PKM2 Is Overexpressed in Glioma Tissues, and Its Inhibition Highly Increases Late Apoptosis in U87MG Cells With Low-density Specificity.

BACKGROUND/AIM: Pyruvate kinase M2 (PKM2) functions as an important rate-limiting enzyme in aerobic glycolysis and is involved in tumor initiation and progression. However, there are few studies on the correlation between PKM2 expression and its role in glioma. MATERIALS AND METHODS: PKM2 expression was immunohistochemically examined in human brain tumor samples. Furthermore, we studied the effects of two PKM2 inhibitors (shikonin and compound 3K) on the U87MG glioma cell line. RESULTS: PKM2 was overexpressed in most glioma tissues when compared to controls. Interestingly, glioma-adjacent tissues from showed slight PKM2 overexpression. This suggests that PKM2 overexpression maybe an important trigger factor for glioma tumorigenesis. We found that the PKM2 inhibitor shikonin was effective against U87MG cells at a relatively low dose and was largely dependent on low cellular density compared to the effects of the anticancer drug vincristine. Shikonin highly increased late-apoptosis of U87MG cells. We also demonstrated that autophagy was involved in the increase in late-apoptosis levels caused by shikonin. Although vincristine treatment led to a high level of G2-phase arrest in U87MG cells, shikonin did not increase G2 arrest. Co-treatment with two PKM2 inhibitors, shikonin and compound 3K, increased the inhibitory effects. CONCLUSION: Combination therapy with PKM2 inhibitors together might be more effective than combination therapy with anticancer drugs. Our findings encourage the application of PKM2-targeting in gliomas, and lay the foundation for the development of PKM2 inhibitors as promising antitumor agents for glioma.

miR-146a-5p/TXNIP axis attenuates intestinal ischemia-reperfusion injury by inhibiting autophagy via the PRKAA/mTOR signaling pathway.

Autophagy is being increasingly recognized as an important regulator of intestinal ischemia-reperfusion(I/R)injury, but its exact role is still debated. Emerging evidence suggests that miR-146a-5p is involved in the initiation and development of I/R injury, but its role in intestinal I/R injury remains unclear. The present study generated an intestinal I/R mouse model and an oxygen glucose deprivation/reoxygenation (OGD/R) Caco-2 cell model and found that autophagy was increased and contributed to the intestinal injury and cell death induced by I/R and OGD/R. In addition, in both I/R and OGD/R models, the miR-146a-5p expression level was decreased and accompanied by an increase in TXNIP expression. By transfecting cells with an miR-146a-5p inhibitor or mimic, we observed that miR-146a-5p inhibits autophagy during OGD/R by targeting TXNIP; this was confirmed by the dual luciferase reporter gene assay. Additionally, through overexpression and knockdown cell lines, we established that TXNIP regulates autophagy during intestinal I/R via the PRKAA/mTOR pathway. The interaction between TXNIP and p-PRKAA was verified by immunofluorescence co-localization and immunoprecipitation assays. Moreover, we confirmed that TXNIP is indispensable for miR-146a-5p-mediated cell protection. Finally, we observed that miR-146a-5p overexpression inhibits autophagy and attenuates intestinal I/R injury via the PRKAA/mTOR pathway by targeting TXNIP in vivo. In conclusion, this study highlights the role of miR-146a-5p in regulating autophagy by targeting TXNIP, suggesting that miR-146a-5p may be a novel drug target for intestinal I/R therapy.

FGF binding protein 3 is required for spinal cord motor neuron development and regeneration in zebrafish.

