A systematic review for prevention of cisplatin-induced nephrotoxicity using different hydration protocols and meta-analysis for magnesium hydrate supplementation.

BACKGROUND: Nephrotoxicity remains the most serious side effect of cisplatin therapy. Cisplatin-induced nephrotoxicity (CIN) limits the use of this drug and affects up to 20% of patients. Several possible interventions such as magnesium supplementation may prevent CIN. This study aimed to review different types of hydration protocols and we conducted a meta-analysis of magnesium supplementation to understand its effect in protecting against CIN. METHODS: A search of the PubMed, Embase, and Cochrane databases was performed. Trials were eligible if they enrolled patients who received cisplatin and different hydration protocols to prevent CIN. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the efficacy of different protocols. RESULTS: We initially identified 1113 different studies and included 33 of them which met the selection criteria. A meta-analysis of 11 retrospective studies that examined magnesium supplementation during hydration showed that this treatment provided significant protection against CIN (OR = 0.22, 95% CI = 0.14 to 0.35). CONCLUSION: There has been uncertainty regarding the best method to prevent CIN. Our results highlight the potentially protective effect of magnesium supplementation during hydration. This study is registered in PROSPERO, CRD42020212682.

Improved active disturbance rejection speed control for autonomous driving of high-speed train based on feedforward compensation.

Due to the complex and changeable train operation environment and the unstable and time-varying parameters, accurate modeling is limited. Therefore, a modified active disturbance rejection control algorithm based on feedforward compensation (FC-MADRC) is proposed targeting the speed control problem of trains under the circumstances of external disturbances, which reduces the dependence on the train model. Firstly, the state space equation is established based on the single-particle mathematical model of the train, and all the running resistances are regarded as disturbances. Secondly, the FC-MADRC algorithm is designed. Based on the terminal attractor function and the novel Sigmoid function, an improved tracking differentiator (ITD) is designed. An improved fal (nsfal) function with better smoothness is constructed by using the properties of the Dirac δ function, and an ameliorative nonlinear state error feedback (ANLSEF) and a modified extended state observer (IESO) are designed based on the nsfal function. Furthermore, based on the thought of PID, the integral term of error is introduced into ANLSEF for the nonlinear operation to reduce the steady-state error of train speed tracking. In order to promote the robustness and control accuracy of the system, the feedforward compensation term and disturbance compensation term are combined to perform dynamic compensation for disturbances in real time. Finally, the simulation is carried out with CRH380A train data. The results indicate that compared with conventional ADRC and 2DOF-PID, FC-MADRC has the more vital anti-disturbance ability and higher tracking accuracy. FC-MADRC has the advantages of solid anti-disturbance, fast response, and high tracking accuracy. Under the premise of external disturbance, it can still achieve accurate speed tracking under different road conditions.

Roles of noncoding RNAs in septic acute kidney injury.

Septic acute kidney injury (SAKI) is one of the most common and life-threatening complications of sepsis. Patients with SAKI have increased mortality. However, the underlying pathogenesis is unclear, and the treatment targeting SAKI is unsatisfactory. Thus, identifying optimal biomarkers for SAKI diagnosis and treatment is an urgent requisite. Accumulating evidence indicates that noncoding RNAs (ncRNAs) are involved in the occurrence and progression of SAKI. In the present review, we summarized the studies of ncRNAs in SAKI, including microRNAs (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs). The ncRNAs are divided into protective and damage factors according to their role in SAKI, and their expression patterns, functions, and molecular mechanisms were elaborated. Next, we proposed that ncRNAs have the potential to be diagnostic and prognostic biomarkers for SAKI and as new therapeutic targets. This review aimed to provide a comprehensive overview of ncRNAs in SKAI and explored the clinical value of ncRNAs as ideal biomarkers of SAKI.

Bispecific antibody targeting both B7-H3 and PD-L1 exhibits superior antitumor activities.

