Creatine for Exercise and Sports Performance, with Recovery Considerations for Healthy Populations.

Creatine is one of the most studied and popular ergogenic aids for athletes and recreational weightlifters seeking to improve sport and exercise performance, augment exercise training adaptations, and mitigate recovery time. Studies consistently reveal that creatine supplementation exerts positive ergogenic effects on single and multiple bouts of short-duration, high-intensity exercise activities, in addition to potentiating exercise training adaptations. In this respect, supplementation consistently demonstrates the ability to enlarge the pool of intracellular creatine, leading to an amplification of the cell's ability to resynthesize adenosine triphosphate. This intracellular expansion is associated with several performance outcomes, including increases in maximal strength (low-speed strength), maximal work output, power production (high-speed strength), sprint performance, and fat-free mass. Additionally, creatine supplementation may speed up recovery time between bouts of intense exercise by mitigating muscle damage and promoting the faster recovery of lost force-production potential. Conversely, contradictory findings exist in the literature regarding the potential ergogenic benefits of creatine during intermittent and continuous endurance-type exercise, as well as in those athletic tasks where an increase in body mass may hinder enhanced performance. The purpose of this review was to summarize the existing literature surrounding the efficacy of creatine supplementation on exercise and sports performance, along with recovery factors in healthy populations.

Bi- and tri-valent T cell engagers deplete tumour-associated macrophages in cancer patient samples.

BACKGROUND: Tumour-associated macrophages (TAMs) are often implicated in cancer progression but can also exert anti-tumour activities. Selective eradication of cancer-promoting (M2-like) TAM subsets is a highly sought-after goal. Here, we have devised a novel strategy to achieve selective TAM depletion, involving the use of T cell engagers to direct endogenous T cell cytotoxicity towards specific M2-like TAMs. To avoid "on-target off-tumour" toxicities, we have explored localising expression of the T cell engagers to the tumour with enadenotucirev (EnAd), an oncolytic adenovirus in Phase I/II clinical trials. METHOD: A panel of bi- and tri-valent T cell engagers (BiTEs/TriTEs) was constructed, recognising CD3ε on T cells and CD206 or folate receptor ß (FRß) on M2-like macrophages. Initial characterisation of BiTE/TriTE activity and specificity was performed with M1- and M2-polarised monocyte-derived macrophages and autologous lymphocytes from healthy human peripheral blood donors. T cell engagers were inserted into the genome of EnAd, and oncolytic activity and BiTE secretion assessed with DLD-1 tumour cells. Clinically-relevant ex vivo models (whole malignant ascites from cancer patients) were employed to assess the efficacies of the free- and virally-encoded T cell engagers. RESULTS: T cells activated by the CD206- and FRß-targeting BiTEs/TriTEs preferentially killed M2- over M1-polarised autologous macrophages, with EC50 values in the nanomolar range. A TriTE with bivalent CD3ε binding - the first of its kind - demonstrated enhanced potency whilst retaining target cell selectivity, whereas a CD28-containing TriTE elicited non-specific T cell activation. In immunosuppressive malignant ascites, both free and EnAd-encoded T cell engagers triggered endogenous T cell activation and IFN-γ production, leading to increased T cell numbers and depletion of CD11b+CD64+ ascites macrophages. Strikingly, surviving macrophages exhibited a general increase in M1 marker expression, suggesting microenvironmental repolarisation towards a pro-inflammatory state. CONCLUSIONS: This study is the first to achieve selective depletion of specific M2-like macrophage subsets, opening the possibility of eradicating cancer-supporting TAMs whilst sparing those with anti-tumour potential. Targeted TAM depletion with T cell engager-armed EnAd offers a powerful therapeutic approach combining direct cancer cell cytotoxicity with reversal of immune suppression.

Amyloid Plaque in the Human Brain Can Decompose from Aß(1-40/1-42) by Spontaneous Nonenzymatic Processes.

