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Let \(G\) be a simple graph of order \(n\) with eigenvalues \(\lambda_1,\lambda_2,\cdots,\lambda_n\) and normalized Laplacian eigenvalues \(\mu_1,\mu_2,\cdots,\mu_n\). The Estrada index and normalized Laplacian Estrada index are defined as \(EE(G)=\sum_{k=1}^ne^{\lambda_k}\) and \(\mathcal{L}EE(G)=\sum_{k=1}^ne^{\mu_k-1}\), respectively. We establish upper and lower bounds to \(EE\) and \(\mathcal{L}EE\) for edge-independent random graphs, containing the classical Erdös-Rényi graphs as special cases.

Species within Fusarium are of global agricultural, medical, and food/feed safety concern and have been extensively characterized. However, accurate identification of species is challenging and usually requires DNA sequence data. FUSARIUM-ID ( http://isolate.fusariumdb.org/blast.php ) is a publicly available database designed to support the identification of Fusarium species using sequences of multiple phylogenetically informative loci, especially the highly informative ∼680-bp 5′ portion of the translation elongation factor 1-alpha (TEF1) gene that has been adopted as the primary barcoding locus in the genus. However, FUSARIUM-ID v.1.0 and 2.0 had several limitations, including inconsistent metadata annotation for the archived sequences and poor representation of some species complexes and marker loci. Here, we present FUSARIUM-ID v.3.0, which provides the following improvements: (i) additional and updated annotation of metadata for isolates associated with each sequence, (ii) expanded taxon representation in the TEF1 sequence database, (iii) availability of the sequence database as a downloadable file to enable local BLAST queries, and (iv) a tutorial file for users to perform local BLAST searches using either freely available software, such as SequenceServer, BLAST+ executable in the command line, and Galaxy, or the proprietary Geneious software. FUSARIUM-ID will be updated on a regular basis by archiving sequences of TEF1 and other loci from newly identified species and greater in-depth sampling of currently recognized species.

Purpose: Women diagnosed with ductal carcinoma in situ (DCIS) face confusion and uncertainty about treatment options. The objective of this study was to determine whether there are differences in decisional conflict about treatment by age and race/ethnicity. Methods: A cross-sectional survey was conducted of women (age ≥ 18) diagnosed with DCIS enrolled at Kaiser Permanente of Southern California. The Decisional Conflict Scale (DCS) measured personal perceptions of decision uncertainty, values clarity, and effective decision-making. We used a multivariable regression to study whether age, race, and ethnicity were associated with patient-reported DCS. Results: Forty-five percent (N = 1395) of women who received the online survey, participated. The mean age was 56 (± 9.6) years, the majority were white. Compared to women younger than 50, women aged 60–69 reported lower overall DCS scores (-5.4; 95% CI -1.5 to -9.3). Women > 70 had lower values clarity scores (-9.0; 95% CI -2.8 to -15.2) about their treatment compared to women aged 50–59 and 60–69 (-7.1; 95% CI -2.9 to -11.3 and − 7.2; 95% CI -2.9 to -11.5) and likewise, lower effective decision-making scores (-5.4; 95% CI -1.7 to -9.2 and − 5.2; 95% CI -1.4 to -9.0) compared to women 70). Age based tailored discussions about treatment options, health education, and supportive decision-making interventions/tools may reduce decision conflict in future DCIS patients.

Extracellular vesicles (EVs) are biomarkers and modifiers of human disease. EVs secreted by insulin-responsive tissues like skeletal muscle (SkM) and white adipose tissue (WAT) contribute to metabolic health and disease but the relative abundance of EVs from these tissues has not been directly examined. Human Protein Atlas data and directly measuring EV secretion in mouse SkM and WAT using an ex vivo tissue explant model confirmed that SkM tissue secretes more EVs than WAT. Differences in EV secretion between SkM and WAT were not due to SkM contraction but may be explained by differences in tissue metabolic capacity. We next examined how many EVs secreted from SkM tissue ex vivo and in vivo are myofiber-derived. To do this, a SkM myofiber-specific dual fluorescent reporter mouse was created. Spectral flow cytometry revealed that SkM myofibers are a major source of SkM tissue-derived EVs ex vivo and EV immunocapture indicates that ∼5% of circulating tetraspanin-positive EVs are derived from SkM myofibers in vivo. Our findings demonstrate that 1) SkM secretes more EVs than WAT, 2) many SkM tissue EVs are derived from SkM myofibers, and 3) SkM myofiber-derived EVs reach the circulation in vivo. These findings advance our understanding of EV secretion between metabolically active tissues and provide direct evidence that SkM myofibers secrete EVs that can reach the circulation in vivo.

