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Skin grafting is often the only treatment for skin trauma when large areas of tissue are affected. This surgical intervention damages the deeper dermal layers of the skin with implications for wound healing and a risk of scar development. Photobiomodulation (PBM) therapy modulates biological processes in different tissues, with a positive effect on many cell types and pathways essential for wound healing. This study investigated the effect of fluorescent light energy (FLE) therapy, a novel type of PBM, on healing after skin grafting in a dermal fibrotic mouse model. Split-thickness human skin grafts were transplanted onto full-thickness excisional wounds on nude mice. Treated wounds were monitored, and excised xenografts were examined to assess healing and pathophysiological processes essential for developing chronic wounds or scarring. Results demonstrated that FLE treatment initially accelerated re-epithelialization and rete ridge formation, while later reduced neovascularization, collagen deposition, myofibroblast and mast cell accumulation, and connective tissue growth factor expression. While there was no visible difference in gross morphology, we found that FLE treatment promoted a balanced collagen remodeling. Collectively, these findings suggest that FLE has a conceivable effect at balancing healing after skin grafting, which reduces the risk of infections, chronic wound development, and fibrotic scarring.

Objectives: Intra-pancreatic fat deposition (IPFD) is suspected to be associated with various medical conditions. This study aimed to assess pancreatic fat content in lean and obese individuals, characterize obese individuals with and without IPFD, and explore the underlying mechanisms., Materials and Methods: Sixty-two obese individuals without diabetes and 35 lean controls underwent magnetic resonance imaging (MRI) using proton density fat fraction (PDFF) maps to evaluate pancreatic and hepatic fat content, and visceral adipose tissue (VAT) content. Pancreatic fibrosis was explored by T1 relaxation time and MR elastography (MRE) measurements. Associations between pancreatic fat, measures of obesity and metabolic syndrome were examined using uni- and multivariate regression analyses., Results: Pancreatic PDFF was higher in obese than in lean controls (median 8.0%, interquartile range (6.1;13.3) % vs 2.6(1.7;3.9)%, p  < 0.001). Obese individuals with IPFD (PDFF ≥6.2%) had higher waist circumference (114.0 ± 12.5 cm vs 105.2 ± 8.7 cm, p  = 0.007) and VAT (224.9(142.1; 316.1) cm 2 vs 168.2(103.4; 195.3) cm 2 , p  < 0.001) than those without. In univariate analysis, pancreatic PDFF in obese individuals correlated with BMI ( r  = 0.27, p  = 0.03), waist circumference ( r  = 0.44, p  < 0.001), VAT ( r  = 0.37, p  = 0.004), hepatic PDFF ( r  = 0.25, p  = 0.046) and diastolic blood pressure ( r  = 0.32, p  = 0.01). However, in multivariate analysis, only VAT was associated to pancreatic fat content. MRI measures of pancreatic fibrosis indicated no evident fibrosis in relation to increased pancreatic fat content., Conclusions: Pancreatic fat content was increased in obese individuals compared with lean controls and predominantly correlated with the amount of visceral adipose tissue. Pancreatic fat content was not clearly linked to measures of pancreatic fibrosis.

Chronic kidney disease (CKD) poses a significant health burden worldwide. Especially, obesity-induced chronic kidney disease (OCKD) is associated with a lack of accuracy in disease diagnostic methods. The identification of reliable biomarkers for the early diagnosis and monitoring of CKD and OCKD is crucial for improving patient outcomes. Extracellular vesicles (EVs) have emerged as potential biomarkers in the context of CKD. In this review, we focused on the role of EVs as potential biomarkers in CKD and OCKD and developed a comprehensive list of EV membrane proteins that could aid in the diagnosis and monitoring of the disease. To assemble our list, we employed a multi-step strategy. Initially, we conducted a thorough review of the literature on EV protein biomarkers in kidney diseases. Additionally, we explored papers investigating circulating proteins as biomarkers in kidney diseases. To further refine our list, we utilized the EV database Vesiclepedia.org to evaluate the qualifications of each identified protein. Furthermore, we consulted the Human Protein Atlas to assess the localization of these candidates, with a particular focus on membrane proteins. By integrating the information from the reviewed literature, Vesiclepedia.org, and the Human Protein Atlas, we compiled a comprehensive list of potential EV membrane protein biomarkers for CKD and OCKD. Overall, our review underscores the potential of EVs as biomarkers in the field of CKD research, providing a foundation for future studies aimed at improving CKD and OCKD diagnosis and treatment., Competing Interests: Author NG was employed by the company GECORP. Author HG-K was employed by the company AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Garcia, Gonzalez-King, Mellergaard, Nair, Salomon and Handberg.)

