Abstract
This study explores if unhealthy lipoprotein distribution (LPD) impairs the anabolic and amino acid sensing responses to whey-protein feeding. Thus, if impairment of such anabolic response ...to protein consumption is seen by the LPD this may negatively affect the skeletal muscle mass. Muscle protein synthesis (MPS) was measured by puromycin labeling in Apolipoprotein E knockout (Apoe KO), characterized by an unhealthy LPD, and wild type mice post-absorptive at 10 and 20 weeks, and post-prandial after whey-protein feeding at 20 weeks. Hypertrophy signaling and amino acid sensing mechanisms were studied and gut microbiome diversity explored. Surprisingly, whey-protein feeding did not affect MPS. p-mTOR and p-4E-BP1 was increased 2 h after whey-protein feeding in both genotypes, but with general lower levels in Apoe KO compared to wild type. At 20 weeks of age, Apoe KO had a greater mRNA-expression for SNAT2, CD98, ATF4 and GCN2 compared to wild type. These responses were not associated with gut microbiota compositional differences. Regardless of LPD status, MPS was similar in Apoe KO and wild type. Surprisingly, whey-protein did not stimulate MPS. However, Apoe KO had lower levels of hypertrophy signaling, was amino acid deprived, and had impaired amino acid sensing mechanisms.
The ‘Latching Orthogonal Cage/Key pRoteins’ (LOCKR) system is a de novo designed protein Switch and Key. The Switch functions to cage a functional protein sequence, such as a degron, in the off or ...“locked” state. Upon Key addition, the Switch is turned on or “unlocked,” which exposes the degron, simultaneously degrading the Switch and any proteins fused to the Switch via the proteasome. Key expression can be tailored for tissue‐specific expression, which gives LOCKR exquisite spatial protein control. LOCKR provides novel post‐translational control over a protein of interest but has not yet been applied to a multicellular organism. We are developing the LOCKR technology for conditional protein depletion in the model organism C. elegans. Although genetic RNAi and similar gene editing techniques benefit from ease of delivery and can target essentially any gene, they have various limitations, including 1) slow protein turnover can prohibit use of RNAi; 2) genetic perturbation of essential genes via genetic deletion prevents characterization of later functions. The degronLOCKR system requires two parts: 1) a gene fused to the ‘degronSwitch’ and 2) an inducer peptide (the Key). The Switch can be fused to any gene of interest using CRISPR‐Cas9 gene editing. We fused degronSwitch to dhc‐1 (dynein heavy chain), an essential cytoplasmic motor protein along with mScarlet and a 3xFLAG tag to aid in protein quantitation via microscopic and Western analysis. We also cloned a negative control, which lacks degronSwitch, to analyze the effect of inserting the Switch in vivo. To complement the degronSwitch, we designed six tissue‐specific Keys for Mos‐Single Copy Insertion (MosSCI), which allows for spatial control of degronSwitch protein degradation. We predict that ubiquitous expression of Key with the degronSwitch fused to dhc‐1 will result in embryonic lethality, whereas germline‐specific Key will permit viability but result in meiotic defects. These phenotypes are easily identifiable and serve as effective proof of concept. Future work includes expanding the LOCKR system to incorporate temporal control of the degronSwitch fusion protein by toggling the Key on and off using heat‐shock promoters and RNAi against the Key. This work establishes a novel and tissue‐specific method for regulating protein function in an animal model.
Altered coagulation has been reported in people living with HIV (PLWH) with ongoing viral replication and may predispose to cardiovascular diseases. However, less is known about coagulation in PLWH ...with undetectable viral replication. In a cross-sectional observational study, we investigated whether HIV infection with undetectable viral replication is independently associated with activated partial thromboplastin time (APTT) and coagulation factor II-VII-X concentrations out of reference. Logistic regression analyses were used to assess the association of HIV infection with APTT and coagulation factor II-VII-X, after adjusting for age, sex, smoking status, alcohol consumption, BMI, diabetes and hsCRP. 936 PLWH with undetectable viral replication from the Copenhagen Co-morbidity in HIV infection study (COCOMO-study) and 2955 uninfected controls were included. Higher prevalence of short APTT was found in PLWH compared to controls (13.5% vs. 7.6%, P < 0.001). Furthermore, higher prevalence of low coagulation factor II-VII-X was found in PLWH than in controls (9.6% vs. 7.4%, P = 0.022). HIV was independently associated with short APTT (adjusted odds ratio (aOR) 2.3 (95% CI 1.7-2.9), P < 0.001) and low coagulation factor II-VII-X (aOR 1.4 (95% CI 1.0-1.9), P = 0.046). Few participants among PLWH and controls had both short APTT and low coagulation factor II-VII-X, 2.1% vs. 0.8%, respectively. We found evidence of both procoagulant (short APTT) and anticoagulant (low coagulation factor II-VII-X) alterations in PLWH with undetectable viral replication, and our findings suggest that two different coagulation phenotypes exist in participants with treated HIV infection.
