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Plastic mismanagement and its subsequent pollution by rapid economic development and urbanisation pose significant challenges for modern world society. Notwithstanding one of the main sources for macro plastic leakage into the ocean from land, precise assessment of plastic pollution origins from Southeast Asia is yet to be clearly examined. In order to make informed decisions and prioritise areas of improvement it is required to better understand the waste leakage dynamics at the local level. In this work, the Waste Flow Diagram (WFD) was applied to understand the sources and fates of plastics leaking from the solid waste management system for the case of Tuy Hoa City, Phu Yen, Vietnam. The study shows scenarios of leakage into the aquatic environment ranging from 0.8 to 2.7 kg/cap/year, which originates mainly from the collection system. Targeted improvements to this stage of the service could reduce leakages and the overall environmental impacts of mismanaged plastic waste. The results of this study show the necessity and importance of having up to date and reliable data to better inform stakeholders and service planning, facilitating efficient action against plastic pollution. As the first peer-reviewed scientific article critically applying the WFD, this work highlights the steps and challenges of the methodology and critically analyses different methodological pathways., Competing Interests: Declaration of competing interest 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., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The ability of tumour cells to thrive in harsh microenvironments depends on the utilization of nutrients available in the milieu. Here we show that pancreatic cancer-associated fibroblasts (CAFs) regulate tumour cell metabolism through the secretion of acetate, which can be blocked by silencing ATP citrate lyase (ACLY) in CAFs. We further show that acetyl-CoA synthetase short-chain family member 2 (ACSS2) channels the exogenous acetate to regulate the dynamic cancer epigenome and transcriptome, thereby facilitating cancer cell survival in an acidic microenvironment. Comparative H3K27ac ChIP-seq and RNA-seq analyses revealed alterations in polyamine homeostasis through regulation of SAT1 gene expression and enrichment of the SP1-responsive signature. We identified acetate/ACSS2-mediated acetylation of SP1 at the lysine 19 residue that increased SP1 protein stability and transcriptional activity. Genetic or pharmacologic inhibition of the ACSS2-SP1-SAT1 axis diminished the tumour burden in mouse models. These results reveal that the metabolic flexibility imparted by the stroma-derived acetate enabled cancer cell survival under acidosis via the ACSS2-SP1-SAT1 axis., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Objectives: This scoping review aims to examine the caregiving experiences of Korean American caregivers of persons with dementia., Methods: A comprehensive electronic search was conducted within 5 databases (PubMed, CINAHL, Web of Science, Embase, PsycINFO-ProQuest) for papers published from 01/01/00 -01/24/22. Seventeen articles met the inclusion criteria. Thematic analysis was used to summarize key findings from these papers., Results: Most Korean American dementia caregivers were immigrants and wives/daughters/daughters-in-law. Two themes emerged: 1) how Korean American caregivers perceived their caregiving experiences, and 2) how Korean American caregivers perceived their caregiving support services. Korean American caregivers often experience poor mental health and burden. Social support and familism were found to be two of the most important factors that determine their attitudes toward caregiving. Most reported barriers to utilizing public services. Challenges in finding culturally relevant resources were common., Conclusions: Dementia caregiving is a significant public health problem facing Korean Americans. Recommendations for future research are provided.

In this study, the stability of the total nitrogen removal efficiency (TNRE) was modeled using an artificial neural network (ANN)-based binary classification model for the anaerobic ammonium oxidation (AMX) process under saline conditions. The TNRE was stabilized to 80.2 ± 11.4% at the final phase under the salinity of 1.0 ± 0.02%. The results of terminal restriction fragment length polymorphism (T-RFLP) analysis showed the predominance of Candidatus Jettenia genus. Real-time quantitative PCR analysis revealed the average abundance of Ca. Jettenia and Kuenenia spp. increased in 3.2 ± 5.4 × 10 8 and 2.0 ± 2.2 × 10 5 copies/mL, respectively. The prediction accuracy using operational parameters with data augmentation was 88.2%. However, integration with T-RFLP and real-time qPCR signals improved the prediction accuracy by 97.1%. This study revealed the feasible application of machine learning and biomolecular signals to the stability prediction of the AMX process under increased salinity., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Neoadjuvant systemic therapy is a preferred treatment approach for a number of tumor types due to many potential advantages over upfront surgery, including tumor downstaging, early treatment of micrometastatic disease, and providing an in vivo test of tumor biology. For colon cancer, current standard of care is upfront surgery followed by adjuvant systemic therapy in high-risk patients. Concerns about inaccurate radiological staging and tumor progression during preoperative treatment, as well the lack of randomized data demonstrating benefit, are among the reasons for the limited use of neoadjuvant therapy in this disease. Locally advanced colon cancer, defined as primary colon cancer with direct invasion into the adjacent structures or extensive regional lymph node involvement, is not always amenable to pathological complete resection, and when attempted it comes with high incidence of postoperative morbidity and mortality because of the required multivisceral resection. Clinical trials of neoadjuvant chemotherapy for colon cancer to date have been promising with downstaging of disease and higher rates of R0 resection. Here, we report a case of a patient with locally advanced, unresectable, mismatch repair deficient sigmoid colon cancer who was treated with neoadjuvant chemoimmunotherapy followed by surgical resection leading to a complete pathologic response after preoperative systemic chemoimmunotherapy.