Importance: Therapies that target and aid the host immune defense to repel cancer cells or invading pathogens are rapidly emerging. Antibiotic resistance is among the largest threats to human health globally. Staphylococcus aureus ( S. aureus ) is the most common bacterial infection, and it poses a challenge to the healthcare system due to its significant ability to develop resistance toward current available therapies. In long-term infections, S. aureus further adapt to avoid clearance by the host immune defense. In this study, we discover a new interaction that allows S. aureus to avoid elimination by the immune system, which likely supports its persistence in the host. Moreover, we find that blocking the specific receptor (PD-1) using antibodies significantly relieves the S. aureus -imposed inhibition. Our findings suggest that therapeutically targeting PD-1 is a possible future strategy for treating certain antibiotic-resistant staphylococcal infections., Competing Interests: The authors declare no conflict of interest.

Non-alcoholic fatty liver disease (NAFLD) is a liver disorder that has become a global health concern due to its increasing prevalence. There is a need for reliable biomarkers to aid in the diagnosis and prognosis of NAFLD. Extracellular vesicles (EVs) are promising candidates in biomarker discovery, as they carry proteins that reflect the pathophysiological state of the liver. In this review, we developed a list of EV proteins that could be used as diagnostic biomarkers for NAFLD. We employed a multi-step strategy that involved reviewing and comparing various sources of information. Firstly, we reviewed papers that have studied EVs proteins as biomarkers in NAFLD and papers that have studied circulating proteins as biomarkers in NAFLD. To further identify potential candidates, we utilized the EV database Vesiclepedia.org to qualify each protein. Finally, we consulted the Human Protein Atlas to search for candidates' localization, focusing on membrane proteins. By integrating these sources of information, we developed a comprehensive list of potential EVs membrane protein biomarkers that could aid in diagnosing and monitoring NAFLD. In conclusion, our multi-step strategy for identifying EV-based protein biomarkers for NAFLD provides a comprehensive approach that can also be applied to other diseases. The protein candidates identified through this approach could have significant implications for the development of non-invasive diagnostic tests for NAFLD and improve the management and treatment of this prevalent liver disorder.

Objective: We recently demonstrated that upper-body rowing exercise (UBROW) improved aerobic fitness in individuals with spinal cord injury (SCI), with no effect on traditional cardiometabolic risk factors. Here, we tested the hypothesis that the exercise-induced increase in aerobic fitness was maintained at 6-month (6M) follow-up., Design: Six-month follow-up., Setting: University/hospital., Participants: Seventeen wheelchair-dependent participants with SCI., Interventions: 12-week of exercise training (UBROW) or control (CON)., Outcome Measures: Aerobic fitness (PO peak and V̇O 2peak ), body composition, blood pressure, and blood biomarkers of cardiometabolic risk were assessed at 6M follow-up and compared to baseline (BL) and immediately post-intervention (12-week). Minutes of mild, moderate, and heavy intensity leisure time physical activity (LTPA) were assessed by self-report., Results: Fourteen participants returned at 6M follow-up (CON, n  = 6; UBROW, n  = 8). In UBROW, PO peak (median (Q1-Q3)) increased from BL (70 W (37-84)) to 12-week (77 W (58-109), P = 0.01) and 6M follow-up (81 W (51-96), P = 0.01), with no difference between 12-week and 6M follow-up (P = 0.21). Similarly, V̇O 2peak increased from BL (15.4 ml/kg/min (10.5-19.4)) to 12-week (16.6 ml/kg/min (12.8-21.3), P = 0.01) with no difference between 12-week and 6M follow-up (16.3 ml/kg/min (12.9-19.7), P = 0.74). No differences were found in CON for either PO peak (P = 0.22) or V̇O 2peak (P = 0.27). There were no changes over time in traditional cardiometabolic risk factors or for minutes of different LTPA intensities., Conclusion: We demonstrate that improvements in aerobic fitness are maintained for at least six months after completion of a 12-week exercise intervention, supporting the use of periodic exercise interventions to boost aerobic fitness level in individuals with SCI. Trial registration: ClinicalTrials.gov identifier: NCT04390087..

