Protocol for isolating amnion cells from human and non-human primate placenta for flow cytometry and transcriptomics.

The amnion is a thin layer of fetal origin in contact with the amniotic fluid which plays a key role at the feto-maternal interface during pregnancy. Here, we present a protocol for isolation of human and Rhesusmacaque amnion cells. We describe steps for tissue dissection, cell isolation for flow cytometry analysis, and RNA isolation for RNA sequencing library preparation and analysis. This protocol can provide insights into altered immunological pathways during intrauterine infections to develop new therapeutic strategies. For complete details on the use and execution of this protocol, please refer to Presicce et al.1.

Effect of Strength-Based Resilience on Patient's Length of Stay at the Renfrew Center for Eating Disorders.

Objective: Throughout past decades, physicians have sought to understand factors that contribute to severity of an eating disorder (ED). There is a potential relationship between patients' resilience and the recovery course of their disorder. The objective of this study is to examine the correlation between resilience, measured by indicators of mindfulness and restraint, and length of stay (LOS) at Renfrew Center for Eating Disorders. Materials and Methods: Data were obtained from Renfrew's database. Secondary analysis was conducted from this database. The database included women aged 13-75 years admitted to Renfrew. The database excluded males and individuals of ages <13 or >75 years old. Resilience was analyzed through Southampton Mindfulness Questionnaire (SMQ) and ED restraint. The dependent variable, LOS at Renfrew, was analyzed by multivariable linear regression, and multivariable logistic regression for LOS >45 days. Results: A sample of 2901 subjects was analyzed. There were significant associations between increased mindfulness scores and decreased restraint scores and a decreased LOS at Renfrew. For every 13-point increase in SMQ, the LOS was associated with a decrease by ∼1 day, and for every 1-point decrease in the restraint score (increased restraint), the LOS was associated with a decrease by ∼1 day. Higher restraint scores were independently associated with an increase in likelihood of LOS >45 days by 22.8%. Conclusions: The Renfrew data support the relationship between a patient's resilience and LOS. This information holds promise for future treatment approaches to improve strength-based modalities in patients suffering from EDs.

Lower resource utilization for patients with healed diabetic foot ulcers during participation in a prevention program with foot temperature monitoring.

INTRODUCTION: We assessed the impact of a diabetic foot ulcer prevention program incorporating once-daily foot temperature monitoring on hospitalizations, emergency department and outpatient visits, and rates of diabetic foot ulcer recurrence and lower extremity amputations for patients with recently healed foot ulcers. RESEARCH DESIGN AND METHODS: In this retrospective analysis of real-world data, we enrolled 80 participants with a healed diabetic foot ulcer in a year-long foot ulcer recurrence prevention program. Four outpatient centers within a large integrated healthcare system in the USA contributed to enrollment. We evaluated diabetic foot-related outcomes and associated resource utilization for participants during three periods: the 2 years before the program, the year during the program, and after the program ended. We reported unadjusted resource utilization rates during the program and the periods before and after it. We then adjusted rates of outcomes in each phase using an interrupted time series approach, explicitly controlling for overall trends in resource utilization and recurrence during the three periods. RESULTS: Our unadjusted data showed high initial rates of resource utilization and recurrence before enrollment in the program, followed by lower rates during the program, and higher rates of resource utilization and similar rates of recurrence in the period following the end of the program. The adjusted data showed lower rates of hospitalizations (relative risk reduction (RRR)=0.52; number needed to treat (NNT)=3.4), lower extremity amputations (RRR=0.71; NNT=6.4), and outpatient visits (RRR=0.26; absolute risk reduction (ARR)=3.5) during the program. We also found lower rates of foot ulcer recurrence during the program in the adjusted data, particularly for wounds with infection or greater than superficial depth (RRR=0.91; NNT=4.4). CONCLUSIONS: We observed lower rates of healthcare resource utilization for high-risk participants during enrollment in a diabetic foot prevention program incorporating once-daily foot temperature monitoring. TRIAL REGISTRATION NUMBER: NCT04345016.

Mental Health Service Use in Depressed Military Personnel: A Systematic Review.

