Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few oncogenic human viruses known to date. Its large genome encodes more than 85 proteins and includes both unique viral proteins as well ...as proteins conserved amongst herpesviruses. KSHV ORF20 is a member of the herpesviral core UL24 family, but the function of ORF20 and its role in the viral life cycle is not well understood. ORF20 encodes three largely uncharacterized isoforms, which we found were localized predominantly in the nuclei and nucleoli. Quantitative affinity purification coupled to mass spectrometry (q-AP-MS) identified numerous specific interacting partners of ORF20, including ribosomal proteins and the interferon-stimulated gene product (ISG) oligoadenylate synthetase-like protein (OASL). Both endogenous and transiently transfected OASL co-immunoprecipitated with ORF20, and this interaction was conserved among all ORF20 isoforms and multiple ORF20 homologs of the UL24 family in other herpesviruses. Characterization of OASL interacting partners by q-AP-MS identified a very similar interactome to that of ORF20. Both ORF20 and OASL copurified with 40S and 60S ribosomal subunits, and when they were co-expressed, they associated with polysomes. Although ORF20 did not have a global effect on translation, ORF20 enhanced RIG-I induced expression of endogenous OASL in an IRF3-dependent but IFNAR-independent manner. OASL has been characterized as an ISG with antiviral activity against some viruses, but its role for gammaherpesviruses was unknown. We show that OASL and ORF20 mRNA expression were induced early after reactivation of latently infected HuARLT-rKSHV.219 cells. Intriguingly, we found that OASL enhanced infection of KSHV. During infection with a KSHV ORF20stop mutant, however, OASL-dependent enhancement of infectivity was lost. Our data have characterized the interaction of ORF20 with OASL and suggest ORF20 usurps the function of OASL to benefit KSHV infection.
The recent Natural Killer (NK) cell maturation model postulates that CD34+ hematopoietic stem cells (HSC) first develop into CD56bright NK cells, then into CD56dimCD57− and finally into terminally ...maturated CD56dimCD57+. The molecular mechanisms of human NK cell differentiation and maturation however are incompletely characterized. Here we present a proteome analysis of distinct developmental stages of human primary NK cells, isolated from healthy human blood donors. Peptide sequencing was used to comparatively analyze CD56bright NK cells versus CD56dim NK cells and CD56dimCD57− NK cells versus CD56dimCD57+ NK cells and revealed distinct protein signatures for all of these subsets. Quantitative data for about 3400 proteins were obtained and support the current differentiation model. Furthermore, 11 donor-independently, but developmental stage specifically regulated proteins so far undescribed in NK cells were revealed, which may contribute to NK cell development and may elucidate a molecular source for NK cell effector functions.
Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization. Upon activation of human primary NK cells, both proteins are recruited into the immune synapse (NKIS), where they colocalize with myosin IIa.
The interferon‐inducible transmembrane (Ifitm/Fragilis) genes encode homologous proteins that are induced by IFNs. Here, we show that IFITM proteins regulate murine CD4+ Th cell differentiation. ...Ifitm2 and Ifitm3 are expressed in wild‐type (WT) CD4+ T cells. On activation, Ifitm3 was downregulated and Ifitm2 was upregulated. Resting Ifitm‐family‐deficient CD4+ T cells had higher expression of Th1‐associated genes than WT and purified naive Ifitm‐family‐deficient CD4+ T cells differentiated more efficiently to Th1, whereas Th2 differentiation was inhibited.
Ifitm‐family‐deficient mice, but not Ifitm3‐deficient mice, were less susceptible than WT to induction of allergic airways disease, with a weaker Th2 response and less severe disease and lower Il4 but higher Ifng expression and IL‐27 secretion. Thus, the Ifitm family is important in adaptive immunity, influencing Th1/Th2 polarization, and Th2 immunopathology.
The interferon‐inducible transmembrane (IFITM) proteins influence Th1/Th2 polarization and promote Th2 differentiation. In absence of IFITM proteins, CD4 T‐cell differentiation is biased towards Th1, by a cell‐autonomous mechanism. IFITM‐deficient mice are less susceptible to induction of allergic airways disease by inhibition of the Th2 response and inflammation.
