PG Synthesis Research Articles

A simplified method for preventing postmortem alterations of brain prostanoids for true in situ level quantification

Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.

Reduced peptidoglycan synthesis capacity impairs growth of E. coli at high salt concentration.

Gram-negative bacteria have a thin peptidoglycan layer between the cytoplasmic and outer membranes protecting the cell from osmotic challenges. Hydrolases of this structure are needed to cleave bonds to allow the newly synthesized peptidoglycan strands to be inserted by synthases. These enzymes need to be tightly regulated and their activities coordinated to prevent cell lysis. To better understand this process in Escherichia coli, we probed the genetic interactions of mrcA (encodes PBP1A) and mrcB (encodes PBP1B) with genes encoding peptidoglycan amidases and endopeptidases in envelope stress conditions. Our extensive genetic interaction network analysis revealed relatively few combinations of hydrolase gene deletions with reduced fitness in the absence of PBP1A or PBP1B, showing that none of the amidases or endopeptidases is strictly required for the functioning of one of the class A PBPs. This illustrates the robustness of the peptidoglycan growth mechanism. However, we discovered that the fitness of ∆mrcB cells is significantly reduced under high salt stress and in vitro activity assays suggest that this phenotype is caused by a reduced peptidoglycan synthesis activity of PBP1A at high salt concentration.IMPORTANCEEscherichia coli and many other bacteria have a surprisingly high number of peptidoglycan hydrolases. These enzymes function in concert with synthases to facilitate the expansion of the peptidoglycan sacculus under a range of growth and stress conditions. The synthases PBP1A and PBP1B both contribute to peptidoglycan expansion during cell division and growth. Our genetic interaction analysis revealed that these two penicillin-binding proteins (PBPs) do not need specific amidases, endopeptidases, or lytic transglycosylases for function. We show that PBP1A and PBP1B do not work equally well when cells encounter high salt stress and demonstrate that PBP1A alone cannot provide sufficient PG synthesis activity under this condition. These results show how the two class A PBPs and peptidoglycan hydrolases govern cell envelope integrity in E. coli in response to environmental challenges and particularly highlight the importance of PBP1B in maintaining cell fitness under high salt conditions.

Mutation of rsmC gene from Serratia marcescens QBN using CRISPR-Cas9 to enhance prodigiosin synthesis

Prodigiosin is a bright red pigment, which is produced by various gram-negative and gram positive bacteria include S. marcescens, Hahella chejuensis, Vibrio psychroerythrus, Streptomyces coelicolor and many marine bacteria such as Pseudomonas sp, Vibrio sp. Prodigiosin has garnered considerable interest among the scientific community due to its immunosuppressive and anticancer properties against various drug-resistant cancer cell lines. In this study, we utilized the biosynthetic pathway of prodigiosin (Pg), using CRISPR/Cas9 gene editing techniques to eliminate the gene inhibiting Pg synthesis, known as rsmC, aiming to enhance the ability to synthesize Pg from S. marcescens strains. The rsmC gene of S. marcescens QBN was randomly mutated using the CRISPR-Cas9 system with the pCasPA vector (containing Cas9) and pCRISPR (carrying the spacer segment and sgRNA synthesis). A 20-nucleotide spacer segment was selected on rsmCpreceding the TGG triplet at the 3' end (PAM, the recognition site of Cas9) and inserted into pCRISPR between two BsaI sites. Following the binding of sgRNA to Cas9, mutations occurred in the nucleotides of the rsmC gene, resulting in the cleavage of the double-stranded DNA at the targeted site. A sequence homologous to the rsmC gene, lacking 100 nucleotides, was introduced into the cell and will involve the cleaved DNA's repair mechanism. The mutated rsmC strain of S. marcescens QBN synthesized Pg at 216 - 231 mg/L, higher than the original strain (180 mg/L). Furthermore, HPLC results and 1H spectral analysis showed that the synthesized red pigment from the mutated strain S. marcescens QBN was prodigiosin. These findings provide a basis for further improving the productivity of Pg synthesis in the S. marcescens QBN strain and serve as a basis for editing genes in other microorganisms using the CRISPR-Cas9 system.

