'Is there any point in me doing this?' Views and experiences of women in the Diabetes and Antenatal Milk Expressing (DAME) trial.

The Diabetes and Antenatal Milk Expressing (DAME) randomised controlled trial (RCT) was conducted in 2011-2015, at six sites in Melbourne, Australia to explore the effect of advising women with diabetes in pregnancy to express breast milk from 36 weeks gestation. Infants whose mothers were randomised to express in pregnancy were more likely to be exclusively breast milk fed during their hospital stay, and there was no evidence of harm. This paper explores women's views and experiences of antenatal expressing. In this two-arm RCT, 635 women with diabetes in pregnancy who were otherwise of low medical risk were randomised at 36-37 weeks gestation to usual care (not expressing, n = 316), or the intervention, where women were advised to hand express for 10 min twice daily until birth (n = 319). Semistructured face-to-face interviews were conducted with 10 women who expressed antenatally. They were asked about their experiences of antenatal expressing, including how they felt about the overall experience, the amount of breast milk they expressed, making time to express, and their experience of breastfeeding. Thematic analysis of the in-depth interviews identified six themes: (1) learning and adapting expressing, (2) feelings and sensations associated with expressing, (3) support, (4) dis/empowerment, (5) health, and (6) the value of breast milk. Women had both positive and negative experiences of antenatal expressing. If health professionals are advising antenatal expressing to women, it is important they understand the range of outcomes and experiences.

Sequential assignment of NMR spectra of peptides at natural isotopic abundance with zero- and ultra-low-field total correlation spectroscopy (ZULF-TOCSY).

A novel method dubbed ZULF-TOCSY results from the combination of Zero and Ultra-Low Field (ZULF) with high-field, high-resolution NMR, leading to a generalization of the concept of total correlation spectroscopy (TOCSY). ZULF-TOCSY is a new building block for NMR methods, which has the unique property that the polarization is evenly distributed among all NMR-active nuclei such as 1H, 13C, 15N, 31P, etc., provided that they belong to the same coupling network, and provided that their relaxation is not too fast at low fields, as may occur in macromolecules. Here, we show that ZULF-TOCSY correlations can be observed for peptides at natural isotopic abundance, such as the protected hexapeptide Boc-Met-enkephalin. The analysis of ZULF-TOCSY spectra readily allows one to make sequential assignments, thus offering an alternative to established heteronuclear 2D experiments like HMBC. For Boc-Met-enkephalin, we show that ZULF-TOCSY allows one to observe all expected cross-peaks between carbonyl carbons and α-CH protons, while the popular HMBC method provides insufficient information.

The antibacterial activity and modes of LI-F type antimicrobial peptides against Bacillus cereus in vitro.

AIMS: LI-Fs are a family of highly potent cyclic lipodepsipeptide antibiotics with a broad antimicrobial spectrum (Gram-positive bacteria and fungi). In this study, LI-F-type antimicrobial peptides (AMP-jsa9) composing of LI-F03a, LI-F03b, LI-F04a, LI-F04b and LI-F05b were isolated from Paenibacillus polymyxa JSA-9. To better understand the antimicrobial mechanism of AMP-jsa9, the potency and action(s) of AMP-jsa9 against Bacillus cereus were examined. METHODS AND RESULTS: Flow cytometry, confocal laser microscopy, scanning electron microscopy, transmission electron microscopy (TEM) and atomic force microscopy observation, as well as determination of peptidoglycan and cell wall-associated protein and other methods were used. The results indicate that AMP-jsa9 exhibits strong, broad-spectrum antimicrobial activity. Moreover, AMP-jsa9 targets the cell wall and membrane of B. cereus to impair membrane integrity, increase membrane permeability and enhance cytoplasm leakage (e.g. K+ , protein, nucleic acid). This leads to bacterial cells with irregular, withered and coarse surfaces. In addition, AMP-jsa9 is also able to bind to DNA and break down B. cereus biofilms. CONCLUSIONS: In this study, the action mechanism of LI-Fs against B. cereus was clarified in details. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide a theoretical basis for utilizing AMP-jsa9 or similar analogues as natural and effective preservatives in the food and feed industries. These efforts could also stimulate research activities interested in understanding the specific effects of other antimicrobial agents.

Antinociceptive effect of [Met5]enkephalin semicarbazide is not affected by dipeptidyl carboxypeptidase-I.

