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Background In vivo muscle protein synthesis rates are typically assessed by measuring the incorporation rate of stable isotope labelled amino acids in skeletal muscle tissue collected from vastus lateralis muscle. It remains to be established whether muscle protein synthesis rates in the vastus lateralis are representative of muscle protein synthesis rates of other muscle groups. We hypothesized that post-absorptive muscle protein synthesis rates differ between vastus lateralis and rectus abdominis, pectoralis major, or temporalis muscle in vivo in humans.Methods Twenty-four patients (62 +/- 3 years, 42% female), scheduled to undergo surgery, participated in this study and underwent primed continuous intravenous infusions with l-[ring-C-13(6)]-phenylalanine. During the surgical procedures, serum samples were collected, and muscle tissue was obtained from the vastus lateralis as well as from the rectus abdominis, pectoralis major, or temporalis muscle. Fractional mixed muscle protein synthesis rates (%/h) were assessed by measuring the incorporation of l-[ring-C-13(6)]-phenylalanine into muscle tissue protein.Results Serum l-[ring-C-13(6)]-phenylalanine enrichments did not change throughout the infusion period. Post-absorptive muscle protein synthesis rates calculated based upon serum l-[ring-C-13(6)]-phenylalanine enrichments did not differ between vastus lateralis and rectus abdominis (0.032 +/- 0.004 vs. 0.038 +/- 0.003%/h), vastus lateralis and pectoralis major, (0.025 +/- 0.003 vs. 0.022 +/- 0.005%/h) or vastus lateralis and temporalis (0.047 +/- 0.005 vs. 0.043 +/- 0.005%/h) muscle, respectively (P > 0.05). When fractional muscle protein synthesis rates were calculated based upon tissue-free l-[ring-C-13(6)]-phenylalanine enrichments as the preferred precursor pool, muscle protein synthesis rates were significantly higher in rectus abdominis (0.089 +/- 0.008%/h) compared with vastus lateralis (0.054 +/- 0.005%/h) muscle (P < 0.01). No differences were observed between fractional muscle protein synthesis rates in vastus lateralis and pectoralis major (0.046 +/- 0.003 vs. 0.041 +/- 0.008%/h) or vastus lateralis and temporalis (0.073 +/- 0.008 vs. 0.083 +/- 0.011%/h) muscle, respectively.Conclusions Post-absorptive muscle protein synthesis rates are higher in rectus abdominis when compared with vastus lateralis muscle. Post-absorptive muscle protein synthesis rates do not differ between vastus lateralis and pectoralis major or temporalis muscle. Protein synthesis rates in muscle tissue samples obtained during surgery do not necessarily represent a good proxy for appendicular skeletal muscle protein synthesis rates.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and can trigger devastating disease in immune-suppressed patients. Cytotoxic lymphocytes (CD8+ T cells and NK cells) control HCMV infection by releasing interferon-γ and five granzymes (GrA, GrB, GrH, GrK, GrM), which are believed to kill infected host cells through cleavage of intracellular death substrates. However, it has recently been demonstrated that the in vivo killing capacity of cytotoxic T cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we demonstrate that (primary) cytotoxic lymphocytes can block HCMV dissemination independent of host cell death, and interferon-α/β/γ. Prior to killing, cytotoxic lymphocytes induce the degradation of viral immediate-early (IE) proteins IE1 and IE2 in HCMV-infected cells. Intriguingly, both IE1 and/or IE2 are directly proteolyzed by all human granzymes, with GrB and GrM being most efficient. GrB and GrM cleave IE1 after Asp398 and Leu414, respectively, likely resulting in IE1 aberrant cellular localization, IE1 instability, and functional impairment of IE1 to interfere with the JAK-STAT signaling pathway. Furthermore, GrB and GrM cleave IE2 after Asp184 and Leu173, respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data indicate that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV infection, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic infection., Author summary Human cytomegalovirus (HCMV) is the leading viral cause of congenital defects, can trigger disease in immune-compromised patients, and plays roles in cancer development. Cytotoxic lymphocytes kill HCMV-infected cells via releasing a set of five cytotoxic serine proteases called granzymes. However, the killing capacity of cytotoxic cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we show that cytotoxic lymphocytes can also use granzymes to inhibit HCMV replication in absence of cell death. All five granzymes cleave and inactivate both viral immediate-early (IE1/2) proteins, which are essential players for initiating HCMV infection. Our data support the model that cytotoxic cells employ granzymes to dampen HCMV replication prior to accumulation of sufficient hits to kill the infected cell.