Paternal genetic effects of cadmium exposure during pregnancy on hormone synthesis disorders in ovarian granulosa cells of offspring

The aim of this study was to investigate the paternal genetic intergenerational and transgenerational genetic effects of cadmium (Cd) exposure during pregnancy on estradiol (E2) and progesterone (Pg) synthesis in the ovarian granulosa cells (GCs) of offspring. Pregnant SD rats were intragastrically exposed to CdCl2 (0, 0.5, 2.0, 8.0 mg/kg) from days 1 to 20 to produce the F1 generation, F1 males were mated with newly purchased females to produce the F2 generation, and the F3 generation was obtained in the same way. Using this model, Cd-induced hormone synthesis disorders in GCs of F1 have been observed [8]. In this study, altered serum E2 and Pg levels in both F2 and F3 generations showed a nonmonotonic dose‒response relationship. In addition, hormone synthesis-related genes (Star, Cyp11a1, Cyp17a1, Cyp19a1, Sf-1) and miRNAs were observed to be altered in both F2 and F3. No differential changes in DNA methylation modifications of hormone synthesis-related genes were observed, and only the Adcy7 was hypomethylated. In summary, paternal genetic intergenerational and transgenerational effects exist in ovarian GCs E2 and Pg synthesis disorders induced by Cd during pregnancy. In F2, the upregulation of StAR and CYP11A1, and changes in the miR-27a-3p, miR-27b-3p, and miR-146 families may be important, while changes in the miR-10b-5p and miR-146 families in F3 may be important.

1432. Exploring Cell Wall Targets to Overcome Mycobacterium tuberculosis (Mtb): Ceftriaxone (CRO) Inhibits LdtMt2, a Major Peptidoglycan (PG) Synthase

Abstract Background Drug-resistant tuberculosis (DR TB) is a deadly, difficult-to-treat infection, and new treatment strategies are needed. Despite the wide success of β-lactams (BLs), DR TB guidelines only include meropenem (MEM) and imipenem (IPM), given with clavulanate (CLA). BlaC, the Mtb β-lactamase, hydrolyzes CRO less efficiently than other cephems and β-lactamase inhibitors improve the in vitro susceptibility of Mtb to CRO. Surprisingly, CRO has not been evaluated in DR TB clinical studies. Moreover, the mechanisms by which CRO disrupts Mtb PG synthesis are not well characterized. CRO inhibits LdtMt1­, but activity against LdtMt2, an important Mtb PG synthase, is unknown. To explore this knowledge gap, we examined CRO inhibition of LdtMt2. In addition, we investigated if combining CRO with MEM or IPM would lower minimum inhibitory concentrations (MICs) more than each agent alone. Methods A panel of Mtb isolates was selected for susceptibility testing with a broth microdilution method. Timed electrospray ionization-mass spectrometry (ESI-MS) and inhibition kinetic assays were performed. Results CRO MICs ranged 0.25 to 16 µg/mL and lowered ≤ 0.06 to 2 µg/mL with CLA (Table 1). Fractional inhibitory concentration indices for CRO + MEM or IPM was < 0.5 for six isolates, suggesting synergy. ESI-MS captured CRO-LdtMt2­ acyl-complexes at timepoints 5 to 120 min, and a 158 Da fragment loss was observed; MEM and IPM were unchanged (Table 2, Figure 1). When LdtMt2 was co-incubated with MEM and CRO together, only MEM complexes were captured. Interestingly, Kiapp­ with CRO (0.07 ± 0.01 µM) was comparable to that with MEM (0.09 ± 0.01 µM). Figure 1Mass spectrometry chromatograms with LdtMt2 (A) alone incubated with ceftriaxone at (B) 5 min, (C) 15 min, (D) 30 min, (E) 120 min. [Ldt¬Mt2] = 13.2 µM and [ceftriaxone] = 264 µM. Changes in MW are also listed in Table 1. (F) shows the structure of ceftriaxone with the proposed leaving group in red, leaving the remaining bound structure accounting for the observed change in MW. Conclusion CRO was effective in lowering MICs with MEM, IPM, and CLA for our Mtb isolates. Based upon ESI-MS, we found that CRO forms a stable complex with LdtMt2 and the R2 side chain is eliminated, while kinetic observations support inhibition of LdtMt2 by CRO (Figure 2). Previous work found MEM and IPM also inhibit multiple other PG synthases (e.g., PonA1, LdtMt1, LdtMt3). We hypothesize that CRO + MEM/IPM inhibits the growth of Mtb by the combined inactivation of multiple cell wall enzymes. Our observations support the further exploration of the notion of “target redundancy” as an approach to treat multidrug-resistant mycobacteria with BLs. Figure 2.Ceftriaxone-LdtMt2 adduct formation leading to release of the R2 side group Disclosures Krisztina Papp-Wallace, Ph.D, Merck: Grant/Research Support|Venatorx: Grant/Research Support|Wockhardt: Advisor/Consultant Robert A. Bonomo, MD, NIH VA: Grant/Research Support|VenatoRx Merck Wockhardt Cystic Fibrosis Foundation: Grant/Research Support.