Background: Radiation therapy (RT) has a suboptimal effect in patients with pancreatic ductal adenocarcinoma (PDAC) due to intrinsic and acquired radioresistance (RR). Comprehensive bioinformatics and microarray analysis revealed that cholesterol biosynthesis (CBS) is involved in the RR of PDAC. We now tested the inhibition of the CBS pathway enzyme, farnesyl diphosphate synthase (FDPS), by zoledronic acid (Zol) to enhance radiation and activate immune cells., Methods: We investigated the role of FDPS in PDAC RR using the following methods: in vitro cell-based assay, immunohistochemistry, immunofluorescence, immunoblot, cell-based cholesterol assay, RNA sequencing, tumouroids (KPC-murine and PDAC patient-derived), orthotopic models, and PDAC patient's clinical study., Findings: FDPS overexpression in PDAC tissues and cells (P < 0.01 and P < 0.05) is associated with poor RT response and survival (P = 0.024). CRISPR/Cas9 and pharmacological inhibition (Zol) of FDPS in human and mouse syngeneic PDAC cells in conjunction with RT conferred higher PDAC radiosensitivity in vitro (P < 0.05, P < 0.01, and P < 0.001) and in vivo (P < 0.05). Interestingly, murine (P = 0.01) and human (P = 0.0159) tumouroids treated with Zol+RT showed a significant growth reduction. Mechanistically, RNA-Seq analysis of the PDAC xenografts and patients-PBMCs revealed that Zol exerts radiosensitization by affecting Rac1 and Rho prenylation, thereby modulating DNA damage and radiation response signalling along with improved systemic immune cells activation. An ongoing phase I/II trial (NCT03073785) showed improved failure-free survival (FFS), enhanced immune cell activation, and decreased microenvironment-related genes upon Zol+RT treatment., Interpretation: Our findings suggest that FDPS is a novel radiosensitization target for PDAC therapy. This study also provides a rationale to utilize Zol as a potential radiosensitizer and as an immunomodulator in PDAC and other cancers., Funding: National Institutes of Health (P50, P01, and R01)., Competing Interests: Declaration of interests Dr. Surinder K. Batra is one of the co-founders of Sanguine Diagnostics and Therapeutics, Inc. All other authors disclosed no potential conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Growing interest is evident in longitudinal mixed methods research, particularly fully longitudinal mixed methods designs in which both quantitative and qualitative data are collected concurrently for the duration of the study. Fully longitudinal mixed methods designs are particularly relevant for research on dynamic phenomena because of their ability to illuminate both quantitative and qualitative dimensions of change in real time as the phenomenon of interest changes. However, these are complex research designs and their data-intense nature makes them potentially burdensome for study participants, challenging for research teams, and costly for funding agencies. Despite growing use, the methodological literature on fully longitudinal mixed methods research is sparse and little guidance is available for researchers considering this approach. We address this gap by describing our experience with the design and implementation of a fully longitudinal mixed methods study of a dynamic phenomenon, namely, family caregiving during cancer treatment. We describe important questions and key decisions confronted while developing the research proposal, proactive strategies for study implementation, and implementation realities encountered while the study was in progress. On the basis of insights gained through real-world experience, we offer three guiding principles for researchers undertaking such a study. First, align the study design with the nature of the dynamics in the phenomenon of interest. Second, plan from the start when and how the integration of the longitudinal quantitative and qualitative data will occur. Third, employ implementation strategies that take into account the practical aspects of repeated contacts with study participants for an extended period., (© 2021 Wiley Periodicals LLC.)

Background: There are many potential treatment options for patients with early stage hepatocellular carcinoma (HCC) and practice patterns vary widely. This project aimed to use a Delphi conference to generate consensus regarding the management of small resectable HCC., Methods: A base case was established with review by members of AHPBA Research Committee. The Delphi panel of experts reviewed the literature and scored clinical case statements to identify areas of agreement and disagreement. Following initial scoring, discussion was undertaken, questions were amended, and scoring was repeated. This cycle was repeated until no further likelihood of reaching consensus existed., Results: The panel achieved agreement or disagreement consensus regarding 27 statements. The overarching themes included that resection, ablation, transplantation, or any locoregional therapy as a bridge to transplant were all appropriate modalities for early or recurrent HCC. For larger lesions, consensus was reached that radiofrequency ablation and microwave ablation were not appropriate treatments., Conclusion: Using a validated system for identifying consensus, an expert panel agreed that multiple treatment modalities are appropriate for early stage HCC. These consensus guidelines are intended to help guide physicians through treatment modalities for early HCC; however, clinical decisions should continue to be made on a patient-specific basis., (Copyright © 2020 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Extracellular vesicles (EVs) play important roles in cell-cell communication. In budding yeast ( Saccharomyces cerevisiae ), EVs function as carriers to transport cargo proteins into the periplasm for storage during glucose starvation. However, intracellular organelles that synthesize these EV-associated cargo proteins have not been identified. Here, we investigated whether cytoplasmic organelles-called intracellular vesicle clusters (IVCs)-serve as sites for the synthesis of proteins targeted for secretion as EV-associated proteins. Using proteomics, we identified 377 IVC-associated proteins in yeast cells grown under steady-state low-glucose conditions, with the largest group being involved in protein translation. Isolated IVCs exhibited protein synthesis activities that required initiation and elongation factors. We have also identified 431 newly synthesized proteins on isolated IVCs. Expression of 103Q-GFP, a foreign protein with a long polyglutamine extension, resulted in distribution of this protein as large puncta that co-localized with IVC markers, including fructose-1,6-bisphosphatase (FBPase) and the vacuole import and degradation protein Vid24p. We did not observe this pattern in cycloheximide-treated cells or in cells lacking VID genes, required for IVC formation. The induction of 103Q-GFP on IVCs adversely affected total protein synthesis in intact cells and on isolated IVCs. This expression also decreased levels of EV-associated cargo proteins in the extracellular fraction without affecting the number of secreted EVs. Our results provide important insights into the functions of IVCs as sites for the synthesis of EV-associated proteins targeted for secretion to the periplasm., (© 2020 Winters et al.)

Exosomes are small vesicles secreted by a variety of cell types under physiological and pathological conditions. When Saccharomyces cerevisiae are grown in low glucose, small vesicles carrying more than 300 proteins with diverse biological functions are secreted. Upon glucose addition, secreted vesicles are endocytosed that requires cup-shaped organelles containing the major eisosome protein Pil1p at the rims. We aim to identify genes that regulate the function of cup-shaped organelles in vesicle endocytosis. In cells lacking either VID27 or VID21, Pil1p distribution was altered and cup-shaped organelles became elongated with narrower openings. Change in shape reduced the number of vesicles in the deeper areas and impaired vesicle endocytosis. Vid21p and Vid27p were localized to vesicle clusters and interacted with other Vid proteins. In the absence of these genes, these vesicles failed to aggregate and were secreted. Vid21p and Vid27p are required for the aggregation and retention of vesicles that contain Vid proteins in the cytoplasm. Increased vesicles near the plasma membrane in mutant strains correlate with an increased Pil1p movement resulting in the fusion of cup-shaped organelles. We conclude that the shape of vesicle-containing organelles is critical for their functions in vesicle endocytosis.