Archivists or museums often use “curiosity cabinets” to present certain collections – an apt unifying concept for this work, which combines a heretofore unknown metal/diazoalkane reactivity mode and a rare type of base-promoted ligand rearrangement.

La depresión es un problema de salud y discapacidad en la población, se ha observado un incremento en las cifras de las personas que la padecen durante los últimos años, ya que es una patología en salud mental que no se le ha dado la relevancia necesaria durante su diagnóstico, tratamiento y seguimiento en los ámbitos sanitario, social y educativo que se requiere. Es por ello que el proceso enfermero (PE) representa un eje metodológico fundamental de enfermería para prestar cuidados que serán necesarios implementar de manera especializada e individualizada en el paciente con Trastorno de Depresión Mayor (TDM).

For a variety of purposes, it is of interest to embed metals in cagelike

OBJECTIVE Pig-to-primate renal xenotransplantation is plagued by early antibody-mediated graft loss which precludes clinical application of renal xenotransplantation. We evaluated whether temporary complement inhibition with anti-C5 antibody Tesidolumab could minimize the impact of early antibody-mediated rejection in rhesus monkeys receiving pig kidneys receiving costimulatory blockade-based immunosuppression. METHODS Double (Gal and Sda) and triple xenoantigen (Gal, Sda, and SLA I) pigs were created using CRISPR/Cas. Kidneys from DKO and TKO pigs were transplanted into rhesus monkeys that had the least reactive crossmatches. Recipients received anti-C5 antibody weekly for 70 days, and T cell depletion, anti-CD154, mycophenolic acid, and steroids as baseline immunosuppression (n = 7). Control recipients did not receive anti-C5 therapy (n = 10). RESULTS Temporary anti-C5 therapy reduced early graft loss secondary to antibody-mediated rejection and improved graft survival (P < 0.01). Deleting class I MHC (SLA I) in donor pigs did not ameliorate early antibody-mediated rejection (table). Anti-C5 therapy did not allow for the use of tacrolimus instead of anti-CD154 (table), prolonging survival to a maximum of 62 days. CONCLUSION Inhibition of the C5 complement subunit prolongs renal xenotransplant survival in a pig to non-human primate model.

This paper describes for the first time the community perception and awareness of sea grapes (Caulerpa spp.) and sea grape farming, its lean and peak seasons, harvest volume and production, prices in market outlets as well as factors affecting the distribution in selected regions in the Philippines through a multi-region socio-economic survey. The majority of the respondents were aware of the presence of sea grapes in their areas since the 1980s but uninformed of their potential to be cultivated. The dry season (January–April) was perceived to be the peak season of sea grapes, both in culture and wild, in the different areas studied with Mactan, Cebu as the top producer, generating an estimated 42,222 kg per month. In market outlets, Calapan and Puerto Galera exhibited the highest market price for sea grapes (PhP 155–160 kg−1) (USD 3.23–3.33 kg−1) in the last 4 years. Socio-economic factors like gender, civil status, education, and income also affected the harvest distribution. The estimated annual income generated via farming and wild harvesting is valued at around PhP 2,259,456 (USD 47,702) during peak season. This study highlights how sea grapes are regarded as an economically important commodity in the Philippines and should be conserved to ensure availability and sustainable production. Furthermore, this research could serve as a basis for policymakers and scientists in addressing challenges and gaps in Caulerpa industry.

