The role of PKC in X-ray-induced megakaryocyte apoptosis and thrombocytopenia.

Thrombocytopenia is a critical complication after radiation therapy and exposure. Dysfunction of megakaryocyte development and platelet production are key pathophysiological stages in ionizing radiation (IR)-induced thrombocytopenia. Protein kinase C (PKC) plays an important role in regulating megakaryocyte development and platelet production. However, it remains unclear how PKC regulates IR-induced megakaryocyte apoptosis. In this study, we found that pretreatment of PKC pan-inhibitor Go6983 delayed IR-induced megakaryocyte apoptosis, and inhibited IR-induced mitochondrial membrane potential and ROS production in CMK cells. Moreover, suppressing PKC activation inhibited cleaved caspase3 expression and reduced p38 phosphorylation levels, and IR-induced PKC activation might be regulated by p53. In vivo experiments confirmed that Go6983 promoted platelet count recovery after 21 days of 3 Gy total body irradiation. Furthermore, Go6983 reduced megakaryocyte apoptosis, increased the number of megakaryocyte and polyploid formation in bone marrow, and improved the survival rate of 6 Gy total body irradiation. In conclusion, our results provided a potential therapeutic target for IR-induced thrombocytopenia.

Diagnosis Value of the Blood Urea Nitrogen-to-Creatinine Ratio in Determining the Need for Intervention of Acute Upper Gastrointestinal Bleeding.

INTRODUCTION: The blood urea nitrogen (BUN)-to-creatinine (Cr) ratio (BUN/Cr ratio) may be used to evaluate the need for intervention of acute upper gastrointestinal bleeding (AUGIB). This study aimed to explore the predictive value of the BUN/Cr ratio in the need for intervention of AUGIB. METHODS: This retrospective observational study included patients with AUGIB in the hospital's emergency department between August 2019 and May 2023. The patients were grouped according to whether they underwent an intervention for AUGIB. Patients treated between August 2019 and May 2022 were selected as the training set and the others as the validation set. RESULTS: A total of 466 patients (328 males, 138 females) with AUGIB were enrolled in the intervention group (n = 167) and the no-intervention group (n = 299). In the training set, multivariable logistic regression showed that the BUN/Cr ratio (odds ratio [OR]: 1.013, 95% confidence interval [CI]: 1.003-1.023, p = 0.009), hemoglobin (OR: 0.989, 95% CI: 0.981-0.997, p = 0.010), and a previous history of esophageal variceal bleeding (OR: 6.898, 95% CI: 3.989-11.929, p < 0.001) were independently associated with intervention for AUGIB. The area under receiver operating characteristic curve of BUN/Cr ratio and the prediction model based on logistic regression to predict the need for intervention of AUGIB were 0.604 (95% CI: 0.544-0.664) and 0.759 (95% CI: 0.706-0.812) in the training set and 0.634 (95% CI: 0.529, 0.740) and 0.708 (95% CI: 0.609, 0.806) in the validation set, respectively. CONCLUSION: The BUN/Cr ratio was associated with the need for AUGIB intervention. Combining it with other parameters might improve its diagnostic value to predict the need for intervention of AUGIB.

X-rays Stimulate Granular Secretions and Activate Protein Kinase C Signaling in Human Platelets.

X-rays can induce morphological as well as functional changes in cells. Platelets are anuclear cellular fragments originating from megakaryocytes and are the major regulators in hemostasis and thrombosis. Platelet products are irradiated to avoid medical complications associated with platelet transfusion. So far, gamma, UV, and laser radiation have been used for this purpose. However, scientists are divided about the effects of radiation on platelet quality. The present study was designed to explore the possible effects of X-rays in washed human platelets and understand the molecular mechanism behind them. In the present study, we exposed washed human platelets to 10 or 30 Gy X-rays at 0.25 Gy/min. Flow cytometry, aggregometry, and western blot were performed to investigate the effect of X-rays on platelet degranulation, integrin activation, platelet aggregation, and apoptosis. It was found that X-rays immediately induced granular secretions with no effect on GP IIb/IIIa activation. Not surprisingly, due to granule secretions in irradiated platelets, platelet aggregation was significantly reduced. In contrast to granular secretions and platelet aggregation, X-rays induced mitochondrial transmembrane potential depolarization in a time-dependent manner to induce apoptosis and activated protein kinase C (PKC) signaling. This study revealed and explained the molecular mechanism activated by X-rays in washed human platelets. Here we also introduced Gö 6983, a PKC inhibitor, as an agent that counteracts X-ray-induced changes and maintains the integrity of platelets.