Objective: The objective of this study was to test the hypothesis that distal pancreatectomy (DP) without intraperitoneal drainage does not affect the frequency of grade 2 or higher grade complications., Background: The use of routine intraperitoneal drains during DP is controversial. Prior to this study, no prospective trial focusing on DP without intraperitoneal drainage has been reported., Methods: Patients undergoing DP for all causes at 14 high-volume pancreas centers were preoperatively randomized to placement of a drain or no drain. Complications and their severity were tracked for 60 days and mortality for 90 days. The study was powered to detect a 15% positive or negative difference in the rate of grade 2 or higher grade complications. All data were collected prospectively and source documents were reviewed at the coordinating center to confirm completeness and accuracy., Results: A total of 344 patients underwent DP with (N = 174) and without (N = 170) the use of intraperitoneal drainage. There were no differences between cohorts in demographics, comorbidities, pathology, pancreatic duct size, pancreas texture, or operative technique. There was no difference in the rate of grade 2 or higher grade complications (44% vs. 42%, P = 0.80). There was no difference in clinically relevant postoperative pancreatic fistula (18% vs 12%, P = 0.11) or mortality (0% vs 1%, P = 0.24). DP without routine intraperitoneal drainage was associated with a higher incidence of intra-abdominal fluid collection (9% vs 22%, P = 0.0004). There was no difference in the frequency of postoperative imaging, percutaneous drain placement, reoperation, readmission, or quality of life scores., Conclusions: This prospective randomized multicenter trial provides evidence that clinical outcomes are comparable in DP with or without intraperitoneal drainage.

Background: Excessive alcohol (EtOH) consumption causes an imbalance in protein metabolism. EtOH impairs protein synthesis in C2C12 myoblasts via a FoxO1-AMPK-TSC2-mTORC1 pathway and also induces protein degradation. As the underlying regulatory signaling cascades for these processes are currently poorly defined, we tested the hypothesis that alcohol-induced autophagy is mediated via activation of the PIK3C3 complex that is regulated by FoxO1-AMPK., Methods: C2C12 myoblasts were incubated with EtOH for various periods of time, and autophagy pathway-related proteins were assessed by Western blotting and immunoprecipitation. Expression of targeted genes was suppressed using electroporation of specific siRNAs and chemical inhibitors., Results: Incubation of C2C12 myoblasts with 100 mM EtOH increased the autophagy markers LC3B-II and ATG7, whereas levels of SQSTM1/p62 decreased. The lysosomal inhibitor bafilomycin A1 caused a similar response, although there was no additive effect when combined with EtOH. EtOH altered ULK1 S555 and S757 phosphorylation in a time- and AMPK-dependent manner. The activation of AMPK and ULK1 was associated with increased BECN1 (S93, S14) and PIK3C3/VPS34 (S164) phosphorylation as well as increased total ATG14 and PIK3C3. These changes promoted formation of the ATG14-AMBRA1-BECN1-PIK3C3 proautophagy complex that is important in autophagosome formation. EtOH-induced changes were not associated with increased production of PtdIns3P, which may be due to enhanced PIK3C3 complex binding with 14-3-3θ. Reduction of AMPK using siRNA suppressed the stimulatory effect of EtOH on BECN1 S93, BECN1 S14, and PIK3C3 S164 phosphorylation in a time-dependent manner. Likewise, knockdown of AMPK or chemical inhibition of FoxO1 attenuated phosphorylation of ULK1 at both residues. Knockdown of ULK1 or BECN1 antagonized the effect of EtOH on LC3B-II, SQSTM1, and ATG7 protein expression., Conclusions: EtOH-induced autophagy is mediated through changes in phosphorylation and interaction of various PIK3C3 complex components. This, in turn, is regulated either directly via FoxO1-AMPK or indirectly via the FoxO1-AMPK-ULK1 signaling cascade in a mTORC1-independent or mTORC1-dependent manner., (Copyright © 2017 by the Research Society on Alcoholism.)

Human papillomavirus (HPV) is the most common sexually transmitted agent worldwide and is etiologically linked to several cancers, including cervical and genital cancers. NKG2D, an activating receptor expressed by NK cells, plays an important role in cancer immune-surveillance. We analyzed the impact of a NKG2D gene variant, rs1049174, on the incidence of HPV-related cancers in Vietnamese patients and utilized various molecular approaches to elucidate the mechanisms of NKG2D receptor regulation by rs1049174. In a group of 123 patients with HPV+ anogenital cancers, the low cytotoxicity allele LNK was significantly associated with increased cancer susceptibility (p = 0.016). Similar results were also observed in a group of 153 women with cervical cancer (p = 0.05). In functional studies, NK cells from individuals with LNK genotype showed a lower NKG2D expression and displayed less efficient NKG2D-mediated functions than NK cells with HNK genotype. Notably, the rs1049174 variant occurs within a targeting site for miR-1245, a negative regulator of NKG2D expression. Compared with the higher cytotoxicity allele HNK, the LNK allele was more efficiently targeted by miR-1245 and thus determined lower NKG2D expression in NK cells with the LNK genotype. The NKG2D variants may influence cancer immunosurveillance and thus determine susceptibility to various malignancies, including HPV-induced cancers.

A variety of stressors including alcohol (EtOH) are known to induce collagen production and fibrotic diseases. Matrix metalloproteinases (MMP) play an important role in regulating fibrosis, but little is known regarding the relationship between EtOH and MMPs. In addition, the signaling cascades involved in this process have not been elucidated. We have identified the MMP Adamts1 as a target of EtOH regulation. To characterize the function of Adamts1, we examined EtOH-induced alterations in collagen I and elastin protein levels in C2C12 myocytes. Incubation of myocytes with 100 mM EtOH decreased elastin and increased collagen content, respectively, and these changes were associated with increased O-GLcNAc modification of Adamts1. Conversely, silencing of Adamts1 by siRNA blocked the adverse effects of EtOH on collagen and elastin levels. Similar results were obtained after treatment with a pharmacological inhibitor of MMP. Changes in collagen were due, at least in part, to a decreased interaction of Adamts1 with its endogenous inhibitor TIMP3. The AMPK inhibitor compound C blocked the EtOH-induced stimulation of collagen and O-GLcNAc Adamts1 protein. Changes in AMPK appear linked to FoxO1, since inhibition of FoxO1 blocked the effects of EtOH on AMPK phosphorylation and O-GLcNAc levels. These FoxO-dependent modifications were associated with an upregulation of the FoxO1 transcription target sestrin 3, as well as increased binding of sestrin 3 with AMPK. Collectively, these data indicate that EtOH regulates the collagen I and elastin content in an Adamts1-dependent manner in myocytes. Furthermore, Adamts1 appears to be controlled by the FoxO1-sestrin 3-AMPK signaling cascade., (© 2014 Wiley Periodicals, Inc.)

