Hybrid capture shotgun sequencing detected unexpected viruses in the cerebrospinal fluid of children with acute meningitis and encephalitis.

PURPOSE: Investigation of undiagnosed cases of infectious neurological diseases, especially in the paediatric population, remains a challenge. This study aimed to enhance understanding of viruses in CSF from children with clinically diagnosed meningitis and/or encephalitis (M/ME) of unknown aetiology using shotgun sequencing enhanced by hybrid capture (HCSS). METHODS: A single-centre prospective study was conducted at Sant Joan de Déu University Hospital, Barcelona, involving 40 M/ME episodes of unknown aetiology, recruited from May 2021 to July 2022. All participants had previously tested negative with the FilmArray Meningitis/Encephalitis Panel. HCSS was used to detect viral nucleic acid in the patients' CSF. Sequencing was performed on Illumina NovaSeq platform. Raw sequence data were analysed using CZ ID metagenomics and PikaVirus bioinformatics pipelines. RESULTS: Forty episodes of M/ME of unknown aetiology in 39 children were analysed by HCSS. A significant viral detection in 30 CSF samples was obtained, including six parechovirus A, three enterovirus ACD, four polyomavirus 5, three HHV-7, two BKV, one HSV-1, one VZV, two CMV, one EBV, one influenza A virus, one rhinovirus, and 13 HERV-K113 detections. Of these, one sample with BKV, three with HHV-7, one with EBV, and all HERV-K113 were confirmed by specific PCR. The requirement for Intensive Care Unit admission was associated with HCSS detections. CONCLUSION: This study highlights HCSS as a powerful tool for the investigation of undiagnosed cases of M/ME. Data generated must be carefully analysed and reasonable precautions must be taken before establishing association of clinical features with unexpected or novel virus findings.

A deep intronic variant in MME causes autosomal recessive Charcot-Marie-Tooth neuropathy through aberrant splicing.

BACKGROUND: Loss-of-function variants in MME (membrane metalloendopeptidase) are a known cause of recessive Charcot-Marie-Tooth Neuropathy (CMT). A deep intronic variant, MME c.1188+428A>G (NM_000902.5), was identified through whole genome sequencing (WGS) of two Australian families with recessive inheritance of axonal CMT using the seqr platform. MME c.1188+428A>G was detected in a homozygous state in Family 1, and in a compound heterozygous state with a known pathogenic MME variant (c.467del; p.Pro156Leufs*14) in Family 2. AIMS: We aimed to determine the pathogenicity of the MME c.1188+428A>G variant through segregation and splicing analysis. METHODS: The splicing impact of the deep intronic MME variant c.1188+428A>G was assessed using an in vitro exon-trapping assay. RESULTS: The exon-trapping assay demonstrated that the MME c.1188+428A>G variant created a novel splice donor site resulting in the inclusion of an 83 bp pseudoexon between MME exons 12 and 13. The incorporation of the pseudoexon into MME transcript is predicted to lead to a coding frameshift and premature termination codon (PTC) in MME exon 14 (p.Ala397ProfsTer47). This PTC is likely to result in nonsense mediated decay (NMD) of MME transcript leading to a pathogenic loss-of-function. INTERPRETATION: To our knowledge, this is the first report of a pathogenic deep intronic MME variant causing CMT. This is of significance as deep intronic variants are missed using whole exome sequencing screening methods. Individuals with CMT should be reassessed for deep intronic variants, with splicing impacts being considered in relation to the potential pathogenicity of variants.

Impact of Limited Enhanced Recovery Pathway for Cardiac Surgery: A Single-Institution Experience.