IMPORTANCE: Patients with nonneovascular age-related macular degeneration (AMD) are encouraged to use the Amsler grid test for self-assessment to facilitate early diagnosis. The test is widely ...recommended, suggesting a belief that it signals worsening AMD, warranting its use in home monitoring. OBJECTIVE: To systematically review studies of the diagnostic test accuracy of the Amsler grid in the diagnosis of neovascular AMD and to perform diagnostic test accuracy meta-analyses. DATA SOURCES: A systematic literature search was conducted in 12 databases for relevant titles from database inception until May 7, 2022. STUDY SELECTION: Studies included those with groups defined as having (1) neovascular AMD and (2) either healthy eyes or eyes with nonneovascular AMD. The index test was the Amsler grid. The reference standard was ophthalmic examination. After removal of obviously irrelevant reports, 2 authors (J.B. and M.S.) independently screened the remaining references in full text for potential eligibility. Disagreements were resolved by a third author (Y.S.). DATA EXTRACTION AND SYNTHESIS: Two authors (J.B. and I.P.) independently extracted all data and evaluated quality and applicability of eligible studies using the Quality Assessment of Diagnostic Accuracy Studies 2. Disagreements were resolved by a third author (Y.S.). MAIN OUTCOMES AND MEASURES: Sensitivity and specificity of the Amsler grid for detecting neovascular AMD with comparators being either healthy control participants or patients with nonneovascular AMD. RESULTS: Of 523 records screened, 10 studies were included with a total of 1890 eyes (mean participant age ranging from 62 to 83 years). Sensitivity and specificity to diagnose neovascular AMD were 67% (95% CI, 51%-79%) and 99% (95% CI, 85%-100%), respectively, when comparators were healthy control participants and 71% (95% CI, 60%-80%) and 63% (95% CI, 49%-51%), respectively, when control participants were patients with nonneovascular AMD. Overall, potential sources of bias were low across studies. CONCLUSIONS AND RELEVANCE: Although the Amsler grid is easy and inexpensive to use for detection of metamorphopsia, its sensitivity may be at levels typically not recommended for monitoring. Coupling this lower sensitivity with only moderate specificity to identify neovascular AMD in a population at risk, these findings suggest that such patients typically should be encouraged to undergo ophthalmic examination regularly, regardless of any results of Amsler grid self-assessment.
The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in ...plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL’E’ domains of VWF. Binding between the FVIII a3 region and VWF TIL’E’ was here observed using NMR spectroscopy, where chemical shift changes were localized to two β-sheet regions on the edge of TIL’E’ upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL’E’ as well as mutants of TIL’E’, which further highlights the importance of the β-sheet region of TIL’E’ for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the β-sheet region of TIL’E’ is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex.
•The outbreak in Germany’s gay hotspot Berlin included 222 cases, most self-identifying as MSM.•Hepatitis A vaccination is recommended and free of charge for MSM in Germany.•The vaccination coverage ...was low among MSM, in particular among young and foreign-born.•Various measures were implemented targeting the MSM community, physicians, and the public.•Increased vaccination uptake shows the success of the interventions.
To describe the characteristics of a large hepatitis A virus (HAV) outbreak among men who have sex with men (MSM) in Berlin and to assess the impact of measures implemented.
Cases of laboratory-confirmed, symptomatic HAV infection notified in Berlin, Germany between August 2016 and February 2018 were analysed using routine and enhanced surveillance data including genotyping results. Several studies involving different groups of participants were conducted to further investigate the outbreak, including surveys on knowledge and practices of HAV vaccination among physicians and vaccination coverage and determinants of vaccination status among MSM. The measures implemented were categorized by target group in a Gantt chart. To assess their impact, health insurance data on HAV vaccination uptake were analysed, comparing Berlin and other federal states.
During the outbreak period, a total of 222 cases were reported (of which 91 were sequence-confirmed), with a peak in case numbers in January 2017. Physicians were aware of the existing vaccination recommendations, but vaccination coverage among 756 MSM was low, with 32.7% being completely vaccinated and 17.3% being incompletely vaccinated before 2017. HAV vaccination before 2017 was associated with being born in Germany (odds ratio 2.36) and HIV-positive (odds ratio 1.80). HAV monovalent vaccination uptake increased by 164% from 2016 to 2017 among males in Berlin, compared to 7% in other federal states.