Background: Ethanol (EtOH) decreases muscle protein synthesis, and this is associated with reduced mammalian target of rapamycin complex (mTORC)1 and increased mTORC2 activities. In contrast, phospholipase D (PLD) and its metabolite phosphatidic acid (PA) positively regulate mTORC1 signaling, whereas their role in mTORC2 function is less well defined. Herein, we examine the role that PLD and PA play in EtOH-mediated mTOR signaling., Methods: C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. For PA experiments, cells were pretreated with the drug for 25 minutes followed by 50-minute incubation with PA in the presence or absence of EtOH. The phosphorylation state of various proteins was assessed by immunoblotting. Protein-protein interactions were determined by immunoprecipitation and immunoblotting. PLD activity was measured using the Amplex Red PLD assay kit. PA concentrations were determined with a total PA assay kit., Results: PA levels and PLD activity increased in C2C12 myocytes exposed to EtOH (100 mM). Increased PLD activity was blocked by inhibitors of AMP-activated protein kinase (AMPK) (compound C) and phosphoinositide 3-kinase (PI3K) (wortmannin). Likewise, suppression of PLD activity with CAY10594 prevented EtOH-induced Akt (S473) phosphorylation. PLD inhibition also enhanced the binding of Rictor to mSin1 and the negative regulatory proteins Deptor and 14-3-3. Addition of PA to myocytes decreased Akt phosphorylation, but changes in mTORC2 activity were not associated with altered binding of complex members and 14-3-3. PA increased S6K1 phosphorylation, with the associated increase in mTORC1 activity being regulated by reduced phosphorylation of AMPKα (T172) and its target tuberous sclerosis protein complex (TSC)2 (S1387). This resulted in increased Rheb and RagA/RagC GTPase interactions with mTOR, as well as suppression of mTORC2., Conclusions: EtOH-induced increases in PLD activity and PA may partially counterbalance the adverse effects of this agent. EtOH and PA regulate mTORC1 via a PI3K/AMPK/TSC2/PLD signaling cascade. PA stimulates mTORC1 function and suppresses activation of mTORC2 as part of an mTORC1/2 feedback loop., (Copyright © 2013 by the Research Society on Alcoholism.)

The powerful activating receptor NKG2D is expressed by natural killer (NK) cells and promotes cytotoxic lysis of cancer cells expressing NKG2D ligands (NKG2D-Ls). We report the effective induction of NKG2D-Ls, achieved with the naturally occurring polyphenol resveratrol, in a broad range of leukemia cells. In this study, resveratrol upregulated the NKG2D-Ls MHC class I chain-related proteins MICA and MICB, and UL16-binding proteins ULBP1, ULBP2, and ULBP3 in most of the leukemia cells analyzed. Ligand upregulation induced by resveratrol was impaired by pharmacological and genetic disruption of ataxia-telangiectasia mutated kinase, the main regulator of NKG2D-L expression. Leukemia cells treated with resveratrol were more susceptible to killing by NK cells than untreated cells, and the enhanced cytotoxicity of NK cells was blocked by treatment of NK cells with anti-NKG2D mAbs. Interestingly, resveratrol consistently upregulated the NKG2D receptor expression and enhanced NKG2D-mediated functions in resting NK cells obtained from healthy individuals. Therefore, resveratrol has attractive immunotherapeutic potential., (© 2013 Japanese Cancer Association.)

Alcoholism and acquired immune deficiency syndrome are associated with severe muscle wasting. This impairment in nitrogen balance arises from increased protein degradation and a decreased rate of protein synthesis. The regulation of protein synthesis is a complex process involving alterations in the phosphorylation state and protein-protein interaction of various components of the translation machinery and mammalian target of rapamycin (mTOR) complexes. This review describes mechanisms that regulate protein synthesis in cultured C2C12 myocytes following exposure to either alcohol or human immunodeficiency virus antiretroviral drugs. Particular attention is given to the upstream regulators of mTOR complexes and the downstream targets which play an important role in translation. Gaining a better understanding of these molecular mechanisms could have important implications for preventing changes in lean body mass in patients with catabolic conditions or illnesses.

Leucine (Leu) and insulin both stimulate muscle protein synthesis, albeit at least in part via separate signaling pathways. While alcohol (EtOH) suppresses insulin-stimulated protein synthesis in cultured myocytes, its ability to disrupt Leu signaling and Rag GTPase activity has not been determined. Likewise, little is known regarding the interaction of EtOH and Leu on the AMPK/TSC2/Rheb pathway. Treatment of myocytes with EtOH (100 mM) decreased protein synthesis, whereas Leu (2 mM) increased synthesis. In combination, EtOH suppressed the anabolic effect of Leu. The effects of EtOH and Leu were associated with coordinate changes in the phosphorylation state of mTOR, raptor, and their downstream targets 4EBP1 and S6K1. As such, EtOH suppressed the ability of Leu to activate these signaling components. The Rag signaling pathway was activated by Leu but suppressed by EtOH, as evidenced by changes in the interaction of Rag proteins with mTOR and raptor. Overexpression of constitutively active (ca)RagA and caRagC increased mTORC1 activity, as determined by increased S6K1 phosphorylation. Furthermore, the caRagA-caRagC heterodimer blocked the inhibitory effect of EtOH. EtOH and Leu produced differential effects on AMPK signaling. EtOH enhanced AMPK activity, resulting in increased TSC2 (S1387) and eEF2 phosphorylation, whereas Leu had the opposite effect. EtOH also decreased the interaction of Rheb with mTOR, and this was prevented by Leu. Collectively, our results indicate that EtOH inhibits the anabolic effects that Leu has on protein synthesis and mTORC1 activity by modulating both Rag GTPase function and AMPK/TSC2/Rheb signaling.

Background: The mammalian target of rapamycin (mTOR) kinase controls cell growth, proliferation, and metabolism through 2 distinct multiprotein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2, and mTORC1., Methods: C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. Levels of various mTORC2 proteins and mRNA were assessed by immunoblotting and real-time PCR, respectively, while protein-protein interactions were determined by immunoprecipitation and immunoblotting. An in vitro mTORC2 kinase activity assay was performed using Akt as a substrate. The rate of protein synthesis was determined by (35) S-methionine/cysteine incorporation into cellular protein., Results: EtOH (100 mM) increased the protein and mRNA levels of the mTORC2 components rictor, mSin1, proline-rich repeat protein 5, and Deptor. There was also an increased association of these proteins with mTOR. EtOH increased the in vitro kinase activity of mTORC2, and this was correlated with decreased binding of rictor with 14-3-3 and Deptor. Reduced rictor phosphorylation at T1135 by EtOH was most likely due to decreased S6K1 activity. Knockdown of rictor elevated mTORC1 activity, as indicated by increased S6K1 phosphorylation and protein synthesis. Likewise, there were decreased amounts and/or phosphorylation levels of various mTORC1 and mTORC2 components including raptor, proline-rich Akt substrate 40 kDa, mSin1, Deptor, and GβL. Activated PP2A was associated with decreased Akt and eukaryotic elongation factor 2 phosphorylation. Collectively, our results provide evidence of a homeostatic balance between the 2 mTOR complexes following EtOH treatments in myoblasts., Conclusions:   EtOH increased the activity of mTORC2 by elevating levels of various components and their interaction with mTOR. Decreased rictor phosphorylation at T1135 acts as mTORC1-dependent feedback mechanisms, functioning in addition to the insulin receptor substrate-I/PI3K signaling pathway to regulate protein synthesis., (Copyright © 2011 by the Research Society on Alcoholism.)