BackgroundDiagnosis of perioperative myocardial infarction (PMI) after coronary artery bypass grafting (CABG) is fraught with complexity since it is primarily based on a single cut-off value for cardiac troponin (cTn) that is exceeded in over 90% of CABG patients, including non-PMI patients. In this study we applied an unsupervised statistical modeling approach to uncover clinically relevant cTn release profiles post-CABG, including PMI, and used this to improve diagnostic accuracy of PMI.MethodsIn 624 patients that underwent CABG, cTnT concentration was serially measured up to 24 h post aortic cross clamping. 2857 cTnT measurements were available to fit latent class linear mixed models (LCMMs).ResultsFour classes were found, described by: normal, high, low and rising cTnT release profiles. With the clinical diagnosis of PMI as golden standard, the rising profile had a diagnostic accuracy of 97%, compared to 83% for an optimally chosen cut-off and 21% for the guideline recommended cut-off value.ConclusionClinically relevant subgroups, including patients with PMI, can be uncovered using serially measured cTnT and a LCMM. The LCMM showed superior diagnostic accuracy of PMI. A rising cTnT profile is potentially a better criterion than a single cut-off value in diagnosing PMI post-CABG.

A call to implement 21st-century change drivers within teacher preparation is evidenced within policy and research. The use of technology-enhanced methods of supervision where virtual observations of pre-service or in-service teachers are performed by university supervisors or mentors has an established and growing research base that demonstrates improvements in pre- and in-service teacher competencies, as well as programmatic benefits, such as time and cost savings and the ability to supervise remotely. To answer this call, the researcher investigated the strategic implementation of an asynchronous video-based remote supervision model within a university-based teacher preparation program from the perspective of education specialist teacher candidates and their cooperating teachers. Results suggest a video-based remote supervision method is feasible and equally effective or preferred over traditional face-to-face observations by teacher candidates and cooperating teachers for self-reflection, professional growth, and convenience. Time and cost savings resulting from reduced travel for the supervisor are analyzed and discussed. The model and methods presented in this study may be replicated to enable strategic scaling of video-based remote supervision within other teacher preparation programs, particularly those serving candidates in remote or rural areas.

Highlights • Different registration methods for position verification of oesophageal cancer. • Clipbox around PTV and vertebrae with soft tissue match was best suited. • Tumour location influences the required setup margins. • A large variety of anatomical changes is revealed when using kV-CBCT. • GTV Reduction and diaphragm position changes were most common anatomical changes., Purpose To evaluate different registration methods, setup margins and number of corrections for CBCT-based position verification for oesophageal cancer and to evaluate anatomical changes during the course of radiotherapy treatment. Methods From 50 patients, 440 CBCT-scans were registered automatically using a soft tissue or bone registration algorithm and compared to the clinical match. Moreover, relevant anatomical changes were monitored. A sub-analysis was performed to evaluate if tumour location influenced setup variations. Margin calculation was performed and the number of setup corrections was estimated. Results were compared to a patient group previously treated with MV-EPID based position verification. Results CBCT-based setup variations were smaller than EPID-based setup variations, resulting in smaller setup margins of 5.9 mm (RL), 7.5 mm (CC) and 4.7 mm (AP) versus 6.0 mm, 7.8 mm and 5.5 mm, respectively. A reduction in average number of setup corrections per patient was found from 0.75 to 0.36. From all automatically registered CBCT-scans, a clipbox around PTV and vertebras combined with soft tissue registration resulted in the smallest setup margins of 5.9 mm (RL), 7.7 mm (CC), 4.8 mm (AP) and smallest average number of corrections of 0.38. For distally located tumours, a setup margin of 7.7 mm (CC) was required compared to 5.6 mm for proximal tumours. Reduction of GTV volume, heart volume and change in diaphragm position were observed in 16, 10 and 15 patients, respectively. Conclusions CBCT-based set-up variations are smaller than EPID-based variations and vary according to tumour location. When using kV-CBCT a large variety of anatomical changes is revealed, which cannot be observed with MV-EPID.

