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Introduction: Individuals with focal segmental glomerular sclerosis (FSGS) typically undergo kidney biopsy only once, which limits the ability to characterize kidney cell gene expression over time. Methods: We used single-cell RNA sequencing (scRNA-seq) to explore disease-related molecular signatures in urine cells from subjects with FSGS. We collected 17 urine samples from 12 FSGS subjects and captured these as 23 urine cell samples. The inflammatory signatures from renal epithelial and immune cells were evaluated in bulk gene expression data sets of FSGS and minimal change disease (MCD) (The Nephrotic Syndrome Study Network [NEPTUNE] study) and an immune single-cell data set from lupus nephritis (Accelerating Medicines Partnership). Results: We identified immune cells, predominantly monocytes, and renal epithelial cells in the urine. Further analysis revealed 2 monocyte subtypes consistent with M1 and M2 monocytes. Shed podocytes in the urine had high expression of marker genes for epithelial-to-mesenchymal transition (EMT). We selected the 16 most highly expressed genes from urine immune cells and 10 most highly expressed EMT genes from urine podocytes as immune signatures and EMT signatures, respectively. Using kidney biopsy transcriptomic data from NEPTUNE, we found that urine cell immune signature and EMT signature genes were more highly expressed in FSGS biopsies compared with MCD biopsies. Conclusion: The identification of monocyte subsets and podocyte expression signatures in the urine samples of subjects with FSGS suggests that urine cell profiling might serve as a diagnostic and prognostic tool in nephrotic syndrome. Furthermore, this approach may aid in the development of novel biomarkers and identifying personalized therapies targeting particular molecular pathways in immune cells and podocytes.

Human papillomavirus (HPV) causes virtually all cervical cancers and many cancers at other anatomical sites in both men and women. However, only 12 of 448 known HPV types are currently classified as carcinogens, and even the most carcinogenic type — HPV16 — only rarely leads to cancer. HPV is therefore necessary but insufficient for cervical cancer, with other contributing factors including host and viral genetics. Over the last decade, HPV whole genome sequencing has established that even fine-scale within-type HPV variation influences precancer/cancer risks, and that these risks vary by histology and host race/ethnicity. In this review, we place these findings in the context of the HPV life cycle and evolution at various levels of viral diversity: between-type, within-type, and within-host. We also discuss key concepts necessary for interpreting HPV genomic data, including features of the viral genome; events leading to carcinogenesis; the role of APOBEC3 in HPV infection and evolution; and methodologies that use deep (high-coverage) sequencing to characterize within-host variation, as opposed to relying on a single representative (consensus) sequence. Given the continued high burden of HPV-associated cancers, understanding HPV carcinogenicity remains important for better understanding, preventing, and treating cancers attributable to infection.

Objective To determine the cost of achieving a live birth after first transfer using highly purified human menotropin (HP-hMG) or recombinant follicle-stimulating hormone (FSH) for controlled ovarian stimulation in predicted high-responder patients in the Menopur in Gonadotropin-releasing hormone Antagonist Single Embryo Transfer–High Responder (MEGASET-HR) trial. Design Cost minimization analysis of trial results. Setting Thirty-one fertility centers. Patient(s) Six hundred and nineteen women with serum antimullerian hormone ≥5 ng/mL. Intervention(s) Controlled ovarian stimulation with HP-hMG or recombinant FSH in a gonadotropin-releasing hormone (GnRH) antagonist assisted reproduction cycle where fresh transfer of a single blastocyst was performed unless ovarian response was excessive whereupon all embryos were cryopreserved and patients could undergo subsequent frozen blastocyst transfer within 6 months of randomization. Main Outcome Measure(s) Mean cost of achieving live birth after first transfer (fresh or frozen). Result(s) First-transfer efficacy, defined as live birth after first fresh or frozen transfer, was 54.5% for HP-hMG and 48.0% for recombinant FSH (difference 6.5%). Average cost to achieve a live birth after first transfer (fresh or frozen) was lower with HP-hMG compared with recombinant FSH. For fresh transfers, the cost was lower with HP-hMG compared with recombinant FSH. The average cost to achieve a live birth after first frozen transfer was also lower in patients treated with HP-hMG compared with recombinant FSH. Conclusion(s) Treatment of predicted high-responders with HP-hMG was associated with lower cost to achieve a live birth after first transfer compared with recombinant FSH. Clinical Trial Registration Number NCT02554279.