Background: There is a paucity of data on the trends in discharge disposition for patients undergoing hepatic resection for malignancy., Aim: To analyse the national trends in discharge disposition after hepatic resection for malignancy., Methods: The National Inpatient Sample (NIS) database was queried (1993 to 2005) to identify patients that underwent hepatic resection for malignancy and analyse the discharge status (home, home health or rehabilitation/skilled facility)., Results: A weighted total of 74,520 patients underwent hepatic resection of whom, 53,770 patients had a principal diagnosis of malignancy. The overall mortality improved from 6.3% to 3.4%. After excluding patients that died in the post-operative period and those with incomplete discharge status, 45,583 patients were included. The proportion of patients that had acute care needs preventing them from being discharged home without assistance increased from 10.9% in 1993 to 19.5% in 2005. While there was an increase in the number of patients discharged to home health care during this time (8.9% to 13.8%), there was a larger increase in the proportion of patients that were discharged to a rehabilitation or skilled nursing facility (2% to 5.7%). Despite a decrease in the mortality rates, there was no improvement in rate of patients discharged home without assistance over the period of the study., Conclusions: The results of the present study demonstrate that after hepatic resection, a significant proportion of patients will need assistance upon discharge. This information needs to be included in patient counselling during pre-operative risk and benefit assessment., (© 2011 International Hepato-Pancreato-Biliary Association.)

The use of systemically absorbed drugs in the gravid and in the lactating patient is of concern to the dentist. This article reviews concerns for the health and safety of the mother, developing fetus, and neonate involving local anesthetics. The available literature on the use of local anesthetics for dentistry in the pregnant and postpartum patient is also reviewed. In addition, the physiology of the pregnant and postpartum woman is discussed because this is essential to understanding potential interplay with local anesthesia and the stress of a dental appointment., (Copyright © 2010. Published by Elsevier Inc.)

The heme peroxidase enzyme myeloperoxidase (MPO) is released by activated neutrophils and monocytes, where it uses hydrogen peroxide (H(2)O(2)) to catalyze the production of the potent oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) from halide and pseudohalide (SCN(-)) ions. These oxidants have been implicated as key mediators of tissue damage in many human inflammatory diseases including atherosclerosis, asthma, rheumatoid arthritis, cystic fibrosis and some cancers. It is shown here that acetaminophen (paracetamol), a phenol-based drug with analgesic and antipyretic actions, is an efficient inhibitor of HOCl and HOBr generation by isolated MPO-H(2)O(2)-halide systems. With physiological halide concentrations, acetaminophen concentrations required for 50% inhibition of oxidant formation (IC(50)) were 77+/-6microM (100mMCl(-)) and 92+/-2microM (100mMCl(-) plus 100microMBr(-)), as measured by trapping of oxidants with taurine. The IC(50) for inhibition of HOCl generation by human neutrophils was ca. 100microM. These values are lower than the maximal therapeutic plasma concentrations of acetaminophen (< or =150microM) resulting from typical dosing regimes. Acetaminophen did not diminish superoxide generation by neutrophils, as measured by lucigenin-dependent chemiluminescence. Inhibition of HOCl production was associated with the generation of fluorescent acetaminophen oxidation products, consistent with acetaminophen acting as a competitive substrate of MPO. Inhibition by acetaminophen was maintained in the presence of heparan sulfate and extracellular matrix, materials implicated in the sequestration of MPO at sites of inflammation in vivo. Overall, these data indicate that acetaminophen may be an important modulator of MPO activity in vivo., (2009 Elsevier Inc. All rights reserved.)

The mTORC1 protein kinase complex consists of mTOR, raptor, mLST8/GbetaL and PRAS40. Previously, we reported that mTOR plays an important role in regulating protein synthesis in response to alcohol (EtOH). However, the mechanisms by which EtOH regulates mTORC1 activity have not been established. Here, we investigated the effect of EtOH on the phosphorylation and interaction of components of mTORC1 in C2C12 myocytes. We also examined the specific role that PRAS40 plays in this process. Incubation of myocytes with EtOH (100 mM, 24 h) increased raptor and PRAS40 phosphorylation. Likewise, there were increased levels of the PRAS40 upstream regulators Akt and IRS-1. EtOH also caused changes in mTORC1 protein-protein interactions. EtOH enhanced the binding of raptor and PRAS40 with mTOR. These alterations occurred in concert with increased binding of 14-3-3 to raptor, while the PRAS40 and 14-3-3 interaction was not affected. The shRNA knockdown (KD) of PRAS40 decreased protein synthesis similarly to EtOH. PRAS40 KD increased raptor phosphorylation and its association with 14-3-3, whereas decreased GbetaL-mTOR binding. The effects of EtOH and PRAS40 KD were mediated by AMPK. Both factors increased in vitro AMPK activity towards the substrate raptor. In addition, KD enhanced the activity of AMPK towards TSC2. Collectively, our results indicate that EtOH stabilizes the association of raptor, PRAS40, and GbetaL with mTOR, while likewise increasing the interaction of raptor with 14-3-3. These data suggest a possible mechanism for the inhibitory effects of EtOH on mTOR kinase activity and protein synthesis in myocytes.

HIV anti-retroviral drugs decrease protein synthesis, although the underlying regulatory mechanisms of this process are not fully established. Therefore, we investigated the effects of the HIV protease inhibitor lopinavir (LPV) on protein metabolism. We also characterized the mechanisms that mediate the effects of this drug on elongation factor-2 (eEF2), a key component of the translational machinery. Treatment of C2C12 myocytes with LPV produced a dose-dependent inhibitory effect on protein synthesis. This effect was observed at 15 min and was maintained for at least 4 h. Mechanistically, LPV increased the phosphorylation of eEF2 and thereby decreased the activity of this protein. Increased phosphorylation of eEF2 was associated with increased activity of its upstream regulators AMP-activated protein kinase (AMPK) and eEF2 kinase (eEF2K). Both AMPK and eEF2K directly phosphorylated eEF2 in an in vitro kinase assay suggesting two distinct paths lead to eEF2 phosphorylation. To verify this connection, myocytes were treated with the AMPK inhibitor compound C. Compound C blocked eEF2K and eEF2 phosphorylation, demonstrating that LPV affects eEF2 activity via an AMPK-eEF2K dependent pathway. In contrast, incubation of myocytes with rottlerin suppressed eEF2K, but not eEF2 phosphorylation, suggesting that eEF2 can be regulated independent of eEF2K. Finally, LPV did not affect PP2A activity when either eEF2 or peptide was used as the substrate. Collectively, these results indicate that LPV decreases protein synthesis, at least in part, via inhibition of eEF2. This appears regulated by AMPK which can act directly on eEF2 or indirectly via the action of eEF2K., ((c) 2008 Wiley-Liss, Inc.)