INTRODUCTION: Major depression is a leading cause of morbidity in military personnel and an important impediment to operational readiness in military organizations. Although treatment options are available, a large proportion of individuals with depression do not access mental health services. Quantifying and closing this treatment gap is a public health priority. However, the scientific literature on the major depression treatment gap in military organizations has never been systematically reviewed. METHODS: We systematically searched the EMBASE, MEDLINE, and PsychINFO databases for studies measuring recent mental health service use in personnel serving in the armed forces of a Five-Eye country (Australia, Canada, New Zealand, the United Kingdom, or the United States). We excluded studies conducted with retired veterans. Because of the substantial heterogeneity in included studies, we did not pool their results. Instead, we computed median period prevalence of mental health service use. RESULTS: Twenty-eight studies were included in the systematic review; 12 had estimated mental health service use in personnel with depression, and another 16 had estimated mental health service use in personnel with depression or another mental health disorder. The period prevalence of mental health service use in depressed military personnel ranged from 20 to 75% in 12 included studies, with a median of 48%, over 2-12 months. The other 16 studies yielded similar conclusions; they reported period prevalence of mental health service use in personnel with any mental health disorder ranging from 14 to 75%, with a median of 36%, over 1-12 months. The median was higher in studies relying on diagnostic interviews to identify depressed personnel, compared to studies relying on screening tools (60% vs. 44%). CONCLUSIONS: There is a large treatment gap for major depression in particular, and for mental health disorders in general, among military personnel. However, our results highlight the association between the use of measurement tools and treatment gaps: estimated treatment gaps were larger when depressed patients were identified by screening tools instead of diagnostic interviews. Researchers should be wary of overestimating the mental health treatment gap when using screening tools in future studies.

Management of Osteomyelitis and Bone Loss in the Diabetic Charcot Foot and Ankle.

Bone loss and destruction due to diabetic Charcot neuroarthropathy (CN) and osteomyelitis of the foot and ankle is a challenging clinical condition when lower extremity preservation is considered. Resection and excision of osteomyelitis and associated nonviable soft tissue can lead into large osseous and soft tissue defects that will most likely need the utilization of bone grafting and subsequent arthrodesis for stability and anatomic alignment. In the diabetic population with peripheral neuropathy, osseous instability can lead to subsequent lower extremity deformity, ulceration, infection and/or amputation. This article reviews the surgical approach in the presence of diabetic CN and concomitant osteomyelitis.

Will Aerosol Hygroscopicity Change with Biodiesel, Renewable Diesel Fuels and Emission Control Technologies?

The use of biodiesel and renewable diesel fuels in compression ignition engines and aftertreatment technologies may affect vehicle exhaust emissions. In this study two 2012 light-duty vehicles equipped with direct injection diesel engines, diesel oxidation catalyst (DOC), diesel particulate filter (DPF), and selective catalytic reduction (SCR) were tested on a chassis dynamometer. One vehicle was tested over the Federal Test Procedure (FTP) cycle on seven biodiesel and renewable diesel fuel blends. Both vehicles were exercised over double Environmental Protection Agency (EPA) Highway fuel economy test (HWFET) cycles on ultralow sulfur diesel (ULSD) and a soy-based biodiesel blend to investigate the aerosol hygroscopicity during the regeneration of the DPF. Overall, the apparent hygroscopicity of emissions during nonregeneration events is consistently low (κ < 0.1) for all fuels over the FTP cycle. Aerosol emitted during filter regeneration is significantly more CCN active and hygroscopic; average κ values range from 0.242 to 0.439 and are as high as 0.843. Regardless of fuel, the current classification of "fresh" tailpipe emissions as nonhygroscopic remains true during nonregeneration operation. However, aftertreatment technologies such as DPF, will produce significantly more hygroscopic particles during regeneration. To our knowledge, this is the first study to show a significant enhancement of hygroscopic materials emitted during DPF regeneration of on-road diesel vehicles. As such, the contribution of regeneration emissions from a growing fleet of diesel vehicles will be important.

Medical Imaging in Differentiating the Diabetic Charcot Foot from Osteomyelitis.

Diabetic Charcot neuroarthropathy (DCN) poses a great challenge to diagnose in the early stages and when plain radiographs do not depict any initial signs of osseous fragmentation or dislocation in a setting of a high clinical index of suspicion. Medical imaging, including magnetic resonance imaging, computed tomography, and advanced bone scintigraphy, has its own unique clinical indications when treating the DCN with or without concomitant osteomyelitis. This article reviews different clinical case scenarios for choosing the most accurate medical imaging in differentiating DCN from osteomyelitis.

Circular External Fixation as a Primary or Adjunctive Therapy for the Podoplastic Approach of the Diabetic Charcot Foot.

