IL-10 Signaling Remodels Adipose Chromatin Architecture to Limit Thermogenesis and Energy Expenditure.

Signaling pathways that promote adipose tissue thermogenesis are well characterized, but the limiters of energy expenditure are largely unknown. Here, we show that ablation of the anti-inflammatory cytokine IL-10 improves insulin sensitivity, protects against diet-induced obesity, and elicits the browning of white adipose tissue. Mechanistic studies define bone marrow cells as the source of the IL-10 signal and adipocytes as the target cell type mediating these effects. IL-10 receptor alpha is highly enriched in mature adipocytes and is induced in response to differentiation, obesity, and aging. Assay for transposase-accessible chromatin sequencing (ATAC-seq), ChIP-seq, and RNA-seq reveal that IL-10 represses the transcription of thermogenic genes in adipocytes by altering chromatin accessibility and inhibiting ATF and C/EBPß recruitment to key enhancer regions. These findings expand our understanding of the relationship between inflammatory signaling pathways and adipose tissue function and provide insight into the physiological control of thermogenesis that could inform future therapy.

Aster Proteins Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Mammalian Cells.

The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.

CLSTN3ß enforces adipocyte multilocularity to facilitate lipid utilization.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.

Progerin forms an abnormal meshwork and has a dominant-negative effect on the nuclear lamina.

Progerin, the protein that causes Hutchinson-Gilford progeria syndrome, triggers nuclear membrane (NM) ruptures and blebs, but the mechanisms are unclear. We suspected that the expression of progerin changes the overall structure of the nuclear lamina. High-resolution microscopy of smooth muscle cells (SMCs) revealed that lamin A and lamin B1 form independent meshworks with uniformly spaced openings (~0.085 µm2). The expression of progerin in SMCs resulted in the formation of an irregular meshwork with clusters of large openings (up to 1.4 µm2). The expression of progerin acted in a dominant-negative fashion to disrupt the morphology of the endogenous lamin B1 meshwork, triggering irregularities and large openings that closely resembled the irregularities and openings in the progerin meshwork. These abnormal meshworks were strongly associated with NM ruptures and blebs. Of note, the progerin meshwork was markedly abnormal in nuclear blebs that were deficient in lamin B1 (~50% of all blebs). That observation suggested that higher levels of lamin B1 expression might normalize the progerin meshwork and prevent NM ruptures and blebs. Indeed, increased lamin B1 expression reversed the morphological abnormalities in the progerin meshwork and markedly reduced the frequency of NM ruptures and blebs. Thus, progerin expression disrupts the overall structure of the nuclear lamina, but that effect-along with NM ruptures and blebs-can be abrogated by increased lamin B1 expression.

Carboxyl-terminal sequences in APOA5 are important for suppressing ANGPTL3/8 activity.

Apolipoprotein AV (APOA5) lowers plasma triglyceride (TG) levels by binding to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppressing its capacity to inhibit lipoprotein lipase (LPL) catalytic activity and its ability to detach LPL from binding sites within capillaries. However, the sequences in APOA5 that are required for suppressing ANGPTL3/8 activity have never been defined. A clue to the identity of those sequences was the presence of severe hypertriglyceridemia in two patients harboring an APOA5 mutation that truncates APOA5 by 35 residues ("APOA5Δ35"). We found that wild-type (WT) human APOA5, but not APOA5Δ35, suppressed ANGPTL3/8's ability to inhibit LPL catalytic activity. To pursue that finding, we prepared a mutant mouse APOA5 protein lacking 40 C-terminal amino acids ("APOA5Δ40"). Mouse WT-APOA5, but not APOA5Δ40, suppressed ANGPTL3/8's capacity to inhibit LPL catalytic activity and sharply reduced plasma TG levels in mice. WT-APOA5, but not APOA5Δ40, increased intracapillary LPL levels and reduced plasma TG levels in Apoa5-/- mice (where TG levels are high and intravascular LPL levels are low). Also, WT-APOA5, but not APOA5Δ40, blocked the ability of ANGPTL3/8 to detach LPL from cultured cells. Finally, an antibody against a synthetic peptide corresponding to the last 26 amino acids of mouse APOA5 reduced intracapillary LPL levels and increased plasma TG levels in WT mice. We conclude that C-terminal sequences in APOA5 are crucial for suppressing ANGPTL3/8 activity in vitro and for regulating intracapillary LPL levels and plasma TG levels in vivo.