Vascular tissue engineering of the middle layer of natural arteries requires contractile smooth muscle cells (SMC) which can be differentiated from adipose-derived mesenchymal stem cells (ASC) by ...treatment with transforming growth factor-
β
, sphingosylphosphorylcholine and bone morphogenetic protein-4 (TSB). Since mechanical stimulation may support or replace TSB-driven differentiation, we investigated its effect plus TSB-treatment on SMC orientation and contractile protein expression. Tubular fibrin scaffolds with incorporated ASC or pre-differentiated SMC were exposed to pulsatile perfusion for 10 days with or without TSB. Statically incubated scaffolds served as controls. Pulsatile incubation resulted in collagen-I expression and orientation of either cell type circumferentially around the lumen as shown by alpha smooth muscle actin (
α
SMA), calponin and smoothelin staining as early, intermediate and late marker proteins. Semi-quantitative Westernblot analyses revealed strongly increased
α
SMA and calponin expression by either pulsatile (12.48-fold;
p
< 0.01 and 38.15-fold;
p
= 0.07) or static incubation plus TSB pre-treatment (8.91-fold;
p
< 0.05 and 37.69-fold;
p
< 0.05). In contrast, contractility and smoothelin expression required both mechanical and TSB stimulation since it was 2.57-fold increased (
p
< 0.05) only by combining pulsatile perfusion and TSB. Moreover, pre-differentiation of ASC prior to pulsatile perfusion was not necessary since it could not further increase the expression level of any marker.
The generation of cellularized bioartificial blood vessels resembling all three layers of the natural vessel wall with physiological morphology and cell alignment is a long pursued goal in vascular ...tissue engineering. Simultaneous culture of all three layers under physiological mechanical conditions requires highly sophisticated perfusion techniques and still today remains a key challenge. Here, three-layered bioartificial vessels based on fibrin matrices were generated using a stepwise molding technique. Adipose-derived stem cells (ASC) were differentiated to smooth muscle cells (SMC) and integrated in a compacted tubular fibrin matrix to resemble the
tunica media
. The
tunica adventitia
-equivalent containing human umbilical vein endothelial cells (HUVEC) and ASC in a low concentration fibrin matrix was molded around it. Luminal seeding with HUVEC resembled the
tunica intima
. Subsequently, constructs were exposed to physiological mechanical stimulation in a pulsatile bioreactor for 72 h. Compared to statically incubated controls, mechanical stimulation induced physiological cell alignment in each layer: Luminal endothelial cells showed longitudinal alignment, cells in the
media
-layer were aligned circumferentially and expressed characteristic SMC marker proteins. HUVEC in the
adventitia
-layer formed longitudinally aligned microvascular tubes resembling
vasa vasorum
capillaries. Thus, physiologically organized three-layered bioartificial vessels were successfully manufactured by stepwise fibrin molding with subsequent mechanical stimulation.
Globally, 296 million people are infected with hepatitis B virus (HBV), and approximately one million people die annually from HBV-related causes, including liver cancer. Although there is a ...preventative vaccine and antiviral therapies suppressing HBV replication, there is no cure. Intensive efforts are under way to develop curative HBV therapies. Currently, only a few biomarkers are available for monitoring or predicting HBV disease progression and treatment response. As new therapies become available, new biomarkers to monitor viral and host responses are urgently needed. In October 2020, the International Coalition to Eliminate Hepatitis B Virus (ICE-HBV) held a virtual and interactive workshop on HBV biomarkers endorsed by the International HBV Meeting. Various stakeholders from academia, clinical practice and the pharmaceutical industry, with complementary expertise, presented and participated in panel discussions. The clinical utility of both classic and emerging viral and immunological serum biomarkers with respect to the course of infection, disease progression, and response to current and emerging treatments was appraised. The latest advances were discussed, and knowledge gaps in understanding and interpretation of HBV biomarkers were identified. This Roadmap summarizes the strengths, weaknesses, opportunities and challenges of HBV biomarkers.