Recent multidrug resistance in

Poor solubility is the main drawback of the direct industrial exploitation of chitin, the second most abundant biopolymer after cellulose. Chemical methods are conventional to solubilize chitin from natural sources. Enzymatic hydrolysis of soluble chitinous substrates is a promising approach to obtain value-added by-products, such as N-acetylglucosamine units or low molecular weight chito-oligomers. Protein display on the bacterial membrane remains attractive to produce active enzymes anchored to a biological surface. The Lpp-OmpA system, a gene fusion of the Lpp signal sequence with the OmpA transmembrane region, represents the traditional system for targeting enzymes to the E. coli surface. EhCHT1, the amoebic chitinase, exhibits an efficient endochitinolytic activity and significant biochemical features, such as stability over a wide range of pH values. Using an extended Lpp-OmpA system as a protein carrier, we engineered E. coli to express the catalytic domain of EhCHT1 on the surface and assess the endochitinase activity as a trait. Engineered bacteria showed a consistent hydrolytic rate over a typical substrate, suggesting that the displayed enzyme has operational stability. This study supports the potential of biomembrane-associated biocatalysts as a reliable technology for the hydrolysis of soluble chitinous substrates.

Species within

IntroductionThe shape of the glucose curve during an oral glucose tolerance test (OGTT) reflects β-cell function in populations without diabetes but has not been as well studied in those with diabetes. A monophasic shape has been associated with higher risk of diabetes, while a biphasic pattern has been associated with lower risk. We sought to determine if phenotypic or metabolic characteristics were associated with glucose response curve shape in adults with type 2 diabetes treated with metformin alone.Research design and methodsThis is a cross-sectional analysis of 3108 metformin-treated adults with type 2 diabetes diagnosed ResultsThe monophasic profile was the most common (67.8% monophasic, 5.5% biphasic, 26.7% continuous rise). The monophasic subgroup was younger, more likely male and white, and had higher body mass index (BMI), while the continuous rise subgroup was more likely female and African American/black. HOMA2-S and fasting glucose did not differ among the subgroups. The biphasic subgroup had the highest early, late, and total insulin and C peptide responses (all pConclusionsBased on the large multiethnic GRADE cohort, sex, race, age, and BMI were found to be important determinants of the shape of the glucose response curve. A pattern of a continuously rising glucose at 2 hours reflected reduced β-cell function and may portend increased glycemic failure rates.Trial registration numberNCT01794143.

Phosphatases are hydrolytic enzymes that cleave the phosphoester bond of numerous substrates containing phosphorylated residues. The typical classification divides them into acid or alkaline depending on the pH at which they have optimal activity. The histidine phosphatase (HP) superfamily is a large group of functionally diverse enzymes characterized by having an active-site His residue that becomes phosphorylated during catalysis. HP enzymes are relevant biomolecules due to their current and potential application in medicine and biotechnology. Entamoeba histolytica, the causative agent of human amoebiasis, contains a gene (EHI_146950) that encodes a putative secretory acid phosphatase (EhHAPp49), exhibiting sequence similarity to histidine acid phosphatase (HAP)/phytase enzymes, i.e., branch-2 of HP superfamily. To assess whether it has the potential as a biocatalyst in removing phosphate groups from natural substrates, we studied the EhHAPp49 structural and functional features using a computational-experimental approach. Although the combined outcome of computational analyses confirmed its structural similarity with HP branch-2 proteins, the experimental results showed that the recombinant enzyme (rEhHAPp49) has negligible HAP/phytase activity. Nonetheless, results from supplementary activity evaluations revealed that rEhHAPp49 exhibits Mg2+-dependent alkaline pyrophosphatase activity. To our knowledge, this study represents the first computational-experimental characterization of EhHAPp49, which offers further insights into the structure–function relationship and the basis for future research.

Additional file 2. Theoretical models applied in the FIVALIN project by project activities. List of project activities where Theoretical models are applied.

Additional file 4. INFORMATION SHEET AND INFORMED CONSENT FIVALIN STUDY.

Additional file 1. Strategies to train new staff and assure consistency of the intervention. List of strategies to ensure intervention consistency and train new staff.

Additional file 3. Physical Activity Unified-7 item Screener (PAU-7S). English version of Physical Activity Unified– 7 items Screener (PAU-7S) administered to children.