Inverse effects of APOC2 and ANGPTL4 on the conformational dynamics of lid-anchoring structures in lipoprotein lipase.

The lipolytic processing of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL) is crucial for the delivery of dietary lipids to the heart, skeletal muscle, and adipose tissue. The processing of TRLs by LPL is regulated in a tissue-specific manner by a complex interplay between activators and inhibitors. Angiopoietin-like protein 4 (ANGPTL4) inhibits LPL by reducing its thermal stability and catalyzing the irreversible unfolding of LPL's α/ß-hydrolase domain. We previously mapped the ANGPTL4 binding site on LPL and defined the downstream unfolding events resulting in LPL inactivation. The binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 protects against LPL unfolding. The binding site on LPL for an activating cofactor, apolipoprotein C2 (APOC2), and the mechanisms by which APOC2 activates LPL have been unclear and controversial. Using hydrogen-deuterium exchange/mass spectrometry, we now show that APOC2's C-terminal α-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2's binding site on LPL overlaps with that for ANGPTL4, but their effects on LPL conformation are distinct. In contrast to ANGPTL4, APOC2 increases the thermal stability of LPL and protects it from unfolding. Also, the regions of LPL that anchor the lid are stabilized by APOC2 but destabilized by ANGPTL4, providing a plausible explanation for why APOC2 is an activator of LPL, while ANGPTL4 is an inhibitor. Our studies provide fresh insights into the molecular mechanisms by which APOC2 binds and stabilizes LPL-and properties that we suspect are relevant to the conformational gating of LPL's active site.

Intracapillary LPL levels in brown adipose tissue, visualized with an antibody-based approach, are regulated by ANGPTL4 at thermoneutral temperatures.

Lipoprotein lipase (LPL) is secreted into the interstitial spaces by parenchymal cells and then transported into capillaries by GPIHBP1. LPL carries out the lipolytic processing of triglyceride (TG)-rich lipoproteins (TRLs), but the tissue-specific regulation of LPL is incompletely understood. Plasma levels of TG hydrolase activity after heparin injection are often used to draw inferences about intravascular LPL levels, but the validity of these inferences is unclear. Moreover, plasma TG hydrolase activity levels are not helpful for understanding LPL regulation in specific tissues. Here, we sought to elucidate LPL regulation under thermoneutral conditions (30 °C). To pursue this objective, we developed an antibody-based method to quantify (in a direct fashion) LPL levels inside capillaries. At 30 °C, intracapillary LPL levels fell sharply in brown adipose tissue (BAT) but not heart. The reduced intracapillary LPL levels were accompanied by reduced margination of TRLs along capillaries. ANGPTL4 expression in BAT increased fourfold at 30 °C, suggesting a potential explanation for the lower intracapillary LPL levels. Consistent with that idea, Angptl4 deficiency normalized both LPL levels and TRL margination in BAT at 30 °C. In Gpihbp1-/- mice housed at 30 °C, we observed an ANGPTL4-dependent decrease in LPL levels within the interstitial spaces of BAT, providing in vivo proof that ANGPTL4 regulates LPL levels before LPL transport into capillaries. In conclusion, our studies have illuminated intracapillary LPL regulation under thermoneutral conditions. Our approaches will be useful for defining the impact of genetic variation and metabolic disease on intracapillary LPL levels and TRL processing.

The lipoprotein lipase that is shuttled into capillaries by GPIHBP1 enters the glycocalyx where it mediates lipoprotein processing.

Lipoprotein lipase (LPL), the enzyme that carries out the lipolytic processing of triglyceride-rich lipoproteins (TRLs), is synthesized by adipocytes and myocytes and secreted into the interstitial spaces. The LPL is then bound by GPIHBP1, a GPI-anchored protein of endothelial cells (ECs), and transported across ECs to the capillary lumen. The assumption has been that the LPL that is moved into capillaries remains attached to GPIHBP1 and that GPIHBP1 serves as a platform for TRL processing. In the current studies, we examined the validity of that assumption. We found that an LPL-specific monoclonal antibody (mAb), 88B8, which lacks the ability to detect GPIHBP1-bound LPL, binds avidly to LPL within capillaries. We further demonstrated, by confocal microscopy, immunogold electron microscopy, and nanoscale secondary ion mass spectrometry analyses, that the LPL detected by mAb 88B8 is located within the EC glycocalyx, distant from the GPIHBP1 on the EC plasma membrane. The LPL within the glycocalyx mediates the margination of TRLs along capillaries and is active in TRL processing, resulting in the delivery of lipoprotein-derived lipids to immediately adjacent parenchymal cells. Thus, the LPL that GPIHBP1 transports into capillaries can detach and move into the EC glycocalyx, where it functions in the intravascular processing of TRLs.