Clinical application of PD-1 and PD-L1 monoclonal antibodies (mAbs) is hindered by their relatively low response rates and the occurrence of drug resistance. Co-expression of B7-H3 with PD-L1 has been found in various solid tumors, and combination therapies that target both PD-1/PD-L1 and B7-H3 pathways may provide  additional therapeutic benefits. Up to today, however, no bispecific antibodies targeting both PD-1 and B7-H3 have reached the clinical development stage. In this study, we generated a stable B7-H3×PD-L1 bispecific antibody (BsAb) in IgG1-VHH format by coupling a humanized IgG1 mAb against PD-L1 with a humanized camelus variable domain of the heavy-chain of heavy-chain antibody (VHH) against human B7-H3. The BsAb exhibited favorable thermostability, efficient T cell activation, IFN-γ production, and antibody-dependent cell-mediated cytotoxicity (ADCC). In a PBMC humanized A375 xenogeneic tumor model, treatment with BsAb (10 mg/kg, i.p., twice a week for 6 weeks) showed enhanced antitumor activities compared to monotherapies and, to some degree, combination therapies. Our results suggest that targeting both PD-1 and B7-H3 with BsAbs increases their specificities to B7-H3 and PD-L1 double-positive tumors and induces a synergetic effect. We conclude that B7-H3×PD-L1 BsAb is favored over mAbs and possibly combination therapies in treating B7-H3 and PD-L1 double-positive tumors.

Thrombospondin 1 Promotes Endoplasmic Reticulum Stress and Apoptosis in HK-2 Cells by Upregulating ATF6-CHOP.

OBJECTIVE: The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1 (TSP1) in human renal tubular epithelial cells (HK-2 cells). METHODS: HK-2 cells were exposed to high concentrations of glucose (HG). The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) was administered by transfecting TSP1 or an empty vector to explore the mechanism of the endoplasmic reticulum response regulated by TSP1 and stress in renal cell apoptosis. The effects of TSP1 and 4-PBA on the proliferation and apoptosis of HK-2 cells under HG conditions were assessed using Cell counting kit-8 and flow cytometry. Western blotting was used to detect the apoptosis- and endoplasmic reticulum stress-related protein expression regulated by TSP1 and 4-PBA. RESULTS: HG treatment induced high cell apoptosis, abundantly expressed TSP1 level and restrained viability in HK-2 cells. Overexpression of TSP1 significantly inhibited the proliferation of and facilitated apoptosis of HK-2 cells under HG conditions. Administration of endoplasmic reticulum stress inhibitor 4-PBA after overexpression of TSP1 antagonized the inhibitory proliferation and promoted apoptosis rate in HG-triggered HK-2 cells induced by TSP1 overexpression. 4-PBA treatment significantly hindered the expression of endoplasmic reticulum stress markers, such as PERK, ATF4, ATF6, p-eIF2α, IRE1, CHOP and XBP1, suggesting that the administration of 4-PBA was successful. CONCLUSION: Overexpression of TSP1 activated endoplasmic reticulum stress by regulating the ATF6-CHOP axis. TSP1 restrained cell proliferation, and promoted apoptosis and endoplasmic reticulum stress by activating the ATF6-CHOP axis.

Age- and Sex-Specific Reference Values of Estimated Glomerular Filtration Rate in Chinese Population.

BACKGROUND: Due to an aging population, prevalence and mortality of CKD continue to increase. Current CKD definition has been challenged recently. Age- and sex-specific reference values of estimated glomerular filtration rate (eGFR) in China are still lacking. METHODS: Age- and sex-stratified, randomly selected inhabitants received a health examination and an inquest into medical history. The GFR was estimated using CKD-EPI equation. We calculated means with ±1.96 times of standard deviation and 2.5th, 97.5th percentiles of eGFR per 5-year age-group. Some of their GFRs were measured by the Gates method (99mTc-DTPA renal scintigraphy) and estimated by cystatin C-based equation. RESULTS: The cohort study included 17,037 male and 9,304 female Chinese persons aged 18-99 years. A reference population of apparently healthy subjects was selected by excluding persons with known hypertension, diabetes, cardiovascular, or renal diseases. This healthy cohort study included 12,231 male subjects and 6,463 female subjects. The mean eGFR was higher in the female than that in the male who were younger than 40-year (122 mL/min/1.73 m2 vs. 111 mL/min/1.73 m2). In these apparently healthy persons, GFR declined approximately 0.8 mL/min/year. The lower limit of eGFR (2.5th percentile or mean minus 1.96 times of standard deviation) was <60 or 45 mL/min/1.73 m2 at the age of ≥40 or 65 years old, respectively. CONCLUSION: The mean eGFR was higher in young females. GFR declined approximately 0.8 mL/min/year. The lower bound of eGFR was <60 mL/min/1.73 m2 or 45 mL/min/1.73 m2 at the age of ≥40 or ≥65 years, respectively. Our study provides age- and sex-specific reference values of GFR in a Chinese population.