Background and Aims: OxLDL modulates innate and adaptive immunity, and extracellular vesicles (EVs) released from both non-immune and immune cells are proposed key players in atherosclerosis development. In the present study, we aimed to investigate EVs expressing markers related to adaptive immunity-driven inflammation and endothelial activation/dysfunction in hypercholesterolemic patients., Methods: EVs were phenotyped in thirty patients with familial hypercholesterolemia (FH) and twenty-three healthy controls using the Extracellular Vesicle (EV) Array with antibodies targeting proteins expressed on B and T cells, and endothelial cells., Results: FH patients had a higher atherosclerotic burden, as determined by the mean carotid intima-media thickness (IMT) (0.64 ± 0.12 mm vs. 0.58 ± 0.07 mm; p = 0.033), higher oxLDL levels (p < 0.0001), and showed increased levels of EV-specific markers: CD9 (p = 0.017), CD63 (p = 0.045), CD81 (p = 0.003), Annexin V (p = 0.018), and EV markers related to adaptive/lymphocyte immunity: CD28 (p = 0.034), CD4 (p = 0.049), CD152 (p = 0.029), LFA-1 (p = 0.024), and endothelial function: CD62E (p = 0.032), CD144 (p = 0.018), tPA (p = 0.017), CD31 (p = 0.024). Linear regression revealed a positive relationship between carotid IMT and several of the increased markers observed within the FH group, including CD9 (β = 0.33; p = 0.022), CD63 (β = 0.35; p 225 = 0.026), CD28 (β = 0.37; p = 0.026), CD4 (β = 0.40; p = 0.025), CD152 (β = 0.41; p = 0.017), LFA-1 (β = 0.42; p = 0.014) and CD62E (β = 0.38; p = 0.024)., Conclusion: EVs associated with adaptive immunity and endothelial dysfunction are elevated in FH patients, and several markers related to a higher atherosclerotic burden., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Purpose: This study assessed the effects of upper-body rowing exercise on cardiorespiratory fitness, traditional cardiometabolic risk factors, and vascular health in individuals with spinal cord injury (SCI)., Methods: Seventeen male and female adults with chronic (> 1 yr) motor-complete and incomplete SCI (level of injury: C4-L3) were randomized to control (CON, n = 9) or exercise (UBROW, n = 8). Participants in UBROW performed 12-week, 3 weekly sessions of 30-min upper-body ergometer rowing exercise, complying with current exercise guidelines for SCI. Cardiorespiratory fitness ([Formula: see text]O 2peak ), traditional risk factors (lipid profile, glycemic control) as well as inflammatory and vascular endothelium-derived biomarkers (derived from fasting blood samples) were measured before and after 6 (6W) and 12 weeks (12W). Brachial artery resting diameter and flow-mediated dilation (FMD) were determined by ultrasound as exploratory outcomes., Results: UBROW increased [Formula: see text]O 2peak from baseline (15.1 ± 5.1 mL/kg/min; mean ± SD) to 6W (16.5 ± 5.3; P < 0.01) and 12W (17.5 ± 6.1; P < 0.01). UBROW increased resting brachial artery diameter from baseline (4.80 ± 0.72 mm) to 12W (5.08 ± 0.91; P < 0.01), with no changes at 6W (4.96 ± 0.91), and no changes in CON. There were no significant time-by-group interactions in traditional cardiometabolic blood biomarkers, or in unadjusted or baseline diameter corrected FMD. Explorative analyses revealed inverse correlations between changes (∆12W-baseline) in endothelin-1 and changes in resting diameter (r = - 0.56) and FMD% (r = - 0.60), both P < 0.05., Conclusion: These results demonstrate that 12 weeks of upper-body rowing complying with current exercise guidelines for SCI improves cardiorespiratory fitness and increases resting brachial artery diameter. In contrast, the exercise intervention had no or only modest effects on traditional cardiometabolic risk factors. The study was registered at Clinicaltrials.gov (N-20190053, May 15, 2020)., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: The purpose of this study was to evaluate different renal proton density fat fraction (PDFF) analysis approaches. Additionally, we assessed renal fat in obese individuals and lean individuals., Methods: This was a retrospective observational case-control study. Twenty-eight obese individuals and 14 lean controls underwent MRI with multi-point Dixon technique for PDFF maps. The following renal PDFF image analysis approaches were performed and compared: (1) five circular regions of interest (ROIs) in six slices, (2) three circular ROIs in one slice, (3) freehand segmentation of renal parenchyma in one slice, and (4) freehand segmentation of renal parenchyma avoiding the renal border in one slice. Furthermore, renal PDFF was compared between obese and lean individuals., Results: Methods 1, 2, and 4 were positively correlated (r ≥ 0.498, p ≤ 0.001). Renal PDFF values varied more with regards to ROI placement within slices than mean PDFF between slices. Using all methods, the obese individuals had significantly higher renal PDFF values compared with the lean controls., Conclusion: Renal PDFF should be measured covering large areas of the kidney while excluding artifacts. This can be achieved using multiple circular ROIs. Increased lipid accumulation in the kidneys was related to obesity., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In this descriptive pilot study, we aim to establish a porcine Staphylococcus aureus skin infection model by subcutaneous injection (s.c.) of the porcine S54F9 S. aureus strain in the groin area. Six pigs were used in the study: Five pigs were injected with S. aureus, inocula ranging from 7 × 10 3 to 5 × 10 7 colony-forming units per kg bodyweight; one pig was injected with saline exclusively. Lesions were recorded up to 6 days postinoculation using clinical evaluation, ultrasound evaluation, microbiology, flow cytometry, and pathology. Inoculation gave rise to lesions ranging from localized skin infection, that is, minute histological changes, intracellular infection, and macroscopic abscess formation with sequestration of soft tissue, to generalized infection and development of disseminated intravascular coagulation necessitating euthanasia only 10 h after inoculation. Ultrasound assessment of maximum width and characteristics was not able to disclose the progress of the local infection. Flow cytometry and immunohistochemistry revealed the participation of γδT cells in the immune response. In conclusion, we did see a graded inflammatory response associated with the dose of s.c. inoculated bacteria, which may be useful for studying, in particular, the interaction of bacteria and inflammatory mononuclear cell populations. It needs to be investigated if the model is discriminatory and robust., (© 2020 Scandinavian Societies for Medical Microbiology and Pathology.)