Fibroblast growth factor binding protein 3 (Fgfbp3) have been known to be crucial for the process of neural proliferation, differentiation, migration, and adhesion. However, the specific role and the molecular mechanisms of fgfbp3 in regulating the development of motor neurons remain unclear. In this study, we have investigated the function of fgfbp3 in morphogenesis and regeneration of motor neuron in zebrafish. Firstly, we found that fgfbp3 was localized in the motor neurons and loss of fgfbp3 caused the significant decrease of the length and branching number of the motor neuron axons, which could be partially rescued by fgfbp3 mRNA injection. Moreover, the fgfbp3 knockdown (KD) embryos demonstrated similar defects of motor neurons as identified in fgfbp3 knockout (KO) embryos. Furthermore, we revealed that the locomotion and startle response of fgfbp3 KO embryos were significantly restricted, which were partially rescued by the fgfbp3 overexpression. In addition, fgfbp3 KO remarkably compromised axonal regeneration of motor neurons after injury. Lastly, the malformation of motor neurons in fgfbp3 KO embryos was rescued by overexpressing drd1b or neurod6a, respectively, which were screened by transcriptome sequencing. Taken together, our results provide strong cellular and molecular evidence that fgfbp3 is crucial for the axonal morphogenesis and regeneration of motor neurons in zebrafish.

Biophysical analysis of the Mycobacteria tuberculosis peptide binding protein DppA reveals a stringent peptide binding pocket.

The peptide binding protein DppA is an ABC transporter found in prokaryotes that has the potential to be used as drug delivery tool for hybrid antibiotic compounds. Understanding the motifs and structures that bind to DppA is critical to the development of these bivalent compounds. This study focused on the biophysical analysis of the MtDppA from M. tuberculosis. Analysis of the crystal structure revealed a SVA tripeptide was co-crystallized with the protein. Further peptide analysis demonstrated MtDppA shows very little affinity for dipeptides but rather preferentially binds to peptides that are 3-4 amino acids in length. The structure-activity relationships (SAR) between MtDppA and tripeptides with varied amino acid substitutions were evaluated using thermal shift, SPR, and molecular dynamics simulations. Efforts to identify novel ligands for use as alternative scaffolds through the thermal shift screening of 35,000 compounds against MtDppA were unsuccessful, indicating that the MtDppA binding pocket is highly specialized for uptake of peptides. Future development of compounds that seek to utilize MtDppA as a drug delivery mechanism, will likely require a tri- or tetrapeptide component with a hydrophobic -non-acidic peptide sequence.

A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

Placental Expression of Bile Acid Transporters in Intrahepatic Cholestasis of Pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-related condition characterized by increased maternal circulating bile acids (BAs) having adverse fetal effects. We investigated whether the human placenta expresses specific regulation patterns to prevent fetal exposition to harmful amounts of BAs during ICP. Using real-time quantitative PCR, we screened placentae from healthy pregnancies (n = 12) and corresponding trophoblast cells (n = 3) for the expression of 21 solute carriers and ATP-binding cassette transporter proteins, all acknowledged as BA- and/or cholestasis-related genes. The placental gene expression pattern was compared between healthy women and ICP patients (n = 12 each). Placental SLCO3A1 (OATP3A1) gene expression was significantly altered in ICP compared with controls. The other 20 genes, including SLC10A2 (ASBT) and EPHX1 (EPOX, mEH) reported for the first time in trophoblasts, were comparably abundant in healthy and ICP placentae. ABCG5 was undetectable in all placentae. Placental SLC10A2 (ASBT), SLCO4A1 (OATP4A1), and ABCC2 mRNA levels were positively correlated with BA concentrations in ICP. Placental SLC10A2 (ASBT) mRNA was also correlated with maternal body mass index. We conclude that at the transcriptional level only a limited response of BA transport systems is found under ICP conditions. However, the extent of the transcriptional response may also depend on the severity of the ICP condition and the magnitude by which the maternal BA levels are increased.

ChREBP deficiency alleviates apoptosis by inhibiting TXNIP/oxidative stress in diabetic nephropathy.