OBJECTIVES: Enhanced recovery pathway (ERP) refers to extensive multidisciplinary, evidence-based pathways used to facilitate recovery after surgery. The authors assessed the impact that limited ERP protocols had on outcomes in patients undergoing cardiac surgery at their institution. DESIGN: A retrospective cohort study. SETTING: This study was a single-institution study conducted at a university hospital. PARTICIPANTS: Patients undergoing open adult cardiac surgery. INTERVENTIONS: Enhanced recovery pathways limited to preoperative, intraoperative, and postoperative management of pain, atrial fibrillation prevention, and nutrition optimization were implemented. MEASUREMENTS AND MAIN RESULTS: A total of 1,058 patients were included in this study. There were 374 patients in each pre- and post-ERP cohort after propensity matching, with no significant baseline differences between the 2 cohorts. Compared to the matched patients in the pre-ERP group, patients in the post-ERP group had decreased total ventilation hours (6.8 v 7.8, p = 0.006), less use of postoperative opioid analgesics as determined by total morphine milligram equivalent (32.5 v 47.5, p < 0.001), and a decreased rate of postoperative atrial fibrillation (23.3% v 30.5%, p = 0.032). Post-ERP patients also experienced less subjective pain and postoperative nausea and drowsiness as compared to their matched pre-ERP cohorts. CONCLUSIONS: Limited ERP implementation resulted in significantly improved perioperative outcomes. Patients additionally experienced less postoperative pain despite decreased opioid use. Implementation of ERP, even in a limited format, is a promising approach to improving outcomes in patients undergoing cardiac surgery.

Use of intraoperative technology in total knee arthroplasty is not associated with reductions in postoperative pain.

PURPOSE: Our systematic review and meta-analysis sought to assess how technology-assistance impacts (1) post-operative pain and (2) opioid use in patients undergoing primary total knee arthroplasty (TKA). METHODS: Four online databases were queried for studies published up to October 2021 that reported on pain and opioid usage between technology-assisted and manual TKA (mTKA) patients. Mantel-Haenszel (M-H) models were utilized to calculate pooled mean difference (MDs) and 95% confidence interval (CIs). Subgroup analyses were conducted to isolate robotic-arm assisted (RAA) and computed-assisted navigation (CAN) cohorts. Risk of bias was assessed for all included non-randomized studies with the Methodological Index for Non-Randomized Studies (MINORS) tool. For the randomized control trials included in our study, the Detsky scale was applied. RESULTS: Our analysis included 31 studies, reporting on a total of 761,300 TKAs (mTKA: n = 753,554; Computer-Assisted Navigation (CAN): n = 1,309; Robotic-Arm Assisted (RAA): n = 6437). No differences were demonstrated when evaluating WOMAC (MD: 0.00, 95% CI - 0.69 to 0.69; p = 1.00), KSS (MD: 0.01, 95% CI - 1.46 to 1.49; p = 0.99), KOOS (MD - 2.91, 95% CI - 6.17 to 0.34; p = 0.08), and VAS (MD - 0.54, 95% CI - 1.01 to - 0.007; p = 0.02) pain scores between cohorts. There was mixed evidence regarding how opioid consumption differed between TKA techniques. CONCLUSION: The present analysis demonstrated no difference in terms of pain across a variety of utilized patient-reported pain measurements. However, there were mixed results regarding how opioid consumption varied between manual and technology-assisted cohorts, particularly in the immediate post-operative period. LEVEL OF EVIDENCE: III.

Rapid Adoption of Telemedicine Increases Opioid Prescribing in Orthopedic Surgery.

Background: The COVID-19 pandemic led to health care practitioners utilizing new technologies to deliver health care, including telemedicine. The purpose of this study was to examine the effect of rapidly proliferative use of video visits on opioid prescribing to orthopedic patients at a large academic health system that had existing procedure-specific opioid prescribing guidelines. Methods: This IRB-exempt study examined 651 opioid prescriptions written to patients who had video (visual and audio), telephone (audio only), or in-person encounters at our institution from March 1 to June 1, 2020 and compared them with 963 prescriptions written during the same months in 2019. Prescriptions were converted into daily milligram morphine equivalents (MMEs) to facilitate direct comparison. Chi-square testing was used to compare categorical data, whereas analysis of variance and Mann-Whitney tests were used to compare numerical data between groups. Statistical significance was set at <0.05. Results: Six hundred fifty-one of 1,614 prescriptions analyzed (40.3%) occurred during the pandemic. Patients prescribed opioids during video visits were prescribed 53.3 ± 37 MME, significantly higher than in-person (p = 0.002) or audio visits (p < 0.001) before or during the pandemic. Prepandemic, significantly higher MME were prescribed for in-person versus audio only visits (41.6 ± 89 vs. 30.2 ± 28 MME; p = 0.026); during the pandemic, there was no difference between these groups (p = 0.91). Significantly higher MME were prescribed by Nurse Practitioners and Physician Associates versus MD or DO prescribers for both time periods (51.3 ± 109 vs. 27.9 ± 42 MME; p < 0.001; 42.9 ± 70 vs. 28.2 ± 42 MME; p < 0.001). Conclusion: During crisis and with new technology, we should be vigilant about prescribing of opioid analgesics. Despite well-established protocols, patients received significantly higher MME through video than for other encounter types, including in-person encounters. In addition, significantly higher MME were prescribed by mid-level prescribers compared with DOs or MDs. Institutions should ensure these prescribers are involved during creation of opioid prescribing protocols after orthopedic surgery.