A protein of capillary endothelial cells, GPIHBP1, is crucial for plasma triglyceride metabolism.

GPIHBP1, a protein of capillary endothelial cells (ECs), is a crucial partner for lipoprotein lipase (LPL) in the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1, which contains a three-fingered cysteine-rich LU (Ly6/uPAR) domain and an intrinsically disordered acidic domain (AD), captures LPL from within the interstitial spaces (where it is secreted by parenchymal cells) and shuttles it across ECs to the capillary lumen. Without GPIHBP1, LPL remains stranded within the interstitial spaces, causing severe hypertriglyceridemia (chylomicronemia). Biophysical studies revealed that GPIHBP1 stabilizes LPL structure and preserves LPL activity. That discovery was the key to crystallizing the GPIHBP1-LPL complex. The crystal structure revealed that GPIHBP1's LU domain binds, largely by hydrophobic contacts, to LPL's C-terminal lipid-binding domain and that the AD is positioned to project across and interact, by electrostatic forces, with a large basic patch spanning LPL's lipid-binding and catalytic domains. We uncovered three functions for GPIHBP1's AD. First, it accelerates the kinetics of LPL binding. Second, it preserves LPL activity by inhibiting unfolding of LPL's catalytic domain. Third, by sheathing LPL's basic patch, the AD makes it possible for LPL to move across ECs to the capillary lumen. Without the AD, GPIHBP1-bound LPL is trapped by persistent interactions between LPL and negatively charged heparan sulfate proteoglycans (HSPGs) on the abluminal surface of ECs. The AD interrupts the HSPG interactions, freeing LPL-GPIHBP1 complexes to move across ECs to the capillary lumen. GPIHBP1 is medically important; GPIHBP1 mutations cause lifelong chylomicronemia, and GPIHBP1 autoantibodies cause some acquired cases of chylomicronemia.

APOA5 deficiency causes hypertriglyceridemia by reducing amounts of lipoprotein lipase in capillaries.

Apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia in mice and humans. For years, the cause remained a mystery, but the mechanisms have now come into focus. Here, we review progress in defining APOA5's function in plasma triglyceride metabolism. Biochemical studies revealed that APOA5 binds to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppresses its ability to inhibit the activity of lipoprotein lipase (LPL). Thus, APOA5 deficiency is accompanied by increased ANGPTL3/8 activity and lower levels of LPL activity. APOA5 deficiency also reduces amounts of LPL in capillaries of oxidative tissues (e.g., heart, brown adipose tissue). Cell culture experiments revealed the likely explanation: ANGPTL3/8 detaches LPL from its binding sites on the surface of cells, and that effect is blocked by APOA5. Both the low intracapillary LPL levels and the high plasma triglyceride levels in Apoa5-/- mice are normalized by recombinant APOA5. Carboxyl-terminal sequences in APOA5 are crucial for its function; a mutant APOA5 lacking 40-carboxyl-terminal residues cannot bind to ANGPTL3/8 and lacks the ability to change intracapillary LPL levels or plasma triglyceride levels in Apoa5-/- mice. Also, an antibody against the last 26 amino acids of APOA5 reduces intracapillary LPL levels and increases plasma triglyceride levels in wild-type mice. An inhibitory ANGPTL3/8-specific antibody functions as an APOA5-mimetic reagent, increasing intracapillary LPL levels and lowering plasma triglyceride levels in both Apoa5-/- and wild-type mice. That antibody is a potentially attractive strategy for treating elevated plasma lipid levels in human patients.

Quantification of lipoprotein lipase in mouse plasma with a sandwich enzyme-linked immunosorbent assay.