Comparison between the Beta-2 Microglobulin-Based Equation and the CKD-EPI Equation for Estimating GFR in CKD Patients in China: ES-CKD Study.

BACKGROUND: Beta-2 microglobulin (B2M) and cystatin C are novel glomerular filtration markers that have a stronger association with adverse outcomes than creatinine. The B2M-based glomerular filtration rate (GFR) estimating equation was built in 2016. Several new creatinine and cystatin C equations were developed in 2019 in China. However, external validation of these new equations remains to be seen. METHODS: This is a prospective cohort study. The equations were validated in a population totaling 830 participants (median age 62 years). These equations include the B2M-based equation (built in 2016), three CKD-EPI equations (built in 2009 and 2012), three Yang-Du equations (C-CKD-EPIscr, C-CKD-EPIcys, and C-CKD-EPIscr-cys equations, all of which were Chinese-modified CKD-EPI equations developed by Yang et al. in 2019), and a Xiangya equation (a creatinine-based equation built in the Third Xiangya Hospital in 2019). The estimated GFR (eGFR) calculated separately by 8 equations (B2M GFR, CKD-EPIscr, CKD-EPIcys, CKD-EPIscr-cys, C-CKD-EPIscr, C-CKD-EPIcys, C-CKD-EPIscr-cys, and Xiangya equations) was compared with the reference GFR (rGFR) measured by the 99mTc-DTPA renal dynamic imaging method. Participants were divided into CKD stage 1-5 specific subgroups. The primary outcomes of this study were bias, precision (interquartile range of difference, IQR), and accuracy (the proportion of eGFR within 30% of rGFR [P30] and root mean square error [RMSE]) of eGFR versus rGFR. RESULTS: The B2M-based equation was worse than CKD-EPI equations and Yang-Du equations in most outcomes. CKD-EPIscr and C-CKD-EPIscr equations had a larger area under the receiver operating characteristic curve (ROCAUC). The CKD-EPIscr equation had the highest sensitivity (83.3%) and the Xiangya equation the highest specificity (89.5%) to diagnose CKD. The bias was the lowest in CKD-EPIcys and C-CKD-EPIscr-cys equations by median and mean difference (1.23 and -1.42, respectively). The Xiangya equation yielded the highest bias by both median and mean difference (8.29 and 6.52, respectively). The C-CKD-EPIscr equation was the most accurate with the highest P30 value (68.1%) and most precise with the lowest IQR (19). The Xiangya equation had the best RMSE (lowest RMSE, 0.56), and gave the best performance in the CKD stage 2 subgroup. The C-CKD-EPIscr-cys equation achieved the lowest bias in CKD stage 3-5 (p = 0.663, 0.104, and 0.130, respectively, compared with rGFR). CONCLUSION: The B2M-based equation was worse than CKD-EPI and Yang-Du equations on the whole. CKD-EPIcys and C-CKD-EPIscr-cys equations had the lowest bias, whereas the Xiangya equation yielded the highest bias. The Xiangya equation gave the best performance in the CKD stage 2 subgroup, while the C-CKD-EPIscr-cys equation achieved the lowest bias in CKD stage 3-5. Further work to improve the performance of the GFR estimating equation is needed.

Identification of a novel homozygous nonsense variant in a Chinese patient with ethylmalonic encephalopathy and a genotype-phenotype spectrum review.

Ethylmalonic encephalopathy (EE) is a rare and devastating neurodegenerative disease caused by mutations in the ETHE1 gene. It is characterized by early-onset encephalopathy, chronic diarrhea, petechiae, orthostatic acrocyanosis, and high levels of methylsuccinic, lactic, and ethylmalonic acids in body fluids. In this study, we report a patient with EE, who was identified through newborn screening, and the diagnosis was confirmed by targeted next-generation sequencing (NGS). The patient displayed recurrent petechiae, intermittent jaundice, protracted diarrhea, and extensive developmental regression. Genetic testing identified a homozygous nonsense variant, c.295C > T (p. Q99*), in the ETHE1 gene. A review of all known ETHE1 variants observed in other EE patients was conducted. This revealed the current difficulties in EE diagnosis. Besides, it also showed that patients with truncated variants of ETHE1 might exhibit pathological symptoms earlier and present more severe manifestations. Finally, a novel nonsense variant was identified, which supported and expanded our current knowledge of the variant spectrum for ETHE1. This novel variant also deepened our understanding of the genotype-phenotype associations that occur in EE patients.