Introduction: The use of photobiomodulation has been proposed to improve wound healing for the last two decades. Recent development in photobiomodulation has led to the development of a novel biophotonic platform that utilizes fluorescent light energy (FLE) within the visible spectrum of light for healing of skin inflammation and wounds., Materials and Methods: In this article, FLE was used in preliminary analysis on 18 case studies of acute second-degree burns and in a pilot study using an ex vivo human skin model. Efficacy of FLE on wound healing and tissue remodeling was evaluated by monitoring improvements in the treated tissues, assessing pain for the patients, and by performing human genome microarray analysis of FLE-treated human skin samples., Results: Healing was reported for all 18 patients treated with FLE for acute second-degree burns without reported adverse effects or development of infections. Furthermore, preliminary ex vivo skin model data suggest that FLE impacts different cellular pathways including essential immune-modulatory mechanisms., Conclusions: The results presented in this article are encouraging and suggest that FLE balances different stages of wound healing, which opens the door to initiating randomized controlled clinical trials for establishing the efficacy of FLE treatment in different phases of wound healing of second-degree burns., (© The Association of Military Surgeons of the United States 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

SCFAs are primarily produced in the colon by bacterial fermentation of nondigestible carbohydrates. Besides providing energy, SCFAs can suppress development of colon cancer. The mechanism, however, remains elusive. Here, we demonstrate that the SCFA propionate upregulates surface expression of the immune stimulatory NKG2D ligands, MICA/B by imposing metabolic changes in dividing cells. Propionate-mediated MICA/B expression did not rely on GPR41/GPR43 receptors but depended on functional mitochondria. By siRNA-directed knockdown, we could further link phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis to propionate regulation of MICA/B expression. Moreover, knockdown of Rictor and specific mTOR inhibitors implicated mTORC2 activity with metabolic changes that control MICA/B expression. SCFAs are precursors to short-chain acyl-CoAs that are used for histone acylation thereby linking the metabolic state to chromatin structure and gene expression. Propionate increased the overall acetylation and propionylation and inhibition of lysine acetyltransferases (KATs) that are responsible for adding acyl-CoAs to histones reduced propionate-mediated MICA/B expression, suggesting that propionate-induced acylation increases MICA/B expression. Notably, propionate upregulated MICA/B surface expression on colon cancer cells in an acylation-dependent manner; however, the impact of mitochondrial metabolism on MICA/B expression was different in colon cancer cells compared with Jurkat cells, suggesting that continuous exposure to propionate in the colon may provide an enhanced capacity to metabolize propionate. Together, our findings support that propionate causes metabolic changes resulting in NKG2D ligand surface expression, which holds potential as an immune activating anticancer therapy., (© 2020 Federation of American Societies for Experimental Biology.)

