficacy was determined by measuring parameters within the peripheral blood, liver, spleen, and bone marrow. We found that G6 significantly reduced extramedullary hematopoiesis in the liver and splenomegaly. In the bone marrow, G6 significantly reduced pathogenic Jak/STAT signaling by 53%, megakaryocytic hyperplasia by 70%, and the Jak2 mutant burden by 68%. Furthermore, G6 significantly improved the myeloid to erythroid ratio and significantly reversed the myelofibrosis. Collectively, these results indicate that G6 is efficacious in Jak2-V617F-mediated myelofibrosis, and given its bone marrow efficacy, it may alter the natural history of this disease.

RNA-Seq Analysis Reveals Altered Expression of Cell Adhesion-Related Genes Following PZR Knockout in Lung Cancer Cells.

Protein zero related (PZR) serves as a substrate and anchor protein for SHP-2, the product of the proto-oncogene PTPN11 that is frequently mutated in cancers. The expression level of PZR is elevated in various cancers, which is correlated with an unfavorable prognosis. The role of PZR in lung cancer is not fully studied. To investigate how PZR affects signaling pathways involved in LUAD development, we utilized the CRISPR technology to knock out PZR expression in SPC-A1 lung adenocarcinoma cells and then conducted RNA sequencing to profile the transcriptome. Our results showed that 226 genes exhibited differential expressions in PZR-knockout SPC-A1 cells vs wild-type cells. Many of the genes encode proteins involved in cell adhesion, migration, actin cytoskeleton organization, and regulation of cell shape. Furthermore, our experimental data showed that PZR-knockout SPC-A1 cells displayed faster attachment to tissue culture dishes and slower detachment from the dishes upon EDTA treatment. The data suggest an important role of PZR in cell-matrix interaction and may provide new insights into the signaling events that regulate cancer development.