Trends of Opioid Usage in Surgical Patients in a Small Community Hospital: Analysis of Patient Data Between 2017 and 2021.

Purpose: The purpose of our study was to quantify and analyze the annual opioid usage in surgical patients at Wood County Hospital (WCH) between 2017 and 2021. Methods: In this retrospective study, patient data between 2017 and 2021 was analyzed to determine the oral morphine milligram equivalent (MME) of opioids used in surgical patients at WCH. Annual MME prescribed per admission was compared each year using one-way ANOVA followed by Tukey post hoc test. Similarly, the annual use of intravenous (IV) acetaminophen for surgical patients per admission was also calculated and analyzed using the one-way ANOVA followed by Tukey post hoc test. Results: Compared to the year 2017 (42.0 ± 3.6), a statistically significant decrease in opioid usage per surgical admission (mean±SEM of MME) was observed during the years 2018 (32.6 ± 1.4; P = .04), 2019 (30.4 ± 1.2; P = .01), and 2021 (30.8 ± 1.9; P = .01). An analysis of individual opioid use revealed a trend toward lower fentanyl and hydromorphone usage each year since 2017. A significant decrease in the annual morphine usage (mean±SEM of MME) for surgical patients was observed during both 2020 (14.4 ± 0.9; P = .05) and 2021 (14.0 ± 0.7; P = .05) compared to the year 2017 (22.1 ± 2.4). Finally, compared to the year 2017, a statistically significant decrease (P < .05) in the annual use of oxycodone (MME) and IV acetaminophen (mg) for pain management in surgical patients was observed from 2018 to 2021. Conclusion: Our analysis reveals a significant decrease in opioid usage per surgical admission at WCH over 2017 to 2021 indicating a positive impact of the various opioid stewardship measures implemented at the hospital.

Can pain be improved with retention of the posterior cruciate ligament during total knee arthroplasty?

PURPOSE: The purpose of the current study was to determine if differences exist between cruciate-retaining (CR) and posterior-stabilized (PS) implant articulations for total knee arthroplasty (TKA) with regards to early post-operative pain. METHODS: We retrospectively reviewed patients who underwent primary TKA, with the same TKA implant design, at our institution between January 2018 and July 2021. Patients were stratified based on whether they received a CR or non-constrained PS (PSnC) articulation and propensity score matched in a 1:1 ratio. A sub-analysis matching patient who received a constrained PS implant (PSC) to those undergoing CR TKA and PSnC TKA was also carried out. Opioid dosages were converted to morphine milligram equivalents (MME). RESULTS: 616 patients after CR TKA were matched 1:1 to 616 patients with a PSnC implant. There were no significant differences between demographic variables. There were no statistically significant differences in opioid usage measured by MME on post-operative day (POD) 0 (p = 0.171), POD1 (p = 0.839), POD2 (p = 0.307), or POD3 (p = 0.138); VAS pain scores (p = 0.175); or 90-day readmission rate for pain (p = 0.654). A sub-analysis of CR versus PSC TKA demonstrated no significant differences in opioid usage on POD0 (p = 0.765), POD1 (p = 0.747), POD2 (p = 0.564), POD3 (p = 0.309); VAS pain scores (p = 0.293); and 90-day readmission rate for pain (p > 0.9). CONCLUSION: Our analysis demonstrated no significant difference in post-operative VAS pain scores and MME usage based on implant. The results suggest that neither the type of articulation or constraint used for primary TKA has a significant impact on immediate post-operative pain and opioid consumption. LEVEL III EVIDENCE: Retrospective Cohort Study.