Type 3 Inositol 1,4,5-Trisphosphate Receptor is a Crucial Regulator of Calcium Dynamics Mediated by Endoplasmic Reticulum in HEK Cells.

Being the largest the Ca2+ store in mammalian cells, endoplasmic reticulum (ER)-mediated Ca2+ signalling often involves both Ca2+ release via inositol 1, 4, 5-trisphosphate receptors (IP3R) and store operated Ca2+ entries (SOCE) through Ca2+ release activated Ca2+ (CRAC) channels on plasma membrane (PM). IP3Rs are functionally coupled with CRAC channels and other Ca2+ handling proteins. However, it still remains less well defined as to whether IP3Rs could regulate ER-mediated Ca2+ signals independent of their Ca2+ releasing ability. To address this, we generated IP3Rs triple and double knockout human embryonic kidney (HEK) cell lines (IP3Rs-TKO, IP3Rs-DKO), and systemically examined ER Ca2+ dynamics and CRAC channel activity in these cells. The results showed that the rate of ER Ca2+ leakage and refilling, as well as SOCE were all significantly reduced in IP3Rs-TKO cells. And these TKO effects could be rescued by over-expression of IP3R3. Further, results showed that the diminished SOCE was caused by NEDD4L-mediated ubiquitination of Orai1 protein. Together, our findings indicate that IP3R3 is one crucial player in coordinating ER-mediated Ca2+ signalling.

The SOS2-SCaBP8 Complex Generates and Fine-Tunes an AtANN4-Dependent Calcium Signature under Salt Stress.

Calcium signals act as universal second messengers that trigger many cellular processes in animals and plants, but how specific calcium signals are generated is not well understood. In this study, we determined that AtANN4, a putative calcium-permeable transporter, and its interacting proteins, SCaBP8 and SOS2, generate a calcium signal under salt stress, which initially activates the SOS pathway, a conserved mechanism that modulates ion homeostasis in plants under salt stress. After activation, SCaBP8 promotes the interaction of protein kinase SOS2 with AtANN4, which enhances its phosphorylation by SOS2. This phosphorylation of AtANN4 further increases its interaction with SCaBP8. Both the interaction with and phosphorylation of AtANN4 repress its activity and alter calcium transients and signatures in HEK cells and plants. Our results reveal how downstream targets are required to create a specific calcium signal via a negative feedback regulatory loop, thereby enhancing our understanding of the regulation of calcium signaling.

Preparation process optimization of pig bone collagen peptide-calcium chelate using response surface methodology and its structural characterization and stability analysis.

In this study, alcalase and neutrase were used in combination to prepare collagen peptides with high calcium binding ability. The optimal conditions for the preparation of peptide-calcium chelate (mass ratio of peptide/calcium of 4.5:1 for 40 min at 50 °C and pH 9) were determined by response surface methodology (RSM), under which a calcium chelating rate of 78.38% was obtained. The results of Ultraviolet-Visible (UV-Vis), fluorescence and Fourier transform infrared (FT-IR) spectra synthetically indicated that calcium could be chelated by carboxyl oxygen and amino nitrogen atoms of collagen peptides, thus forming peptide-calcium chelate. The chelate was stable at various temperatures and pH values, and exhibited excellent stability in the gastrointestinal environment, which could promote calcium absorption in human gastrointestinal tract. Moreover, Caco-2 cell monolayer model was used to investigate the effect of peptide-calcium chelate on promoting calcium absorption. Results showed that peptide-calcium chelate could significantly improve calcium transport in Caco-2 cell monolayer and reverse the inhibition of calcium absorption by phosphate and phytate. The findings provide a scientific basis for developing new calcium supplements and the high-value utilization of pig bone.

Brief Report: Interleukin-17A-Dependent Asymmetric Stem Cell Divisions Are Increased in Human Psoriasis: A Mechanism Underlying Benign Hyperproliferation.