To support in vivo and in vitro studies of intravascular triglyceride metabolism in mice, we created rat monoclonal antibodies (mAbs) against mouse LPL. Two mAbs, mAbs 23A1 and 31A5, were used to develop a sandwich ELISA for mouse LPL. The detection of mouse LPL by the ELISA was linear in concentrations ranging from 0.31 ng/ml to 20 ng/ml. The sensitivity of the ELISA made it possible to quantify LPL in serum and in both pre-heparin and post-heparin plasma samples (including in grossly lipemic samples). LPL mass and activity levels in the post-heparin plasma were lower in Gpihbp1-/- mice than in wild-type mice. In both groups of mice, LPL mass and activity levels were positively correlated. Our mAb-based sandwich ELISA for mouse LPL will be useful for any investigator who uses mouse models to study LPL-mediated intravascular lipolysis.

Tool to Resolve Distortions in Elemental and Isotopic Imaging.

Nanoscale secondary ion mass spectrometry (NanoSIMS) makes it possible to visualize elements and isotopes in a wide range of samples at a high resolution. However, the fidelity and quality of NanoSIMS images often suffer from distortions because of a requirement to acquire and integrate multiple image frames. We developed an optical flow-based algorithm tool, NanoSIMS Stabilizer, for all-channel postacquisition registration of images. The NanoSIMS Stabilizer effectively deals with the distortions and artifacts, resulting in a high-resolution visualization of isotope and element distribution. It is open source with an easy-to-use ImageJ plugin and is accompanied by a Python version with GPU acceleration.

Performance evaluation of the high-throughput quantitative Alinity m BK virus assay.

BK virus (BKV) infection or reactivation in immunocompromised individuals can lead to adverse health consequences including BKV-associated nephropathy (BKVAN) in kidney transplant patients and BKV-associated hemorrhagic cystitis (BKV-HC) in allogeneic hematopoietic stem cell transplant recipients. Monitoring BKV viral load plays an important role in post-transplant patient care. This study evaluates the performance of the Alinity m BKV Investigational Use Only (IUO) assay. The linearity of the Alinity m BKV IUO assay had a correlation coefficient of 1.000 and precision of SD ≤ 0.25 Log IU/mL for all panel members tested (2.0-7.3 Log IU/mL). Detection rate at 50 IU/mL was 100%. Clinical plasma specimens tested comparing Alinity m BKV IUO to ELITech MGB Alert BKV lab-developed test (LDT) on the Abbott m2000 platform using specimen extraction protocols for DNA or total nucleic acid (TNA) resulted in coefficient of correlation of 0.900 and 0.963, respectively, and mean bias of 0.03 and -0.54 Log IU/mL, respectively. Alinity m BKV IUO compared with Altona RealStar BKV and Roche cobas BKV assays demonstrated coefficient of correlation of 0.941 and 0.980, respectively, and mean bias of -0.47 and -0.31 Log IU/mL, respectively. Urine specimens tested on Alintiy m BKV IUO and ELITech BKV LDT using TNA specimen extraction had a coefficient of correlation of 0.917 and mean bias of 0.29 Log IU/mL. The Alinity m BKV IUO assay was performed with high precision across the dynamic range and correlated well with other available BKV assays. IMPORTANCE: BK virus (BKV) in transplant patients can lead to adverse health consequences. Viral load monitoring is important in post-transplant patient care. This study evaluates the Alinity m BKV assay with currently available assays.

Polymyalgia rheumatica shows metabolomic alterations that are further altered by glucocorticoid treatment: Identification of metabolic correlates of fatigue.