Permeability enhancement of Kv1.2 potassium channel by a terahertz electromagnetic field.

As biomolecules vibrate and rotate in the terahertz band, the biological effects of terahertz electromagnetic fields have drawn considerable attention from the physiological and medical communities. Ion channels are the basis of biological electrical signals, so studying the effect of terahertz electromagnetic fields on ion channels is significant. In this paper, the effect of a terahertz electromagnetic field with three different frequencies, 6, 15, and 25 THz, on the Kv1.2 potassium ion channel was investigated by molecular dynamics simulations. The results show that an electromagnetic field with a 15 THz frequency can significantly enhance the permeability of the Kv1.2 potassium ion channel, which is 1.7 times higher than without an applied electric field. By analyzing the behavior of water molecules, it is found that the electromagnetic field with the 15 THz frequency shortens the duration of frozen and relaxation processes when potassium ions pass through the channel, increases the proportion of the direct knock-on mode, and, thus, enhances the permeability of the Kv1.2 potassium ion channel.

Effect of THz Waves of Different Orientations on K+ Permeation Efficiency in the KcsA Channel.

Potassium (K) channels show the highest variability and most frequent alterations in expression in many tumor types, and modulation of K+ channels may represent a new window for cancer therapy. In previous work, we found that a terahertz (THz) field incident along the z-axis with a frequency of 51.87 THz increased the ion flux through K+ channels. In practice, it is difficult to ensure that the incident electromagnetic (EM) wave is strictly parallel to the direction of channel ion flow. In this paper, we found by changing the direction of the applied electric field that the EM wave of a specific frequency has the largest ion flux when the incident direction is along the ion flow, and the smallest ion flux when the incident direction is perpendicular to the ion flow, and that overall the EM wave of this frequency enhances the ion flow of the K+ channel. Changes in the direction of the applied field at a specific frequency affect the stability of the φ dihedral angle of the GLY77 residue and alter the ion permeation mechanism in the selectivity filter (SF) region, thus affecting the ion flux. Therefore, this frequency can be used to modulate K+ fluxes by THz waves to cause rapid apoptosis in potassium-overloaded tumor cells. This approach consequently represents an important tool for the treatment of cancer and is expected to be applied in practical therapy.

Effect of Terahertz Electromagnetic Field on the Permeability of Potassium Channel Kv1.2.

In this paper, the influence of external terahertz electromagnetic fields with different frequencies of 4 THz, 10 THz, 15 THz, and 20 THz on the permeability of the Kv1.2 voltage-gated potassium ion channel on the nerve cell membrane was studied using the combined model of the "Constant Electric Field-Ion Imbalance" method by molecular dynamics. We found that although the applied terahertz electric field does not produce strong resonance with the -C=O groups of the conservative sequence T-V-G-Y-G amino acid residue of the selective filter (SF) of the channel, it would affect the stability of the electrostatic bond between potassium ions and the carbonyl group of T-V-G-Y-G of SF, and it would affect the stability of the hydrogen bond between water molecules and oxygen atoms of the hydroxyl group of the 374THR side chain at the SF entrance, changing the potential and occupied states of ions in the SF and the occurrence probability of the permeation mode of ions and resulting in the change in the permeability of the channel. Compared with no external electric field, when the external electric field with 15 THz frequency is applied, the lifetime of the hydrogen bond is reduced by 29%, the probability of the "soft knock on" mode is decreased by 46.9%, and the ion flux of the channel is activated by 67.7%. Our research results support the view that compared to "direct knock-on", "soft knock-on" is a slower permeation mode.

Suppression of phosphodiesterase IV enzyme by roflumilast ameliorates cognitive dysfunction in aged rats after sevoflurane anaesthesia via PKA-CREB and MEK/ERK pathways.