The balance between asymmetric and symmetric stem cell (SC) divisions is key to tissue homeostasis, and dysregulation of this balance has been shown in cancers. We hypothesized that the balance between asymmetric cell divisions (ACDs) and symmetric cell divisions (SCDs) would be dysregulated in the benign hyperproliferation of psoriasis. We found that, while SCDs were increased in squamous cell carcinoma (SCC) (human and murine), ACDs were increased in the benign hyperproliferation of psoriasis (human and murine). Furthermore, while sonic hedgehog (linked to human cancer) and pifithrinα (p53 inhibitor) promoted SCDs, interleukin (IL)-1α and amphiregulin (associated with benign epidermal hyperproliferation) promoted ACDs. While there was dysregulation of the ACD:SCD ratio, no change in SC frequency was detected in epidermis from psoriasis patients, or in human keratinocytes treated with IL-1α or amphiregulin. We investigated the mechanism whereby immune alterations of psoriasis result in ACDs. IL17 inhibitors are effective new therapies for psoriasis. We found that IL17A increased ACDs in human keratinocytes. Additionally, studies in the imiquimod-induced psoriasis-like mouse model revealed that ACDs in psoriasis are IL17A-dependent. In summary, our studies suggest an association between benign hyperproliferation and increased ACDs. This work begins to elucidate the mechanisms by which immune alteration can induce keratinocyte hyperproliferation. Altogether, this work affirms that a finely tuned balance of ACDs and SCDs is important and that manipulating this balance may constitute an effective treatment strategy for hyperproliferative diseases. Stem Cells 2017;35:2001-2007.

Effects of escitalopram with a Chinese traditional compound Jiuweizhenxin-keli on mismatch negativity and P50 in patients with major depressive disorders.

OBJECTIVE: The objective of this study was to investigate the therapeutic effects of escitalopram in conjunction with Jiuweizhenxin-keli on neuroelectrophysiology in patients with major depressive disorders (MDD). PATIENTS AND METHODS: Patients with depressive episode of MDD according to the criteria of Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, were randomly assigned to Esc group (30 patients) receiving escitalopram treatment and JK group (30 patients) treated with a combination of escitalopram and Jiuweizhenxin-keli. The healthy control (HC) group (30 persons with normal health condition) served as control. All groups were subject to examination of 24-item Hamilton Depression Rating Scale and Hamilton Anxiety Scale, mismatch negativity (MMN), and sensory gating potential P50 (SG-P50) of event-related potentials. Data were collected at three different time points: baseline (before treatment) and week 2 and week 6 post treatment. RESULTS: At baseline, all electrophysiological parameters of patients with MDD were significantly higher than those of HCs. After treatment, in the Esc group, MMN latency, S2-P50 amplitude, and S2-P50/S1-P50 amplitude ratio decreased; however, the decrements were not statistically significant compared to either baseline or the HC group. Also, no significant changes were observed in the percentage of individuals whose S2-P50/S1-P50 ≥0.5 in the Esc group. On the other hand, in the JK group after a 6-week treatment, MMN latency (206.35±32.14 ms) was significantly shorter than that of the Esc group (219.57±36.51 ms), S2-P50 amplitude (7.27±4.85 µV) reduced significantly compared with the baseline level (10.21±4.10 µV), the percentage of individuals whose S2-P50/S1-P50 ≥0.5 in the JK group greatly decreased and this was not significantly different compared to that of the HC group (P≥0.05). CONCLUSION: Neuroplasticity of patients with MDD is apparently disturbed, characterized by aberrant MMN latency and SG-P50-related event-related potential parameters. A combination of escitalopram and Jiuweizhenxin-keli treatment can markedly restore these neuroelectrophysiological features, and thus, could be a novel therapeutic solution for improving the impaired neuroplasticity of MDD patients.

Targeting ALDH1 to decrease tumorigenicity, growth and metastasis of human melanoma.