The anti-inflammatory cytokine IL-22 exhibited a proinflammatory effect in the ovary aiding with the parturition of ovoviviparous black rockfish (Sebastes schlegelii)

IntroductionAs a unique type of immunological process, pregnancy and subsequent parturition are associated with a series of inflammatory events. As an anti-inflammatory cytokine, IL-22 has been proven to participate in the parturition process in mammals. However, less is known about ovoviviparous teleosts. To investigate the role of IL-22 in fish parturition, black rockfish, an ovoviviparous teleost, was used. This fish is unique in that it provides over 60% nutrition supply for over 50 thousand embryos at the same time though a placenta-like structure during pregnancy while delivering all fries in two hours.MethodsSequence alignment, phylogenetic tree analysis and homology modeling were performed on IL-22 and its receptor. In situ hybridization demonstrated ovarian localization of il22 and il22r1. The protein of rIL-22 was obtained through the prokaryotic expression. RNA-seq analysis was performed on black rockfish ovarian cells treated with rIL-22.ResultsIn situ hybridization results showed that both il22 and il22ra1 were localized in the perinatal follicle layer and embryonic envelope. The il22 expression level was significantly increased during parturition compared to before and after parturition (P<0.05). To further understand the mechanism of IL-22 in parturition, rIL-22 was obtained. The downstream variation genes in primary cultured ovarian cells in the perinatal period were analyzed according to the transcriptomic results. A total of 168 differentially expressed genes (DEGs) were identified in the rIL-22 group compared with the PBS control group (padj < 0.05 and |log2FoldChange| 1). Of those, 134 DEGs were identified in the LPS and rIL-22 treatment groups, including chemokine, cytokine, and PG synthesis. 34 DEGs were mainly identified in immune-related pathways and reorganization of the cytoskeleton responded only to rIL-22 stimulation but not LPS. KEGG pathways mainly included immune response, PG synthesis, cell death and angiogenesis (p value < 0.05).DiscussionTaken together, our results indicated that IL-22 plays an important role in the parturition of black rockfish by upregulating PG synthesis and increasing chemokine, proinflammatory factor, as well as PG synthetase levels.

Revealing novel synergistic defense and acid tolerant performance of Escherichia coli in response to organic acid stimulation.

