Divergent expiratory braking activity of costal and crural diaphragm.

BACKGROUND: There is increasing clinical interest in understanding the contribution of the diaphragm in early expiration, especially during mechanical ventilation. However, current experimental evidence is limited, so essential activity of the diaphragm during expiration and diaphragm segmental differences in expiratory activity, are unknown. OBJECTIVES: To determine if: 1) the diaphragm is normally active into expiration during spontaneous breathing and hypercapnic ventilation, 2) expiratory diaphragmatic activity is distributed equally among the segments of the diaphragm, costal and crural. METHODS: In 30 spontaneously breathing male and female canines, awake without confounding anesthetic, we measured directly both inspiratory and expiratory electrical activity (EMG), and corresponding mechanical shortening, of costal and crural diaphragm, during room air and hypercapnia. RESULTS: During eupnea, costal and crural diaphragm are active into expiration, showing significant and distinct expiratory activity, with crural expiratory activity greater than costal, for both magnitude and duration. This diaphragm segmental difference diverged further during progressive hypercapnic ventilation: crural expiratory activity progressively increased, while costal expiratory activity disappeared. CONCLUSION: The diaphragm is not passive during expiration. During spontaneous breathing, expiratory activity -"braking"- of the diaphragm is expressed routinely, but is not equally distributed. Crural muscle "braking" is greater than costal muscle in magnitude and duration. With increasing ventilation during hypercapnia, expiratory activity -"braking"- diverges notably. Crural expiratory activity greatly increases, while costal expiratory "braking" decreases in magnitude and duration, and disappears. Thus, diaphragm expiratory "braking" action represents an inherent, physiological function of the diaphragm, distinct for each segment, expressing differing neural activation.

Expiratory and inspiratory action of transversus abdominis during eupnea and hypercapnic ventilation.

BACKGROUND: Recently, there is interest in the clinical importance of monitoring abdominal muscles during respiratory failure. The clinical interpretation relies on the assumption that expiration is a passive physiologic process and, since diaphragm and abdomen are arranged in series, any inward motion of the abdominal wall represents a sign of diaphragm dysfunction. However, previous studies suggest transversus abdominis might be active even during eupnea and is preferentially recruited over the other abdominal muscles. OBJECTIVE: 1) Is transversus abdominis normally recruited during eupnea? 2) What is the degree of activation of transversus abdominis during hypercapnia? 3) Does the end-inspiratory length of transversus abdominis change during hypercapnia, while diaphragm function is normal? METHODS: In 30 spontaneously breathing canines, awake without confounding anesthetic, we measured directly both electrical activity and corresponding mechanical length and shortening of transversus abdominis during eupnea and hypercapnia. RESULTS: Transversus abdominis is consistently recruited during eupnea. During hypercapnia, transversus abdominis recruitment is progressive and significant. Throughout hypercapnia, transversus abdominis baseline end-inspiratory length is not constant: baseline length decreases progressively throughout hypercapnia. After expiration, into early inspiration, transversus abdominis shows a consistent neural mechanical post -expiratory expiratory activity (PEEA) at rest, which progressively increases during hypercapnia. CONCLUSION: Transversus abdominis is an obligatory expiratory muscle, reinforcing the fundamental principle expiration is not a passive process. Beyond expiration, during hypercapnic ventilation, transversus abdominis contributes as an "accessory inspiratory muscle" into the early phase of inspiration. Clinical monitoring of abdominal wall motion during respiratory failure may be confounded by action of transversus abdominis.

Parasternal intercostal, costal, and crural diaphragm neural activation during hypercapnia.

The parasternal intercostal is an obligatory inspiratory muscle working in coordination with the diaphragm, apparently sharing a common pathway of neural response. This similarity has attracted clinical interest, promoting the parasternal as a noninvasive alternative to the diaphragm, to monitor central neural respiratory output. However, this role may be confounded by the distinct and different functions of the costal and crural diaphragm. Given the anatomic location, parasternal activation may significantly impact the chest wall via both mechanical shortening or as a "fixator" for the chest wall. Either mechanical function of the parasternal may also impact differential function of the costal and crural. The objectives of the present study were, during eupnea and hypercapnia, 1) to compare the intensity of neural activation of the parasternal with the costal and crural diaphragm and 2) to examine parasternal recruitment and changes in mechanical action during progressive hypercapnia, including muscle baseline length and shortening. In 30 spontaneously breathing canines, awake without confounding anesthetic, we directly measured the electrical activity of the parasternal, costal, and crural diaphragm, and the corresponding mechanical shortening of the parasternal, during eupnea and hypercapnia. During eupnea and hypercapnia, the parasternal and costal diaphragm share a similar intensity of neural activation, whereas both differ significantly from crural diaphragm activity. The shortening of the parasternal increases significantly with hypercapnia, without a change in baseline end-expiratory length. In conclusion, the parasternal shares an equivalent intensity of neural activation with the costal, but not crural, diaphragm. The parasternal maintains and increases its active inspiratory shortening during augmented ventilation, despite high levels of diaphragm recruitment. Throughout hypercapnic ventilation, the parasternal contributes mechanically; it is not relegated to chest wall fixation.NEW & NOTEWORTHY This investigation directly compares neural activation of the parasternal intercostal muscle with the two distinct segments of the diaphragm, costal and crural, during room air and hypercapnic ventilation. During eupnea and hypercapnia, the parasternal intercostal muscle and costal diaphragm share a similar neural activation, whereas they both differ significantly from the crural diaphragm. The parasternal intercostal muscle maintains and increases active inspiratory mechanical action with shortening during ventilation, even with high levels of diaphragm recruitment.