Staphylococcus aureus is among the leading causes of bacterial infections worldwide. The pathogenicity and establishment of S. aureus infections are tightly linked to its ability to modulate host immunity. Persistent infections are often associated with mutant staphylococcal strains that have decreased susceptibility to antibiotics; however, little is known about how these mutations influence bacterial interaction with the host immune system. Here, we discovered that clinical S. aureus isolates activate human monocytes, leading to cell-surface expression of immune stimulatory natural killer group 2D (NKG2D) ligands on the monocytes. We found that expression of the NKG2D ligand ULBP2 (UL16-binding protein 2) is associated with bacterial degradability and phagolysosomal activity. Moreover, S. aureus -induced ULBP2 expression was linked to altered host cell metabolism, including increased cytoplasmic (iso)citrate levels, reduced glycolytic flux, and functional mitochondrial activity. Interestingly, we found that the ability of S. aureus to induce ULBP2 and proinflammatory cytokines in human monocytes depends on a functional ClpP protease in S. aureus These findings indicate that S. aureus activates ULBP2 in human monocytes through immunometabolic mechanisms and reveal that clpP inactivation may function as a potential immune evasion mechanism. Our results provide critical insight into the interplay between the host immune system and S. aureus that has evolved under the dual selective pressure of host immune responses and antibiotic treatment. Our discovery of an immune stimulatory pathway consisting of human monocyte-based defense against S. aureus suggests that targeting the NKG2D pathway holds potential for managing persistent staphylococcal infections., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Mellergaard et al.)

Immune surveillance of cancer cells is facilitated by the Natural Killer Group 2D (NKG2D) receptor expressed by different lymphocyte subsets. It recognizes NKG2D ligands that are rarely expressed on healthy cells, but upregulated by tumorigenesis, presenting a target for immunological clearance. The molecular mechanisms responsible for NKG2D ligand regulation remain complex. Here we report that cancer cell metabolism supports constitutive surface expression of the NKG2D ligand MHC class I chain-related proteins A (MICA). Knockout of the N -glycosylation gene N -acetylglucosaminyltransferase V (MGAT5) in HEK293 cells induced altered metabolism and continuous high MICA surface expression. MGAT5 knockout cells were used to examine the association of cell metabolism and MICA expression through genetic, pharmacological and metabolic assays. Findings were verified in cancer cell lines. Cells with constitutive high MICA expression showed enhanced spare respiratory capacity and elevated mitochondrial efflux of citrate, determined by extracellular flux analysis and metabolomics. MICA expression was reduced by inhibitors of mitochondrial function, FCCP and etomoxir e.g., and depended on conversion of citrate to acetyl-CoA and oxaloacetate by ATP citrate lyase, which was also observed in several cancer cell types. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis revealed that upregulated MICA transcription was associated with an open chromatin structure at the MICA transcription start site. We identify mitochondria and cytoplasmic citrate as key regulators of constitutive MICA expression and we propose that metabolic reprogramming of certain cancer cells facilitates MICA expression and NKG2D-mediated immune recognition., (Copyright © 2020 Møller, Mellergaard, Madsen, Bermejo, Jepsen, Hansen, Høgh, Aldana, Desler, Rasmussen, Sustarsic, Gerhart-Hines, Daskalaki, Wheelock, Hiron, Lin, O’Callaghan, Wandall, Andresen and Skov.)