The combustion of pure H

Estrada JL, Bautista NS, Dionisio-Sese ML. 2020. Morphological variation of two common sea grapes (Caulerpa lentillifera and Caulerpa racemosa) from selected regions in the Philippines. Biodiversitas 21: 1823-1832. Seagrapes, locally known in the Philippines as “lato” or “ar-arusip”, are economically important macroalgae belonging to the edible species of the genus Caulerpa. This study characterized and compared distinct populations of sea grapes from selected regions in the Philippines and described the influence of physicochemical parameters of seawater on their morphology. Morphometric, cluster and principal component analyses showed that morphological plasticity exists in sea grapes species (Caulerpa lentillifera and Caulerpa racemosa) found in different sites in the Philippines. These are evident in morphometric parameters namely, assimilator height, space between assimilators, ramulus diameter and number of rhizoids on stolon wherein significant differences were found. This evident morphological plasticity was analyzed in relation to physicochemical parameters of the seawater. Assimilator height of C. racemosa is significantly associated and highly influenced by water depth, salinity, temperature and dissolved oxygen whereas for C. lentillifera depth and salinity are the significant influencing factors. Highest assimilator height of sea grapes was found in Coron and Culion in Palawan due to higher salinity and temperature while in Mactan, Cebu, it is primarily attributed to lower depth and higher salinity values.

Poor solubility is the main drawback of the direct industrial exploitation of chitin, the second most abundant biopolymer after cellulose. Chemical methods are conventional to solubilize chitin from natural sources. Enzymatic hydrolysis of soluble chitinous substrates is a promising approach to obtain value-added by-products, such as N-acetylglucosamine units or low molecular weight chito-oligomers. Protein display on the bacterial membrane remains attractive to produce active enzymes anchored to a biological surface. The Lpp-OmpA system, a gene fusion of the Lpp signal sequence with the OmpA transmembrane region, represents the traditional system for targeting enzymes to the E. coli surface. EhCHT1, the amoebic chitinase, exhibits an efficient endochitinolytic activity and significant biochemical features, such as stability over a wide range of pH values. Using an extended Lpp-OmpA system as a protein carrier, we engineered E. coli to express the catalytic domain of EhCHT1 on the surface and assess the endochitinase activity as a trait. Engineered bacteria showed a consistent hydrolytic rate over a typical substrate, suggesting that the displayed enzyme has operational stability. This study supports the potential of biomembrane-associated biocatalysts as a reliable technology for the hydrolysis of soluble chitinous substrates.

Cancer incidence rates vary regionally among American Indians (AIs) and Latinos. The goal of this was to identify areas of research necessary to reduce cancer health disparities in AIs and Latinos, the two major racial/ethnic minority groups in Arizona. In an effort to better understand cancer health disparities, cancer incidence rates in AIs and Latinos in Arizona were compared to non-Hispanic Whites (NHWs). Age-adjusted incidence rates (per 100,000) were obtained from the Arizona Cancer Registry and the North American Association of Central Cancer Registries. Spearman’s rank test was used to examine correlation between county-level cancer incidence rates and socio-demographic factors. AIs and Latinos had lower incidence rates of screening for detectable cancers than NHWs. Among older men (age ≥65), however, AIs and Latinos had similar prostate cancer incidence rates to NHWs. Some of less common cancers, such as kidney, stomach, liver, and gallbladder, were more frequently diagnosed in AIs and Latinos than NHWs. AIs and Latinos were more likely to be diagnosed with advanced cancer stage, except for cervical cancer. Correlations between prostate and breast cancer incidence rates and percent urban residents as well as correlations between incidence rates of these two cancer types and population size were significantly positive. Poverty levels were inversely correlated with colorectal and lung cancer incidence rates. Our review of cancer incidence rates suggests that socio-demographic factors, such as population size (rural/urban) and poverty levels, have influenced cancer detection and incidence rates in Arizona.

Spanish-only speaking residents in the United States face barriers to receiving potentially life-saving 911 interventions such as Telephone -cardiopulmonary resuscitation (TCPR) instructions. Since 2015, 911 dispatchers have placed an increased emphasis on rapid identification of potential cardiac arrest. The purpose of this study was to describe the utilization and timing of the 911 system during suspected out-of-hospital cardiac arrest (OHCA) by Spanish-speaking callers in Metropolitan Phoenix, Arizona.The dataset consisted of suspected OHCA from 911 centers from October 10, 2010 through December 31, 2013. Review of audio TCPR process data included whether the need for CPR was recognized by telecommunicators, whether CPR instructions were provided, and the time elements from call receipt to initiation of compressions.A total of 3398 calls were made to 911 for suspected OHCA where CPR was indicated. A total of 39 (1.2%) were determined to have a Spanish language barrier. This averages to 18 calls per year with a Spanish language barrier during the study period, compared with 286 OHCAs expected per year among this population. The average time until telecommunicators recognized CPR need was 87.4s for the no language barrier group compared to 160.6s for the Spanish-language barrier group (p0.001).Time to CPR instructions started was significantly different between these groups (144.4s vs 231.3s, respectively) (p0.001), as was time to first compression, (174.4s vs. 290.9s, respectively) (p0.001).Our study suggests that Hispanic callers under-utilize the 911 system, and when they do call 911, there are significant delays in initiating CPR.