Summary Background The increasing prevalence of overweight and obesity needs effective approaches for weight loss in primary care and community settings. We compared weight loss with standard ...treatment in primary care with that achieved after referral by the primary care team to a commercial provider in the community. Methods In this parallel group, non-blinded, randomised controlled trial, 772 overweight and obese adults were recruited by primary care practices in Australia, Germany, and the UK. Participants were randomly assigned with a computer-generated simple randomisation sequence to receive either 12 months of standard care as defined by national treatment guidelines, or 12 months of free membership to a commercial programme (Weight Watchers), and followed up for 12 months. The primary outcome was weight change over 12 months. Analysis was by intention to treat (last observation carried forward LOCF and baseline observation carried forward BOCF) and in the population who completed the 12-month assessment. This trial is registered, number ISRCTN85485463. Findings 377 participants were assigned to the commercial programme, of whom 230 (61%) completed the 12-month assessment; and 395 were assigned to standard care, of whom 214 (54%) completed the 12-month assessment. In all analyses, participants in the commercial programme group lost twice as much weight as did those in the standard care group. Mean weight change at 12 months was −5·06 kg (SE 0·31) for those in the commercial programme versus −2·25 kg (0·21) for those receiving standard care (adjusted difference −2·77 kg, 95% CI −3·50 to −2·03) with LOCF; −4·06 kg (0·31) versus −1·77 kg (0·19; adjusted difference −2·29 kg, −2·99 to −1·58) with BOCF; and −6·65 kg (0·43) versus −3·26 kg (0·33; adjusted difference −3·16 kg, −4·23 to −2·11) for those who completed the 12-month assessment. Participants reported no adverse events related to trial participation. Interpretation Referral by a primary health-care professional to a commercial weight loss programme that provides regular weighing, advice about diet and physical activity, motivation, and group support can offer a clinically useful early intervention for weight management in overweight and obese people that can be delivered at large scale. Funding Weight Watchers International, through a grant to the UK Medical Research Council.
This paper explores the interplay of trade credit and short-term bank loans between 1980 and 2012 in Japan. Many of the issues discussed for the U.S. remain largely unexplored for Japan, yet Japan is ...an interesting country study because of the particular features of its financial sector and its experience of extended zero interest rates. This paper makes three contributions: (1) We develop a new approach to using the Bank of Japan
tankan
survey, matched with individual firm-level data, to explore the impact of perceived business conditions and credit constraints on trade credit. (2) We divide the 30 years into sub-periods to gain a more granular view of the trade credit-bank loan relationship over time. (3) We use firm-level data to explore the relationship between trade credit and bank loans for listed firms in distress. We find that trade credit was a bank loan substitute during the upswing of the 1980s, but a complement (driven by the transaction motive) during the stagnation of the 1990s and 2000s. At the firm level, trade credit is contingent on the financial health of the borrower. For firms in distress, in the 1980s and 1990s trading partners extended trade credit after the main bank had come to the rescue. In contrast, in the early 2000s, both main banks and trading partners have reduced support of firms in distress, removing any substitution effect. Trade credit discriminates at the micro-level, which explains why previous research has yielded ambiguous results on the substitute versus complement question.
The role of tobacco smoke exposure in the development and persistence of asthma and rhinoconjunctivitis through childhood into adolescence is unclear.
We assessed the associations of parental smoking ...from fetal life through adolescence with asthma and rhinoconjunctivitis during childhood and adolescence.
We analyzed data for 10,860 participants of five European birth cohort studies from the Mechanisms of the Development of Allergy (MeDALL) consortium. Parental smoking habits and health outcomes (early transient, persistent, and adolescent-onset asthma and rhinoconjunctivitis) were based on questionnaires covering the period from pregnancy to 14-16 y of age. Data were combined and analyzed using a one-stage and two-stage individual participant data meta-analysis.
Overall, any maternal smoking during pregnancy tended to be associated with an increased odds of prevalent asthma adjusted odds ratio (aOR)=1.19 (95% CI: 0.98, 1.43), but not prevalent rhinoconjunctivitis aOR=1.05 (95% CI: 0.90, 1.22), during childhood and adolescence. In analyses with phenotypes related to age of onset and persistence of disease, any maternal smoking during pregnancy was associated with early transient asthma aOR=1.79 (95% CI: 1.14, 2.83). Maternal smoking of ≥10 cigarettes/day during pregnancy was associated with persistent asthma aOR=1.66 (95% CI: 1.29, 2.15) and persistent rhinoconjunctivitis aOR=1.55 (95% CI, 1.09, 2.20). Tobacco smoke exposure during fetal life, infancy, childhood, and adolescence was not associated with adolescent-onset asthma or rhinoconjunctivitis.
Findings from this combined analysis of five European birth cohorts strengthen evidence linking early exposure to tobacco smoke with asthma during childhood and adolescence. Children with high early-life exposure were more likely than unexposed children to have early transient and persistent asthma and persistent rhinoconjunctivitis. https://doi.org/10.1289/EHP2738.
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