Cells with aldehyde dehydrogenase activity (ALDH+) are the most tumorigenic cells in many cancers, including melanoma, making ALDH a candidate therapeutic target. We examined the effects of chemical inhibition of ALDH1 on the response of human melanoma xenografts to chemotherapy and the effects of ALDH1A1 RNA silencing on melanoma growth and metastasis. Addition of ALDH1 inhibitors (e.g. diethylaminobenzaldehyde) to dacarbazine chemotherapy, not only reduced tumor growth in vivo, but also resulted in a significant decrease in the number of residual cells capable of tumorigenesis. shRNA depletion of ALDH1A1 in melanoma cells resulted not only in a significant delay in appearance of xenograft melanomas and reduction in growth, but also significantly decreased the number of metastases and metastatic burden after lateral tail vein injections in mice. In summary, ALDH1 inhibition in combinatorial therapy with dacarbazine reduced the number of residual tumorigenic cells post-therapy and ALDH1A1 depletion had marked inhibitory effects on both melanoma growth and metastasis. These findings suggest that ALDH1 inhibition may not only be able to provide a therapeutic advantage in melanoma treatment, but may also prevent rapid relapse after therapy, as residual tumorigenic cells are fewer and metastatic ability is diminished.

Effects of polarizability on the structural and thermodynamics properties of [Cnmim][Gly] ionic liquids (n = 1-4) using EEM/MM molecular dynamic simulations.

An extended electronegativity equalization method/molecular mechanics (EEM/MM) model for ionic liquids is used to investigate the structures and properties of 1-alkyl-3-methylimidazolium glycine ionic liquids [Cnmim][Gly] (n = 1-4) with alkyl substituents of different lengths. The EEM/MM model describes the electrostatic interactions of atoms and their changes in different ambient environments. This property is the most outstanding characteristic of the model. EEM parameters (i.e., valence electronegativities and valence hardness parameters) are calibrated using linear regression and least-squares methods, which can accurately predict the gas-phase properties of [Cnmim](+), [Gly](-), and [Cnmim][Gly] ion pairs. We utilize the EEM/MM force field to systematically investigate the effects of polarizability on the accuracy of [Cnmim][Gly] properties predicted through the molecular dynamic simulations. EEM/MM explicitly describes the atom-based polarizability of [Cnmim][Gly]; thus, the densities, enthalpies of vaporization, self-diffusion coefficients, and conductivities of the [Cnmim][Gly] are consistent with the experimental values. The calculated radial distribution functions provide a mechanistic understanding of the effects of polarizability on ionic aggregations in amino acid ionic liquids. The effects of alkyl chain length on the diffusion coefficient and conductivity are also discussed.

Conditional deletion of FAK in mice endothelium disrupts lung vascular barrier function due to destabilization of RhoA and Rac1 activities.

Loss of lung-fluid homeostasis is the hallmark of acute lung injury (ALI). Association of catenins and actin cytoskeleton with vascular endothelial (VE)-cadherin is generally considered the main mechanism for stabilizing adherens junctions (AJs), thereby preventing disruption of lung vascular barrier function. The present study identifies endothelial focal adhesion kinase (FAK), a nonreceptor tyrosine kinase that canonically regulates focal adhesion turnover, as a novel AJ-stabilizing mechanism. In wild-type mice, induction of ALI by intraperitoneal administration of lipopolysaccharide or cecal ligation and puncture markedly decreased FAK expression in lungs. Using a mouse model in which FAK was conditionally deleted only in endothelial cells (ECs), we show that loss of EC-FAK mimicked key features of ALI (diffuse lung hemorrhage, increased transvascular albumin influx, edema, and neutrophil accumulation in the lung). EC-FAK deletion disrupted AJs due to impairment of the fine balance between the activities of RhoA and Rac1 GTPases. Deletion of EC-FAK facilitated RhoA's interaction with p115-RhoA guanine exchange factor, leading to activation of RhoA. Activated RhoA antagonized Rac1 activity, destabilizing AJs. Inhibition of Rho kinase, a downstream effector of RhoA, reinstated normal endothelial barrier function in FAK-/- ECs and lung vascular integrity in EC-FAK-/- mice. Our findings demonstrate that EC-FAK plays an essential role in maintaining AJs and thereby lung vascular barrier function by establishing the normal balance between RhoA and Rac1 activities.

The CD44+ ALDH+ population of human keratinocytes is enriched for epidermal stem cells with long-term repopulating ability.