The selective removal of copper and lead ions from aqueous solutions by diethylenetriamine (DETA)-functionalized polymeric adsorbent was investigated. The adsorbent was prepared by amination of the micro-beads synthesized from glycidyl methacrylate and trimethylolpropane trimethacrylate co-polymerization (denoted as P-DETA). In the single metal species system (only copper or lead ions present), P-DETA was found to adsorb copper ions or lead ions significantly (with a slightly higher adsorption uptake capacity for lead ions than copper ions). However, P-DETA displayed an excellent selectivity in the adsorption of copper ions over lead ions in the binary metal species system (with both copper and lead ions present). It was also found that initially (or previously) adsorbed lead ions on P-DETA were displaced, even completely, by subsequently adsorbed copper ions from the solution but the case was not vice versa. The greater electronegativity of copper ions than lead ions was identified as the major factor that caused P-DETA to selectively adsorb copper ions over lead ions during competitive adsorption in the binary metal species system. It was speculated that the displacement of already adsorbed lead ions on P-DETA by subsequently adsorbed copper ions was through an adjacent attachment and repulsion mechanism. P-DETA has been shown to have the potential to be used as an effective adsorbent for the removal as well as selective recovery of heavy metal ions in water or wastewater treatment.

Ethanol decreases protein synthesis in cells, although the underlying regulatory mechanisms of this process are not fully established. In the present study incubation of C2C12 myocytes with 100 mm EtOH decreased protein synthesis while markedly increasing the phosphorylation of eukaryotic elongation factor 2 (eEF2), a key component of the translation machinery. Both mTOR and MEK pathways were found to play a role in regulating the effect of EtOH on eEF2 phosphorylation. Rapamycin, an inhibitor of mammalian target of rapamycin, and the MEK inhibitor PD98059 blocked the EtOH-induced phosphorylation of eEF2, whereas the p38 MAPK inhibitor SB202190 had no effect. Unexpectedly, EtOH decreased the phosphorylation and activity of the eEF2 upstream regulator eEF2 kinase. Likewise, treatment of cells with the inhibitor rottlerin did not block the stimulatory effect of EtOH on eEF2, suggesting that eEF2 kinase (eEF2K) does not play a role in regulating eEF2. In contrast, increased eEF2 phosphorylation was correlated with an increase in AMP-activated protein kinase (AMPK) phosphorylation and activity. Compound C, an inhibitor of AMPK, suppressed the effects of EtOH on eEF2 phosphorylation but had no effect on eEF2K, indicating that AMPK regulates eEF2 independent of eEF2K. Finally, EtOH decreased protein phosphatase 2A activity when either eEF2 or AMPK was used as the substrate. Thus, this later action may partially account for the increased phosphorylation of eEF2 in response to EtOH and the observed sensitivity of AMPK to rapamycin and PD98059 treatments. Collectively, the induction of eEF2 phosphorylation by EtOH is controlled by an increase in AMPK and a decrease in protein phosphatase 2A activity.

Background: Alcohol and the antiretroviral drug indinavir (Ind) decrease protein synthesis in skeletal muscle under in vivo and in vitro conditions. The goal of the present study was to identify signaling mechanisms responsible for the inhibitory effect of ethanol (EtOH) and Ind on protein synthesis., Methods: C2C12 mouse myocytes were incubated with EtOH, Ind, or a combination of both for 24 hours. The rate of protein synthesis was determined by [35S]methionine/cysteine incorporation into cellular protein. Phosphorylation of eukaryotic initiation and elongation factors were quantitated by Western blot analysis to identify potential mechanisms for regulating translation., Results: Treatment of myocytes with Ind or EtOH for 24 hours decreased protein synthesis by 19 and 22%, respectively, while a 35% decline was observed in cells treated simultaneously with both agents. Mechanistically, treatment with EtOH or Ind decreased the phosphorylation of the S6 ribosomal protein, and this reduction was associated with decreased S6K1 and p90rsk phosphorylation. Ethanol also decreased the phosphorylation of ERK1/2, mTOR, and 4EBP1, while Ind only suppressed ERK1/2 phosphorylation. Both agents inhibited the phosphorylation of Mnk1 and its upstream regulator p38 MAPK, and they decreased the amount of the active eukaryotic initiation factor (eIF) 4G/eIF4E complex. Finally, EtOH and/or Ind increased phosphorylation of the eukaryotic elongation factor (eEF)-2 by 1.6- to 6-fold. The effects of these agents were not additive, although the combination did exert a greater effect on S6K1 and eEF2 phosphorylation., Conclusions: Ethanol and Ind decreased protein synthesis in myocytes and this response was associated with changes in the phosphorylation of proteins that regulate translation initiation and elongation.

Background: Sentinel lymph node (SLN) biopsy combined with microstaging-associated immunohistochemical staining for cytokeratin more accurately assigns patients to their corresponding diagnostic stage. The purpose of this study was to compare the survival outcomes of node-negative patients who received an SLN biopsy with historical control data of node-negative patients who received routine complete axillary lymph node dissection (CALND) in the pre-SLN biopsy era., Methods: Under institutional review board approval, 2458 node-negative invasive breast cancer patients between the ages of 25 and 94 years (mean, 60 years) were treated at our institution from January 1986 to May 2004. Of these 2458 patients, 604 (25%) were evaluated with CALND, whereas 1854 (75%) were evaluated with SLN biopsy. All were treated according to the current stage-specific guidelines. Kaplan-Meier graphs of overall survival and disease-free survival were constructed for each group of patients, and the two groups were compared by using the log-rank test., Results: Overall survival and disease-free survival for the CALND and SLN biopsy groups did not differ significantly (P = .98). The average number of lymph nodes extracted in the pre-SLN biopsy group was 18, whereas the average number of SLNs extracted in the post-SLN biopsy group was 3., Conclusions: The survival rate among node-negative breast cancer patients who received an SLN biopsy alone has proven to have no significant difference (P = .98) from the survival rate among node-negative patients who received a CALND. SLN biopsy alone should replace CALND as the primary tool for axillary staging of breast cancer in node-negative patients.