Dipeptidyl carboxypeptidase-I is an enzyme involved in the biological degradation of enkephalins. It has been suggested that C-terminal amidation of enkephalins enhances their resistance to dipeptidyl carboxypeptidase-I-mediated biodegradation. In this study, a novel [Met5]enkephalin amide (MEA) analogue [Met5]enkephalin (ME)-semicarbazide synthesized by another laboratory in our group was assessed for its antinociceptive effects compared with ME-ethylamide, MEA and ME, using tail flick test. To protect the administered drugs from biodegradation, rats were pretreated with peptidase inhibitors including amastatin, phosphoramidon and captopril. Then captopril (dipeptidyl carboxypeptidase-I inhibitor) was deleted from the peptidase inhibitors' combination for evaluating in vivo resistance of the synthetic drugs to dipeptidyl carboxypeptidase-I. According to the results, ME-semicarbazide and MEA were resistant enough to dipeptidyl carboxypeptidase-I to exert their strong antinociception following intrathecal administration even in the absence of captopril, whereas the antinociceptive effects produced by ME-ethylamide (10 nmol) were abolished in rats not pretreated with captopril, indicating that significant amounts of the ME-ethylamide were degraded by dipeptidyl carboxypeptidase-I. Replacement of the amide moiety of MEA with semicarbazide provides a new ME derivative, with high analgesic effects as well as more resistance to dipeptidyl carboxypeptidase-I-mediated biodegradation.

An in silico approach to study the role of epitope order in the multi-epitope-based peptide (MEBP) vaccine design.

With different countries facing multiple waves, with some SARS-CoV-2 variants more deadly and virulent, the COVID-19 pandemic is becoming more dangerous by the day and the world is facing an even more dreadful extended pandemic with exponential positive cases and increasing death rates. There is an urgent need for more efficient and faster methods of vaccine development against SARS-CoV-2. Compared to experimental protocols, the opportunities to innovate are very high in immunoinformatics/in silico approaches, especially with the recent adoption of structural bioinformatics in peptide vaccine design. In recent times, multi-epitope-based peptide vaccine candidates (MEBPVCs) have shown extraordinarily high humoral and cellular responses to immunization. Most of the publications claim that respective reported MEBPVC(s) assembled using a set of in silico predicted epitopes, to be the computationally validated potent vaccine candidate(s) ready for experimental validation. However, in this article, for a given set of predicted epitopes, it is shown that the published MEBPVC is one among the many possible variants and there is high likelihood of finding more potent MEBPVCs than the published candidates. To test the same, a methodology is developed where novel MEBP variants are derived by changing the epitope order of the published MEBPVC. Further, to overcome the limitations of current qualitative methods of assessment of MEBPVC, to enable quantitative comparison and ranking for the discovery of more potent MEBPVCs, novel predictors, Percent Epitope Accessibility (PEA), Receptor specific MEBP vaccine potency (RMVP), MEBP vaccine potency (MVP) are introduced. The MEBP variants indeed showed varied MVP scores indicating varied immunogenicity. Further, the MEBP variants with IDs, SPVC_446 and SPVC_537, had the highest MVP scores indicating these variants to be more potent MEBPVCs than the published MEBPVC and hence should be preferred candidates for immediate experimental testing and validation. The method enables quicker selection and high throughput experimental validation of vaccine candidates. This study also opens the opportunity to develop new software tools for designing more potent MEBPVCs in less time.

Angiotensin-converting enzyme gates brain circuit-specific plasticity via an endogenous opioid.

Angiotensin-converting enzyme (ACE) regulates blood pressure by cleaving angiotensin I to produce angiotensin II. In the brain, ACE is especially abundant in striatal tissue, but the function of ACE in striatal circuits remains poorly understood. We found that ACE degrades an unconventional enkephalin heptapeptide, Met-enkephalin-Arg-Phe, in the nucleus accumbens of mice. ACE inhibition enhanced µ-opioid receptor activation by Met-enkephalin-Arg-Phe, causing a cell type-specific long-term depression of glutamate release onto medium spiny projection neurons expressing the Drd1 dopamine receptor. Systemic ACE inhibition was not intrinsically rewarding, but it led to a decrease in conditioned place preference caused by fentanyl administration and an enhancement of reciprocal social interaction. Our results raise the enticing prospect that central ACE inhibition can boost endogenous opioid signaling for clinical benefit while mitigating the risk of addiction.