The degradation of long-lived proteins in the body is an important aspect of aging, and much of the breakdown is due to the intrinsic instability of particular amino acids. In this study, peptides were examined to discover if spontaneous nonenzymatic reactions could be responsible for the composition of Alzheimer's (AD) plaque in the human brain. The great majority of AD plaque consists of N-terminally truncated versions of Aß(1-40/1-42), with the most abundant peptide commencing with Glu (residue 3 in Aß1-40/1-42) that is present as pyroGlu. Several Asp residues are racemized in Aß plaque, with residue 1 being predominantly l-isoAsp and peptide bond cleavage next to Ser 8 is also evident. In peptides, loss of the two N-terminal amino acids as a diketopiperazine was demonstrated at pH 7. For the Aß N-terminal hexapeptide, AspAlaGluPheArgHis, this resulted in the removal of AspAla diketopiperazine and the generation of Glu as the new N-terminal residue. The Glu cyclized readily to pyroGlu. This pathway was altered significantly by zinc, which promoted pyroGlu formation but decreased AspAla diketopiperazine release. Zinc also facilitated cleavage on the N-terminal side of Ser 8. Racemization of the original N-terminal Asp to l-isoAsp was also detected and loss of one amino acid from the N-terminus. These data are therefore entirely consistent with plaque in the human brain forming from deposition of Aß(1-40/1-42) and, over time, decomposing spontaneously. Since amyloid plaque is present in the human brain for years prior to the onset of AD, gradual spontaneous changes to the polypeptides within it will alter its properties and those of the oligomers that can diffuse from it. Such incremental changes in composition may therefore contribute to the origin of AD-associated cytotoxicity.

Dysregulation of inter-photoreceptor retinoid-binding protein (IRBP) after induced Müller cell disruption.

Reduced expression of a ~150 kDa protein was unexpectedly observed while investigating Norrin protein in a transgenic murine model in which Müller cells can be selectively and inducibly disrupted. Isolation of this unknown protein via ion exchange and hydrophobic interaction chromatography followed by Tandem mass spectrometry identified it as Inter-photoreceptor retinoid-binding protein (IRBP). Significantly reduced IRBP mRNA expression was observed at the early and late stages after Müller cell disruption. IRBP protein expression was also consistently reduced to 5.7% of the control level as early as 1 week after Müller cell disruption. This down-regulation of IRBP was accompanied by focal hyperfluorescent dots and cytotoxic N-retinylidene-N-retinylethanolamine (A2E) accumulation. In vitro treatment of cone photoreceptor cell lines with conditioned medium collected from stressed Müller cells suggested that Müller cells regulated photoreceptors expression of IRBP via secreted factor(s). In vivo studies suggested that one of these secreted factors was tumour necrosis factor alpha (TNFα). These findings suggest that dysregulation of IRBP expression caused by Müller cell dysfunction may be an important early event in photoreceptor degeneration in some retinal diseases. This study reports down-regulation of inter-photoreceptor retinoid-binding protein (IRBP) in photoreceptors and retinoid cycle derangement after Müller cell disruption in a transgenic mouse model. The findings indicate that Müller cells communicate with photoreceptors in response to stress by secreting soluble protein factor(s). We propose that down-regulation of IRBP may represent an early and novel pathogenic mechanism in degenerative retinal diseases.

Neostigmine Administered With MoviPrep Improves Bowel Preparation for Elective Colonoscopy in Patients With Spinal Cord Injury: A Randomized Study.