Combined antiretroviral drug regimens have promoted clinical, immunologic, and virologic improvements in AIDS patients. Nevertheless, these therapies are associated with derangements in lipid and carbohydrate metabolism. In this study, we examined the effects of a representative protease inhibitor (nelfinavir), a nonnucleoside reverse transcriptase inhibitor (nevirapine), and a nucleoside reverse transcriptase inhibitor (zidovudine) on protein synthesis in skeletal muscle cells. To examine these processes, C2C12 myocytes were treated with increasing concentrations of nelfinavir, nevirapine, or zidovudine for 1 or 2 days, and rates of protein synthesis were determined by measuring [35S]methionine/cysteine incorporation into cellular proteins. Treatment of myocytes with therapeutic concentrations of nelfinavir, nevirapine, or zidovudine for 48 hr decreased protein synthesis by 14-20%. An approximately 60% decline was observed in cells treated with higher concentrations of nevirapine or nelfinavir. In contrast, the basal rate of protein synthesis was not affected when cells were incubated with these compounds for 24 hr. Therapeutic concentrations of nelfinavir and nevirapine did not impair the anabolic effect of insulin on protein synthesis. However, zidovudine suppressed the stimulatory effect of insulin. The decreased protein synthesis induced by nelfinavir and zidovudine was associated with decreases in the phosphorylation of the S6 ribosomal protein (rpS6) and the repressor binding protein 4EBP1, while the inhibitory effect of nevirapine was mainly associated with a decline in phosphorylated 4EBP1. In conclusion, nelfinavir, nevirapine, and zidovudine treatments decreased protein synthesis in myocytes and this effect was correlated with a reduction in the phosphorylation level of proteins that regulate translation initiation.

The HIV protease inhibitor indinavir adversely impairs carbohydrate and lipid metabolism, whereas its influence on protein metabolism under in vivo conditions remains unknown. The present study tested the hypothesis that indinavir also decreases basal protein synthesis and impairs the anabolic response to insulin in skeletal muscle. Indinavir was infused intravenously for 4 h into conscious rats, at which time the homeostasis model assessment of insulin resistance was increased. Indinavir decreased muscle protein synthesis by 30%, and this reduction was due to impaired translational efficiency. To identify potential mechanisms responsible for regulating mRNA translation, several eukaryotic initiation factors (eIFs) were examined. Under basal fasted conditions, there was a redistribution of eIF4E from the active eIF4E.eIF4G complex to the inactive eIF4E.4E-BP1 complex, and this change was associated with a marked decrease in the phosphorylation of 4E-BP1 in muscle. Likewise, indinavir decreased constitutive phosphorylation of eIF4G and mTOR in muscle, but not S6K1 or the ribosomal protein S6. In contrast, the ability of a maximally stimulating dose of insulin to increase the phosphorylation of PKB, 4E-BP1, S6K1, or mTOR was not altered 20 min after intravenous injection. Indinavir increased mRNA expression of the ubiquitin ligase MuRF1, but the plasma concentration of 3-methylhistidine remained unaltered. These indinavir-induced changes were associated with a marked reduction in the plasma testosterone concentration but were independent of changes in plasma levels of IGF-I, corticosterone, TNF-alpha, or IL-6. In conclusion, indinavir acutely impairs basal protein synthesis and translation initiation in skeletal muscle but, in contrast to muscle glucose uptake, does not impair insulin-stimulated signaling of protein synthetic pathways.

Anti-retroviral therapy promotes clinical, immunologic, and virologic improvement in human immunodeficiency virus-infected patients. Whereas this therapy adversely affects carbohydrate and lipid metabolism, the effects of anti-retroviral drugs on muscle protein synthesis and degradation have not been reported. To examine these processes, we treated C2C12 myocytes with increasing concentrations of the protease inhibitor indinavir for 1 or 2 days. Treatment of myocytes with a therapeutic concentration of indinavir (20 microM) for 24 h decreased basal protein synthesis by 18%, whereas a 42% decline was observed after 48 h. A similar decrement, albeit quantitatively smaller, was detected with other protease inhibitors. Indinavir did not alter the rate of proteolysis. Likewise, indinavir did not impair the anabolic effect of insulin-like growth factor-I on protein synthesis. Mechanistically, indinavir decreased the phosphorylation of the S6 ribosomal protein (rpS6), and this reduction was associated with a decreased phosphorylation of p70S6 kinase and p90rsk as well as the upstream regulators ERK1/2 and MEK1/2. Indinavir also decreased the phosphorylation of Mnk1 and its upstream effectors, p38 MAPK and ERK1/2. Indinavir did not affect the phosphorylation of mTOR or 4E-BP1, but it did decrease the amount of the active eukaryotic initiation factor eIF4G-eIF4E complex. In conclusion, indinavir decreased protein synthesis in myocytes. This decrease was associated with the disruption of the ERK1/2 and p38 MAPK pathways and a reduction in both the level of functional eIF4F complex and rpS6 phosphorylation.

Growth hormone (GH) stimulates insulin-like growth factor I (IGF-I) synthesis in both liver and muscle. During sepsis, proinflammatory cytokines inhibit GH action in liver, but it is unknown whether sepsis also produces GH resistance in muscle. Sepsis was induced by cecal ligation and puncture, and 18 h later the effect of GH on signal transducer and activator of transcription (STAT) phosphorylation and IGF-I mRNA content was assessed in rat gastrocnemius and liver. The relative abundance of phosphorylated (p)STAT5a, pSTAT5b, pSTAT3, and pSTAT1 was increased in liver from control rats after GH. Sepsis alone also increased hepatic pSTAT5a, pSTAT3, and pSTAT1. Sepsis dramatically impaired the ability of GH to stimulate the phosphorylation of STAT5a and -5b, as well as to increase IGF-I mRNA in liver. In muscle from control rats, GH increased pSTAT5a and -5b, whereas content of pSTAT3 and pSTAT1 was not affected. Sepsis increased basal content of pSTAT3 but not pSTAT5a, pSTAT5b, or pSTAT1 in muscle. The GH-induced increase of pSTAT5a and -5b in muscle from septic rats was not inhibited, suggesting that muscle was not GH resistant. In contrast to these changes in pSTAT5, the ability of GH to increase IGF-I mRNA was completely absent in muscle from septic rats. Because the suppressor of cytokine signaling (SOCS) proteins may function as negative regulators of GH signaling, we examined the content of these proteins. Sepsis produced small (30-50%), albeit statistically significant, increases in SOCS-1, -2, and -3 protein in muscle. In contrast to muscle, the SOCS proteins in the liver did not change under the various experimental conditions, suggesting that these proteins are not responsible for the impaired phosphorylation of STAT5 by GH. In conclusion, sepsis produces GH resistance in both muscle and liver, with the locus of this impairment in muscle differing from that in liver and being independent of a defect in STAT5 phosphorylation.