Numerous techniques have been described for surgical management of the diabetic Charcot foot. External fixation has become a main surgical tool for the reconstructive foot and ankle surgeon when dealing with the ulcerated diabetic Charcot foot. In the presence of an open wound and/or osteomyelitis, staged reconstruction with circular external fixation becomes ideal for salvage of the diabetic lower extremity. Also, circular external fixation can provide simultaneous compression and stabilization, correct the underlying osseous or soft tissue deformities, and surgically offload the diabetic Charcot foot. This article describes a variety of circular external fixation applications for the diabetic Charcot foot.

Mutations in two pollen self-incompatibility factors in geographically marginal populations of Solanum habrochaites impact mating system transitions and reproductive isolation.

PREMISE OF THE STUDY: Self-incompatibility (SI) is a mechanism that prevents inbreeding in many plant species. The mutational breakdown of SI occurs frequently, yet relatively little is known about the evolutionary steps involved in the progressive loss of pistil and pollen SI function. METHODS: In Solanaceae, SI is the S-RNase-based gametophytic type. We used SI and SC populations of the wild tomato species Solanum habrochaites to study natural variation for two pollen SI factors: a Cullin1 (CUL1) protein and an S-locus F-box protein (SLF-23). Pollen compatibility was assessed on an allotriploid tester line encoding an S-RNase recognized by SLF-23. Both pollen factors are required for compatibility on this tester line. Complementation tests and gene sequencing were used to identify mutations in CUL1 or SLF-23. KEY RESULTS: We detected loss-of-function mutations in CUL1 and/or SLF-23 in SC populations collected near the northern and southern geographic margins of this taxon's natural range. Nonmarginal SC and all SI accessions expressed mostly functional alleles of these pollen factors. Comparison of the CUL1 sequences identified several shared deletion mutations present in both northern and southern margin SC accessions. CONCLUSIONS: Loss-of-function mutations in CUL1 and SLF-23 likely became fixed relatively late during SI to SC transitions, after loss of pistil SI function. Mutations in CUL1 establish unilateral incompatibility with SI populations and strengthen reproductive isolation. Point mutations common to northern and southern SC biotypes likely derive from shared ancestral variants found in more central SI populations.

Components of Particle Emissions from Light-Duty Spark-Ignition Vehicles with Varying Aromatic Content and Octane Rating in Gasoline.

Typical gasoline consists of varying concentrations of aromatic hydrocarbons and octane ratings. However, their impacts on particulate matter (PM) such as black carbon (BC) and water-soluble and insoluble particle compositions are not well-defined. This study tests seven 2012 model year vehicles, which include one port fuel injection (PFI) configured hybrid vehicle, one PFI vehicle, and six gasoline direct injection (GDI) vehicles. Each vehicle was driven on the Unified transient testing cycle (UC) using four different fuels. Three fuels had a constant octane rating of 87 with varied aromatic concentrations at 15%, 25%, and 35%. A fourth fuel with higher octane rating, 91, contained 35% aromatics. BC, PM mass, surface tension, and water-soluble organic mass (WSOM) fractions were measured. The water-insoluble mass (WIM) fraction of the vehicle emissions was estimated. Increasing fuel aromatic content increases BC emission factors (EFs) of transient cycles. BC concentrations were higher for the GDI vehicles than the PFI and hybrid vehicles, suggesting a potential climate impact for increased GDI vehicle production. Vehicle steady-state testing showed that the hygroscopicity of PM emissions at high speeds (70 mph; κ > 1) are much larger than emissions at low speeds (30 mph; κ < 0.1). Iso-paraffin content in the fuels was correlated to the decrease in WSOM emissions. Both aromatic content and vehicle speed increase the amount of hygroscopic material found in particle emissions.

Evaluating the Effects of Aromatics Content in Gasoline on Gaseous and Particulate Matter Emissions from SI-PFI and SIDI Vehicles.

We assessed the emissions response of a fleet of seven light-duty gasoline vehicles for gasoline fuel aromatic content while operating over the LA92 driving cycle. The test fleet consisted of model year 2012 vehicles equipped with spark-ignition (SI) and either port fuel injection (PFI) or direct injection (DI) technology. Three gasoline fuels were blended to meet a range of total aromatics targets (15%, 25%, and 35% by volume) while holding other fuel properties relatively constant within specified ranges, and a fourth fuel was formulated to meet a 35% by volume total aromatics target but with a higher octane number. Our results showed statistically significant increases in carbon monoxide, nonmethane hydrocarbon, particulate matter (PM) mass, particle number, and black carbon emissions with increasing aromatics content for all seven vehicles tested. Only one vehicle showed a statistically significant increase in total hydrocarbon emissions. The monoaromatic hydrocarbon species that were evaluated showed increases with increasing aromatic content in the fuel. Changes in fuel composition had no statistically significant effect on the emissions of nitrogen oxides (NOx), formaldehyde, or acetaldehyde. A good correlation was also found between the PM index and PM mass and number emissions for all vehicle/fuel combinations with the total aromatics group being a significant contributor to the total PM index followed by naphthalenes and indenes.