Postoperative cognitive dysfunction (POCD) is a prevalent disorder after anaesthesia in the elderly patients. Roflumilast (RF), a phosphodiesterase 4 (PDE-4) inhibitor, could improve cognition with no side effects. Here, we sought to explore the efficacy of RF in the improvement of cognitive dysfunction caused by sevoflurane (Sev). Sprague-Dawley rats were anaesthetized, and the hippocampal neurons were treated with Sev to develop in vivo and in vitro POCD models, followed by RF administration. The mechanism of the PKA-CREB and MEK/ERK pathways in the pathogenesis of POCD was explored. Sev impaired the cognitive functions of rats, significantly reduced cyclic adenosine monophosphate (cAMP) concentrations and blocked the PKA-CREB and MEK/ERK pathways. Moreover, the Sev-treated rats and neurons exhibited enhanced apoptosis and reactive oxygen species (ROS). After treatment with RF, rats had better learning and memory function, and the activity of neurons in hippocampus and cortex was improved. Loss-of-function assay indicated that PKA-CREB and MEK/ERK signalling impairment reduced cAMP levels and promoted apoptosis and ROS in rat hippocampus and neurons. Generally, RF promotes neuronal activity in rats after Sev treatment by maintaining cAMP levels and sustaining the activation of PKA-CREB and MEK/ERK pathways. This might offer novel sights for POCD therapy.

Endoscopic follow-up of mucosal defect after hot versus cold snare polypectomy in animal model.

BACKGROUND AND AIM: Cold snare polypectomy (CSP) has received increasing attention in recent years, but few studies have assessed defect repair after polypectomy. Therefore, we compared the repair of mucosal defect after CSP and hot snare polypectomy (HSP) in a rabbit model. METHODS: Resection of normal colonic mucosa using both HSP and CSP were performed in 40 male New Zealand white rabbits by an experienced endoscopist. Follow-up colonoscopy was performed after 7 and 15 days by another endoscopist. We assessed mucosal defect repair, status of healing, scar formation, and intraoperative or delayed complications (including perforation and bleeding). RESULTS: Eight animals died of intraoperative or delayed perforation; follow-up colonoscopy was performed in 32 animals. On follow-up colonoscopy at 7 days after operation, 78.1% cases in the CSP group showed healing of mucosal defect compared with none in the HSP group (P < 0.001); mucosal repair score in the CSP group was significantly higher than HSP group (P < 0.001). On follow-up colonoscopy at 15 days, mucosal defect after CSP had completely healed in all cases (100%) versus 96.9% after HSP (P = 0.313). Among these healed defects, scar formation was observed in 2 of 32 cases in the CSP group compared with 19 of 31 in the HSP group (P < 0.001). Intraoperative perforation rate was significantly higher in the HSP group (15% vs 2.5%; P = 0.048). CONCLUSIONS: Mucosal defect repair after CSP is quicker compared with HSP and is more likely to result in scarless healing. HSP is more likely to cause perforation in the thin colon walls.

Regulation of Ion Permeation of the KcsA Channel by Applied Midinfrared Field.

Ion transport molecules are involved in many physiological and pathological processes and are considered potential targets for cancer treatment. In the large family of ion transport molecules, potassium (K) ion channels, as surface-expressed proteins, show the highest variability and most frequent expression changes in many tumor types. The key to exploring the permeation of K+ through potassium channels lies in the conserved sequence TVGYG, which is common in the selectivity filter (SF) region of all potassium channels. We found that the K+ flux significantly increased with the help of a specific frequency terahertz electromagnetic wave (51.87 THz) in the KcsA channel using a molecular dynamics combined model through the combined simulation of the constant electric field method and ion imbalance method. This frequency has the strongest absorption peak in the infrared spectrum of -C=O groups in the SF region. With the applied electric field of 51.87 THz, the Y78 residue at the S1 site of the SF has a smaller vibration amplitude and a more stable structure, which enables the K+ to bind closely with the carbonyl oxygen atoms in the SF and realize ion conduction in a more efficient direct Coulomb knock-on.

Exploring boron applications in modern agriculture: A structure-activity relationship study of a novel series of multi-substitution benzoxaboroles for identification of potential fungicides.

Several boron-containing small molecules have been approved by the US FDA to treat human diseases. We explored potential applications of boron-containing compounds in modern agriculture by pursuing multiple research and development programs. Here, we report a novel series of multi-substitution benzoxaboroles (1-36), a compound class that we recently reported as targeting geranylgeranyl transferase I (GGTase I) and thereby inhibiting protein prenylation (Kim et al., 2020). These compounds were designed, synthesized, and tested against the agriculturally important fungal pathogens Mycosphaerella fijiensis and Colletotrichum sublineolum in a structure-activity relationship (SAR) study. Compounds 13, 28, 30, 34 and 36 were identified as active leads with excellent antifungal MIC95 values in the range of 1.56-3.13 ppm against M. fijiensis and 0.78-3.13 ppm against C. sublineolum.