Objective: Elimination of food calories as heat could help redress the excess accumulation of metabolic energy exhibited as obesity. Prior studies have focused on the induction of thermogenesis in beige and brown adipose tissues as the application of this principle, particularly because the β-adrenergic environment associated with thermogenic activation has been shown to have positive health implications. The counterpoint to this strategy is the regulation of heat loss; we propose that mammals with inefficient heat conservation will require more thermogenesis to maintain body temperature. Methods: Surface temperature thermography and rates of trans-epidermal water loss were integrated to profile the total heat transfer of genetically-engineered and genetically variable mice. Results: These data were incorporated with energy expenditure data to generate a biophysical profile to test the significance of increased rates of evaporative cooling. Conclusions: We show that mouse skins vary considerably in their heat retention properties, whether because of naturally occurring variation (SKH-1 mice), or genetic modification of the heat-retaining lipid lamellae (SCD1, DGAT1 or Agouti Ay obese mice). In particular, we turn attention to widely different rates of evaporative cooling as the result of trans-epidermal water loss; higher rates of heat loss by evaporative cooling leads to increased demand for thermogenesis. We speculate that this physiology could be harnessed to create an energy sink to assist with strategies aimed at treating metabolic diseases. Keywords: Trans-epidermal water loss, Evaporative cooling, Epidermal barrier, Syndecan-1, Obesity, Mouse skin, Thermogenesis, Dermal white adipose tissue, Energy expenditure, Brown adipose tissue

Recycling of wastewater provides a substantial solution to the global issue of water scarcity and high water use in aquaculture. However, this sustainable way of wastewater use has not been given much attention and exploration. This study focused on the consumer preference for fish grown in treated wastewater as well as the effect of aeration on the growth performance and economic benefit of African catfish (Clarias gariepinus) grown in treated wastewater. Two hundred (200) respondents from two communities (Chirapatre and Gyinyase) near the wastewater treatment plant in Kumasi were interviewed to determine their willingness to accept and pay for African catfish grown in treated wastewater. For the growth trial, a total of 600 fish (of average initial weight 39.12g) were stocked in two maturation ponds with 4 h (3:00am–7:00am) of aeration daily. The trial lasted for 12 weeks and variables monitored included the survival, growth performance (weight gain, specific growth rate, and yield) and water quality. Fish cultured in non-aerated wastewater ponds (NWFPs) under similar conditions as in aerated wastewater-fed ponds (AWFPs) served as control. The results indicated most important considerations for consumers in their choice of fish to consume were in order of importance; food safety, freshness of fish, taste and packaging. The proximity of consumers to the treatment plant, the price of fish, religion, and age and whether or not they were fish consumers affected their willingness to pay for African catfish grown in the treated wastewater significantly. For the growth trial, dissolved oxygen concentrations in the aerated ponds were significantly higher than in the NWFPs and this led to more than a doubling of the growth rates in the African catfish grown in the AWFPs (189.10g ± 11.32) as compared to the NWFPs (90.70g ± 11.59). The pond aeration improved fish growth significantly (p < 0.0098). On economic benefit, the aerated system yielded profits of 618.83 (€103.13) as compared to a loss of 104.99 (€17.50), which was incurred in the non-aerated ponds. Education of the consumers on the process of wastewater treatment and establishment of food safety guidelines will therefore be recommended to increase consumer interest in consuming fish from the treated wastewater., wastewater, African catfish, wastewater ponds