Controlling charge states of peptides through inductive electrospray ionization mass spectrometry.

A novel ionization device for controlling the charge states of peptides based on an inductive elecrospray ionization technique was developed. This ion source keeps the major capabilities of electrospray ionization (ESI) which is compatible with liquid separation techniques (such as liquid chromatography (LC) and capillary electrophoresis (CE)) and can be potentially used to control the charge states of peptides accurately by simply varying the AC voltage applied. In comparison with conventional ESI, inductive ESI successfully simplifies the mass spectrum by reducing the charge states of peptide to a singly charged one, as well as eliminating the adduct ions.

[Bovine adrenal medulla 22 attenuates hyperalgesia in the early phase of complete Freund's adjuvant-induced inflammation in rats].

The present study investigated the effects of intrathecal (i.t.) application of bovine adrenal medulla 22 (BAM22), an endogenous opioid peptide potently activating opioid receptors and sensory neuron-specific receptor (SNSR), on a model of complete Freund's adjuvant (CFA)-induced inflammatory pain. Unilateral, but not bilateral, inflammatory pain was induced by intraplantar (i.pl.) injection of CFA in one side, as indicated by the shortened paw withdrawal latency and the increased edema of paw. Paw withdrawal latency test, paw edema determination and immunohistochemistry were used in CFA-induced inflammatory pain model after i.t. administration of BAM22 or saline. It was found that administration of BAM22 dose-dependently attenuated CFA-induced hyperalgesia and edema, and resumed antinociceptive effects against thermal stimulation in behavioral test. In 10 nmol BAM22 group, paw withdrawal latency was resumed to 83.2% of normal, and edema increased only by 60% of normal at 48 h. The potency of BAM22 was 33.5% of maximal possible effect (MPE) at 24 h, and the antinociception persisted for at least 1 h. Furthermore, i.t. treatment of 10 nmol BAM22 evidently decreased the expressions of CFA-evoked neuronal nitric oxide synthase (nNOS)-positive cells and calcitonin gene-related peptide (CGRP)-immunoreactivity positive nerve fibers by 25.6% (P<0.01) and 25.2% (P<0.001) compared with saline group, respectively, at L3-L5 segments of the spinal cord. Small and medium CGRP-positive cells were 57.4% and 35.2% in dorsal root ganglion (DRG) in 10 nmol BAM22 group, respectively, which were remarkably lower than those in saline group (P<0.001). The present study suggests that BAM22 relieves CFA-induced thermal hyperalgesia in the early phase and resumes antinociceptive effects through down-regulation of nNOS and CGRP expressions in DRG and spinal cord, which is possibly mediated via SNSR.

Computational structure-activity study directs synthesis of novel antitumor enkephalin analogs.

The capability of a Support Vector Machines QSAR model to predict the antiproliferative ability of small peptides was evaluated by screening a virtual library of enkephalin-like analogs modified by incorporation of the (R,S)-(1-adamantyl)glycine (Aaa) residue. From an initial set of 390 compounds, the peptides, Tyr-Aaa-Gly-Phe-Met (2), Tyr-Aaa-Gly-Phe-Phe (3), Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5) were selected, synthesized and their antitumor activity was tested and compared to that of Met-enkephalin (1). The antiproliferative activity correlated with the computational prediction and with the foldamer-forming ability of the studied peptides. The most active compounds were the hydrophobic peptides, Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5), having a greater propensity to adopt folded structures than the other peptides.

Analogues of both Leu- and Met-enkephalin containing a constrained dipeptide isostere prepared from a Baylis-Hillman adduct.

An efficient route was developed for the synthesis of the Fmoc-protected dipeptide 4, isostere of Gly-Gly containing an alpha-methylene beta-amino acid; the conformationally restricted analogues of Leu-enkephalin, 3a, and Met-enkephalin, 3b, respectively, were prepared by changing 4 for Gly(2)-Gly(3) in the native compounds 3a and 3b whose biological activities were significantly lower than the parent compounds.

Opioids in the hypothalamic paraventricular nucleus stimulate ethanol intake.