AIMS: In the present study, we investigated the effect of carbohydrate responsive element binding protein (ChREBP) on the TXNIP/oxidative stress and apoptosis in diabetic nephropathy. METHODS: ChREBP-/- mice (8-week old) were produced using the CRISPR/Cas9 gene editing approach. Diabetes was induced in C57BL/6 mice with streptozotocin. HK-2 cells was transfected with plasmid containing either ChREBP shRNA or TXNIP siRNA. RESULTS: Renal expression of ChREBP and thioredoxin-interacting protein (TXNIP) was increased in patients with type 2 diabetes mellitus (T2DM) and diabetic mice. ChREBP deficiency improved renal function, apoptosis as well as endoplasmic reticulum (ER) stress in diabetic mice. In addition, ChREBP deficiency prevented expression levels of TXNIP and NADPH oxidase 4 (Nox4), 8-hydroxydeoxyguanosine (8-OHdG) and heme oxygenase-1 (HO-1) in diabetic kidneys. The increased urinary 8-OHdG level induced by diabetes was also attenuated in ChREBP deficiency mice. Similarly, HG was shown to induce ChREBP expression and nuclear translocation in HK-2 cells. HG-induced apoptosis was inhibited by transfection of ChREBP shRNA plasmid. Moreover, we found that knockdown of ChREBP suppressed HG-induced TXNIP and Nox4 expression, reactive oxygen species (ROS) generation and ER stress in HK-2 cells. Furthermore, TXNIP knockdown effectively abrogated HG-induced apoptosis in HK-2 cells. CONCLUSIONS: These results suggest that ChREBP deficiency prevents diabetes-induced apoptosis via inhibiting oxidative stress and ER stress, highlighting ChREBP as a potential therapy target for diabetic nephropathy.

GRHL3 activates FSCN1 to relax cell-cell adhesions between migrating keratinocytes during wound reepithelialization.

The migrating keratinocyte wound front is required for skin wound closure. Despite significant advances in wound healing research, we do not fully understand the molecular mechanisms that orchestrate collective keratinocyte migration. Here, we show that, in the wound front, the epidermal transcription factor Grainyhead like-3 (GRHL3) mediates decreased expression of the adherens junction protein E-cadherin; this results in relaxed adhesions between suprabasal keratinocytes, thus promoting collective cell migration and wound closure. Wound fronts from mice lacking GRHL3 in epithelial cells (Grhl3-cKO) have lower expression of Fascin-1 (FSCN1), a known negative regulator of E-cadherin. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on wounded keratinocytes shows decreased wound-induced chromatin accessibility near the Fscn1 gene in Grhl3-cKO mice, a region enriched for GRHL3 motifs. These data reveal a wound-induced GRHL3/FSCN1/E-cadherin pathway that regulates keratinocyte-keratinocyte adhesion during wound-front migration; this pathway is activated in acute human wounds and is altered in diabetic wounds in mice, suggesting translational relevance.

Streptococcus pyogenes TrxSR Two-Component System Regulates Biofilm Production in Acidic Environments.

Bacteria form biofilms for their protection against environmental stress and produce virulence factors within the biofilm. Biofilm formation in acidified environments is regulated by a two-component system, as shown by studies on isogenic mutants of the sensor protein of the two-component regulatory system in Streptococcus pyogenes. In this study, we found that the LiaS histidine kinase sensor mediates biofilm production and pilus expression in an acidified environment through glucose fermentation. The liaS isogenic mutant produced biofilms in a culture acidified by hydrochloric acid but not glucose, suggesting that the acidified environment is sensed by another protein. In addition, the trxS isogenic mutant could not produce biofilms or activate the mga promoter in an acidified environment. Mass spectrometry analysis showed that TrxS regulates M protein, consistent with the transcriptional regulation of emm, which encodes M protein. Our results demonstrate that biofilm production during environmental acidification is directly under the control of TrxS.

Development of a process for large scale production of PfRH5 in E. coli expression system.

The Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) has recently shown great promise to be developed as a vaccine candidate to prevent blood-stage malaria. However, because of its molecular complexity, most previous efforts were focused on expressing PfRH5 in its native and soluble form. Here, we describe the E. coli expression of full-length PfRH5 as inclusion bodies (IBs), followed by its high cell density fermentation at 1, 5 and 30 L scale. Denatured full-length PfRH5 was purified using a two-step chromatography process before being refolded using design of experiments (DoE). Refolded PfRH5 was further purified using size exclusion chromatography (SEC), recovering high purity antigen with an overall yield of 102 mg/L from fermentation cell harvest. Purified PfRH5 was further characterized using orthogonal analytical methods, and a short-term stability study revealed -80 °C as an optimum storage temperature. Moreover, refolded, and purified PfRH5, when formulated with adjuvant Glucopyranosyl A lipid stable emulsion (GLA-SE), elicited high antibody titers in BALB/c mice, proving its potential to neutralize the blood-stage malarial parasite. Here, we establish an E. coli-based process platform for the large-scale cGMP production of full-length PfRH5, enabling global malaria vaccine development efforts.

Estradiol exerts a neuroprotective effect on SH-SY5Y cells through the miR-106b-5p/TXNIP axis.

Alzheimer's disease (AD) is a neurodegenerative disease. Thioredoxin and thioredoxin-interacting protein (TXNIP) complexes help sustain cell oxidation/reduction balance. In the present study, we verified the neuroprotective role of estradiol against amyloid-beta 42 in SH-SY5Y cells through inhibiting TXNIP expression, promoting cell viability and DNA synthesis ability, inhibiting cell apoptosis, and affecting caspase and Bax/Bcl-2 apoptotic signaling. miR-106b-5p could bind to TXNIP 3'-untranslated region to inhibit the expression level of TXNIP. Within SH-SY5Y cells, miR-106b-5p inhibition repressed cell viability and DNA synthesis ability and promoted cell apoptosis through caspase and Bax/Bcl-2 apoptotic signaling, while miR-106b-5p overexpression or TXNIP knockdown exerted the opposite effects on SH-SY5Y cells; TXNIP knockdown remarkably attenuated the roles of miR-106b-5p inhibition. In conclusion, estradiol treatment on SH-SY5Y cells downregulates TXNIP expression and upregulates miR-106b-5p expression. miR-106b-5p exerts a neuroprotective effect on SH-SY5Y cells by promoting cell proliferation and inhibiting cell apoptosis through targeting TXNIP.

Expression, Purification and Characterization of a ZIP Family Transporter from Desulfovibrio vulgaris.

The ZIP family transport zinc ions from the extracellular medium across the plasma membrane or from the intracellular compartments across endomembranes, which play fundamental roles in metal homeostasis and are broadly involved in physiological and pathological processes. Desulfovibrio is the predominant sulphate-reducing bacteria in human colonic microbiota, but also a potential choice for metal bioremediation. while, there are no published studies describing the zinc transporters from Desulfovibrio up to now. In this study, we obtained for the first time a heterologously expressed ZIP homolog from Desulfovibrio vulgaris, termed dvZip. The purified dvZip was reconstituted into proteoliposomes, and confirmed its zinc transport ability in vitro. By combining topology prediction, homology modeling and phylogenetic approaches, we also noticed that dvZip belongs to the GufA and probably have 8 transmembrane α-helical segments (TM 1-8) in which both termini are located on the extracellular, with TM2, 4, 5 and 7 create an inner bundle. We believe that purification and characterization of zinc (probably also cadmium) transporters from Desulfovibrio vulgaris such as dvZip could shed light on understanding of metal homeostasis of Desulfovibrio and provided protein products for future detailed function and structural studies.

Thioredoxin interacting protein regulates age-associated neuroinflammation.