Akt-mediated platelet apoptosis and its therapeutic implications in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count which can cause fatal hemorrhage. ITP patients with antiplatelet glycoprotein (GP) Ib-IX autoantibodies appear refractory to conventional treatments, and the mechanism remains elusive. Here we show that the platelets undergo apoptosis in ITP patients with anti-GPIbα autoantibodies. Consistent with these findings, the anti-GPIbα monoclonal antibodies AN51 and SZ2 induce platelet apoptosis in vitro. We demonstrate that anti-GPIbα antibody binding activates Akt, which elicits platelet apoptosis through activation of phosphodiesterase (PDE3A) and PDE3A-mediated PKA inhibition. Genetic ablation or chemical inhibition of Akt or blocking of Akt signaling abolishes anti-GPIbα antibody-induced platelet apoptosis. We further demonstrate that the antibody-bound platelets are removed in vivo through an apoptosis-dependent manner. Phosphatidylserine (PS) exposure on apoptotic platelets results in phagocytosis of platelets by macrophages in the liver. Notably, inhibition or genetic ablation of Akt or Akt-regulated apoptotic signaling or blockage of PS exposure protects the platelets from clearance. Therefore, our findings reveal pathogenic mechanisms of ITP with anti-GPIbα autoantibodies and, more importantly, suggest therapeutic strategies for thrombocytopenia caused by autoantibodies or other pathogenic factors.

Structural Analysis of Binding Determinants of Salmonella typhimurium Trehalose-6-phosphate Phosphatase Using Ground-State Complexes.

Trehalose-6-phosphate phosphatase (T6PP) catalyzes the dephosphorylation of trehalose 6-phosphate (T6P) to the disaccharide trehalose. The enzyme is not present in mammals but is essential to the viability of multiple lower organisms as trehalose is a critical metabolite, and T6P accumulation is toxic. Hence, T6PP is a target for therapeutics of human pathologies caused by bacteria, fungi, and parasitic nematodes. Here, we report the X-ray crystal structures of Salmonella typhimurium T6PP (StT6PP) in its apo form and in complex with the cofactor Mg2+ and the substrate analogue trehalose 6-sulfate (T6S), the product trehalose, or the competitive inhibitor 4-n-octylphenyl α-d-glucopyranoside 6-sulfate (OGS). OGS replaces the substrate phosphoryl group with a sulfate group and the glucosyl ring distal to the sulfate group with an octylphenyl moiety. The structures of these substrate-analogue and product complexes with T6PP show that specificity is conferred via hydrogen bonds to the glucosyl group proximal to the phosphoryl moiety through Glu123, Lys125, and Glu167, conserved in T6PPs from multiple species. The structure of the first-generation inhibitor OGS shows that it retains the substrate-binding interactions observed for the sulfate group and the proximal glucosyl ring. The OGS octylphenyl moiety binds in a unique manner, indicating that this subsite can tolerate various chemotypes. Together, these findings show that these conserved interactions at the proximal glucosyl ring binding site could provide the basis for the development of broad-spectrum therapeutics, whereas variable interactions at the divergent distal subsite could present an opportunity for the design of potent organism-specific therapeutics.

Cistanches alleviates sevoflurane-induced cognitive dysfunction by regulating PPAR-γ-dependent antioxidant and anti-inflammatory in rats.

This study aimed to investigate the protective effects and underlying mechanisms of cistanche on sevoflurane-induced aged cognitive dysfunction rat model. Aged (24 months) male SD rats were randomly assigned to four groups: control group, sevoflurane group, control + cistanche and sevoflurane + cistanche group. Subsequently, inflammatory cytokine levels were measured by ELISA, and the cognitive dysfunction of rats was evaluated by water maze test, open-field test and the fear conditioning test. Three days following anaesthesia, the rats were killed and hippocampus was harvested for the analysis of relative biomolecules. The oxidative stress level was indicated as nitrite and MDA concentration, along with the SOD and CAT activity. Finally, PPAR-γ antagonist was used to explore the mechanism of cistanche in vivo. The results showed that after inhaling the sevoflurane, 24- but not 3-month-old male SD rats developed obvious cognitive impairments in the behaviour test 3 days after anaesthesia. Intraperitoneal injection of cistanche at the dose of 50 mg/kg for 3 consecutive days before anaesthesia alleviated the sevoflurane-induced elevation of neuroinflammation levels and significantly attenuated the hippocampus-dependent memory impairments in 24-month-old rats. Cistanche also reduced the oxidative stress by decreasing nitrite and MDA while increasing the SOD and CAT activity. Moreover, such treatment also inhibited the activation of microglia. In addition, we demonstrated that PPAR-γ inhibition conversely alleviated cistanche-induced protective effect. Taken together, we demonstrated that cistanche can exert antioxidant, anti-inflammatory, anti-apoptosis and anti-activation of microglia effects on the development of sevoflurane-induced cognitive dysfunction by activating PPAR-γ signalling.