OBJECTIVE: In polymyalgia rheumatica (PMR), glucocorticoids (GCs) relieve pain and stiffness, but fatigue may persist. We aimed to explore the effect of disease, GCs and PMR symptoms in the metabolite signatures of peripheral blood from patients with PMR or the related disease, giant cell arteritis (GCA). METHODS: Nuclear magnetic resonance spectroscopy was performed on serum from 40 patients with untreated PMR, 84 with new-onset confirmed GCA, and 53 with suspected GCA who later were clinically confirmed non-GCA, and 39 age-matched controls. Further samples from PMR patients were taken one and six months into glucocorticoid therapy to explore relationship of metabolites to persistent fatigue. 100 metabolites were identified using Chenomx and statistical analysis performed in SIMCA-P to examine the relationship between metabolic profiles and, disease, GC treatment or symptoms. RESULTS: The metabolite signature of patients with PMR and GCA differed from that of age-matched non-inflammatory controls (R2 > 0.7). There was a smaller separation between patients with clinically confirmed GCA and those with suspected GCA who later were clinically confirmed non-GCA (R2 = 0.135). In PMR, metabolite signatures were further altered with glucocorticoid treatment (R2 = 0.42) but did not return to that seen in controls. Metabolites correlated with CRP, pain, stiffness, and fatigue (R2 ≥ 0.39). CRP, pain, and stiffness declined with treatment and were associated with 3-hydroxybutyrate and acetoacetate, but fatigue did not. Metabolites differentiated patients with high and low fatigue both before and after treatment (R2 > 0.9). Low serum glutamine was predictive of high fatigue at both time points (0.79-fold change). CONCLUSION: PMR and GCA alter the metabolite signature. In PMR, this is further altered by glucocorticoid therapy. Treatment-induced metabolite changes were linked to measures of inflammation (CRP, pain and stiffness), but not to fatigue. Furthermore, metabolite signatures distinguished patients with high or low fatigue.

Actomyosin is the main driver of interkinetic nuclear migration in the retina.

Progenitor cell nuclei in the rapidly expanding epithelium of the embryonic vertebrate central nervous system undergo a process called interkinetic nuclear migration (IKNM). Movements of IKNM are generally believed to involve smooth migration of nuclei from apical to basal and back during the G1 and G2 phases of the cell cycle, respectively. Yet, this has not been formally demonstrated, nor have the molecular mechanisms that drive IKNM been identified. Using time-lapse confocal microscopy to observe nuclear movements in zebrafish retinal neuroepithelial cells, we show that, except for brief apical nuclear translocations preceding mitosis, IKNM is stochastic rather than smooth and directed. We also show that IKNM is driven largely by actomyosin-dependent forces as it still occurs when the microtubule cytoskeleton is compromised but is blocked when MyosinII activity is inhibited.

Increased expression of LAP2ß eliminates nuclear membrane ruptures in nuclear lamin-deficient neurons and fibroblasts.

Defects or deficiencies in nuclear lamins cause pathology in many cell types, and recent studies have implicated nuclear membrane (NM) ruptures as a cause of cell toxicity. We previously observed NM ruptures and progressive cell death in the developing brain of lamin B1-deficient mouse embryos. We also observed frequent NM ruptures and DNA damage in nuclear lamin-deficient fibroblasts. Factors modulating susceptibility to NM ruptures remain unclear, but we noted low levels of LAP2ß, a chromatin-binding inner NM protein, in fibroblasts with NM ruptures. Here, we explored the apparent link between LAP2ß and NM ruptures in nuclear lamin-deficient neurons and fibroblasts, and we tested whether manipulating LAP2ß expression levels would alter NM rupture frequency. In cortical plate neurons of lamin B1-deficient embryos, we observed a strong correlation between low LAP2ß levels and NM ruptures. We also found low LAP2ß levels and frequent NM ruptures in neurons of cultured Lmnb1-/- neurospheres. Reducing LAP2ß expression in Lmnb1-/- neurons with an siRNA markedly increased the NM rupture frequency (without affecting NM rupture duration), whereas increased LAP2ß expression eliminated NM ruptures and reduced DNA damage. Consistent findings were observed in nuclear lamin-deficient fibroblasts. Reduced LAP2ß expression increased NM ruptures, whereas increased LAP2ß expression virtually abolished NM ruptures. Increased LAP2ß expression nearly abolished NM ruptures in cells subjected to mechanical stress (an intervention that increases NM ruptures). Our studies showed that increasing LAP2ß expression bolsters NM integrity in nuclear lamin-deficient cells and markedly reduces NM rupture frequency.

The intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding.

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's α/ß-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including ß2, ß3-α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on ß3-α3 and progress to ß5 and ß4-α4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's α/ß-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

Multicenter Evaluation of the DiaSorin Molecular Simplexa Congenital CMV Direct PCR Test on Neonatal Saliva and Urine Specimens.