Immune system hypersensitivity is believed to contribute to mental frailty in the elderly. Solid evidence indicates NOD-like receptor pyrin domain containing-3 (NLRP3)-inflammasome activation intimately connects aging-associated chronic inflammation (inflammaging) to senile cognitive decline. Thioredoxin interacting protein (TXNIP), an inducible protein involved in oxidative stress, is essential for NLRP3 inflammasome activity. This study aims to find whether TXNIP/NLRP3 inflammasome pathway is involved in senile dementia. According to our studies on sex-matched mice, TXNIP was significantly upregulated in aged animals, paralleled by the NLRP3-inflammasome over-activity leading to enhanced caspase-1 cleavage and IL-1ß maturation, in both sexes. This was closely associated with depletion of the anti-aging and cognition enhancing protein klotho, in aged males. Txnip knockout reversed age-related NLRP3-hyperactivity and enhanced thioredoxin (TRX) levels. Further, TXNIP inhibition along with verapamil replicated TXNIP/NLRP3-inflammasome downregulation in aged animals, with FOXO-1 and mTOR upregulation. These alterations concurred with substantial improvements in both cognitive and sensorimotor abilities. Together, these findings substantiate the pivotal role of TXNIP to drive inflammaging in parallel with klotho depletion and functional decline, and delineate thioredoxin system as a potential target to decelerate senile dementia.

Effect of type 2 diabetes mellitus on placental expression and activity of nutrient transporters and their association with birth weight and neonatal adiposity.

AIMS: Infants born to women with Type 2 Diabetes Mellitus (T2DM) are at risk of being born large for gestational age due to excess fetal fat accretion. Placental nutrient transport determines fetal nutrient availability, impacting fetal growth. The aims of the study were to evaluate the effect of T2DM on placental insulin signaling, placental nutrient transporters and neonatal adiposity. METHODS: Placentas were collected from BMI-matched normoglycemic controls (NGT, n = 9) and T2DM (n = 9) women. Syncytiotrophoblast microvillous (MVM) and basal (BM) plasma membranes were isolated. Expression of glucose (GLUT1, -4), fatty acid (FATP2, -4, -6, FAT/CD36), amino acid (SNAT1, -2, -4, LAT1, -2) transporters, insulin signaling, and System A transporter activity was determined. Neonatal fat mass (%) was measured in a subset of neonates born to T2DM women. RESULTS: GLUT1 protein expression was increased (p = 0.001) and GLUT4 decreased (p = 0.006) in BM from T2DM. MVM FATP6 expression was increased (p = 0.02) and correlated with birth weight in both T2DM and NGT groups (r = 0.65, p = 0.02). BM FATP6 expression was increased (p = 0.01) in T2DM. In MVM of T2DM placentas, SNAT1 expression was increased (p = 0.05) and correlated with birth weight (r = 0.84, p = 0.004); SNAT2 was increased (p = 0.01), however System A transporter activity was not different between groups. MVM LAT1 expression was increased (p = 0.01) in T2DM and correlated with birth weight (r = 0.59, p = 0.04) and neonatal fat mass (r = 0.76, p = 0.06). CONCLUSION: In pregnancies complicated by T2DM placental protein expression of transporters for glucose, amino acids and fatty acids is increased, which may contribute to increased fetal growth and neonatal adiposity.

Expression and Mutation Alterations of ZMYM4 Gene in Gastric and Colonic Cancers.

ZMYM4 is a zinc finger protein, whose cancer-related functions are partially known (cell shape maintenance and cell death). In this study, we analyzed 4 sites of mononucleotide repeats in the coding sequence of ZMYM4 in gastric (GC) and colonic cancers (CC). Seven of the 32 high microsatellite instability (MSI-H) GCs (21.9%) and 23 of 113 MSI-H CCs (20.4%) harbored ZMYM4 frameshift mutations with no significant difference between the 2 organs (P>0.05). There was no ZMYM4 frameshift mutations in microsatellite-stable GCs and CCs. We also identified that 6 of 16 MSI-H CCs (37.5%) exhibited intratumoral heterogeneity of the ZMYM4 frameshift mutations. In both GC and CC with MSI-H, ZMYM4 expression in ZMYM4-mutated cases was significantly lower than that in ZMYM4-nonmutated cases. Our study indicates that ZMYM4 is altered at multiple levels (frameshift mutation, mutational intratumoral heterogeneity, and loss of expression), suggesting their relations with MSI-H GC and CC.