ModelMatcher: A scientist-centric online platform to facilitate collaborations between stakeholders of rare and undiagnosed disease research.

Next-generation sequencing is a prevalent diagnostic tool for undiagnosed diseases and has played a significant role in rare disease gene discovery. Although this technology resolves some cases, others are given a list of possibly damaging genetic variants necessitating functional studies. Productive collaborations between scientists, clinicians, and patients (affected individuals) can help resolve such medical mysteries and provide insights into in vivo function of human genes. Furthermore, facilitating interactions between scientists and research funders, including nonprofit organizations or commercial entities, can dramatically reduce the time to translate discoveries from bench to bedside. Several systems designed to connect clinicians and researchers with a shared gene of interest have been successful. However, these platforms exclude some stakeholders based on their role or geography. Here we describe ModelMatcher, a global online matchmaking tool designed to facilitate cross-disciplinary collaborations, especially between scientists and other stakeholders of rare and undiagnosed disease research. ModelMatcher is integrated into the Rare Diseases Models and Mechanisms Network and Matchmaker Exchange, allowing users to identify potential collaborators in other registries. This living database decreases the time from when a scientist or clinician is making discoveries regarding their genes of interest, to when they identify collaborators and sponsors to facilitate translational and therapeutic research.

An interdisciplinary approach to safe opioid prescribing and administration for surgical patients at an academic medical center.

AIM AND OBJECTIVES: To explore and understand the current practice, perceptions, and knowledge of general surgery trainees, advanced practice providers, and surgical nurses regarding opioid prescribing and administration. To this end, a novel opioid education and training was introduced to educate these practitioners on safe opioid practices in surgical patients. BACKGROUND: National awareness of the opioid epidemic has increased significantly in the last several years. However, there remain a disturbingly high number of opioid prescriptions written in the US indicating a need for improved provider and nurse education. This involves increasing awareness and understanding of national guidelines as well as implementing multi-modal therapy to treat pain. DESIGN: Pre-post-intervention quality improvement project. METHODS: An opioid education and training involving a morphine equivalent daily dosing calculator in the electronic medical record was provided to 26 surgical trainees, eight advanced practice providers and 97 surgical nurses in November 2019. Perceptions, current practice and knowledge were measured using a pre- and post-intervention survey (SQUIRE checklist). RESULTS: The survey results showed a positive clinical change in perception of opioid use in surgical patients following the intervention and a modest decrease in the average morphine equivalent daily dosing at discharge in general and transplant surgery patients. CONCLUSIONS: Effective pain management for surgical patients must be individualised. Safe opioid prescribing should involve an interdisciplinary approach with all members of the team undergoing assessment of their opioid knowledge and prescribing habits, easily accessible training tools and opioid calculators in the electronic medical record. RELEVANCE TO CLINICAL PRACTICE: Our initiative may provide useful information to settings that replicate use of a morphine equivalent daily dosing calculator in the electronic medical record. Utilisation of safe opioid prescribing tools in the electronic medical record and continuing education for providers and nurses can help ensure the safety of surgical patients.

Association between timing of kyphoplasty and opioid prescribing risk after vertebral fracture.

OBJECTIVE: Approximately 550,000 Americans experience vertebral fracture annually, and most receive opioids to treat the resulting pain. Kyphoplasty of the fractured vertebra is a procedural alternative that may mitigate risks of even short-term opioid use. While reports of kyphoplasty's impact on pain scores are mixed, no large-scale data exist regarding opioid prescribing before and after the procedure. This study was conducted to determine whether timing of kyphoplasty following vertebral fracture is associated with duration or intensity of opioid prescribing. METHODS: This retrospective cohort study used 2001-2014 insurance claims data from a single, large private insurer in the US across multiple care settings. Patients were adults with vertebral fractures who were prescribed opioids and underwent balloon-assisted kyphoplasty within 4 months of fracture. Opioid overdose risk was stratified by prescribed average daily morphine milligram equivalents using CDC guidelines. Filled prescriptions and risk categories were evaluated at baseline and 90 days following kyphoplasty. RESULTS: Inclusion criteria were met by 7119 patients (median age 77 years, 71.7% female). Among included patients, 3505 (49.2%) were opioid naïve before fracture. Of these patients, 31.1% had new persistent opioid prescribing beyond 90 days after kyphoplasty, and multivariable logistic regression identified kyphoplasty after 8 weeks as a predictor (OR 1.34, 95% CI 1.02-1.76). For patients previously receiving opioids, kyphoplasty > 4 weeks after fracture was associated with persistently elevated prescribing risk (OR 1.84, 95% CI 1.23-2.74). CONCLUSIONS: New persistent opioid prescribing occurred in nearly one-third of patients undergoing kyphoplasty after vertebral fracture, although early treatment was associated with a reduction in this risk. For patients not naïve to opioids before fracture diagnosis, early kyphoplasty was associated with less persistent elevation of opioid overdose risk. Subsequent trials must compare opioid use by vertebral fracture patients treated via operative (kyphoplasty) and nonoperative (ongoing opioid) strategies before concluding that kyphoplasty lacks value, and early referral for kyphoplasty may be appropriate to avoid missing a window of efficacy.