Fumarate is a tricarboxylic acid cycle metabolite whose intracellular accumulation is linked to inflammatory signaling and development of cancer. In this study, we demonstrate that endogenous fumarate accumulation upregulates surface expression of the immune stimulatory NK group 2, member D (NKG2D) ligands ULBP2 and ULBP5. In agreement with this, accumulation of fumarate by the therapeutic drug dimethyl fumarate (DMF) also promotes ULBP2/5 surface expression. Mechanistically, we found that the increased ULBP2/5 expression was dependent on oxidative stress and the antioxidants N -acetylcysteine and glutathione (GSH) abrogated ULBP2/5 upregulated by DMF. Fumarate can complex with GSH and thereby exhaust cells of functional GSH capacity. In line with this, inhibition of GSH reductase (GR), the enzyme responsible for GSH recycling, promoted ULBP2/5 surface expression. Loss of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) associates with a malignant form of renal cancer characterized by fumarate accumulation and increased production of reactive oxygen species, highlighting fumarate as an oncometabolite. Interestingly, FH-deficient renal cancer cells had low surface expression of ULBP2/5 and were unresponsive to DMF treatment, suggesting that the fumarate-stimulating ULBP2/5 pathway is abrogated in these cells as an immune-evasive strategy. Together, our data show that ULBP2/5 expression can be upregulated by accumulation of fumarate, likely by depleting cells of GSH antioxidant capacity. Given that DMF is an approved human therapeutic drug, our findings support a broader use of DMF in treatment of cancers and inflammatory conditions., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Background: We have previously reported clinical efficacy with a novel form of photobiomodulation-a biophotonic platform inducing fluorescent light energy (FLE)-in both disease-affected and healthy skin; however, the cellular mechanisms remain largely unknown. Objective: This study investigated the cellular mechanism of action of FLE on key skin and immune cells. Methods: We examined the effects of FLE on the clinical presentation of inflammation in a representative patient with acne vulgaris. The effect of FLE and an FLE-mimicking control lamp on collagen production from primary human dermal fibroblast (HDF) cells was assessed in the presence and absence of the proinflammatory cytokine, interferon gamma (IFN-γ). Cytokine production was assessed from HDF and human epidermal keratinocytes (HEK) exposed to M1 macrophage-conditioned media following illumination with either a blue light-emitting diode (LED) or FLE. Finally, the effects of FLE on angiogenesis were assessed in human aortic endothelial (HAE) cells. Results: FLE reduced inflammatory lesions and associated redness in the representative acne patient. Following the resolution of inflammation there was an overall enhancement of the skin's texture. FLE enhanced collagen production from nonstressed HDF cells, decreased the inflammatory profile of HDF and HEK cells, and enhanced angiogenesis in HAE cells. Conclusion: Our results suggest FLE is capable of enhancing collagen production, modulating cutaneous inflammation, and encouraging angiogenesis. While further research is required, our findings have important implications for approaches to treating inflammatory skin conditions and achieving better aesthetic outcomes., Competing Interests: FUNDING:No funding was received for this study. DISCLOSURES:Drs. Edge and Nielsen are employees of FB Dermatology, Ltd./Kleresca®. The other authors have no conflicts of interest relevant to the content of this article.

Daptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. The emergence of daptomycin-nonsusceptible S. aureus isolates during therapy is often associated with multiple genetic changes; however, the relative contributions of these changes to resistance and other phenotypic changes usually remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single-nucleotide polymorphism (SNP) in rpoB (RpoB A477D) that decreased susceptibility not only to daptomycin but also to vancomycin, β-lactams, and rifampin. Furthermore, the rpoB mutant exhibited pleiotropic phenotypes, including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator Spx, and slow growth. A subsequently acquired loss-of-function mutation in clpX partly alleviated the growth defect conferred by the rpoB mutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP in mprF (MprF S295L) known to confer daptomycin nonsusceptibility, and accordingly, this isolate was the only daptomycin-nonsusceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as that of the parental strain, while the level of transcription of the vraSR (cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selected in vivo contribute to daptomycin nonsusceptibility, growth fitness, and virulence traits., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation, and we have previously shown that changes in cellular N-glycosylation are involved in regulation of NKG2D ligand surface expression. The specific mode of regulation through N-glycosylation is, however, unknown. Here we investigated whether direct N-glycosylation of the NKG2D ligand MICA itself is critical for cell surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (Asn(8)) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (Thr(24)) in the extracellular domain of MICA018 was essential for the N-glycosylation dependence, whereas the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation, and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018, and we pinpoint the residues essential for this N-glycosylation dependence. In addition, we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)