BACKGROUND: Poor preparation for elective colonoscopy is exceedingly common in persons with spinal cord injury (SCI). This unsatisfactory outcome is likely due to long-standing difficulty with evacuation and decreased colonic motility, which may result in inadequate responses to conventional bowel preparation regimens. We determined whether the addition of neostigmine to MoviPrep before elective colonoscopy produced a higher percentage of acceptable bowel preparations in patients with SCI. METHODS: Twenty-seven SCI subjects were prospectively randomized to 1 of 2 arms: low-volume polyethylene glycol-electrolyte lavage with ascorbic acid (MoviPrep) or MoviPrep plus neostigmine methylsulfate and glycopyrrolate (MoviPrep+NG); 28 able-bodied subjects received MoviPrep alone. The quality of the cleansing preparation for colonoscopy was determined by gastroenterologists "calibrated" to use the Ottawa Scoring System, with an acceptable Ottawa Score (OS) considered to be ≤3. RESULTS: The administration of MoviPrep alone resulted in suboptimal bowel cleansing in the SCI group compared with the able-bodied group (50% vs. 89% of subjects had an acceptable OS; χ=7.94, P=0.05). However, when NG was added to MoviPrep in the SCI group, it markedly improved the quality of the bowel preparation, with 85% of patients then having an acceptable OS. The use of NG resulted in minimal bloating and distention before bowel evacuation (P=0.0005), and eye and muscle twitching; these were resolved within 1 hour after NG administration. No significant differences were noted among the preparation groups for adenoma detection rate (P=0.41). CONCLUSIONS: The combination of MoviPrep+NG was safe, well tolerated, and an effective approach to prepare the bowel for elective colonoscopy in patients with SCI. The side effects of this preparation were significant compared with the other treatment groups but were considered mild and anticipated.

Comparison between pulsed irrigation enhanced evacuation and polyethylene glycol-electrolyte lavage solution for bowel preparation prior to elective colonoscopy in veterans with spinal cord injury.

BACKGROUND: Poor preparation for elective colonoscopy is common in persons with spinal cord injury (SCI). This unsatisfactory outcome is likely due to long-standing difficulty with evacuation and decreased colonic motility. Our objective was to determine the most effective preparation for elective colonoscopy applying a novel and traditional approach to bowel cleansing. METHODS: Twenty-four subjects with SCI were consented and scheduled to receive one of the two possible arms: pulsed irrigation enhanced evacuation (PIEE) or polyethylene glycol-electrolyte lavage solution (PEG; CoLyte(®)). The quality of the preparation was scored during the colonoscopy by applying the Ottawa scoring system. RESULTS: Patients with SCI who received PIEE tended to have lower Ottawa scores and a higher percentage of acceptable preparations than did those who received PEG; however, the results were not statistically different. CONCLUSION: In this preliminary study in subjects with SCI, neither PIEE nor PEG produced acceptable bowel preparation for elective colonoscopy. Future studies should confirm our findings and consider studying alternative, more efficacious approaches to bowel cleansing prior to colonoscopic procedures in patients with SCI, which should provide better outcomes. Registration number for clinicaltrials.gov: NCT00745095.

Spontaneous cyclization of polypeptides with a penultimate Asp, Asn or isoAsp at the N-terminus and implications for cleavage by aminopeptidase.

A cyclic product that forms spontaneously from peptides that contain a penultimate Asp, Asn or isoAsp residue at the N-terminus has been characterized. This 2,5-diketopiperazine derivative forms under physiological conditions and is stable, showing little degradation even following heating at 60 °C. A mechanism for its formation from Asn and Asp peptides is proposed that involves a succinimide or isoaspartate intermediate. A diketopiperazine-modified peptide was also detected in human lens extracts. Since peptides that contain the diketopiperazine moiety are not readily hydrolysed by leucine aminopeptidase, it is hypothesized that proteins and peptides modified in this way in the body may not readily be digested by the normal proteolytic machinery of cells.

Separate mechanisms for age-related truncation and racemisation of peptide-bound serine.

Some amino acids are particularly susceptible to degradation in long-lived proteins. Foremost among these are asparagine, aspartic acid and serine. In the case of serine residues, cleavage of the peptide bond on the N-terminal side, as well as racemisation, has been observed. To investigate the role of the hydroxyl group, and whether cleavage and racemisation are linked by a common mechanism, serine peptides with a free hydroxyl group were compared to analogous peptides where the serine hydroxyl group was methylated. Peptide bond cleavage adjacent to serine was increased when the hydroxyl group was present, and this was particularly noticeable when it was present as the hydroxide ion. Adjacent amino acid residues also had a pronounced affect on cleavage at basic pH, with the SerPro motif being especially susceptible to scission. Methylation of the serine hydroxyl group abolished truncation, as did insertion of a bulky amino acid on the N-terminal side of serine. By contrast, racemisation of serine occurred to a similar extent in both O-methylated and unmodified peptides. On the basis of these data, it appears that racemisation of Ser, and cleavage adjacent to serine, occur via separate mechanisms. Addition of water across the double bond of dehydroalanine was not detected, suggesting that this mechanism was unlikely to be responsible for conversion of L-serine to D-serine. Abstraction of the alpha proton may account for the majority of racemisation of serine in proteins.

Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates.

Nonenzymatic post-translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC-tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty-four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through ß-elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein-protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between ßB2- & ßB2-, ßA4- & ßA3-, γS- & ßB1-, and ßA4- & ßA4-crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long-lived proteins and that DHA- and DHB-mediated protein crosslinking may be the source of the long-sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long-lived proteins of other tissues leading to protein aggregation diseases.

Age-dependent modification of proteins: N-terminal racemization.

Age-dependent deterioration of long-lived proteins in humans may have wide-ranging effects on health, fitness and diseases of the elderly. To a large extent, denaturation of old proteins appears to result from the intrinsic instability of certain amino acids; however, these reactions are incompletely understood. One method to investigate these reactions involves exposing peptides to elevated temperatures at physiological pH. Incubation of PFHSPSY, which corresponds to a region of human αB-crystallin that is susceptible to age-related modification, resulted in the appearance of a major product. NMR spectroscopy confirmed that this novel peptide formed via racemization of the N-terminal Pro. This phenomenon was not confined to Pro, because peptides with N-terminal Ser and Ala residues also underwent racemization. As N-terminal racemization occurred at 37 °C, a long-lived protein was examined. LC-MS/MS analysis revealed that approximately one third of aquaporin 0 polypeptides in the centre of aged human lenses were racemized at the N-terminal methionine.

Molecular signatures of long-lived proteins: autolytic cleavage adjacent to serine residues.

The centre of the human lens, which is composed of proteins that were synthesized prior to birth, is an ideal model for the evaluation of long-term protein stability and processes responsible for the degradation of macromolecules. By analysing the sequences of peptides present in human lens nuclei, characteristic features of intrinsic protein instability were determined. Prominent was the cleavage on the N-terminal side of serine residues. Despite accounting for just 9% of the amino acid composition of crystallins, peptides with N-terminal Ser represented one-quarter of all peptides. Nonenzymatic cleavage at Ser could be reproduced by incubating peptides at elevated temperatures. Serine residues may thus represent susceptible sites for autolysis in polypeptides exposed to physiological conditions over a period of years. Once these sites are cleaved, other chemical processes result in progressive removal or 'laddering' of amino acid residues from newly exposed N- and C-termini. As N-terminal Ser peptides originated from several crystallins with unrelated sequences, this may represent a general feature of long-lived proteins.

Is protein methylation in the human lens a result of non-enzymatic methylation by S-adenosylmethionine?

Since crystallins in the human lens do not turnover, they are susceptible to modification by reactive molecules over time. Methylation is a major post-translational lens modification, however the source of the methyl group is not known and the extent of modification across all crystallins has yet to be determined. Sites of methylation in human lens proteins were determined using HPLC/mass spectrometry following digestion with trypsin. The overall extent of protein methylation increased with age, and there was little difference in the extent of modification between soluble and insoluble crystallins. Several different cysteine and histidine residues in crystallins from adult lenses were found to be methylated with one cysteine (Cys 110 in γD crystallin) at a level approaching 70%, however, methylation of crystallins was not detected in fetal or newborn lenses. S-adenosylmethionine (SAM) was quantified at significant (10-50 µM) levels in lenses, and in model experiments SAM reacted readily with N-α-tBoc-cysteine and N-α-tBoc-histidine, as well as ßA3-crystallin. The pattern of lens protein methylation seen in the human lens was consistent with non-enzymatic alkylation. The in vitro data shows that SAM can act directly to methylate lens proteins and SAM was present in significant concentrations in human lens. Thus, non-enzymatic methylation of crystallins by SAM offers a possible explanation for this major human lens modification.