Maternal Overweight Downregulates MME (Neprilysin) in Feto-Placental Endothelial Cells and in Cord Blood.

Maternal overweight in pregnancy alters the metabolic environment and generates chronic low-grade inflammation. This affects fetal development and programs the offspring's health for developing cardiovascular and metabolic disease later in life. MME (membrane-metalloendopeptidase, neprilysin) cleaves various peptides regulating vascular tone. Endothelial cells express membrane-bound and soluble MME. In adults, the metabolic environment of overweight and obesity upregulates endothelial and circulating MME. We here hypothesized that maternal overweight increases MME in the feto-placental endothelium. We used primary feto-placental endothelial cells (fpEC) isolated from placentas after normal vs. overweight pregnancies and determined MME mRNA, protein, and release. Additionally, soluble cord blood MME was analyzed. The effect of oxygen and tumor necrosis factor α (TNFα) on MME protein in fpEC was investigated in vitro. Maternal overweight reduced MME mRNA (-39.9%, p < 0.05), protein (-42.5%, p = 0.02), and MME release from fpEC (-64.7%, p = 0.02). Both cellular and released MME protein negatively correlated with maternal pre-pregnancy BMI. Similarly, cord blood MME was negatively associated with pre-pregnancy BMI (r = -0.42, p = 0.02). However, hypoxia and TNFα, potential negative regulators of MME expression, did not affect MME protein. Reduction of MME protein in fpEC and in cord blood may alter the balance of vasoactive peptides. Our study highlights the fetal susceptibility to maternal metabolism and inflammatory state.

Membrane metallo-endopeptidase is dispensable for repair after nerve injury.

Membrane metallo-endopeptidase (MME), also known as neprilysin (NEP), has been of interest for its role in neurodegeneration and pain due to its ability to degrade ß-amyloid and substance-P, respectively. In addition to its role in the central nervous system, MME has been reported to be expressed in the peripheral system, specifically in the inner and outer border of myelinating fibers, in the Schmidt-Lantermann cleft and in the paranodes. Recently, mutations of this gene have been associated with Charcot-Marie-Tooth Type 2 (CMT2). Peripheral nerve morphometry in mice lacking MME previously showed minor abnormalities in aged animals in comparison to CMT2 patients. We found that MME expression was dysregulated after nerve injury in a Neuregulin-1 dependent fashion. We therefore explored the hypothesis that MME may have a role in remyelination. In the naïve state in adulthood we did not find any impairment in myelination in MME KO mice. After nerve injury the morphological outcome in MME KO mice was indistinguishable from WT littermates in terms of axon regeneration and remyelination. We did not find any difference in functional motor recovery. There was a significant difference in sensory function, with MME KO mice starting to recover response to mechanical stimuli earlier than WT. The epidermal reinnnervation, however, was unchanged and this altered sensitivity may relate to its known function in cleaving the peptide substance-P, known to sensitise nociceptors. In conclusion, although MME expression is dysregulated after nerve injury in a NRG1-dependent manner this gene is dispensable for axon regeneration and remyelination after injury.

Properties and functions of adipose tissue macrophages in obesity.