Chronically endurance-trained individuals preserve skeletal muscle mitochondrial gene expression with age but differences within age groups remain.

Maintenance of musculoskeletal function in older adults is critically important for preserving cardiorespiratory function and health span. Aerobic endurance training (ET) improves skeletal muscle metabolic function including age-related declines in muscle mitochondrial function. To further understand the underlying mechanism of enhanced muscle function with ET, we profiled the gene transcription (mRNA levels) patterns by gene array and determined the canonical pathways associated with skeletal muscle aging in a cross-sectional study involving vastus lateralis muscle biopsy samples of four subgroups (young and old, trained, and untrained). We first analyzed the sedentary individuals and then sought to identify the pathways impacted by long-term ET (>4 years) and determined the age effect. We found that skeletal muscle aging in older sedentary adults decreased mitochondrial genes and pathways involved in oxidative phosphorylation while elevating pathways in redox homeostasis. In older adults compared to their younger counterparts who chronically perform ET however, those differences were absent. ET did, however, impact nearly twice as many genes in younger compared to older participants including downregulation of gene transcripts involved in protein ubiquitination and the ERK/MAPK pathways. This study demonstrates that in individuals who are chronically endurance trained, the transcriptional profile is normalized for mitochondrial genes but aging impacts the number of genes that respond to ET including many involved in protein homeostasis and cellular stress.

Assessing the impacts of ethanol and isobutanol on gaseous and particulate emissions from flexible fuel vehicles.

This study investigated the effects of higher ethanol blends and an isobutanol blend on the criteria emissions, fuel economy, gaseous toxic pollutants, and particulate emissions from two flexible-fuel vehicles equipped with spark ignition engines, with one wall-guided direct injection and one port fuel injection configuration. Both vehicles were tested over triplicate Federal Test Procedure (FTP) and Unified Cycles (UC) using a chassis dynamometer. Emissions of nonmethane hydrocarbons (NMHC) and carbon monoxide (CO) showed some statistically significant reductions with higher alcohol fuels, while total hydrocarbons (THC) and nitrogen oxides (NOx) did not show strong fuel effects. Acetaldehyde emissions exhibited sharp increases with higher ethanol blends for both vehicles, whereas butyraldehyde emissions showed higher emissions for the butanol blend relative to the ethanol blends at a statistically significant level. Particulate matter (PM) mass, number, and soot mass emissions showed strong reductions with increasing alcohol content in gasoline. Particulate emissions were found to be clearly influenced by certain fuel parameters including oxygen content, hydrogen content, and aromatics content.

Changes in droplet surface tension affect the observed hygroscopicity of photochemically aged biomass burning aerosol.

This study examines the hygroscopic and surface tension properties as a function of photochemical aging of the aerosol emissions from biomass burning. Experiments were conducted in a chamber setting at the UC-Riverside Center for Environmental Research and Technology (CE-CERT) Atmospheric Processes Lab using two biomass fuel sources, manzanita and chamise. Cloud condensation nuclei (CCN) measurements and off-line filter sample analysis were conducted. The water-soluble organic carbon content and surface tension of the extracted filter samples were measured. Surface tension information was then examined with Köhler theory analysis to calculate the hygroscopicity parameter, κ. Laboratory measurement of biomass burning smoke from two chaparral fuels is shown to depress the surface tension of water by 30% or more at organic matter concentrations relevant at droplet activation. Accounting for surface tension depression can lower the calculated κ by a factor of 2. This work provides evidence for surface tension depression in an important aerosol system and may provide closure for differing sub- and supersaturated κ measurements.

A change of heart: case series of peripartum cardiomyopathy.

Peripartum cardiomyopathy (PPCM) is an uncommon disease of pregnancy, occurring in about 1 in 2000 live births, and is characterized by the development of heart failure, due to left ventricular systolic dysfunction. It is associated with high rates of maternal and neonatal mortality. Cardiac disease is the leading cause of maternal death in the UK: PPCM accounts for about 17% of these. Clinical findings of decompensated heart failure (HF) are often masked by the normal physiological changes seen in pregnancy making the diagnosis challenging. A high index of suspicion is essential-prompting referral for echocardiogram, which is crucial for diagnosis. Favourable prognosis is dependent on the early initiation of HF medications. Although full recovery occurs in around half of cases, left ventricular systolic dysfunction persists in a significant proportion of patients with PPCM and the risk of recurrence in subsequent pregnancies is high. The pathophysiology of PPCM is under intense research. We present four patients with PPCM and a review of the literature. Owing to the diagnostic challenge of PPCM and decompensated HF in pregnant mothers and its high mortality rate without treatment, prompt investigation and referral are key to improving maternal survival.