Like for other somatic tissues, isolation of a pure population of stem cells has been a primary goal in epidermal biology. We isolated discrete populations of freshly obtained human neonatal keratinocytes (HNKs) using previously untested candidate stem cell markers aldehyde dehydrogenase (ALDH) and CD44 as well as the previously studied combination of integrin α6 and CD71. An in vivo transplantation assay combined with limiting dilution analysis was used to quantify enrichment for long-term repopulating cells in the isolated populations. The ALDH(+) CD44(+) population was enriched 12.6-fold for long-term repopulating epidermal stem cells (EpiSCs) and the integrin α6(hi) CD71(lo) population was enriched 5.6-fold, over unfractionated cells. In addition to long-term repopulation, CD44(+) ALDH(+) keratinocytes exhibited other stem cell properties. CD44(+) ALDH(+) keratinocytes had self-renewal ability, demonstrated by increased numbers of cells expressing nuclear Bmi-1, serial transplantation of CD44(+) ALDH(+) cells, and holoclone formation in vitro. CD44(+) ALDH(+) cells were multipotent, producing greater numbers of hair follicle-like structures than CD44(-) ALDH(-) cells. Furthermore, 58% ± 7% of CD44(+) ALDH(+) cells exhibited label-retention. In vitro, CD44(+) ALDH(+) cells showed enhanced colony formation, in both keratinocyte and embryonic stem cell growth media. In summary, the CD44(+) ALDH(+) population exhibits stem cell properties including long-term epidermal regeneration, multipotency, label retention, and holoclone formation. This study shows that it is possible to quantify the relative number of EpiSCs in human keratinocyte populations using long-term repopulation as a functional test of stem cell nature. Future studies will combine isolation strategies as dictated by the results of quantitative transplantation assays, in order to achieve a nearly pure population of EpiSCs.

Apolipoprotein E enhances endothelial-NO production by modulating caveolin 1 interaction with endothelial NO synthase.

Apolipoprotein E (apoE) is widely expressed in mammalian tissues, and one of the important tissue-specific effects is the atheroprotection ascribed to macrophage-derived apoE in the arterial wall. However, underlying mechanisms are not well understood. In this study, using subcellular fractionation, confocal microscopy, and coimmunoprecipitation, we demonstrated that macrophage-derived apoE is internalized by endothelial cells and impacts the subcellular distribution/interaction of caveolin 1 (cav-1) and endothelial NO synthase (eNOS). The addition of apoE disrupts the heteromeric complex formed between cav-1 and eNOS, and increases NO production. Sterol and oxysterol enhance endothelial cav-1/eNOS interaction and suppress NO production, but these effects are reversed by apoE. Silencing endothelial cav-1 attenuates apoE-induced NO production, establishing the importance of the cav-1-eNOS interaction for the increment in endothelial NO produced by apoE. Consistent with these observations, macrophage-derived apoE significantly improves vasodilation to acetylcholine in resistance arteries isolated from adipose tissue of obese humans. We conclude that macrophage-derived apoE enhances endothelial NO production by disrupting the inhibitory interaction of eNOS with cav-1. These results establish a novel mechanism by which apoE modulates endothelial cell function.

CD133 is a marker for long-term repopulating murine epidermal stem cells.

Maintenance, repair, and renewal of the epidermis are thought to depend on a pool of dedicated epidermal stem cells (EpiSCs). Like for many somatic tissues, isolation of a nearly pure population of stem cells is a primary goal in cutaneous biology. We used a quantitative transplantation assay, using injection of keratinocytes into subcutis combined with limiting dilution analysis, to assess the long-term repopulating ability of putative murine EpiSC populations. Putative EpiSC populations were isolated by FACS sorting. The CD133(+) population and the subpopulation of CD133(+) cells that exhibits high mitochondrial membrane potential (DΨm(hi)) were enriched for long-term repopulating EpiSCs versus unfractionated cells (3.9- and 5.2-fold, respectively). Evidence for self-renewal capacity was obtained by serial transplantation of long-term epidermal repopulating units derived from CD133(+) and CD133(+)ΔΨm(hi) keratinocytes. CD133(+) keratinocytes were multipotent and produced significantly more hair follicles than CD133(-) cells. CD133(+) cells were a subset of the previously described integrin α6(+)CD34(+) bulge cell population, and 28.9±8.6% were label-retaining cells. Thus, murine keratinocytes within the CD133(+) and CD133(+)ΔΨm(hi) populations contain EpiSCs that regenerate the epidermis for the long term, are self-renewing, multipotent, and label-retaining cells.