Microglia regulate central nervous system myelin growth and integrity.

Myelin is required for the function of neuronal axons in the central nervous system, but the mechanisms that support myelin health are unclear. Although macrophages in the central nervous system have been implicated in myelin health1, it is unknown which macrophage populations are involved and which aspects they influence. Here we show that resident microglia are crucial for the maintenance of myelin health in adulthood in both mice and humans. We demonstrate that microglia are dispensable for developmental myelin ensheathment. However, they are required for subsequent regulation of myelin growth and associated cognitive function, and for preservation of myelin integrity by preventing its degeneration. We show that loss of myelin health due to the absence of microglia is associated with the appearance of a myelinating oligodendrocyte state with altered lipid metabolism. Moreover, this mechanism is regulated through disruption of the TGFß1-TGFßR1 axis. Our findings highlight microglia as promising therapeutic targets for conditions in which myelin growth and integrity are dysregulated, such as in ageing and neurodegenerative disease2,3.

SAGA1 and SAGA2 promote starch formation around proto-pyrenoids in Arabidopsis chloroplasts.

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated "proto-pyrenoid" Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well-studied pyrenoid, Chlamydomonas reinhardtii [N. Atkinson, Y. Mao, K. X. Chan, A. J. McCormick, Nat. Commun. 11, 6303 (2020)]. Here, we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesized as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, while also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and broadening the avenues for engineering starch morphology.

A SERS-Active Electrospun Polymer Mesh for Spatially Localized pH Measurements of the Cellular Microenvironment.

Extracellular pH (pHe) is an important chemical factor in many cellular processes and disease pathologies. The routine sampling of pHe in vitro could lead to innovative advances in therapeutics. To this end, we have fabricated a novel gold-coated polymer mesh, which facilitates the real-time measurement of pHe via surface-enhanced Raman scattering (SERS). In this proof of concept study, we apply our SERS sensor to measure metabolically induced changes in the pHe of carcinoma-derived cell line HepG2/C3A. We demonstrate that gold-coated polyurethane electrospun nanofibers (AuNF) have strong and reproducible SERS spectra of surface-adsorbed analytes. By functionalizing AuNF with pH-responsive reporter 4-mercaptobenzoic acid (MBA), we have developed an accurate pH SERS sensor for the extracellular microenvironment. We cultured HepG2/C3A on the surface of MBA-AuNF and measured an acidic shift in pHe at the cell-fiber interface. Following exposure to staurosporine, an apoptosis-inducing drug, we observed changes in the HepG2/C3A cellular morphology indicative of controlled cell death, and detected an increase in the pHe of HepG2/C3A. These results demonstrate how subtle changes in pHe, induced by the metabolic activity of cells, can be measured with our novel SERS sensor MBA-AuNF. The excellent pH measurement performance of MBA-AuNF provides a unique platform to study extracellular pH on the microscale and will help to deepen our understanding of pHe in disease pathology.

S-Carboxymethyl-l-cysteine: a multiple dosing study using pharmacokinetic modelling.

S-Carboxymethyl-l-cysteine is a mucolytic agent used as adjunctive therapy in the treatment of respiratory disorders. Various mechanisms of action have been proposed but few studies have attempted to link the required in vitro concentrations with those achieved actually in vivo during clinical therapy.The data from several published studies has been re-analysed by WinNonlin using non-compartmental analysis modelling, Phoenix modelling and Classic PK compartmental modelling for both single (500-1500 mg) and multiple oral administration of the drug.Multiple dose modelling indicated maximum peak concentrations (Cmax) ranging from 1.29 to 11.22 µg/ml and those at steady state (Css(av)) from 1.30 to 8.40 µg/ml. For the standard therapeutic regimen of 3 × 750 mg (2250 mg/day) these values were 1.29-5.22 µg/ml (Cmax) and 1.30-3.50 µg/ml (Css(av)). No accumulation was observed.Hence, only the pharmacodynamic studies reporting significant effects below c.10 µg/ml were likely to occur in vivo and these were mainly gene-related mechanisms. The majority of events, although demonstrable in vitro, required levels much greater than possible to achieve in the clinical situation.Such unappreciated disregard for in vitro-in vivo 'concentration matching' may lead to erroneous conclusions regarding mechanisms of action for many drugs as well as for S-carboxymethyl-l-cysteine.