A 66-year-old woman was admitted with diarrhea, weight loss, slight recurrent abdominal pain, and raised serum amylase and lipase. Lactose intolerance was diagnosed, and treatment was begun. The symptoms diminished. However, slight back pain and elevated serum amylase and lipase levels persisted. A pancreatic tumor was then suspected. Ultrasound, spiral computed tomography (CT), magnetic resonance imaging (MRI), and magnetic resonance cholangiopancreatography (MRCP) examinations were inconclusive. Endoscopic retrograde cholangiopancreatography (ERCP) showed a slight narrowing of the pancreatic duct within the pancreatic body, and endoscopic ultrasound (EUS) revealed a 10 mm intrapancreatic lesion. Finally, intraductal ultrasonography (IDUS) reliably identified a small pancreatic tumor. The tumor was resected, and histology confirmed a well-differentiated adenocarcinoma in situ (UICC stage 0, TisN0M0). This case shows that using high-resolution imaging techniques such as EUS plus IDUS, small malignant pancreatic lesions can be detected at an early stage, when curative action is possible.

Background: Hepatic cysts are detected incidentally in 2.5-5% of the population. Only about 15% of such cysts are symptomatic. Since laparoscopic deroofing for liver cysts was first described in 1991 there have been a number of reports of successful laparoscopic management of hepatic cysts, including the laparoscopic management of complex and parasitic cysts., Methods: A systematic review of English-language articles on the subject appearing in journals through May 2002 was conducted using the Medline database., Results: Only a minority of cystic liver lesions need treatment, and the therapeutic approach is guided by the type of cyst. Laparoscopic deroofing (combined with omentoplasty and/or oversewing) of uncomplicated liver cysts is associated with a recurrence rate of 10-25%, with less morbidity and mortality as compared to open surgery. On the other hand, treatment of complex liver cysts and hydatid cysts by laparoscopy is not considered a standard approach., Conclusion: The laparoscopic approach in uncomplicated but symptomatic liver cysts is effective, with low morbidity and mortality. Additional omentoplasty or oversewing appears to reduce the recurrence rate., (Copyright 2002 S. Karger AG, Basel)

Transient constrictive pericarditis is a rare entity. It is characterized by clinical and echocardiographic features similar to constrictive pericarditis, but is distinguished by its transient nature. This feature is important to recognize for avoiding unnecessary pericardectomy. The case of a patient who presented with acute myopericarditis and typical echocardiographic features of constriction is described. Within weeks, all signs of constriction disappeared spontaneously.

We have previously shown that local application at the time of operation of Staphylococcus aureus, nonviable S. aureus, its cell wall, or S. aureus peptidoglycan accelerates wound healing. We hypothesized that this effect is due to both direct and indirect mechanisms, among which is an increase in the inflammatory response to wounding, resulting in an increase in macrophages, angiogenesis, and fibroblasts. Twenty-seven Sprague-Dawley male rats were anesthetized, and two 7-cm paravertebral skin incisions were made. Four polyvinyl alcohol sponges, two on each side, containing either 100 microliter of isotonic saline or 0.5 mg of nonviable S. aureus or S. aureus peptidoglycan in 100-microliter saline were implanted subcutaneously. Nonviable S. aureus or S. aureus peptidoglycan (860 microgram/cm incision) in 200-microliter saline were inoculated into the incisions at closure. The rats ate a commercial rat chow and drank tap water ad libitum throughout. After days 3 and 7 postwounding, rats were euthanized, and tissues were examined for immunohistochemical features of reparative tissue using ED-1, Factor VIII, and vimentin antibodies, markers for monocyte/macrophages, endothelial cells, and mesenchymal cells (including fibroblasts), respectively. Incisions treated with nonviable S. aureus or S. aureus peptidoglycan showed more macrophages along and deep in the wound tract 7 days postoperatively. Nonviable S. aureus or S. aureus peptidoglycan-treated sponges were surrounded and penetrated by much larger capsules of reparative tissue than saline-treated sponges at both 3 and 7 days. Neutrophil influx was much greater in nonviable S. aureus or S. aureus peptidoglycan-treated sponges, especially in central regions, and there were many more ED-1-stained macrophages in distinct geographic locations, specifically, the more peripheral-cortical areas. Some clustering of macrophages occurred around areas of invasion by reparative tissue into the surrounding subcutaneous fat and within the interstices of the sponges at the interface between reparative tissue and acute inflammatory cells. In contrast, saline-treated sponge reparative tissue had significantly fewer macrophages, much thinner and flimsy reparative tissue, with proportionately fewer macrophages clustering centrally. There were many more mesenchymal cells (notably fibroblasts) and new blood vessels and much more reparative collagen in the nonviable S. aureus or S. aureus peptidoglycan-treated sponges. We conclude that local application of nonviable S. aureus or S. aureus peptidoglycan at wounding induces an increased number and alteration in location of macrophages, increased influx (or proliferation) of mesenchymal cells (notably fibroblasts), and increased angiogenesis and reparative collagen accumulation, as well as increasing the overall acute inflammatory response to wounding.

IgE-binding components in an extract of horse dander were analyzed, especially with regard to the glycoprotein allergens. After SDS-PAGE under reducing conditions and blotting, several of the glycoprotein IgE-binding components, including two distinct bands of 27 and 31 kDa, were detected. Together with several other bands, they were shown to bind to the lectins Sambucus nigra agglutinin (SNA) and Datura stramonium agglutinin (DSA), indicating terminal sialic acid linked alpha 2 --> 6 to galactose, and galactose linked beta 1 --> 4 to N-acetylglucosamine, respectively. Carbohydrate analysis after SDS-PAGE and blotting showed the presence of mannose, galactose, N-acetylglucosamine, and N-acetyl neuraminic acid in the 27-kDa band. The 14.4-kDa glycoprotein component lost its IgE-binding activity after periodate oxidation. Minor differences in allergenic activity in the other glycoprotein allergenic components were also detected, indicating that the carbohydrate part of the molecule seems to play a role in the IgE-binding activity. Two-dimensional electrophoresis and subsequent immunoblotting were performed to estimate the approximate number, molecular mass, and pI of IgE-binding components. This resulted in a cluster of such components on the blot membrane.