Black Americans are at increased risk for preeclampsia. Genetic variants in apolipoprotein L1 (APOL1) account for much of the increased risk for kidney disease in blacks. APOL1 is expressed in human placenta and transgenic mice expressing APOL1 develop preeclampsia. We evaluated the role of APOL1 variants in human preeclampsia. We determined maternal and fetal APOL1 genotypes in black women with preeclampsia in two populations. At Einstein Montefiore Center (EMC) Affiliated Hospitals, we studied 121 pregnancies in black women with preeclampsia. At University of Tennessee Health Science Center (UTHSC), we studied 93 pregnancies in black women with preeclampsia and 793 pregnancies without preeclampsia. We measured serum markers of preeclampsia soluble fms-like tyrosine kinase 1 (sFlt-1), placental growth factor (PlGF), and soluble endoglin (sEng). Fetal APOL1 high-risk (HR) genotype was associated with preeclampsia, with odds ratios at EMC and UTHSC of 1.84 (95% CI 1.11, 2.93) and 1.92 (95% CI 1.05, 3.49), respectively. Maternal APOL1 HR genotype was not associated with preeclampsia. Mothers with the fetal APOL1 HR genotype had more cerebral or visual disturbances (63% versus 37%, p = 0.04). In addition, fetal APOL1 HR genotype was associated with a higher sFLT-1/PlGF ratio at birth (p = 0.04). Fetal APOL1 high-risk genotype increases the risk for preeclampsia, likely by adversely affecting placental function. Further research is needed to assess whether APOL1 genetic testing can predict preeclampsia and improve pregnancy outcomes.

PAK6 is a Group II p21 activated kinase that unlike traditional signal transduction proteins interacts with multiple binding partners including sex-steroid receptors. PAK6 acts as a nodal checkpoint integrating multiple cellular inputs to promote distinct cellular outcomes, some of which are associated with cytoskeletal remodeling. Despite the possibility that PAK6 may couple sex-specific neuronal function and therefore serve as a valuable research, diagnostic and therapeutic target, there is currently no standardized protocol for assessing PAK6 activity in a neuronal cell line. Here, we present a protocol for assessing PAK6 levels in a commonly used neuronal cell line, PC-12. In comparison with other methodology, this approach (1) does not require ex-planted tissue to identify PAK6 in neurons and (2) unlike other protocols which require steroid depleted media for detection of PAK6 in non-neuronal cell lines, such as prostate cancer cell lines, we were easily able to detect PAK6 in PC-12 cells grown in complete, steroid-containing media. Thus the present protocol allows for the efficient detection of native PAK6 in PC-12 cells to expedite targeted basic research of the emerging importance of PAK6 function in the brain as well as to accelerate the identification and isolation of potential therapeutic targets not only in cancerous but brain disease states as well.

The apoC-III proteoform containing two sialic acid residues (apoC-III2) has different in vitro effects on lipid metabolism compared with asialylated (apoC-III0) or the most abundant monosialylated (apoC-III1) proteoforms. Cross-sectional and longitudinal associations between plasma apoC-III proteoforms (by mass spectrometric immunoassay) and plasma lipids were tested in two randomized clinical trials: ACT NOW, a study of pioglitazone in subjects with impaired glucose tolerance (n = 531), and RACED (n = 296), a study of intensive glycemic control and atherosclerosis in type 2 diabetes patients. At baseline, higher relative apoC-III2 and apoC-III2/apoC-III1 ratios were associated with lower triglycerides and total cholesterol in both cohorts, and with lower small dense LDL in the RACED. Longitudinally, changes in apoC-III2/apoC-III1 were inversely associated with changes in triglycerides in both cohorts, and with total and small dense LDL in the RACED. apoC-III2/apoC-III1 was also higher in patients treated with PPAR-γ agonists and was associated with reduced cardiovascular events in the RACED control group. Ex vivo studies of apoC-III complexes with higher apoC-III2/apoC-III1 showed attenuated inhibition of VLDL uptake by HepG2 cells and LPL-mediated lipolysis, providing possible functional explanations for the inverse association between a higher apoC-III2/apoC-III1 and hypertriglyceridemia, proatherogenic plasma lipid profiles, and cardiovascular risk.