Mapping Post-Translational Modifications of de Novo Purine Biosynthetic Enzymes: Implications for Pathway Regulation.

Purines represent a class of essential metabolites produced by the cell to maintain cellular homeostasis and facilitate cell proliferation. In times of high purine demand, the de novo purine biosynthetic pathway is activated; however, the mechanisms that facilitate this process are largely unknown. One plausible mechanism is through intracellular signaling, which results in enzymes within the pathway becoming post-translationally modified to enhance their individual enzyme activities and the overall pathway metabolic flux. Here, we employ a proteomic strategy to investigate the extent to which de novo purine biosynthetic pathway enzymes are post-translationally modified in 293T cells. We identified 7 post-translational modifications on 135 residues across the 6 human pathway enzymes. We further asked whether there were differences in the post-translational modification state of each pathway enzyme isolated from cells cultured in the presence or absence of purines. Of the 174 assigned modifications, 67% of them were only detected in one experimental growth condition in which a significant number of serine and threonine phosphorylations were noted. A survey of the most-probable kinases responsible for these phosphorylation events uncovered a likely AKT phosphorylation site at residue Thr397 of PPAT, which was only detected in cells under purine-supplemented growth conditions. These data suggest that this modification might alter enzyme activity or modulate its interaction(s) with downstream pathway enzymes. Together, these findings propose a role for post-translational modifications in pathway regulation and activation to meet intracellular purine demand.

The short interference RNA (siRNA) targeting NMUR2 relieves nociception in a bone cancer pain model of rat through PKC-ERK and PI3K-AKT pathways.

BACKGROUND/AIM: Bone cancer pain (BCP) causes troubles and burdens to patients globally. Increasing evidence proved that neuromedin U receptor 2 (NMUR2) was involved in pains. Our study was performed to investigate the role of NMUR2 on BCP and the underlying mechanism. METHODS: The rats were raised and BCP rat model was established by injection with Walker 256 cells. The RNA and protein expression levels of NMUR2 in rat neurons-dorsal spinal cord cells, RNdsc cells were detected by qRT-PCR and western blot. The administration with NMUR2 was via intrathecal injection with siRNA to silence NMUR2. The tolerance of rat to pain was measured by mechanical allodynia test and presented by paw withdrawal threshold (PWT) value. The effects on protein kinase C (PKC)/extracellular regulated protein kinases (ERK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signal pathways were examined by western blot. RESULTS: The expression of NMUR2 in both mRNA and protein levels was upregulated in BCP rat model. In addition, siRNA injection significantly decreased the expression of NMUR2 on the 3rd, 7th and 14th day. BCP group revealed lower PWT value compared with control while NMUR2 silence increased the PWT value compared with negative control. The phosphorylation of PKC, ERK, PI3K and AKT was increased in BCP model while was decreased by si-NMUR2. PKC/ERK and PI3K/AKT inhibitor administration increased the PWT value compared with BCP group. CONCLUSION: si-NMUR2 alleviates BCP via inactivation of PKC/ERK and PI3K/AKT signal pathways.

Phosphodiesterase 4 Inhibitor Roflumilast Protects Rat Hippocampal Neurons from Sevoflurane Induced Injury via Modulation of MEK/ERK Signaling Pathway.