OBJECTIVES We have implemented an intraoperative checklist aiming to reduce the incidence of re-exploration for bleeding after cardiac surgery. The present report addresses the results of adopting such a checklist regarding the incidence of postoperative bleeding. METHODS The checklist was implemented by presenting it in several staff meetings of the Catharina Heart Center. Copies of the checklist were presented in every operating room. Data were collected by the Catharina Heart Center, aligned with the 'Meetbaar Beter' data manual and validated by 'Meetbaar Beter' through their data quality system. The incidence of re-exploration for bleeding was analysed in a variable life-adjusted display curve. The patient population operated after the implementation of the checklist was compared with a recent historical population before its implementation. RESULTS From January 2013 through April 2016, 4817 cardiac surgical procedures were performed in our institution. Before May 2015, 3210 procedures were performed (Group 1), complicated by 112 re-exploration for bleeding (3.5%). The 'reoperation for bleeding checklist' was implemented on 1 May 2015. After this date, the number of re-explorations for bleeding decreased to 29 (1.8%) of the 1607 cardiac surgical procedures (Group 2) (P

Elevated cardiac enzymes after coronary artery bypass grafting (CABG) surgery have been identified as a risk factor for worse postoperative outcome. Cardiac enzymes play an important role in the diagnosis of perioperative myocardial infarction. This study aims to investigate the predictive value of aspartate aminotransferase (AST) with respect to early and late mortality after CABG.Patients undergoing isolated CABG in a single center between January 1998 and December 2010 were prospectively enrolled in our database. Patients were arbitrarily divided into 4 groups according to the postoperative AST level: group 1 (AST50 U/L), group 2 (AST = 51 to 100 U/L), group 3 (AST = 101 to 200 U/L), group 4 (AST = 201 to 300 U/L), and group 5 (AST300 U/L). The impact of biomedical variables on early mortality was determined using univariate and multivariate logistic regression analyses. Risk factors for late mortality were identified using Cox proportional hazard regression analyses.The study population consisted of 13,505 patients who underwent isolated CABG. Postoperative AST level was identified as a risk factor for early (odds ratio = 3.6 [2.5 to 5.4], p0.0001) and late mortality (hazard ratio = 1.4 [1.2 to 1.7], p0.001). After correction for other risk factors, AST level was an independent predictor of worse survival.Elevated postoperative AST level is an independent predictor of early and late mortality after CABG. Although it is not a specific indicator for cardiac damage, it can reflect ischemic effects on the other organs as an indirect sign of depressed cardiac function.

Polyoxyethylene sorbate (Tween) compounds were tested to compare their growth stimulation effects on Mycobacterium paratuberculosis. Three low passage and three high passage clinical isolates and ATCC strain 19698 were used. Tween 20, 40, 60, and 80 were tested at concentrations of 0, 0.001, 0.01, 1.0, and 3.0% (w/v) in radiometric broth culture media and in Middlebrook 7H9 agar plates. Scanning and transmission electron microscopy were used to examine cell wall appearance and ultrastructure, respectively. In broth culture, 0.1% (w/v) Tween 60 most dramatically enhanced growth of M. paratuberculosis ATCC strain 19698. The effects of Tween 40 and 80 on growth took a bimodal form, enhancing growth at concentration ranges of 0-0.01% and 0.1-1.0% (w/v) but suppressing growth at concentrations of 0.01-0.1% (w/v). Two of three high passage clinical isolates grew optimally in the presence of 1.0% (w/v) Tween 80, while the remaining high passage isolate and all three low passage isolates grew best in media containing 0.1% (w/v) Tween 80. Colonial morphology of all strains grown on Middlebrook 7H9 agar without Tween 80 was irregular and granular whereas colonies on plate media containing greater than 0.01% (w/v) Tween 80 were entire, smooth, and domed. Scanning electron microscopy also revealed a transition from rough to smooth cell walls with increasing Tween 80 concentration. Transmission electron microscopy showed the presence of low electron dense intracellular vacuoles in Tween 80 grown M. paratuberculosis cells. Thus, Tweens altered colonial morphology, the cell wall surface, and ultrastructure of M. paratuberculosis and stimulated its growth in vitro in a concentration-dependent, and often bimodal, fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Smooth (Sm) and rough (Rg) colonial types of Mycobacterium paratuberculosis ATCC 19698 and two clinical isolates were tested to examine their growth responses in medium containing antimicrobial agents. Susceptibility tests were done in Middlebrook 7H12B medium with and without Tween 80 and one of the following antimicrobial agents: streptomycin, isoniazid, rifampin, ethambutol, ciprofloxacin, and penicillin G. Growth responses in the presence of antimicrobial agents led to the following observations. (i) In the absence of Tween, Rg colony types were more resistant than Sm colony types; (ii) the addition of Tween 80 significantly increased the susceptibility of both Sm and Rg colony types; however, the increase was greater with the Sm colony types. These studies showed that the antimicrobial susceptibility of M. paratuberculosis was significantly affected when Tween 80 was present in either the primary culture medium or the drug susceptibility test medium. In the absence of the perturbing influence of Tween 80, M. paratuberculosis was resistant to the antimicrobial agents tested.