Multiple measures targeting the MSM community, physicians, and public health to increase HAV vaccination uptake were successfully implemented. To prevent future HAV outbreaks, we recommend monitoring vaccination coverage among MSM, promoting awareness of existing recommendations among physicians, and ensuring access for foreign-born and young MSM.
Residual immune dysfunctions, resembling those that occur during normal aging, may persist even in well-treated people with HIV (PWH), and accelerated aging has been proposed. We aimed to determine ...if HIV infection is an independent risk factor for T-cell immune dysfunctions including increased immune activation, senescence and apoptosis. Moreover, in PWH we aimed to identify the associations between age and immune activation, senescence and apoptosis.
We included 780 PWH with suppressed viral replication (<50 copies/mL) and absence of hepatitis B and hepatitis C co-infection and 65 uninfected controls from the Copenhagen Co-morbidity in HIV Infection (COCOMO) Study. Flow cytometry was used to determine T-cell activation (CD38+HLA-DR+), senescence (CD28-CD57+), and apoptosis (CD28-CD95+). T-cell subsets are reported as proportions of CD4+ and CD8+ T-cells. We defined an elevated proportion of a given T-cell subset as above the 75th percentile. Regression models were used to determine the association between HIV status and T-cell subset and in PWH to determine the association between age or HIV-specific risk factors and T-cell subsets. Furthermore, an interaction between HIV status and age on T-cell subsets was investigated with an interaction term in models including both PWH and controls. Models were adjusted for age, sex, BMI, and smoking status.
In adjusted models a positive HIV status was associated with elevated proportions of CD8+ activated (
= 0.009), CD4+ senescent (
= 0.004), CD4+ apoptotic (
= 0.002), and CD8+ apoptotic (
= 0.003) T-cells. In PWH a 10-year increase in age was associated with higher proportions of CD4+ and CD8+ senescent (
= 0.001 and
< 0.001) and CD4+ and CD8+ apoptotic T-cells (
< 0.001 and
< 0.001). However, no interaction between HIV status and age was found. Furthermore, in PWH a CD4+/CD8+ ratio < 1 was associated with elevated proportions of T-cell activation, senescence, and apoptosis.
We found evidence of residual T-cell immune dysfunction in well-treated PWH without HBV or HCV co-infection, and age was associated with T-cell senescence and apoptosis. Our data supports that HIV infection has similar effects as aging on T-cell subsets. However, since no interaction between HIV status and age was found on these parameters, we found no evidence to support accelerated immunological aging in PWH.
Stem cell transplantation is a promising therapeutic approach for several neurological disorders. However, it has yet to fulfill its high expectations, partially due to the lack of a reliable ...noninvasive method for monitoring the biodistribution of the grafted stem cells in vivo. We have used high-resolution magnetic resonance imaging (MRI) at 17.6 T, combined with efficient magnetic labeling of the stem cells with iron oxide nanoparticles, in order to assess the in vivo detection limit in small animal models. Injection of different concentrations of magnetically labeled stem cells in gel phantoms led to significant reductions in image intensity from small cellular clusters of less than 10 cells. To determine the detection limit in vivo, various numbers of both labeled and unlabeled cells were injected stereotactically into the striatum of rats. Significant hypointense signal changes were observed for 100 labeled cells. After injection of approximately 20 labeled cells, signal reduction at the injection site was observed but could not be assigned unambiguously to the cells. Our results show that high-field MRI allows tracking of a minimal number of cells in vivo, well below the number used in previous studies, opening the possibility of gaining new insights into cell migration and differentiation.
Some of the most varied colors in the natural world are created by iridescent nanostructures in bird feathers, formed by layers of melanin-containing melanosomes. The morphology of melanosomes in ...iridescent feathers is known to vary, but the extent of this diversity, and when it evolved, is unknown. We use scanning electron microscopy to quantify the diversity of melanosome morphology in iridescent feathers from 97 extant bird species, covering 11 orders. In addition, we assess melanosome morphology in two Eocene birds, which are the stem lineages of groups that respectively exhibit hollow and flat melanosomes today. We find that iridescent feathers contain the most varied melanosome morphologies of all types of bird coloration sampled to date. Using our extended dataset, we predict iridescence in an early Eocene trogon (cf. Primotrogon) but not in the early Eocene swift Scaniacypselus, and neither exhibit the derived melanosome morphologies seen in their modern relatives. Our findings confirm that iridescence is a labile trait that has evolved convergently in several lineages extending down to paravian theropods. The dataset provides a framework to detect iridescence with more confidence in fossil taxa based on melanosome morphology.
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