BACKGROUND: Specialized hypothalamic systems that increase food intake might also increase ethanol intake. To test this possibility, morphine and receptor-specific opioid agonists were microinjected in the paraventricular nucleus (PVN) of rats that had learned to drink ethanol. To cross-validate the results, naloxone methiodide (m-naloxone), an opioid antagonist, was microinjected with the expectation that it would have the opposite effect of morphine and the specific opioid agonists. METHODS: Sprague-Dawley rats were trained, without sugar, to drink 4 or 7% ethanol and were then implanted with chronic brain cannulas aimed at the PVN. After recovery, those drinking 7% ethanol, with food and water available, were injected with 2 doses each of morphine or m-naloxone. To test for receptor specificity, 2 doses each of the mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-Enkephalin (DAMGO), delta-receptor agonist D-Ala-Gly-Phe-Met-NH2 (DALA), or kappa-receptor agonist U-50,488H were injected. DAMGO was also tested in rats drinking 4% ethanol without food or water available. As an anatomical control for drug reflux, injections were made 2 mm dorsal to the PVN. RESULTS: A main result was a significant increase in ethanol intake induced by PVN injection of morphine. The opposite effect was produced by m-naloxone. The effects of morphine and m-naloxone were exclusively on intake of ethanol, even though food and water were freely available. In the analysis with specific receptor agonists, PVN injection of the delta-agonist DALA significantly increased 7% ethanol intake without affecting food or water intake. This is in contrast to the kappa-agonist U-50,488H, which decreased ethanol intake, and the mu-agonist DAMGO, which had no effect on ethanol intake in the presence or absence of food and water. In the anatomical control location 2 mm dorsal to the PVN, no drug caused any significant changes in ethanol, food, or water intake, providing evidence that the active site was close to the cannula tip. CONCLUSIONS: The delta-opioid receptor agonist in the PVN increased ethanol intake in strong preference over food and water, while the kappa-opioid agonist suppressed ethanol intake. Prior studies show that learning to drink ethanol stimulates PVN expression and production of the peptides enkephalin and dynorphin, which are endogenous agonists for the delta- and kappa-receptors, respectively. These results suggest that enkephalin via the delta-opioid system can function locally within a positive feedback circuit to cause ethanol intake to escalate and ultimately contribute to the abuse of ethanol. This is in contrast to dynorphin via the kappa-opioid system, which may act to counter this escalation. Naltrexone therapy for alcoholism may act, in part, by blocking the enkephalin-triggered positive feedback cycle.

Solution structural investigation and conformation-activity relationship of BAM8-22 by NMR and molecular dynamics simulations.

NMR spectroscopy and molecular dynamics simulations show that BAM8-22 (Val(8)-Gly(9)-Arg(10)-Pro(11)-Glu(12)-Trp(13)-Trp(14)-Met(15)-Asp(16)-Tyr(17)-Gln(18)-Lys(19)-Arg(20)-Tyr(21)-Gly(22)) possesses a relatively well-defined alpha-helix extending from Glu(12) to Arg(20), whereas both termini remain highly flexible in aqueous solution. The conformation-activity relationship of BAM8-22 indicates that the integrity of the well-defined alpha-helical structure is essential but not the sole determining factor for its bioactivity.

Antibacterial action of peptide F1 against colistin resistance E. coli SHP45 (mcr-1).

The emergence of the plasmid-mediated colistin resistance mechanism (mcr-1) makes bacterial resistance to colistin increasingly serious. This mcr-1 mediated bacterial resistance to colicin is conferred primarily through modification of lipid A in lipopolysaccharides (LPS). In our previous research, antimicrobial peptide F1 was derived from Tibetan kefir and has been shown to effectively inhibit the growth of Gram-negative bacteria (E. coli), Gram-positive bacteria (Staphylococcus aureus), and other pathogenic bacteria. Based on this characteristic of antibacterial peptide F1, we speculated that it could inhibit the growth of the colicin-resistant E. coli SHP45 (mcr-1) and not easily produce drug resistance. Studies have shown that antimicrobial peptide F1 can destroy the liposome structure of the phospholipid bilayer by destroying the inner and outer membranes of bacteria, thereby significantly inhibiting the growth of E. coli SHP45 (mcr-1), but without depending on LPS. The results of this study confirmed our hypothesis, and we anticipate that antimicrobial peptide F1 will become a safe antibacterial agent that can assist in solving the problem of drug resistance caused by colistin.

Mu-opioid receptor activation in live cells.