Escherichia coli is an important producer of mono- and di-acids, such as D-lactic acid, itaconic acid, and succinic acid. However, E. coli has limited acid tolerance and requires neutralizers in large-scale fermentation, which leads to increased production costs. Mutagenesis breeding has been shown to be inefficient in improving the acid tolerance of strains. Therefore, it is crucial to analyze the acid resistance mechanism of E. coli. To this end, important regulatory genes and metabolic pathways in the highly evolved acid-resistant E. coli were identified based on transcriptome sequencing. By analyzing the overlap of the genes with significantly different expression levels in the four groups, a synergistic membrane-centric defense mechanism for E. coli against organic acid stress was identified. The mechanism includes four modules: signal perception, energy countermeasures, input conditioning, and envelope reinforcement. In addition, genes related to the ABC transporter pathway, polyketide metabolism, pyrimidine metabolism, and dual-arginine translocation system pathways were found for the first time to be potentially resistant to organic acid stress after overexpression. A new antacid ingredient, RffG, increases the survival rate of E. coli by 4509.6 times. This study provides new clues for improving the performance of acid-tolerant cells and reducing the production cost of industrial organic acid fermentation. KEY POINTS: • Systematic analysis of the mechanism of membrane protein partitioning in E. coli to resist organic acids • TAT system transports correctly folded hydrogenase accessory proteins to resistD-lactic acid stress • Enhanced PG synthesis and weakened hydrolysis to reduce acid penetration into cells • Overexpression of RffG in the polyketide synthesis pathway enhances acid tolerance.

Metabolic regulation by prostaglandin E2 impairs lung group 2 innate lymphoid cell responses.

Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin E2 (PGE2 ). However, the respective roles for the PGE2 receptors EP2 and EP4 (both share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered. The roles of PGE2 receptors EP2 and EP4 on ILC2-mediated lung inflammation were investigated using genetically modified mouse lines and pharmacological approaches in IL-33-induced lung allergy model. The effects of PGE2 receptors and downstream signals on ILC2 metabolic activation and effector function were examined using in vitro cell cultures. Deficiency of EP2 rather than EP4 augments IL-33-induced mouse lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 and EP2 or inhibition of phosphodiesterase markedly restricts IL-33-induced lung ILC2 responses. Mechanistically, PGE2 directly suppresses IL-33-dependent ILC2 activation through the EP2/EP4-cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL-33-dependent ILC2 responses in mice where endogenous PG synthesis or EP2 signaling is blocked but not in mice with intact PGE2 -EP2 signaling. We have defined a mechanism for optimal suppression of mouse lung ILC2 responses by endogenous PGE2 -EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE2 -EP4-cAMP and energy metabolic pathways may provide novel opportunities for treating the ILC2-initiated lung inflammation in asthma and NERD.

Insights Into the Helical Shape Complex of Helicobacter pylori.

One important factor that promotes the colonization of the upper digestive system of the human pathogen Helicobacter pylori is its helical cell shape. The bacteria cell shape is predominantly defined by its peptidoglycan cell wall. In rod-shaped species, PG synthesis is mediated by two dynamic molecular machines that facilitate growth along the perpendicular axis and the septum, called the elongasome and the divisome, respectively. Furthermore, many bacteria evolved additional mechanisms to locally change PG synthesis patterns to generate diverse cell shapes. Recent work characterizing cell shape mutants of Helicobacter pylori revealed a novel mechanism for the generation of a twisted helix from a rod, including PG-modifying enzymes as well as additional proteins such as the bactofilin homolog CcmA or the membrane proteins Csd5 and Csd7. In this study, we investigate the localization and dynamics of CcmA and Csd7 using live-cell imaging. We also address the question of how these change in the presence or absence of the putative interaction partners.

Divergent Effects of Peptidoglycan Carboxypeptidase DacA on Intrinsic β-Lactam and Vancomycin Resistance.