Anatomic all-epiphyseal ACL reconstruction with "inside-out" femoral tunnel placement in immature patients yields high return to sport rates and functional outcome scores a minimum of 24 months after reconstruction.

PURPOSE: To understand if anatomic physeal-sparing ACL reconstruction in the immature host preserves range of motion, permits a return to sports, and avoids limb length discrepancy and accelerated intra-articular degeneration with a cross-sectional radiographic, physical examination and patient-reported outcomes analysis. METHODS: A cross-sectional recall study included 38 patients aged 7-15 who underwent all-epiphyseal ACL reconstruction with hamstring allograft performed by a single surgeon at a large academic medical center. All-epiphyseal reconstructions were performed using a modified Anderson physeal-sparing technique, with the femoral tunnel placed using an "inside-out" technique. Assessments consisted of a physical exam, long leg cassette radiographs, KT-1000 measurements, subjective patient metrics, and magnetic resonance imaging. RESULTS: Thirty-eight (56.7%) of 66 eligible patients returned for in-person clinical and radiographic exams. Patients were 11.4 ± 1.8 years at the time of surgery. Five patients were females (13.2%). Mean follow-up was 5.5 ± 2.4 years. ACL re-injuries occurred in four patients (10.5%), all of whom underwent revision reconstructions. Thirty-three of the remaining 34 (97.1%) patients returned to sports following their reconstruction, and 24 (70.6%) returned to their baseline level of competition. Mean limb length discrepancy (LLD) was 0.2 ± 1.4 cm. Nine patients had an LLD of > 1 cm (26.5%), which occurred at an equivalent age as those with < 1 cm LLD (10.8 ± 2.0 vs. 11.7 ± 1.7, n.s.). Pre-operative Marx scores (13.1 ± 3.5) were not significantly different from post-operative values (12.3 ± 5.1, n.s.). Patients who required ACL revisions had significantly lower Marx scores than those with intact primary grafts (8.3 ± 7.1 vs. 13.4 ± 4.5, p = 0.047). Cohort mean International Knee Documentation Committee (IKDC) score was 89.7 ± 12.7. CONCLUSION: Anatomic all-epiphyseal anatomic ACL reconstruction appears to be useful in patients with significant projected remaining growth, with good return-to-sport outcomes and minimal risk of clinically significant physeal complications. However, given the limited patient recall possible in the present study, further large sample size, high-quality works are necessary to validate our findings. LEVEL OF EVIDENCE: Level IV.

N-acetyltransferase: the practical consequences of polymorphic activity in man.

Over the years, numerous studies have supported the premise that individuals possessing the "slow acetylator" phenotype are more at risk from developing drug side-effects. Most prominent amongst these reports are those concerned with hepatotoxicity and peripheral neuropathy following treatment with isoniazid, lupus-like symptoms during procainamide therapy and experiencing hypersensitivity reactions to the various sulphonamide derivatives. Similarly, "slow acetylators" undergoing heavy exposure to arylamines and related carcinogens are more likely to develop bladder cancer. Contrariwise, there appears a slight risk of "rapid acetylators" developing pancreatic tumours.Other therapeutic agents for which polymorphic N-acetylation plays a minor role in their metabolism have been investigated but any impact of this metabolic difference on clinical efficacy or associated toxicity is still under question. In the search for clues as to the underlying aetiology, patient groups with many disease states have been examined for association with differences in N-acetylation and the majority have provided data that could be interpreted as equivocal. Studies have given contradictory, often opposing, results, calculated risk factors that are (perhaps) just significant but certainly not high, and patients within the cohorts who are always exceptions. Undoubtedly, other as yet unappreciated factors are at play.

Pharmacogenetics and drug metabolism: historical perspective and appraisal.

The events leading up to the discovery of genetically controlled polymorphic metabolism of xenobiotics and pharmaceutical chemicals are briefly summarised with the salient historical features being emphasised. Especial attention has been given to seminal works in the then emerging field.The evolving knowledge of such polymorphic metabolism and its role in the quest for personalised medicine and the individualisation of patient drug therapy are appraised. Opinion is offered as to whether or not the full potential has been exploited and if the practical application of this information may be regarded as a success or failure within the present clinical arena.

Phenylalanine 4-monooxygenase: the "sulfoxidation polymorphism".