Interaction of the mu-opioid receptor (MOP) with selected ligands was investigated in live cells using advanced imaging by confocal laser scanning microscopy integrated with fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. In PC12 cells stably transformed to express the fluorescently labeled MOP-enhanced green fluorescent protein construct, two pools of MOP were identified that could be discriminated by differences in their lateral mobility in the cell membrane. The majority of MOP receptors (80+/-10%) were characterized by a diffusion coefficient D(MOP,1) = (4+/-2) x 10(-11) m(2) s(-1), compared with the slowly moving fraction, D(MOP,2) = (4+/-2) x 10(-12) m(2) s(-1). On stimulation with selected agonists ([D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin, enkephalin-heptapeptide Tyr-Gly-Gly-Phe-Met-Arg-Phe, morphine, and methadone), surface density of the MOP decreased, whereas the lateral mobility increased. In contrast, antagonists (naloxone and naltrexone) "froze" the receptor in the membrane, i.e., increased MOP surface density and decreased lateral mobility. Agonist activation was also accompanied by pronounced changes in the dynamics of plasma membrane lipids, as revealed by the general lipid marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate dye. The results provide new information about MOP activation in live cells at the molecular level, with a special focus on the dynamics of the intricate interplay between this receptor and the surrounding lipids.

Metkephamid, a systemically active analog of methionine enkephalin with potent opioid alpha-receptor activity.

Metkephamid is an analog of methionine enkephalin that retains high affinity for the delta receptor and is a systemically active analgesic. Since it is at least 100 times more potent than morphine as an analgesic when placed directly into the lateral ventricles, and is 30 to 100 times more potent on the delta receptor and yet is roughly equipotent on the mu receptor in vitro, it is concluded that it probably produces analgesia by action on delta receptors as well as, or rather than, on mu receptors. It has less tendency to produce respiratory depression, tolerance, and physical dependence than standard analgesics, and it is presently undergoing clinical trial.

Ethanol modulation of opiate receptors in cultured neural cells.

The mouse neuroblastoma-rat glioma hybrid cell line NG108-15 was used to study the acute and chronic interaction of ethanol with intact neural cells. In the short term, ethanol inhibited opiate receptor binding, but after long-term exposure the cells exhibited an apparent adaptive increase in the number of opiate binding sites; this was reversible when ethanol was withdrawn. High concentrations of ethanol (200 mM) increased opiate binding after 18 to 24 hours, whereas lower concentrations (25 to 50 mM) produced similar changes after 2 weeks. This model system has potential for exploring the cellular and molecular mechanisms underlying ethanol intoxication, tolerance, and withdrawal.

Irreversible ligands with high selectivity toward delta and mu opiate receptors.

Alkylating agents that display strong selectivity for opiate receptor types delta or mu were prepared by appropriate modification of the structures of the strong analgesics fentanyl, etonitazene, and endoethenotetrahydrooripavine. The availability of these substances should facilitate studies of the structural basis of receptor specificity and of the physiologic roles of these receptors.

Processing of proenkephalin is tissue-specific.

Most neuropeptides are synthesized as large precursor proteins. These precursors undergo a maturation process involving several proteolytic events that generate the biologically active peptides. The enzymatic mechanisms underlying this processing are still largely unknown. The processing of the precursor protein proenkephalin was studied in two different bovine tissues, the hypothalamus and adrenal medulla. The high molecular weight enkephalin-containing peptides that accumulate in these two tissues were found to be different, indicating the existence of two processing pathways for this neuropeptide precursor.

Opiates and opioid peptides hyperpolarize locus coeruleus neurons in vitro.

Intracellular recordings were made from locus coeruleus neurons in a brain slice preparation. Opiates and opioid peptides produced a dose-dependent, stereospecific, naloxone-reversible hyperpolarization of the neuronal membrane. This was associated with an increase in membrane conductance.

Potentiation of opiate analgesia and apparent reversal of morphine tolerance by proglumide.

Exogenous cholecystokinin selectively antagonizes opiate analgesia, which suggests that endogenous cholecystokinin may act physiologically as an opiate antagonist and may play a role in opiate tolerance. The use of the selective cholecystokinin antagonist proglumide provided a test of these hypotheses in rats that were either inexperienced with or tolerant to opiates. Proglumide potentiated analgesia produced by morphine and endogenous opiates and seemed to reverse tolerance. These results suggest that endogenous cholecystokinin systems oppose the action of opiates.