ABSTRACTVancomycin and β-lactams are clinically important antibiotics that inhibit the formation of peptidoglycan cross-links, but their binding targets are different. The binding target of vancomycin is d-alanine-d-alanine (d-Ala-d-Ala), whereas that of β-lactam is penicillin-binding proteins (PBPs). In this study, we revealed the divergent effects of peptidoglycan (PG) carboxypeptidase DacA on vancomycin and β-lactam resistance in Escherichia coli and Bacillus subtilis. The deletion of DacA induced sensitivity to most β-lactams, whereas it induced strong resistance toward vancomycin. Notably, both phenotypes did not have a strong association with ld-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and an Lpp outer membrane (OM) lipoprotein. Vancomycin resistance was induced by an increased amount of decoy d-Ala-d-Ala residues within PG, whereas β-lactam sensitivity was associated with physical interactions between DacA and PBPs. The presence of an OM permeability barrier strongly strengthened vancomycin resistance, but it significantly weakened β-lactam sensitivity. Collectively, our results revealed two distinct functions of DacA, which involved inverse modulation of bacterial resistance to clinically important antibiotics, β-lactams and vancomycin, and presented evidence for a link between DacA and PBPs.IMPORTANCE Bacterial PG hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and stress adaptation. Of all the PG hydrolases, the role of PG carboxypeptidases is poorly understood, especially regarding their impacts on antibiotic resistance. We have revealed two distinct functions of PG carboxypeptidase DacA with respect to antibiotic resistance. The deletion of DacA led to sensitivity to most β-lactams, while it caused strong resistance to vancomycin. Our study provides novel insights into the roles of PG carboxypeptidases in the regulation of antibiotic resistance and a potential clue for the development of a drug to improve the clinical efficacy of β-lactam antibiotics.

FtsZ-Ring Regulation and Cell Division Are Mediated by Essential EzrA and Accessory Proteins ZapA and ZapJ in Streptococcus pneumoniae.

The bacterial FtsZ-ring initiates division by recruiting a large repertoire of proteins (the divisome; Z-ring) needed for septation and separation of cells. Although FtsZ is essential and its role as the main orchestrator of cell division is conserved in most eubacteria, the regulators of Z-ring presence and positioning are not universal. This study characterizes factors that regulate divisome presence and placement in the ovoid-shaped pathogen, Streptococcus pneumoniae (Spn), focusing on FtsZ, EzrA, SepF, ZapA, and ZapJ, which is reported here as a partner of ZapA. Epi-fluorescence microscopy (EFm) and high-resolution microscopy experiments showed that FtsZ and EzrA co-localize during the entire Spn cell cycle, whereas ZapA and ZapJ are late-arriving divisome proteins. Depletion and conditional mutants demonstrate that EzrA is essential in Spn and required for normal cell growth, size, shape homeostasis, and chromosome segregation. Moreover, EzrA(Spn) is required for midcell placement of FtsZ-rings and PG synthesis. Notably, overexpression of EzrA leads to the appearance of extra Z-rings in Spn. Together, these observations support a role for EzrA as a positive regulator of FtsZ-ring formation in Spn. Conversely, FtsZ is required for EzrA recruitment to equatorial rings and for the organization of PG synthesis. In contrast to EzrA depletion, which causes a bacteriostatic phenotype in Spn, depletion of FtsZ results in enlarged spherical cells that are subject to LytA-dependent autolysis. Co-immunoprecipitation and bacterial two-hybrid assays show that EzrA(Spn) is in complexes with FtsZ, Z-ring regulators (FtsA, SepF, ZapA, MapZ), division proteins (FtsK, StkP), and proteins that mediate peptidoglycan synthesis (GpsB, aPBP1a), consistent with a role for EzrA at the interface of cell division and PG synthesis. In contrast to the essentiality of FtsZ and EzrA, ZapA and SepF have accessory roles in regulating pneumococcal physiology. We further show that ZapA interacts with a non-ZapB homolog, named here as ZapJ, which is conserved in Streptococcus species. The absence of the accessory proteins, ZapA, ZapJ, and SepF, exacerbates growth defects when EzrA is depleted or MapZ is deleted. Taken together, these results provide new information about the spatially and temporally distinct proteins that regulate FtsZ-ring organization and cell division in Spn.