Introduction and objectives Heart failure is a common comorbidity of COPD and contributes to increased breathlessness and adverse clinical outcomes. Neural respiratory drive (NRD) is closely related to breathlessness intensity in COPD. This study aimed to investigate the impact of comorbid left heart failure (COPD-HF) on NRD in patients with COPD. We hypothesised that NRD would be higher during an inspiratory threshold loading protocol (ITL) in COPD-HF than in COPD patients without left heart failure. Methods COPD and COPD-HF patients underwent incremental ITL at 12%, 24%, 36%, 48% and 60% of maximal inspiratory mouth pressure (PImax). NRD was recorded continuously using 2nd intercostal space transcutaneous electromyography (EMGpara). EMGpara signals were converted to root mean square (RMS), normalised to peak RMS EMGpara during maximal inspiratory manoeuvres (EMGpara%max) and multiplied by respiratory rate to calculate NRD index (NRDI). NRDI in COPD and COPD-HF were compared at each load using mixed effect model repeated measurement analysis. Results 11 COPD patients without left heart failure (mean (SD) age 69(7) years, FEV1%predicted 49.3 (16.4)%, VC%predicted 99.8 (22.0)%, PIMax 55.7 (15.8)cmH2O) and 11 COPD-HF patients (mean (SD) age 72(6) years, FEV1%predicted 54.8 (13.6)%, VC%predicted 86.8 (17.4)%), PIMax 53.1 (30.9)cmH2O) were studied. mMRC dyspnoea scores were higher in COPD-HF (median (IQR) 3 (2 – 4) than in COPD (median (IQR) 2 (1 to 3), p=0.0406). 11/11 COPD patients completed all loads of the ITL protocol to 60% PImax, compared to 4/11 patients in the COPD-HF group (p=0.0039). There were significant fixed effects of diagnosis (p=0.0136), load (p Conclusions Observations of higher levels of NRDI at equivalent inspiratory threshold loads in COPD-HF suggests that heart failure further increases the mechanical load on the respiratory muscles in COPD. Contributions of potential aetiological factors, such as reduced lung and chest wall compliance, require further study.

Introduction & objectives Human diaphragm contractility typically declines with advancing age. Associated changes in human phrenic nerve function are less well-investigated. The recent development of multipair oesophageal electrode catheters allows accurate measurement of the latency and amplitude of the crural diaphragm compound muscle action potential (CMAPdi). The principal aim of this study was to investigate the effect of healthy ageing on CMAPdi amplitude and latency using this method. Methods 20 highly active older adults (HAOA, aged 49 to 80 years), and 16 younger adults (YA, age 20 to 35 years), all male, were instrumented with a multipair oesophageal electrode catheter and a dual oesophageal/gastric pressure transducer. Transdiaphragmatic pressure (TwPdi), crural CMAPdi latency and amplitude were measured following left, right and bilateral anterolateral magnetic phrenic nerve stimulation at 100% of maximum stimulator output and compared between the HAOA and YA groups. Maximal inspiratory mouth pressure (PImax), sniff nasal inspiratory pressure (Sniff Pnasal) sniff oesophageal pressure (Sniff Poes) and sniff transdiaphragmatic pressure (Sniff Pdi) were also measured. Results Bilateral TwPdi was significantly lower in HAOA (median (IQR) bilateral TwPdi HAOA 24.8 (22.3 to 35.1) cmH2O) than in YA (bilateral TwPdi YA 31.1 (28.7 to 39.2) cmH2O, p=0.0152). Right TwPdi was significantly lower in HAOA (HAOA 9.8 (9.2 to 11.1) cmH2O, YA 15.2 (13.5 to 18.3) cmH2O, p=0.0005) but differences in left TwPdi did not reach statistical significance (HAOA 11.6 (8.9 to 13.4) cmH2O, YA 15.6 (12.5 to 17.6) cmH2O, p=0.0814). CMAPdi latencies were significantly greater following both left and right phrenic nerve stimulation in HAOA compared to YA (left CMAPdi latency HAOA= 8.9 (8.5 to 9.3) ms, YA=7.5 (7.2 to 8.1) ms,p Conclusions Healthy ageing is associated with increased phrenic nerve latency, interestingly without decrement in motor unit size. Reference ranges of human phrenic nerve function should be updated with age-specific normal values.