Circular RNA circZNF292 regulates H2 O2 -induced injury in human lens epithelial HLE-B3 cells depending on the regulation of the miR-222-3p/E2F3 axis.

Circular RNAs (circRNAs) play important roles in the pathogenesis of age-related cataract (ARC). CircRNA zinc finger protein 292 (circZNF292, hsa_circ_0004058) is downregulated in ARC lens capsules. Here, we focused on its precise roles in oxidative stress underlying the pathogenesis of ARC. CircZNF292, microRNA (miR)-222-3p, and E2F transcription factor 3 (E2F3) were quantified by quantitative real-time polymerase chain reaction or western blot. Cell viability was assessed by the cell counting kit-8 assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The activities of superoxide dismutase, catalase, and malondialdehyde were measured using the corresponding assay kit. Targeted correlations among circZNF292, miR-222-3p, and E2F3 were verified by the dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. Our data showed that circZNF292 was downregulated in ARC tissues and H2 O2 -treated human lens epithelial B3 (HLE-B3) cells. Increased expression of circZNF292 alleviated H2 O2 -induced cell viability suppression, apoptosis promotion, and oxidative stress enhancement. Mechanistically, circZNF292 directly targeted miR-222-3p, and circZNF292 regulated E2F3 expression through miR-222-3p. MiR-222-3p was a functional mediator of circZNF292 in modulating H2 O2 -induced injury in HLE-B3 cells. Furthermore, reduced level of miR-222-3p ameliorated H2 O2 -induced HLE-B3 cell damage by upregulating E2F3. Our present study demonstrated that increased expression of circZNF292 ameliorated H2 O2 -induced injury in HLE-B3 cells at least in part through the miR-222-3p/E2F3 axis, highlighting a novel insight into the involvement of circRNAs in the pathogenesis of ARC.

Marginally reduced maternal hepatic and splenic ferroportin under severe nutritional iron deficiency in pregnancy maintains systemic iron supply.

The demand for iron is high in pregnancy to meet the increased requirements for erythropoiesis. Even pregnant females with initially iron-replete stores develop iron-deficiency anemia, due to inadequate iron absorption. In anemic females, the maternal iron supply is dedicated to maintaining iron metabolism in the fetus and placenta. Here, using a mouse model of iron deficiency in pregnancy, we show that iron recycled from senescent erythrocytes becomes a predominant source of this microelement that can be transferred to the placenta in females with depleted iron stores. Ferroportin is a key protein in the molecular machinery of cellular iron egress. We demonstrate that under iron deficiency in pregnancy, levels of ferroportin are greatly reduced in the duodenum, placenta and fetal liver, but not in maternal liver macrophages and in the spleen. Although low expression of both maternal and fetal hepcidin predicted ferroportin up-regulation in examined locations, its final expression level was very likely correlated with tissue iron status. Our results argue that iron released into the circulation of anemic females is taken up by the placenta, as evidenced by high expression of iron importers on syncytiotrophoblasts. Then, a substantial decrease in levels of ferroportin on the basolateral side of syncytiotrophoblasts, may be responsible for the reduced transfer of iron to the fetus. As attested by the lowest decrease in iron content among analyzed tissues, some part is retained in the placenta. These findings confirm the key role played by ferroportin in tuning iron turnover in iron-deficient pregnant mouse females and their fetuses.

Downregulation of Zn-transporters along with Fe and redox imbalance causes growth and photosynthetic disturbance in Zn-deficient tomato.