1. Consistent differences in the proportion of an orally administered dose of S-carboxymethyl-l-cysteine subsequently excreted in the urine as S-oxide metabolites were reported 40 years ago. This observation suggested the existence of inter-individual variation in the ability to undertake the enzymatic S-oxygenation of this compound. Pedigree studies and investigations employing twin pairs indicated a genetically controlled phenomenon overlaid with environmental influences. It was reproducible and not related to gender or age.2. Studies undertaken in several healthy volunteer cohorts always provided similar results that were not significantly different when statistically analysed. However, when compared to these healthy populations, a preponderance of subjects exhibiting the characteristic of poor sulfoxidation of S-carboxymethyl-l-cysteine was found within groups of patients suffering from various disease conditions. The most striking of these associations were witnessed amongst subjects diagnosed with neurodegenerative disorders; although, underlying mechanisms were unknown.3. Exhaustive investigation has identified the enzyme responsible for this S-oxygenation reaction as the tetrahydrobiopterin-dependent aromatic amino acid hydroxylase, phenylalanine 4-monooxygenase classically assigned the sole function of converting phenylalanine to tyrosine. The underlying principle is discussed that enzymes traditionally associated solely with intermediary metabolism may have as yet unrecognised alternative roles in protecting the organism from potential toxic assault.

Vagus nerve stimulation (VNS) therapy in patients with treatment resistant depression: A systematic review and meta-analysis.

BACKGROUND: Vagus nerve stimulation (VNS) therapy is approved for treatment-resistant depression (TRD). A recent 5-year comparative study prompted this review of its impact in this very severe population. Previous systematic literature reviews (SLR) cited concerns in terms of missing studies or patient duplication. METHODS: This SLR addressed these criticisms, assessed all outcomes of longer-term adjunctive VNS in all studies, irrespective of TRD severity, comparing where feasible with treatment-as-usual (TAU). We searched for adult VNS+TAU studies (January 1, 2000 to June 24, 2019). Comparative and single-arm studies were eligible. All reported efficacy, safety and quality of life (QOL) outcomes were assessed. Where possible, meta-analysis was used to calculate overall pooled effect estimates across studies at several time points. RESULTS: Of 22 identified studies, there were two randomized controlled (RCT), sixteen single-arm and four non-randomized comparative studies. Numerous depression-specific, safety and QOL measures were reported. Meta-analysis was possible for three efficacy [Montgomery-Asberg Depression Rating Scale, Clinician Global Impression-Improvement, Hamilton Rating Scale for Depression] and three safety [serious adverse events, study drop-outs and all-cause mortality] but no QOL measures. Data beyond 2 years was not poolable. Analyses demonstrated that antidepressant benefits improved to 24 months and safety issues were minimal. Heterogeneity was high and statistically significant. CONCLUSIONS: Despite limitations in the evidence base, our comprehensive summary of VNS+TAU outcomes suggests that this treatment provides improving benefit and hope for this very hard-to-treat chronic population. More comparative TRD studies should describe safety and QOL.

The S-oxidation of S-carboxymethyl-L-cysteine in hepatic cytosolic fractions from BTBR and phenylketonuria enu1 and enu2 mice.

Mice that were heterozygous dominant for the enu1 and enu2 mutation in phenylalanine monooxygenase/phenylalanine hydroxylase (PAH) resulted in hepatic PAH assays for S-carboxymethyl-L-cysteine (SCMC) that had significantly increased calculated Km (wild type (wt)/enu1, 1.84-2.12 fold increase and wt/enu2 a 2.75 fold increase in PAH assays). The heterozygous dominant phenotypes showed a significantly reduced catalytic turnover of SCMC (wt/enu1, 6.11 fold decrease and wt/enu2 an 11.25 fold decrease in calculated Vmax). Finally, these phenotypes also had a significantly reduced clearance, CLE (wt/enu1, 13.02 fold and wt/enu2, a 30.80-30.94 fold decrease) The homozygous recessive phenotype (enu1/enu1) was also found to have significantly increased calculated Km (2.16 fold increase), a significantly reduced calculated Vmax (11.35-12.33 fold decrease) and CLE (24.75-25.00 fold decrease). The enu2/enu2, homozygous recessive phenotype had no detectable PAH activity using SCMC as substrate. The identity of the enzyme responsible for the C-oxidation of L-phenylalanine (L-Phe) and the S-oxidation of SCMC in wt/wt (BTBR) mice was identified using monoclonal antibody and selective chemical inhibitors and was found to be PAH. This in vitro mouse hepatic cytosolic fraction metabolism investigation provides further evidence to support the hypothesis that an individual possessing one variant allele for PAH will result in a poor metaboliser phenotype that is unable to produce significant amounts of S-oxide metabolites of SCMC.