Infections due to Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) are often mild and self-limiting, but more severe cases can develop into hemolytic uremic syndrome (HUS) and hemorrhagic colitis. This case report documents a sporadic case of ischemic colitis likely triggered by EHEC but without manifestations of hemolytic uremic syndrome. From our literature review, we identified only one other case of an EHEC infection presenting as ischemic colitis in the absence of HUS. To our knowledge, this is the first case presentation of EHEC-induced ischemic colitis which did not lead to significant morbidity.

The intestine plays a prominent role in the biosynthesis of triacylglycerol (triglyceride; TAG). Digested dietary TAG is repackaged in the intestine to form the hydrophobic core of chylomicrons, which deliver metabolic fuels, essential fatty acids, and other lipid-soluble nutrients to the peripheral tissues. By controlling the flux of dietary fat into the circulation, intestinal TAG synthesis can greatly impact systemic metabolism. Genes encoding many of the enzymes involved in TAG synthesis have been identified. Among TAG synthesis enzymes, acyl-CoA:monoacylglycerol acyltransferase 2 and acyl-CoA:diacylglycerol acyltransferase (DGAT)1 are highly expressed in the intestine. Their physiological functions have been examined in the context of whole organisms using genetically engineered mice and, in the case of DGAT1, specific inhibitors. An emerging theme from recent findings is that limiting the rate of TAG synthesis in the intestine can modulate gut hormone secretion, lipid metabolism, and systemic energy balance. The underlying mechanisms and their implications for humans are yet to be explored. Pharmacological inhibition of TAG hydrolysis in the intestinal lumen has been employed to combat obesity and associated disorders with modest efficacy and unwanted side effects. The therapeutic potential of inhibiting specific enzymes involved in intestinal TAG synthesis warrants further investigation.

Acyl-CoA:monoacylglycerol acyltransferase (MGAT) 2 catalyzes triacylglycerol (TAG) synthesis, required in intestinal fat absorption. We previously demonstrated that mice without a functional MGAT2-coding gene (Mogat2(-/-)) exhibit increased energy expenditure and resistance to obesity induced by excess calories. One critical question raised is whether lacking MGAT2 during early development is required for the metabolic phenotypes in adult mice. In this study, we found that Mogat2(-/-) pups grew slower than wild-type littermates during the suckling period. To determine whether inactivating MGAT2 in adult mice is sufficient to confer resistance to diet-induced obesity, we generated mice with an inducible Mogat2-inactivating mutation. Mice with adult-onset MGAT2 deficiency (Mogat2(AKO)) exhibited a transient decrease in food intake like Mogat2(-/-) mice when fed a high-fat diet and a moderate increase in energy expenditure after acclimatization. They gained less weight than littermate controls, but the difference was smaller than that between wild-type and Mogat2(-/-) mice. The moderate reduction in weight gain was associated with reduced hepatic TAG and improved glucose tolerance. Similar protective effects were also observed in mice that had gained weight on a high-fat diet before inactivating MGAT2. These findings suggest that adult-onset MGAT2 deficiency mitigates metabolic disorders induced by high-fat feeding and that MGAT2 modulates early postnatal nutrition and may program metabolism later in life.

Bacteriophage T4 encodes orthologs of the proteins Rad50 (gp46) and Mre11 (gp47), which form a heterotetrameric complex (MR) that is responsible for host genome degradation and the processing of DNA ends for recombination-dependent DNA repair. In this chapter, we describe the ensemble methods currently employed by our laboratory to characterize the exonuclease activity of the T4 MR complex. DNA exonucleases play a vital role in maintaining the integrity of DNA through their participation in DNA repair pathways and as proofreaders for DNA polymerases. Methods for quantifying the general features of the exonuclease, and for determining steady-state kinetic parameters (Km, kcat), the polarity of exonuclease activity, and processivity are presented. These methods should be applicable to all DNA exonucleases, and to some extent endonucleases.