The expansion of adipose tissue (AT) in obesity is accompanied by the accumulation of immune cells that contribute to a state of low-grade, chronic inflammation and dysregulated metabolism. Adipose tissue macrophages (ATMs) represent the most abundant class of leukocytes in AT and are involved in the regulation of several regulatory physiological processes, such as tissue remodeling and insulin sensitivity. With progressive obesity, ATMs are key mediators of meta-inflammation, insulin resistance and impairment of adipocyte function. While macrophage recruitment from blood monocytes is a critical component of the generation of AT inflammation, new studies have revealed a role for ATM proliferation in the early stages of obesity and in sustaining AT inflammation. In addition, studies have revealed a more complex range of macrophage activation states than the previous M1/M2 model, and the existence of different macrophage profiles between human and animal models. This review will summarize the current understanding of the regulatory mechanisms of ATM function in relation to obesity, type 2 diabetes, depot of origin, and to other leukocytes such as AT dendritic cells, with hopes of emphasizing the regulatory nodes that can potentially be targeted to prevent and treat obesity-related metabolic disorders.

Preoperative depression, lumbar fusion, and opioid use: an assessment of postoperative prescription, quality, and economic outcomes.

OBJECTIVE Preoperative depression has been linked to a variety of adverse outcomes following lumbar fusion, including increased pain, disability, and 30-day readmission rates. The goal of the present study was to determine whether preoperative depression is associated with increased narcotic use following lumbar fusion. Moreover, the authors examined the association between preoperative depression and a variety of secondary quality indicator and economic outcomes, including complications, 30-day readmissions, revision surgeries, likelihood of discharge home, and 1- and 2-year costs. METHODS A retrospective analysis was conducted using a national longitudinal administrative database (MarketScan) containing diagnostic and reimbursement data on patients with a variety of private insurance providers and Medicare for the period from 2007 to 2014. Multivariable logistic and negative binomial regressions were performed to assess the relationship between preoperative depression and the primary postoperative opioid use outcomes while controlling for demographic, comorbidity, and preoperative prescription drug-use variables. Logistic and log-linear regressions were also used to evaluate the association between depression and the secondary outcomes of complications, 30-day readmissions, revisions, likelihood of discharge home, and 1- and 2-year costs. RESULTS The authors identified 60,597 patients who had undergone lumbar fusion and met the study inclusion criteria, 4985 of whom also had a preoperative diagnosis of depression and 21,905 of whom had a diagnosis of spondylolisthesis at the time of surgery. A preoperative depression diagnosis was associated with increased cumulative opioid use (ß = 0.25, p < 0.001), an increased risk of chronic use (OR 1.28, 95% CI 1.17-1.40), and a decreased probability of opioid cessation (OR 0.96, 95% CI 0.95-0.98) following lumbar fusion. In terms of secondary outcomes, preoperative depression was also associated with a slightly increased risk of complications (OR 1.14, 95% CI 1.03-1.25), revision fusions (OR 1.15, 95% CI 1.05-1.26), and 30-day readmissions (OR 1.19, 95% CI 1.04-1.36), although it was not significantly associated with the probability of discharge to home (OR 0.92, 95% CI 0.84-1.01). Preoperative depression also resulted in increased costs at 1 (ß = 0.06, p < 0.001) and 2 (ß = 0.09, p < 0.001) years postoperatively. CONCLUSIONS Although these findings must be interpreted in the context of the limitations inherent to retrospective studies utilizing administrative data, they provide additional evidence for the link between a preoperative diagnosis of depression and adverse outcomes, particularly increased opioid use, following lumbar fusion.

Improved glycaemia in high-fat-fed neprilysin-deficient mice is associated with reduced DPP-4 activity and increased active GLP-1 levels.