Risk Calculator to Predict 30-Day Readmission After Coronary Artery Bypass: A Strategic Decision Support Tool.

BACKGROUND: Re-admission is an important source of patient dissatisfaction and increased hospital costs. A simple calculator to determine the probability of re-admission may help guide patient dismissal planning. METHODS: Using the national readmissions database (NRD), we identified admissions for isolated primary coronary artery bypass (CABG) and stratified them according to 30-day readmission. Including pre, intra and postoperative variables, we prepared a logistic regression model to determine the probability for re-admission. The model was tested for reliability with boot-strapping and 10-fold cross-validation. RESULTS: From 135,699 procedures, 19,355 were readmitted at least once within 30days of dismissal. Patients who were readmitted were older (67±10 vs 65 ± 10 years, p<0.01), females (32% vs 24%; p<0.01) and had a higher Elixhauser comorbidity score (1.5±1.4 vs 1.1±1.2; p<0.01). Our final model (c- statistic=0.65) consisted of 16 pre and three postoperative factors. End-stage renal disease (OR 1.79 [1.57-2.04]) and length of stay>9days (OR 1.60 [1.52-1.68]) were most prominent indicators for readmission. Compared to Medicaid beneficiaries, those with private insurance (OR 0.62 [0.57-0.68]) and Medicare (OR 0.85 [0.79-0.92]) coverage were less likely to be readmitted. CONCLUSIONS: Our simple 30-days CABG readmission calculator can be used as a strategic tool to help reduce readmissions after coronary artery bypass surgery.

Xenobiotic C-sulfonate derivatives; metabolites or metabonates?

1. The production of sulfate conjugates is a well-known and established pathway within the field of xenobiotic metabolism. In addition to the usual attachment of a sulfonate grouping via an oxygen atom (O-sulfonates) to yield a sulfate conjugate, so-called "N-sulfates" (N-sulfonates) have been reported and "S-sulfates" (S-sulfonates) mooted to exist. 2. The few examples cited in the literature where the sulfur atom of the sulfonate group was attached directly to a carbon atom of the xenobiotic (C-sulfonates) and subsequently excreted as a metabolite have been collated, examined and reviewed. 3. The potential mechanisms of formation of these C-sulfonates are discussed, both biological and chemical; the potential role of the gut microbiome raised and hopefully by highlighting this curiosity further fruitful investigation will be stimulated.

Lung cells support osteosarcoma cell migration and survival.

BACKGROUND: Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined. METHODS: Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment. RESULTS: Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline phosphatase staining. CONCLUSIONS: Lung endothelial HULEC-5a cells are attractants for OS cell migration, proliferation, and survival. The SJSA-1 osteosarcoma cell line demonstrated greater metastatic potential than Saos-2 and U-2 cells. ALDH appears to be involved in the interaction between lung and OS cells, and ALP may be a valuable biomarker for monitoring functional OS changes during metastasis.

A review of patient-reported outcome measures to assess female infertility-related quality of life.

BACKGROUND: Infertility has a negative impact on quality of life (QoL) and well-being of affected individuals and couples. A variety of patient-reported outcome (PRO) measures to assess infertility-related QoL are available; however, there is a concern regarding potential issues with their development methodology, validation and use. This review aimed to i) identify PRO measures used in infertility interventional studies ii) assess validation evidence to identify a reliable, valid PRO measure to assess changes in QoL or treatment satisfaction in clinical studies with female patients following treatment with novel therapies iii) identify potential gaps in evidence for validity. METHODS: A structured literature search of Medline, Embase, and the Cochrane Library (accessed in September 2015) was conducted using pre-defined search terms. The identified publications were reviewed applying eligibility criteria to select interventional female infertility studies using PROs. Infertility-specific PRO measures assessing QoL, treatment satisfaction or psychiatric health, and included in studies by ≥2 research groups were selected and critically reviewed in light of scientific and regulatory guidance (e.g. FDA PRO Guidance for Industry) for evidence of content validity, psychometric strength, and patient acceptability. RESULTS: The literature search and hand-searching yielded 122 publications; 78 unique PRO measures assessing QoL, treatment satisfaction or psychiatric health were identified. Five PRO measures met the selection criteria for detailed review: Fertility Quality of Life (FertiQoL); Fertility Problem Inventory (FPI); Fertility Problem Stress (FPS); Infertility Questionnaire (IFQ); Illness Cognitions Questionnaire adapted for Infertility (ICQ-I). None of the PRO measures met all validation criteria. The FertiQoL was the most widely used infertility-specific PRO measure to assess QoL in interventional studies, with reasonable evidence for adequate content validity, psychometric strength, and linguistic validation. However, gaps in evidence remain including test-retest reliability and thresholds for interpreting clinically important changes. While the FPI demonstrated reasonable evidence for content and psychometric validity, its utility as an outcome measure is limited by a lack of recall period. CONCLUSION: The FertiQoL and the FPI are potentially useful measures of infertility-related QoL in interventional studies. Further research is recommended to address gaps in evidence and confirm both PRO measures as reliable assessments of patient outcomes.