BACKGROUND/AIMS: Sevoflurane, a commonly used volatile anesthetic, recently has been found has neurotoxicity in the central nervous system of neonatal rodents. This study aimed to reveal whether phosphodiesterase 4 (PDE-4) inhibitor roflumilast has protective functions in sevoflurane-induced nerve damage. METHODS: Hippocampal neurons were isolated from juvenile rats, and were exposed to sevoflurane with or without roflumilast treatment. Cell viability and apoptosis were respectively assessed by CCK-8 and flow cytometry. Western blot analysis was performed to detect the protein expressions of apoptosis-related factors, and core factors in MEK/ERK and mTOR signaling pathways. RESULTS: Toxic effects of sevoflurane on hippocampal neurons were observed, as cell viability was reduced, apoptotic cell rate was increased, Bcl-2 was down-regulated, and Bax, cleaved caspase-3 and -9 were up-regulated after 1% sevoflurane exposure for 16 h. Sevoflurane exhibited a temporarily (less than 16 h) inhibitory effect on MEK/ERK pathway, but has no impact on mTOR pathway. Roflumilast promoted the release of cAMP and down-regulated the protein expression of PDE-4. Roflumilast (1 µM) alone has no impact on viability and apoptosis of hippocampal neurons. However, roflumilast increased cell viability and deceased apoptosis in sevoflurane-injured neurons. Besides, roflumilast could recover sevoflurane-induced deactivation of MEK/ERK pathway. CONCLUSION: To conclude, this study demonstrated a neuroprotective role of roflumilast in sevoflurane-induced nerve damage. Roflumilast promoted hippocampal neurons viability, and reduced apoptosis possibly via modulation of MEK/ERK signaling pathway.

Panoramic view of a superfamily of phosphatases through substrate profiling.

Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.

Folate receptor alpha is associated with cervical carcinogenesis and regulates cervical cancer cells growth by activating ERK1/2/c-Fos/c-Jun.

Folate receptor alpha (FRα) is over-expressed in numerous epithelial malignancies, however, the association between FRα and cervical cancer remains unclear. The purpose of this study was to explore the effects of FRα on cervical cancer and its regulation of the ERK signaling pathway. In this case-control study, moderate/strong expression of FRα, phosphorylated ERK1/2 (p-ERK1/2), p-c-Fos, and p-c-Jun proteins was increased with the progressive severity of cervix lesions (P < 0.05). Moreover, the expression levels of p-ERK1/2, p-c-Fos, and p-c-Jun proteins was positively correlated with those of FRα protein in cervical squamous cell carcinoma (SCC) group (P < 0.05). In vitro experiment indicated down-regulation of FRα by siRNA suppressed cell proliferation, promoted cell apoptosis, induced cell cycle arrest at G0/G1 phase, and reduced expression of p-ERK1/2, p-c-Fos, and p-c-Jun proteins. The results suggest that FRα is associated with the progression of cervical cancer and regulates cervical cancer cells growth through phosphorylating ERK1/2, c-Fos, and c-Jun, which are key factors of the ERK signaling pathway. Therefore, FRα may be an effective target for early detection and therapy of cervical cancer.

Secreted IL-1α promotes T-cell activation and expansion of CD11b(+) Gr1(+) cells in carbon tetrachloride-induced liver injury in mice.

Interleukin-1α is mainly expressed on the cell membrane, but can also be secreted during inflammation. The roles of secreted and membrane IL-1α in acute liver inflammation are still not known. Here, we examined the functions of secreted and membrane IL-1α in a mouse model of carbon tetrachloride-induced acute liver injury. We show that secreted IL-1α aggravates liver damage and membrane IL-1α slightly protects mice from liver injury. Further studies showed that secreted IL-1α promotes T-cell activation. It also increased the expansion of CD11b(+) Gr1(+) myeloid cells, which may serve as a negative regulator of acute liver inflammation. Moreover, secreted IL-1α induced IL-6 production from hepatocytes. IL-6 neutralization reduced the proliferation of CD11b(+) Gr1(+) myeloid cells in vivo. CCL2 and CXCL5 expression was increased by secreted IL-1α in vitro and in vivo. Antagonists of the chemokine receptors for CCL2 and CXCL5 significantly reduced the migration of CD11b(+) Gr1(+) myeloid cells. These results demonstrate that secreted and membrane IL-1α play different roles in acute liver injury. Secreted IL-1α could promote T-cell activation and the recruitment and expansion of CD11b(+) Gr1(+) myeloid cells through induction of CCL2, CXCL5, and IL-6. The controlled release of IL-1α could be a critical regulator during acute liver inflammation.

Structure of the trehalose-6-phosphate phosphatase from Brugia malayi reveals key design principles for anthelmintic drugs.

Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.