AIM/HYPOTHESIS: Neprilysin, a widely expressed peptidase, is upregulated in metabolically altered states such as obesity and type 2 diabetes. Like dipeptidyl peptidase-4 (DPP-4), neprilysin can degrade and inactivate the insulinotropic peptide glucagon-like peptide-1 (GLP-1). Thus, we investigated whether neprilysin deficiency enhances active GLP-1 levels and improves glycaemia in a mouse model of high fat feeding. METHODS: Nep +/+ and Nep -/- mice were fed a 60% fat diet for 16 weeks, after which active GLP-1 and DPP-4 activity levels were measured, as were glucose, insulin and C-peptide levels during an OGTT. Insulin sensitivity was assessed using an insulin tolerance test. RESULTS: High-fat-fed Nep -/- mice exhibited elevated active GLP-1 levels (5.8 ± 1.1 vs 3.5 ± 0.8 pmol/l, p < 0.05) in association with improved glucose tolerance, insulin sensitivity and beta cell function compared with high-fat-fed Nep +/+ mice. In addition, plasma DPP-4 activity was lower in high-fat-fed Nep -/- mice (7.4 ± 1.0 vs 10.7 ± 1.3 nmol ml-1 min-1, p < 0.05). No difference in insulin:C-peptide ratio was observed between Nep -/- and Nep +/+ mice, suggesting that improved glycaemia does not result from changes in insulin clearance. CONCLUSIONS/INTERPRETATION: Under conditions of increased dietary fat, an improved glycaemic status in neprilysin-deficient mice is associated with elevated active GLP-1 levels, reduced plasma DPP-4 activity and improved beta cell function. Thus, neprilysin inhibition may be a novel treatment strategy for type 2 diabetes.

A novel Drosophila mitochondrial carrier protein acts as a Mg(2+) exporter in fine-tuning mitochondrial Mg(2+) homeostasis.

The homeostasis of magnesium (Mg(2+)), an abundant divalent cation indispensable for many biological processes including mitochondrial functions, is underexplored. In yeast, the mitochondrial Mg(2+) homeostasis is accurately controlled through the combined effects of importers, Mrs2 and Lpe10, and an exporter, Mme1. However, little is known about this Mg(2+) homeostatic process in multicellular organisms. Here, we identified the first mitochondrial Mg(2+) transporter in Drosophila, the orthologue of yeast Mme1, dMme1, by homologous comparison and functional complementation. dMme1 can mediate the exportation of mitochondrial Mg(2+) when heterologously expressed in yeast. Altering the expression of dMme1, although only resulting in about a 10% change in mitochondrial Mg(2+) levels in either direction, led to a significant survival reduction in Drosophila. Furthermore, the reduced survival resulting from dMme1 expression changes could be completely rescued by feeding the dMME1-RNAi flies Mg(2+)-restricted food or the dMME1-over-expressing flies the Mg(2+)-supplemented diet. Our studies therefore identified the first Drosophila mitochondrial Mg(2+) exporter, which is involved in the precise control of mitochondrial Mg(2+) homeostasis to ensure an optimal state for survival.

The role of ACE2, angiotensin-(1-7) and Mas1 receptor axis in glucocorticoid-induced intrauterine growth restriction.

BACKGROUND: Plasma and urine levels of the potent vasodilator Ang-(1-7) are elevated in mid and late pregnancy and are correlated with elevated placental angiogenesis, fetal blood flow, and rapid fetal growth. We hypothesized that Ang-(1-7), its receptor (Mas1) and the enzymes involved in Ang-(1-7) production (ACE2 and Membrane metallo-endopeptidase; MME) are down regulated in response to glucocorticoid administration contributing to IUGR. METHODS: Pregnant female Sprague-Dawley rats were injected with dexamethasone (DEX; 0.4 mg/kg/day) starting from 14 day gestation (dg) till sacrifice at 19 or 21 dg while control groups were injected with saline (n = 6/group). The gene and protein expression of ACE2, MME, Ang-(1-7) and Mas1 receptor in the placental labyrinth (LZ) and basal zones (BZ) were studied. RESULTS: DEX administration caused a reduction in LZ weight at 19 and 21 dg (p < 0.001). IUGR, as shown by decreased fetal weights, was evident in DEX treated rats at 21 dg (p < 0.01). ACE2 gene expression was elevated in the LZ of control placentas at 21 dg (p < 0.01) compared to 19 dg and DEX prevented this rise at both gene (p < 0.01) and protein levels (p < 0.05). In addition, Ang-(1-7) protein expression in LZ was significantly reduced in DEX treated rats at 21 dg (p < 0.05). On the other hand, Mas1 and MME were upregulated in LZ at 21 dg in both groups (p < 0.05 and p < 0.001, respectively). CONCLUSION: The results of this study indicate that a reduced expression of ACE2 and Ang-(1-7) in the placenta by DEX treatment may be responsible for IUGR and consequent disease programming later in life.