Trimethylamine-The Extracorporeal Envoy.

One of the most widespread and efficient mechanisms that has evolved to enable communication between discrete and spatially separate living organisms is the use of specific chemical messengers. The organoleptic properties of certain molecules, even at concentrations that do not necessarily evoke a conscious response, have been exploited to transmit information across relatively large distances. The trimethylated derivative of ammonia is one such molecule that is ideally suited to this function and several species are known to respond to its presence. This review uniquely collects together and integrates widely dispersed data to show that trimethylamine also may serve a communicatory role in man, with its influence extending outside of the body.

Xenobiotic conjugation with phosphate - a metabolic rarity.

1. Although not unknown, the conjugation of a xenobiotic with phosphate appears a rarity amongst the routes available for foreign compound metabolism. This is especially true in mammals and may be somewhat surprising as conjugation with sulphate, a seemingly similar moiety, is commonplace. 2. Information from the literature, where xenobiotic phosphate conjugates have been described or suggested, has been collated and presented in this article. By bringing together this diverse material, hopefully interest will be generated in this unusual xenobiotic reaction, and perhaps further research undertaken to better understand and delineate the reasons for its relative absence from the xenobiotic scene.

Sulphate absorption across biological membranes.

1. Sulphonation is unusual amongst the common Phase II (condensation; synthetic) reactions experienced by xenobiotics, in that the availability of the conjugating agent, sulphate, may become a rate-limiting factor. This sulphate is derived within the body via the oxygenation of sulphur moieties liberated from numerous ingested compounds including the sulphur-containing amino acids. Preformed inorganic sulphate also makes a considerable contribution to this pool. 2. There has been a divergence of opinion as to whether or not inorganic sulphate may be readily absorbed from the gastrointestinal tract and this controversy still continues in some quarters. Even more so, is the vexing question of potential absorption of inorganic sulphate via the lungs and through the skin. 3. This review examines the relevant diverse literature and concludes that sulphate ions may move across biological membranes by means of specific transporters and, although the gastrointestinal tract is by far the major portal of entry, some absorption across the lungs and the skin may take place under appropriate circumstances.

Phenylalanine monooxygenase and the sulfur oxygenation of S-carboxymethyl-L-cysteine in mice.

1. The extent of sulfoxidation of the drug, S-carboxymethyl-L-cysteine, has been shown to vary between individuals, with this phenomenon being mooted as a biomarker for certain disease states and susceptibilities. Studies in vitro have indicated that the enzyme responsible for this reaction was phenylalanine monooxygenase but to date no in vivo evidence exists to support this assumption. Using the mouse models of mild hyperphenylalaninamia (enu1 PAH variant) and classical phenylketonuria (enu2 PAH variant), the sulfur oxygenation of S-carboxymethyl-L-cysteine has been investigated. 2. Compared to the wild type (wt/wt) mice, both the heterozygous dominant (wt/enu1 and wt/enu2) mice and the homozygous recessive (enu1/enu1 and enu2/enu2) mice were shown to have significantly increased Cmax, AUC(0-180 min) and AUC(0-∞ min) values (15 - to 20-fold higher). These results were primarily attributable to the significantly reduced clearance of S-carboxymethyl-L-cysteine (13 - to 22-fold lower). 3. Only the wild type mice produced measurable quantities of the parent S-oxide metabolites. Those mice possessing one or more allelic variant showed no evidence of blood SCMC (R/S) S-oxides. These observations support the proposition that differences in phenylalanine hydroxylase activity underlie the variation in S-carboxymethyl-L-cysteine sulfoxidation and that no other enzyme is able to undertake this reaction.