Study Objectives To evaluate the contribution of long-term and short-term REM sleep homeostatic processes to REM sleep recovery and the ultradian organization of the sleep wake cycle. Methods Fifteen rats were sleep recorded under a 12:12 LD cycle. Animals were subjected during the rest phase to two protocols (2T2I or 2R2I) performed separately in non-consecutive experimental days. 2T2I consisted of 2 h of total sleep deprivation (TSD) followed immediately by 2 h of intermittent REM sleep deprivation (IRD). 2R2I consisted of 2 h of selective REM sleep deprivation (RSD) followed by 2 h of IRD. IRD was composed of four cycles of 20-min RSD intervals alternating with 10 min of sleep permission windows. Results REM sleep debt that accumulated during deprivation (9.0 and 10.8 min for RSD and TSD, respectively) was fully compensated regardless of cumulated NREM sleep or wakefulness during deprivation. Protocol 2T2I exhibited a delayed REM sleep rebound with respect to 2R2I due to a reduction of REM sleep transitions related to enhanced NREM sleep delta-EEG activity, without affecting REM sleep consolidation. Within IRD permission windows there was a transient and duration-dependent diminution of REM sleep transitions. Conclusions REM sleep recovery in the rat seems to depend on a long-term hourglass process activated by REM sleep absence. Both REM sleep transition probability and REM sleep episode consolidation depend on the long-term REM sleep hourglass. REM sleep activates a short-term REM sleep refractory period that modulates the ultradian organization of sleep states.

Introduction Breathlessness is a cardinal feature of COPD and is frequently distressing and disabling. Thorough assessment of breathlessness is necessary for its effective management, but this is often not achieved in clinical practice. Although no single tool for subjectively measuring breathlessness and disease impact is universally accepted, the questionnaire based modified (m)MRC Dyspnoea Scale and COPD Assessment Test (CAT) scores are widely used in clinical practice and research. Lung function tests and other objective physiological biomarkers in routine clinical practice correlate poorly with patient-reported breathlessness. Neural respiratory drive (NRD), as quantified by transoesophageal diaphragm electromyography (EMGdi), is closely related to breathlessness during cardiopulmonary exercise testing in COPD. Surface parasternal intercostal muscle EMG activity (sEMGpara) can be used as a non-invasive alternative to EMGdi. This study aimed to investigate the relationship between mMRC dyspnoea, and CAT scores, and sEMGpara in COPD patients at rest and when breathing against an inspiratory threshold load. Methods sEMGpara was recorded in 17 COPD patients (mean±SD FEV151.09%±15.1% predicted,14 male, age 70.9±6.6 years), breathing against an inspiratory threshold load at 60% of maximum mouth inspiratory pressure (60% PImax). mMRC dyspnoea and CAT scores were recorded pre-protocol at rest. Mean peak root mean square (RMS) sEMGpara per breath was calculated, and relationships with mMRC dyspnoea and CAT scores were determined by correlation analysis. Results Significant positive correlations were observed between mMRC dyspnoea and sEMGpara at 60%PImax (Spearman r=0.624, p=0.007) and between CAT scores and sEMGpara at 60%PImax (Pearson r=0.497, p=0.042) (figure 1). There were no significant correlations between mMRC dyspnoea score and sEMGpara at rest (Spearman r=−0.048, p=0.853) or FEV1% predicted (r=−0.143, p=0.584), or between CAT score and sEMGpara at rest (Pearson r=0.107, p=0.682) or FEV1% predicted (r=−0.081, p=0.756). Conclusion sEMGpara recorded at 60% PImax, but not at rest, correlated with patient-reported breathlessness and disease impact in COPD. This suggests that sEMGpara recorded at 60% PImax could provide a clinically-useful objective physiological correlate of breathlessness-related quality of life in COPD.