Anti-inflammatory activity of methanolic extract of Ficus hispida dried fruit

Background: Natural products are a valuable resource of novel bioactive metabolites and these products exist in which the anti-inflammatory activity. The present investigation studies the in vivo and in vitro anti-inflammatory activity of methanolic extract of Ficus hispida in rat’s model.Methods: Plant material was extracted with methanol in a Soxhlet extraction apparatus. Indomethacin was used as a standard drug here, which is a known potent inhibitor of PG synthesis. The carrageenin and histamine induced paw oedema were selected to represent models of acute inflammations. The test compounds and standard drugs were administered orally. After 60 minutes paw oedema was induced by giving 0.1 ml of 1% Carrageenan and 0.1 % histamine by sub-plantar administration. Paw volume-Plethysmometer by mercury displacement method, before and after 1 hr to 4 hours of carrageenan and histamine administration. Performed MTT-based cytotoxicity assay of the Ficus hispida on the RAW264.7 cell line to determine the IC50 and calculate the pro-inflammatory cytokines viz, IL-6, IL-1β and TNF-α and compared to the LPS control.Results: The result obtained from the in-vivo study shows that the Ficus hispida has significant anti- inflammatory activity in a dose dependent manner. This effect is similar to that produced by NSAIDS such as Indomethacin. The concentrations of IL-6, IL-1β and TNF-α, secreted by the cells after challenging with bacterial LPS (2 µg/ml) and subsequent treatment with 50 µg Ficus hispida has been found to reduce the production of all the three pro-inflammatory cytokines viz, IL-6, IL-1β and TNF-α as compared to the LPS control. The activity, in fact, is comparable to the standard NSAID Indomethacin.Conclusions: All these findings and phytoconstituents present in the extract could be the possible chemicals involved in the prevention of inflammations.

Approaches to Potentiated Neuroprotective Treatment in the Rodent Model of Ischemic Optic Neuropathy.

Nonarteritic anterior ischemic optic neuropathy (NAION) commonly causes sudden optic nerve (ON)-related vision loss. The rodent NAION model (rAION) closely resembles NAION in presentation and physiological responses. We identified early rAION-associated optic nerve head (ONH) inflammatory gene expression responses and the anti-inflammatory prostaglandin PGJ2’s effects on those responses. We hypothesized that blocking pro-inflammatory prostaglandin (PGE2) production by inhibiting monoacylglycerol lipase or cyclooxygenase activity and co-administering PGJ2 would potentiate RGC survival following ischemic neuropathy. Deep sequencing was performed on vehicle- and PGJ2-treated ONHs 3d post-rAION induction. Results were compared against responses from a retinal ischemia model. Animals were treated with PGJ2 and MAGL inhibitor KML29, or PGJ2 + COX inhibitor meloxicam. RGC survival was quantified by stereology. Tissue PG levels were quantified by ELISA. Gene expression was confirmed by qPCR. PGJ2 treatment nonselectively reduced inflammatory gene expression post-rAION. KML29 did not reduce PGE2 1d post-induction and KML29 alone increased RGC loss after rAION. Combined treatments did not improve ONH edema and RGC survival better than reported with PGJ2 alone. KML29′s failure to suppress PGE2 ocular synthesis, despite its purported effects in other CNS tissues may result from alternative PG synthesis pathways. Neither KML29 nor meloxicam treatment significantly improved RGC survival compared with vehicle. While exogenous PGJ2 has been shown to be neuroprotective, treatments combining PGJ2 with these PG synthesis inhibitors do not enhance PGJ2’s neuroprotection.

Mechanisms of TGFß in prostaglandin synthesis and sperm guidance in Caenorhabditis elegans.

The transparent epidermis of Caenorhabditis elegans makes it an attractive model to study sperm motility and migration within an intact reproductive tract. C elegans synthesize specific F-series prostaglandins (PGFs) that are important for guiding sperm toward the spermatheca. These PGFs are synthesized from polyunsaturated fatty acid (PUFA) precursors, such as arachidonic acid (AA), via a novel pathway, independent of the classical cyclooxygenases (Cox) responsible for most PG synthesis. While the enzyme(s) responsible for PG synthesis has yet to be identified, the DAF-7 TGFß pathway has been implicated in modulating PG levels and sperm guidance. We find that the reduced PGF levels in daf-1 type I receptor mutants are responsible for the sperm guidance defect. The lower level of PGs in daf-1 mutants is due in part to the inaccessibility of AA. Finally, lipid analysis and assessment of sperm guidance in daf-1;daf-3 double mutants suggest DAF-3 suppresses PG production and sperm accumulation at the spermatheca. Our data suggest that DAF-3 functions in the nervous system, and possibly the germline, to affect sperm guidance. The C elegans TGFß pathway regulates many pathways to modulate PG metabolism and sperm guidance. These pathways likely function in the nervous system and possibly the germline.