Zinc (Zn) deficiency hinders growth and development in tomato. This study unveils the responses of how Zn starvation affects physiological and molecular processes in tomato. Zn deficiency negatively affected the biomass, cellular integrity, and chlorophyll synthesis in tomato. Also, Zn deficiency decreased the maximum yield of PSII, photosynthesis performance index and dissipation energy per active reaction center, although the antenna size, trapping energy efficiency and electron transport flux were stable in Zn-starved leaves. Further, Zn shortage caused a substantial reduction in Zn and Fe concentrations in both roots and shoots along with decreased root Fe-reductase activity accompanied by the downregulation of Fe-regulated transporter 1, Zn transporter-like (LOC100037509), and Zn transporter (LOC101255999) genes predicted to be localized in the root plasma membrane. The interactome partners of these Zn transporters are predominantly associated with root-specific metal transporter, ferric-chelate reductase, BHLH transcriptional regulator, and Zn metal ion transporters, suggesting that Zn homeostasis may be tightly linked to the Fe status along with BHLH transcription factor in Zn-deficient tomato. We also noticed elevated O2.- and H2O2 due to Zn deficiency which was consistent with the inefficient antioxidant properties. These findings will be useful in the downstream approach to improve vegetable crops sensitive to Zn-deficiency.

The NKG2D ligand ULBP4 is not expressed by human monocytes.

The C-type lectin-like receptor NKG2D contributes to the immunosurveillance of virally infected and malignant cells by cytotoxic lymphocytes. A peculiar and puzzling feature of the NKG2D-based immunorecognition system is the high number of ligands for this single immunoreceptor. In humans, there are a total of eight NKG2D ligands (NKG2DL) comprising two members of the MIC (MICA, MICB) and six members of the ULBP family of glycoproteins (ULBP1 to ULBP6). While MICA has been extensively studied with regard to its biochemistry, cellular expression and function, very little is known about the NKG2DL ULBP4. This is, at least in part, due to its rather restricted expression by very few cell lines and tissues. Recently, constitutive ULBP4 expression by human monocytes was reported, questioning the view of tissue-restricted ULBP4 expression. Here, we scrutinized ULBP4 expression by human peripheral blood mononuclear cells and monocytes by analyzing ULBP4 transcripts and ULBP4 surface expression. In contrast to MICA, there was no ULBP4 expression detectable, neither by freshly isolated monocytes nor by PAMP-activated monocytes. However, a commercial antibody erroneously indicated surface ULBP4 on monocytes due to a non-ULBP4-specific binding activity, emphasizing the critical importance of validated reagents for life sciences. Collectively, our data show that ULBP4 is not expressed by monocytes, and likely also not by other peripheral blood immune cells, and therefore exhibits an expression pattern rather distinct from other human NKG2DL.

SH3BGRL2 functions as a crucial tumor suppressor in glioblastoma tumorigenesis.

Glioblastoma is the most common and severe primary intrinsic tumor of the central nervous system. Glioblastoma harbors glioma stem cells (GSCs) as it not only possesses self-renewal and differentiation properties but also accounts for significant chemotherapy resistance and recurrence. Thus, targeting GSCs may be essential in overcoming the resistance and recurrence thereby improving GBM treatment. However, the underlying mechanism to sustain GSCs remains largely unknown. Here, we report that SH3 domain binding glutamate-rich protein like 2 (SH3BGRL2) is weakly expressed in glioblastoma multiforme (GBM) and isocitrate dehydrogenase1 (IDH1) wildtype GBM and correlated with glioma patients' poor prognosis. Moreover, ectopic expression of SH3BGRL2 significantly inhibited GBM cell growth, migration, and GSCs self-renewal in vitro as well as tumor growth in vivo. Additionally, we found that SH3BGRL2 suppressed SOX2 and CD133 expression, which are key regulators involved in GSCs self-renewal. Collectively, our findings shed additional light on SH3BGRL2 has potential to serve as a biomarker and a potent therapeutic target for patients with glioma.