Introduction Breathlessness is a dominant, distressing, symptom of chronic obstructive pulmonary disease (COPD) which has a significant impact on quality of life. Neural respiratory drive (NRD), quantified as diaphragm electromyogram (EMGdi) activity, is closely related to breathlessness intensity in COPD. The surface mechanomyogram (sMMG), which can be recorded using skin-surface triaxial accelerometers, measures the lateral vibration of muscle fibres and is considered to be the mechanical counterpart of the surface EMG (sEMG). The principal aim of this study was to investigate the relationship between the sMMG of the diaphragm (sMMGdi) and breathlessness in COPD. We hypothesised that there would be strong positive relationships between sMMGdi and modified Borg (mBorg) breathlessness intensity during an inspiratory threshold loading protocol. Methods Nine COPD patients (7 male, median (IQR) age 70 (64–78) years, BMI 25.6 (24.8–30.1) kg/m2, FEV144.7 (37.1–63.9)%predicted), underwent an incremental inspiratory loading protocol at 12%, 24%, 36%, 48% and 60% of maximum mouth inspiratory pressure (PImax). Left and right sEMGdi and sMMGdi activity were recorded using 2 pairs of sEMG electrodes, and one pair of triaxial accelerometers, positioned over the 7th/8th intercostal spaces in the anterior axillary lines bilaterally. sEMG and sMMG signals were analysed using fixed sample entropy (fSampEn) and the relationships with mBorg breathlessness intensity during each inspiratory load were determined using correlation analysis. Results Moderate to strong correlations were observed between mBorg breathlessness intensity and the sEMG and sMMG signals, expressed as fSampEn, recorded at all sites (table 1). Conclusion The relationships observed between mBorg breathlessness intensity and sMMGdi, and sEMGdi, expressed as fSampEn, demonstrates the potential value of sMMGdi as a complementary tool to sEMGdi for the investigation of breathlessness in COPD. sMMGdi could provide a novel non-invasive physiological correlate of breathlessness intensity in COPD and other chronic respiratory diseases.

The future of solid organ transplantation is challenged by an increasing shortage of available allografts. Xenotransplantation of genetically modified porcine organs offers an answer to this problem. Strategies of genetic modification have ‘humanized’ the porcine model towards clinical relevance. Most notably, these approaches have aimed at either antigen reduction or human transgene expression. The object of this study was to evaluate the relative effects of both antigen reduction and direct complement regulation on the human-anti-porcine complement dependent cytotoxicity response. Genetically modified animals were created through CRISPR/Cas9-directed mutation and human transgene delivery. Pigs doubly deficient in GGTA1 and CMAH genes were compared to pigs of the same background that expressed a human complement regulatory protein (hCRP). A third animal was made deficient in GGTA1, CMAH and B4GalNT2 gene expression. Cells from these animals were subjected to measures of human antibody binding and antibody-mediated complement-dependent cytotoxicity by flow cytometry. Human IgG and IgM antibody binding was unchanged between the double knockout and the transgenic hCRP double knockout pig. IgG and IgM binding was reduced by 49.1 and 43.2 % respectively by silencing the B4GalNT2 gene. Compared to the double knockout, human anti-porcine cytotoxicity was reduced by 8 % with the addition of a hCRP (p = .032); It was reduced by 21 % with silencing the B4GalNT2 gene (p = .012). Conclusions: Silencing the GGTA1, CMAH and B4GalNT2 genes in pigs achieved a significant antigen reduction. Changing the porcine carbohydrate profile effectively mediates human antibody-mediated complement dependent cytoxicity.

BACKGROUND Disrupting the porcine GGTA1 and CMAH genes [double knockout (DKO)] that produce the gal-α(1,3)-gal and N-glycolylneuraminic acid xenoantigens reduces human antibody binding to porcine peripheral blood mononuclear cells. It is important to examine rejection pathways at an organ-specific level. The object of this study is to evaluate the human preformed antibody reactivity against DKO renal microvascular endothelial cells (RMEC) in vitro. METHODS Characteristics of DKO RMEC were analyzed using flow cytometry. Human IgG/M binding to primary RMEC, immortalized RMEC (iRMEC), and iRMEC-deficient in B4GALNT2 genes were examined using flow cytometric crossmatch assay. RESULTS Porcine RMEC expressed gal-α(1,3)-gal, N-glycolylneuraminic acid, and Dolichos biflorus agglutinin glycans recognized by human preexisting antibodies in humans. Antigenicity of DKO RMEC was lower than GGTA1 KO RMEC. The disruption of B4GALNT2 gene in DKO iRMEC further reduced human IgG/IgM binding. CONCLUSIONS Silencing the porcine GGTA1, CMAH, and B4GALNT2 genes is an effective strategy to reduce human preformed antibody binding to RMEC. Porcine RMEC will be a useful reagent for the further study of xenoimmunology.