Can NSAIDs Be Used Safely for Analgesia in Patients with CKD?: CON.

Nonsteroidal anti-inflammatory drugs (NSAIDs) reversibly inhibit cyclooxygenase (COX) and therefore alter PG synthesis in many tissues with a range of effects beyond their intended one. NSAIDs have a number of well-known adverse effects on the kidney, gastrointestinal (GI) tract, and cardiovascular system. Adverse effect profiles vary by drug, in part depending on relative selectivity for the COX-1 and COX-2 isozymes, with more COX-2 selective inhibitors having lower risk of GI bleeding but higher risk of cardiovascular disease. Therefore, all NSAIDs have potentially serious adverse effects, and NSAIDs are a common cause of drug-related emergency hospital admission and drug-related death, from GI bleeding, AKI, and serious cardiovascular events (1,2). International consensus guidelines recommend avoiding NSAIDs in people with eGFR <30 ml/min per 1.73 m2, and to avoid prolonged use in those with eGFR 30–59 ml/min per 1.73 m2 (3). Despite this, NSAIDs are commonly prescribed to people with CKD. One in ten people with CKD in the Chronic Renal Insufficiency Cohort Study were prescribed an NSAID annually, with 24% exposed at some point during 8 years of follow-up. Exposure was common in all subgroups examined, but was somewhat less likely in people with more severe CKD and those seeing nephrologists (4). A systematic review of NSAID use in people with CKD in seven cross-sectional studies found that 8%–21% were currently taking NSAIDs (5). One likely reason for liberal prescribing is that NSAID prescribing has rapidly observable benefits on pain, whereas harms are merely theoretical risks at the point of prescribing. In addition, the prescriber will often not observe the harmful outcome because the patient presents with adverse effects elsewhere in the health care system. Randomized controlled trials examining NSAID effectiveness routinely exclude people with CKD, and often do not evaluate renal outcomes or other potential …

Can NSAIDs Be Used Safely for Analgesia in Patients with CKD?: PRO

Nearly 60% of patients with CKD suffer pain. Of those patients with CKD who have pain, most rate their pain as moderate or severe in intensity. Undermanaged pain is associated with higher rates of mood disorders, maladaptive coping, and decreased quality of life for patients with CKD (1). In general, nonsteroidal anti-inflammatory drugs (NSAIDs) are recommended as first-line for analgesia and also act as antipyretics and anti-inflammatory medications. Estimates indicate that 98 million patients annually are prescribed NSAIDs (2), which likely represents only a fraction of total use given widespread nonprescription access. Epidemiologic studies suggest that 70%–80% of all NSAID users consume over-the-counter products like ibuprofen or naproxen (3,4). In the National Health and Nutrition Examination Survey, routine NSAID use was common in patients with CKD and use increased with increasing CKD severity (3). NSAIDs have well-known adverse effects in CKD patients. These risks can broadly be categorized into affecting the kidneys, gastrointestinal (GI) tract, or cardiovascular system. Among these risks, it is the direct and indirect nephrotoxicity that has primarily led to hesitation with the use of NSAIDs in patients with CKD. In one study, patients with CKD were more likely to receive an opioid than an NSAID or gabapentinoid across the entire spectrum of CKD (5). The highest rates of opioid prescribing were in patients with the lowest GFR. Indirect nephrotoxicity from NSAIDs is linked to altered intraglomerular hemodynamics. NSAIDs inhibit PG synthesis, which decreases afferent arteriolar vasodilation and can reduce glomerular pressure. This is especially prominent in patients with already jeopardized renal perfusion as in shock or intravascular volume depletion. For this reason, use of NSAIDs in acutely ill patients with AKI, acute kidney disease, or in the midst of renal recovery remains ill-advised. Patterns of direct nephrotoxicity include interstitial nephritis, papillary necrosis, and …

Distinct cytoskeletal proteins define zones of enhanced cell wall synthesis in Helicobacter pylori.

Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori, but the molecular mechanisms for generating helical shape remain unclear. The helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod H. pylori. Quantitative 3D microscopy analysis of short pulses with either N-acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape.

Kaempferia galanga L. sebagai Anti-Inflamasi dan Analgetik

Galangal ( Kaempferia galanga l ) is an herb that is often used in everyday life and widely used as a spice, food flavoring, and local tonic ingredients called ‘Jamu’. Galangal is a source of zinc, starch, ethyl ester, paraeumarin, borneaol, minerals, cinnamic acid, and other nutrients that are widely used in traditional medicine applications for a long time, especially in Southeast Asia. Besides that, it has medical functions, one of which is anti-inflammatory. Inflammation is a normal protective response to tissue injuries caused by physical trauma, damaging chemicals or microbiological substances. Inflammation can also be interpreted as the body's effort to activate or damage organisms that attack, eliminate irritants and regulate tissue repair. Signs of inflammation are redness, swelling, heat, and pain. Many chemical drugs are used to prevent inflammation, one of which is that modern drugs commonly used as anti-inflammatory drugs are NSAIDs (Non-Steroid Anti-inflammatory) Mechanism as an anti-inflammatory activity by elevating anti-inflammatory production IL-10, reducing pro-inflammatory IL-6 or TNF-a production, reducing the expression of iNOS and COX-2, inhibition of PG synthesis, Kaempferia galanga L also inhibits several inflammatory mediators TNF-α, IL-1β, and Nitric Oxide (NO) and angiogenesis.

How FtsEX localizes to the Z ring and interacts with FtsA to regulate cell division.

In Escherichia coli, FtsEX, a member of the ABC transporter superfamily, is involved in regulating the assembly and activation of the divisome to couple cell wall synthesis to cell wall hydrolysis at the septum. Genetic studies indicate FtsEX acts on FtsA to begin the recruitment of the downstream division proteins but blocks septal PG synthesis until a signal is received that divisome assembly is complete. However, the details of how FtsEX localizes to the Z ring and how it interacts with FtsA are not clear. Our results show that recruitment of FtsE and FtsX is codependent and suggest that the FtsEX complex is recruited through FtsE interacting with the conserved tail of FtsZ (CCTP), thus adding FtsEX to a growing list of proteins that interacts with the CCTP of FtsZ. Furthermore, we find that the N-terminus of FtsX is not required for FtsEX localization to the Z ring but is required for its functions in cell division indicating that it interacts with FtsA. Taken together, these results suggest that FtsEX first interacts with FtsZ to localize to the Z ring and then interacts with FtsA to promote divisome assembly and regulate septal PG synthesis.

Evaluation of analgesic activity of glucose in Swiss Albino mice

Background: Pain is the first and foremost symptom which alerts us about the underlying diseases, injuries or inflammation. Varied treatments are followed for pain relief worldwide. Nowadays tramadol, a centrally acting opioid analgesic is used widely. There are evidences that sweet substances like sucrose produce analgesia through endogenous opioid system. Sucrose has been proved to produce analgesic effect in healthy neonates and also in animals. Likewise, analgesic effect of glucose has also been studied but only limited no. of studies available.Methods: Swiss Albino mice of either sex (20-30g) were procured from the central animal house of KFMS and R, Coimbatore. Animals were maintained under controlled temperature and light conditions with food and water ad libitum. Mice were kept in the department to get acclimatized. 24 mice were divided into 4 groups (n=6). Drugs were given orally after 12hours of fasting. Group I was the control. Group II received standard-tramadol (40mg/kg). Group III received glucose (200mg/kg). Group IV received glucose (400mg/kg).Results: The latency period of glucose was significant (p&lt;0.001) compared to controls and standard was significant (p&lt;0.001) when compared to glucose by hot plate method.Conclusions: Analgesic activity of glucose may be due to both central and peripheral inhibition of PG synthesis. This has been proved in previous studies. This study showed that glucose can be used as an add-on in non-diabetic patients with better compliance.