The enormous shortage of health workers in sub-Saharan Africa (SSA) is a major contributor to the unacceptably high rates of morbidity and mortality in the region. This is especially true for patients whose illnesses and injuries require time-sensitive interventions. To address the crisis, a number of countries have utilized "task-shifting" in various health disciplines where they call upon other cadres, often nurses, to assume new roles and responsibilities that are not traditionally within their scope of practice. This practice has been shown to increase access, to be cost-effective and of high-quality. A literature review was undertaken to better understand the implications of task-shifting on emergency medical care in Africa. This review demonstrates that, while task-shifting has been used effectively for specific emergency procedures in specialty fields such as obstetrics and surgery, to date there are no studies on the use of task-shifting to treat the acute, undifferentiated patient in SSA. Task shifting is a potential solution to help address the very limited access to emergency care across SSA, but requires further study to ensure effective implementation.

Introduction In June of 2008, Karoli Lwanga ("Nyakibale") Hospital and Global Emergency Care Collaborative (GECC) opened the first functional Emergency Centre (EC) in rural Uganda. GECC is developing a training programme for a new cadre of midlevel Emergency Care Practitioners (ECPs), to increase access to quality emergency care. In order to determine the skills and resources needed, the unique practice demographics and the feasibility of treating patients in this setting must be understood. Methods A descriptive cross-sectional analysis of the first 500 consecutive patient visits in the EC's patient care log was reviewed. Data on demographics, procedures performed, laboratory testing, bedside ultrasounds (USs) performed, radiographs (XRs) ordered, diagnoses, condition upon discharge and disposition were collated. Descriptive statistics were performed. Results Of the first 500 patient visits, there were 275 (55%) male visits and 132 (26.4%) visits for children under five. Procedures were performed in 367 (73.4%) patients. Laboratory testing, XRs and USs were performed in 188 (37.6%), 99 (19.8%) and 45 (7%) patients, respectively. Infectious diseases were diagnosed in 217 (43.4%) patients; traumatic injuries in 140 (28%) patients. Only one patient expired in the ED, and 401 (80.2%) were in good condition after treatment. One person was transferred to another hospital. After treatment, 180 (36%) patients were discharged home. Only five (1.0%) patients went directly to the operating theatre. Conclusions This pilot study describes the patient population, resource and training needs of a rural Emergency Centre in SSA. It demonstrates that acute care providers will be required to evaluate a wide variety of patient complaints, effectively utilise laboratory and radiologic testing, and perform numerous focused treatments and therapies. Specialised training programmes, such as GECC's ECP programme, are needed to create providers able to provide high quality, lifesaving care.

Collection and monitoring of specific indicators may enable dynamic and proactive risk assessment. In fact, such collection, improved by today's advanced information technology systems, may provide real-time information on the overall risk variation. Moreover, monitoring human and organizational factors may allow prevention of potential underlying causes of accidents. Four classes of proactive approaches for dynamic risk assessment are presented in this chapter. They represent four levels of connection to the overall risk picture, from connection assumed on the basis of past accident analysis to dynamic aggregation methodologies. The risk barometer technique is presented as representative example of the latter. Its attention to human and organizational factors is demonstrated not only by real-time collection of specific indicators but also by its capability to support key safety decision-makers in short- and medium-term planning.

Novel mass spectrometric immunoassays (MSIAs) for the isolation and structural characterization of plasma apolipoprotein A-I (apoA-I), apoA-II, and apoE have been developed. The assays combine selective isolation of apolipoprotein species via affinity capture with mass-specific detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In application, plasma (from 50 microl of whole blood drawn from individuals, using finger lancet) was addressed with affinity pipette tips derivatized with antibodies toward the specific apolipoprotein. The time required for each assay was approximately 15 min, less if assays on multiple individuals were performed in parallel. In a brief study of five individuals, several recently reported apoA-II variants were identified and observed consistently in all individuals. Additionally, the apoE phenotype of E3/E3 was observed in three of the individuals, and E2/E3 and E3/E4 observed in the remaining two individuals, the latter of whom suffers from Alzheimer's disease. Overall, the MSIA approach offers a rapid, sensitive, and highly accurate means of profiling apolipoproteins from small volumes of plasma.