A novel mitochondrial carrier protein Mme1 acts as a yeast mitochondrial magnesium exporter.

The homeostasis of magnesium (Mg2+), an abundant divalent cation indispensable for many biological processes including mitochondrial functions, is underexplored. Previously, two mitochondrial Mg2+ importers, Mrs2 and Lpe10, were characterized for mitochondrial Mg2+ uptake. We now show that mitochondrial Mg2+ homeostasis is accurately controlled through the combined effects of previously known importers and a novel exporter, Mme1 (mitochondrial magnesium exporter 1). Mme1 belongs to the mitochondrial carrier family and was isolated for its mutation that is able to suppress the mrs2Δ respiration defect. Deletion of MME1 significantly increased steady-state mitochondrial Mg2+ concentration, while overexpression decreased it. Measurements of Mg2+ exit from proteoliposomes reconstituted with purified Mme1 provided definite evidence for Mme1 as an Mg2+ exporter. Our studies identified, for the first time, a mitochondrial Mg2+ exporter that works together with mitochondrial importers to ensure the precise control of mitochondrial Mg2+ homeostasis.

Multiple mutualist effects on genomewide expression in the tripartite association between Medicago truncatula, nitrogen-fixing bacteria and mycorrhizal fungi.

While all species interact with multiple mutualists, the fitness consequences and molecular mechanisms underlying these interactions remain largely unknown. We combined factorial ecological experiments with genomewide expression analyses to examine the phenotypic and transcriptomic responses of model legume Medicago truncatula to rhizobia and mycorrhizal fungi. We found synergistic effects of these mutualists on plant performance and examined unique features of plant gene expression responses to multiple mutualists. There were genomewide signatures of mutualists and multiple mutualists on expression, with partners often affecting unique sets of genes. Mycorrhizal fungi had stronger effects on plant expression than rhizobia, with 70% of differentially expressed genes affected by fungi. Fungal and bacterial mutualists had joint effects on 10% of differentially expressed genes, including unexpected, nonadditive effects on some genes with important functions such as nutrient metabolism. For a subset of genes, interacting with multiple mutualists even led to reversals in the direction of expression (shifts from up to downregulation) compared to interacting with single mutualists. Rhizobia also affected the expression of several mycorrhizal genes, including those involved in nutrient transfer to host plants, indicating that partner species can also impact each other's molecular phenotypes. Collectively, these data illustrate the diverse molecular mechanisms and transcriptional responses associated with the synergistic benefits of multiple mutualists.

A genome-wide analysis in cluster headache points to neprilysin and PACAP receptor gene variants.

BACKGROUND: Cluster Headache (CH) is a severe primary headache, with a poorly understood pathophysiology. Complex genetic factors are likely to play a role in CH etiology; however, no confirmed gene associations have been identified. The aim of this study is to identify genetic variants influencing risk to CH and to explore the potential pathogenic mechanisms. METHODS: We have performed a genome-wide association study (GWAS) in a clinically well-defined cohort of 99 Italian patients with CH and in a control sample of 360 age-matched sigarette smoking healthy individuals, using the Infinium PsychArray (Illumina), which combines common highly-informative genome-wide tag SNPs and exonic SNPs. Genotype data were used to carry out a genome-wide single marker case-control association analysis using common SNPs, and a gene-based association analysis focussing on rare protein altering variants in 745 candidate genes with a putative role in CH. RESULTS: Although no single variant showed statistically significant association at the genome-wide threshold, we identified an interesting suggestive association (P = 9.1 × 10-6) with a common variant of the PACAP receptor gene (ADCYAP1R1). Furthermore, gene-based analysis provided significant evidence of association (P = 2.5 × 10-5) for a rare potentially damaging missense variant in the MME gene, encoding for the membrane metallo-endopeptidase neprilysin. CONCLUSIONS: Our study represents the first genome-wide association study of common SNPs and rare exonic variants influencing risk for CH. The most interesting results implicate ADCYAP1R1 and MME gene variants in CH susceptibility and point to a role for genes involved in pain processing. These findings provide new insights into the pathogenesis of CH that need further investigation and replication in larger CH samples.