Cytomegalovirus (CMV) is the most common virus associated with congenital infection worldwide and is a major cause of sensorineural hearing loss (SNHL) and developmental delay. Up to 90% of infants with congenital CMV (cCMV) infection are asymptomatic at birth, making the diagnosis challenging. Postnatal diagnosis involves testing newborn saliva and/or urine collected before 21 days of life to confirm cCMV infection. This multicenter study evaluated the performance of the Simplexa Congenital CMV Direct real-time PCR assay for the qualitative detection of CMV in newborn saliva (n = 2,023) and urine (n = 1,797) specimens. Compared to two PCR/bidirectional sequencing assays, the Simplexa Congenital CMV Direct assay demonstrated positive percent agreement (PPA) and negative percent agreement (NPA) of 98.6% and 99.9%, respectively, for saliva samples and a PPA of 97.8% and an NPA of 99.9% for urine specimens. Overall concordance was κ = 0.98 or near perfect compared to the composite reference methods with both sample types. By 95% probit analysis, the limit of detection (LoD) using the AD-169 reference strain was 350 ± 12 copies/mL in urine. The LoDs of saliva swabs in either 1 mL or 3 mL of transport medium were 274 ± 12 copies/mL and 300 ± 14 copies/mL, respectively. The Simplexa Congenital CMV Direct assay can be applied to both saliva and urine specimens collected from newborns less than 21 days of age to rapidly and reliably identify CMV infection.

Clinical Evaluation of a New Molecular Test for the Detection of Organisms Causing Vaginitis and Vaginosis.

In this prospective, observational, method comparison clinical study, the Xpert Xpress MVP test (MVP) was evaluated using both clinician-collected (CVS) and self-collected vaginal swabs (SVS) collected in a clinical setting. The study was conducted at 12 sites, including point-of-care (POC) settings, from geographically diverse locations in the United States. Participants were biologically female patients ≥ 14 years old with signs and/or symptoms of vaginitis/vaginosis. MVP test results for BV were compared to the BD MAX Vaginal Panel (BDVP). Results for Candida group and Candida glabrata and Candida krusei targets (species not differentiated) were assessed relative to yeast culture followed by mass spectrometry for species identification. Trichomonas vaginalis (TV) results were compared relative to a composite method that included results from the BDVP and InPouch TV culture. The investigational test demonstrated high positive percent agreement ranging from 93.6 to 99.0%, and negative percent agreement ranging from 92.1% to 99.8% for both CVS and SVS specimens, indicating it may be a valuable tool for the diagnosis of vaginitis/vaginosis in laboratory and POC settings.

The zinc finger and BTB domain containing protein ZBTB20 regulates plasma triglyceride metabolism by repressing lipoprotein lipase gene transcription in hepatocytes.

BACKGROUND AND AIMS: Lipoprotein lipase (LPL) is responsible for the lipolytic processing of triglyceride-rich lipoproteins, the deficiency of which causes severe hypertriglyceridemia. Liver LPL expression is high in suckling rodents but relatively low at adulthood. However, the regulatory mechanism and functional significance of liver LPL expression are incompletely understood. We have established the zinc finger protein ZBTB20 as a critical factor for hepatic lipogenesis. Here, we evaluated the role of ZBTB20 in regulating liver Lpl gene transcription and plasma triglyceride metabolism. APPROACH AND RESULTS: Hepatocyte-specific inactivation of ZBTB20 in mice led to a remarkable increase in LPL expression at the mRNA and protein levels in adult liver, in which LPL protein was mainly localized onto sinusoidal epithelial cells and Kupffer cells. As a result, the LPL activity in postheparin plasma was substantially increased, and postprandial plasma triglyceride clearance was significantly enhanced, whereas plasma triglyceride levels were decreased. The dysregulated liver LPL expression and low plasma triglyceride levels in ZBTB20-deficient mice were normalized by inactivating hepatic LPL expression. ZBTB20 deficiency protected the mice against high-fat diet-induced hyperlipidemia without causing excessive triglyceride accumulation in the liver. Chromatin immunoprecipitation and gel-shift assay studies revealed that ZBTB20 binds to the LPL promoter in the liver. A luciferase reporter assay revealed that ZBTB20 inhibits the transcriptional activity of LPL promoter. The regulation of LPL expression by ZBTB20 is liver-specific under physiological conditions. CONCLUSIONS: Liver ZBTB20 serves as a key regulator of LPL expression and plasma triglyceride metabolism and could be a therapeutic target for hypertriglyceridemia.