Tipping the balance: the pathophysiology of obesity and type 2 diabetes mellitus.

Obesity plays a major role in the development of type 2 diabetes mellitus, and it has long been accepted that weight loss plays a significant role in diabetes therapy. This weight loss has traditionally been accomplished through lifestyle changes including diet and exercise. What has only more recently gained acceptance is that bariatric surgery may have a role to play in diabetes therapy as well. This article discusses the pathophysiology of type 2 diabetes mellitus and obesity and provides a basic understanding of these diseases, which forms the basis for understanding the importance of weight loss in their treatment.

Endurance exercise as a countermeasure for aging.

OBJECTIVE: We determined whether reduced insulin sensitivity, mitochondrial dysfunction, and other age-related dysfunctions are inevitable consequences of aging or secondary to physical inactivity. RESEARCH DESIGN AND METHODS: Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp and ATP production in mitochondria isolated from vastus lateralis biopsies of 42 healthy sedentary and endurance-trained young (18-30 years old) and older (59-76 years old) subjects. Expression of proteins involved in fuel metabolism was measured by mass spectrometry. Citrate synthase activity, mitochondrial DNA (mtDNA) abundance, and expression of nuclear-encoded transcription factors for mitochondrial biogenesis were measured. SIRT3, a mitochondrial sirtuin linked to lifespan-enhancing effects of caloric restriction, was measured by immunoblot. RESULTS: Insulin-induced glucose disposal and suppression of endogenous glucose production were higher in the trained young and older subjects, but no age effect was noted. Age-related decline in mitochondrial oxidative capacity was absent in endurance-trained individuals. Although endurance-trained individuals exhibited higher expression of mitochondrial proteins, mtDNA, and mitochondrial transcription factors, there were persisting effects of age. SIRT3 expression was lower with age in sedentary but equally elevated regardless of age in endurance-trained individuals. CONCLUSIONS: The results demonstrate that reduced insulin sensitivity is likely related to changes in adiposity and to physical inactivity rather than being an inevitable consequence of aging. The results also show that regular endurance exercise partly normalizes age-related mitochondrial dysfunction, although there are persisting effects of age on mtDNA abundance and expression of nuclear transcription factors and mitochondrial protein. Furthermore, exercise may promote longevity through pathways common to effects of caloric restriction.

Effect of age on cutaneous vasoconstrictor responses to norepinephrine in humans.

To test the hypothesis that cutaneous vasoconstrictor responsiveness to exogenous norepinephrine is reduced in older compared with young subjects, dose-response relations between norepinephrine and skin blood flow were established. Seven doses of norepinephrine (1.10(-8) to 10(-2) log M) were perfused (2 microl/min) intradermally (4 min/dose) using cutaneous microdialysis (2 probes/subject). To account for possible differences in endogenous norepinephrine between groups, one microdialysis probe was perfused with bretylium tosylate to locally block noradrenergic vesicle release before establishing the norepinephrine dose-response relations. Skin blood flow was indexed via laser-Doppler flowmetry directly over both microdialysis probe sites and is expressed as cutaneous vascular conductance (laser-Doppler flux/mean arterial blood pressure). Local skin temperature was maintained at 34 degrees C at both sites throughout the protocol. Dose-response relation between norepinephrine and cutaneous vascular conductance was similar between control and bretylium-pretreated sites in young subjects (EC50 = -5.18 +/- 0.27 and -5.03 +/- 0.27 log M, respectively). In contrast, the dose-response relation was significantly shifted to the right (i.e., a higher dose of norepinephrine was needed to produce the same vasoconstrictor response) in the bretylium-pretreated site in older subjects (EC50 = -5.46 +/- 0.23 and -4.53 +/- 0.23 log M, respectively). Significant increases in EC50 were observed in older compared with young subjects at the bretylium-pretreated but not the control sites. These data indicate that cutaneous vasoconstrictor responsiveness is decreased in older subjects when endogenous release of norepinephrine is antagonized. Furthermore, these findings suggest that differences in presynaptic norepinephrine release between older and younger subjects are profound enough to affect dose-response relations between norepinephrine and cutaneous vascular conductance.