Publisher Summary Biomolecular interaction analysis mass spectrometry (BIA/MS) is a multidimensional methodology for functional and structural protein analysis. Proteins are affinity captured and quantified from a solution via ligands covalently attached on the SPR sensor surface. Because the SPR detection is nondestructive, proteins retrieved on the SPR sensing surface can be further analyzed via mass spectrometry, either directly from the sensor/chip surface or separately, following elution and microrecovery. Detection of attomole amounts of proteins from complex biological mixtures is possible via the combined SPR-MS approach. A chip cutter with a circular heated cutter head is used for excising a chip or plastic mount of a defined circular shape that fits into an appropriately configured MALDI mass spectrometer target. The MALDI matrix is applied to the chip via an aerosol-spraying device. MALDI-TOF mass spectrometry analysis from the chip surface is performed on a custom-made MALDITOF mass spectrometer. The instrument consists of a linear translation stage or ion source capable of precise targeting of each of the flow cells under a focused laser spot.

The increase of n-6 polyunsaturated fatty acids (PUFA) in animal tissues has been proposed as a mechanism of lead (Pb) poisoning through lipid peroxidation or altered eicosanoids metabolism. We have studied fatty acid (FA) composition in liver and brain of mallards (Anas platyrhynchos) feeding for 3 weeks on diets containing combinations of low or high levels of vitamin E (20 or 200 UI/kg) and Pb (0 or 2 g/kg). Saturated FA, n-6 PUFA and total concentrations of FA were higher in livers of Pb-exposed mallards, but not in their brains. The percentage of n-6 PUFA in liver and brain was slightly higher in Pb-exposed mallards. The increase of n-6 PUFA in liver was associated with decreased triglycerides and increased cholesterol in plasma, thus could be in part attributed to feed refusal and fat mobilization. The hepatic ratios between adrenic acid (22:4 n-6) and arachidonic acid (20:4 n-6) or between adrenic acid and linoleic acid (18:2 n-6) were higher in Pb exposed birds, supporting the existing hypothesis of increased fatty acid elongation by Pb. Among the possible consequences of increased n-6 PUFA concentration in tissues, we found increased lipid peroxidation in liver without important histopathological changes, and decreased plasma alkaline phosphatase activity that may reflect altered bone metabolism in birds.

Publisher Summary Biomolecular interaction analysis (BIA) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) are capable of the accurate characterization of biomolecules with extreme speed and sensitivity. Although the two analytical approaches operate on mutually exclusive detection principles (either surface plasmon resonance detection of a refractive index change or the physical determination the molecular mass of a gas-phase ion), they can share a common denominator—the use of affinity interactions in selecting the analyte. Interfacing of the two, thereby creates a unique approach for the investigation of the kinetic parameters of biomolecular interaction (using BIA), and the unambiguous confirmation of the presence of targeted affinity ligands by direct mass analysis. In more recent times, new mass spectrometric approaches for the rapid, sensitive, and accurate characterization of proteins are in development. This chapter discusses some of the findings on the interfacing of biomolecular interaction analysis with mass spectrometry, and the use of enzymatically active—or bioreactive—mass spectrometer probe tips in the characterization of analytes. The use of bioreactive mass spectrometer probe tips to serially digest myoglobin is discussed in the chapter. The object of the serial digestion is to simultaneously view the relative stability of molecule fragments of myoglobin (generated during an initial, limited digestion of the myoglobin under denaturing conditions using pepsin-active tips at low pH), by exposing the fragment set to extensive digestion (using trypsin tips) under renaturing conditions.