A profound thrombocytopenia limits hepatic xenotransplantation in the pig-to-primate model. Porcine livers also have shown the ability to phagocytose human platelets in the absence of immune-mediated injury. Recently, inactivation of the porcine ASGR1 gene has been shown to decrease this phenomenon. Inactivating GGTA1 and CMAH genes has reduced the antibody-mediated barrier to xenotransplantation; herein, we describe the effect that these modifications have on xenogeneic consumption of human platelets in the absence of immune-mediated graft injury.Wild type (WT), ASGR1, GGTA1, and GGTA1CMAH knockout pigs were compared for their xenogeneic hepatic consumption of human platelets. An in vitro assay was established to measure the association of human platelets with liver sinusoidal endothelial cells (LSECs) by immunohistochemistry. Perfusion models were used to measure human platelet uptake in livers from WT, ASGR1, GGTA1, and GGTA1 CMAH pigs.GGTA1, CMAH LSECs exhibited reduced levels of human platelet binding in vitro when compared with GGTA1 and WT LSECs. In a continuous perfusion model, GGTA1 CMAH livers consumed fewer human platelets than GGTA1 and WT livers. GGTA1 CMAH livers also consumed fewer human platelets than ASGR1 livers in a single-pass model.Silencing the porcine carbohydrate genes necessary to avoid antibody-mediated rejection in a pig-to-human model also reduces the xenogeneic consumption of human platelets by the porcine liver. The combination of these genetic modifications may be an effective strategy to limit the thrombocytopenia associated with pig-to-human hepatic xenotransplantation.

Background Thrombocytopenia may represent a significant challenge to the clinical application of solid-organ xenotransplantation. When studied in a pig-to-primate model, consumptive coagulopathy has challenged renal xenografts. New strategies of genetic manipulation have altered porcine carbohydrate profiles to significantly reduce human antibody binding to pig cells. As this process continues to eliminate immunologic barriers to clinical xenotransplantation, the relationship between human platelets and pig organs must be considered. Methods Genetically modified pigs that were created by the CRISPR/Cas9 system with α-1,3-galactosyltransferase (GGTA1) −/− or GGTA1 −/− cytidine monophosphate-N-acetylneuraminic acid hydroxylase −/− phenotype, as well as domestic pigs, were used in this study. Autologous porcine platelets were isolated from donor animal blood collection, and human platelets were obtained from a blood bank. Platelets were fluorescently labeled and in a single-pass model, human, or autologous platelets were perfused through porcine organs at a constant concentration and controlled temperature. Platelet uptake was measured by sampling venous output and measuring sample florescence against input florescence. In vitro study of the interaction between human platelets and porcine endothelial cells was accomplished by immunohistochemical stain and confocal microscopy. Results Differences between human and autologous platelet loss through the porcine kidney were not significant in any genetic background tested (WT P = 0.15, GGTA1 −/− P = 0.12, GGTA1 −/− cytidine monophosphate-N-acetylneuraminic acid hydroxylase −/− P = 0.25). The unmodified porcine liver consumed human platelets in a single-pass model of platelet perfusion in fewer than 10 min. WT suprahepatic inferior vena cava fluoresce reached a maximum of 76% of input fluoresce within the human platelet cohort and was significantly lower than the autologous platelet control cohort ( P = 0.001). Confocal microscopic analysis did not demonstrate a significant association between human platelets and porcine renal endothelial cells compared with porcine liver endothelial positive controls. Conclusions Our results suggest that in the absence of immunologic injury, human platelets respond in a variable fashion to organ-specific porcine endothelial surfaces. Human platelets are not removed from circulation by exposure to porcine renal endothelium but are removed by unmodified porcine hepatic endothelium. Kidneys possessing genetic modifications currently relevant to clinical xenotransplantation failed to consume human platelets in an isolated single-pass model. Human platelets did not exhibit significant binding to renal endothelial cells by in vitro assay.