A Bit Stickier, a Bit Slower, a Lot Stiffer: Specific vs. Nonspecific Binding of Gal4 to DNA.

Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to a subtle interplay of specific and nonspecific interactions that is challenging to quantify. Here, we exploit Reflective Phantom Interface (RPI), a label-free biosensor based on optical reflectivity, to investigate the binding of the N-terminal domain of Gal4, a well-known gene regulator, to double-stranded DNA fragments containing or not its consensus sequence. The analysis of RPI-binding curves provides interaction strength and kinetics and their dependence on temperature and ionic strength. We found that the binding of Gal4 to its cognate site is stronger, as expected, but also markedly slower. We performed a combined analysis of specific and nonspecific binding-equilibrium and kinetics-by means of a simple model based on nested potential wells and found that the free energy gap between specific and nonspecific binding is of the order of one kcal/mol only. We investigated the origin of such a small value by performing all-atom molecular dynamics simulations of Gal4-DNA interactions. We found a strong enthalpy-entropy compensation, by which the binding of Gal4 to its cognate sequence entails a DNA bending and a striking conformational freezing, which could be instrumental in the biological function of Gal4.

Limitations of surface EMG estimate of parasternal intercostal to infer neural respiratory drive.

BACKGROUND: Recently, surface EMG of parasternal intercostal muscle has been incorporated in the "ERS Statement of Respiratory Muscle Testing" as a clinical technique to monitor the neural respiratory drive (NRD). However, the anatomy of the parasternal muscle risks confounding EMG "crosstalk" activity from neighboring muscles. OBJECTIVES: To determine if surface "parasternal" EMG: 1) reliably estimates parasternal intercostal EMG activity, 2) is a valid surrogate expressing neural respiratory drive (NRD). METHODS: Fine wire electrodes were implanted into parasternal intercostal muscle in 20 severe COPD patients along with a pair of surface EMG electrodes at the same intercostal level. We recorded both direct fine wire parasternal EMG (EMGPARA) and surface estimated "parasternal" EMG (SurfEMGpara) simultaneously during resting breathing, volitional inspiratory maneuvers, apnoea with extraneous movement of upper extremity, and hypercapnic ventilation. RESULTS: Surface estimated "parasternal" EMG showed spurious "pseudobreathing" activity without any airflow while real parasternal EMG was silent, during apnoea with body extremity movement. Surface estimated "parasternal" EMG did not faithfully represent real measured parasternal EMG. Surface estimated "parasternal" EMG was significantly less active than directly measured parasternal EMG during all conditions including baseline, inspiratory capacity and hypercapnic ventilation. Bland-Altman analysis showed consistent bias between direct parasternal EMG recording and surface estimated EMG during stimulated breathing. CONCLUSION: Surface "parasternal" EMG does not consistently or reliably express EMG activity of parasternal intercostal as recorded directly by implanted fine wires. A chest wall surface estimate of parasternal intercostal EMG may not faithfully express NRD and is of limited utility as a biomarker in clinical applications.

Distinct neural-mechanical efficiency of costal and crural diaphragm during hypercapnia.

Classic physiology suggests that the two distinct diaphragm segments, costal and crural, are functionally different. It is not known if the two diaphragm muscles share a common neural mechanical activation. We hypothesized that costal and crural diaphragm are recruited differently during hypercapnic stimulated ventilation, and the EMG recordings of the esophageal crural diaphragm segment does not translate to the same level of mechanical shortening for costal and crural segments In 30 spontaneously breathing canines, without confounding anesthetic, we measured directly electrical activity and corresponding mechanical shortening of both the costal and crural diaphragm, at room air and during increasing hypercapnia. During hypercapnic ventilation, the costal diaphragm showed a predominant recruitment over the crural diaphragm. The distinct mechanical contribution of the costal segment was not due to a different level of neural activation between the two muscles as measured by segmental EMG activity. Thus, the two diaphragm segments exhibited a significantly different neural-mechanical relationship.

Scientific mistakes from the agri-food biotech critics.

Critics of the use of advanced biotechnologies in the agri-food sector ("New Breeding Techniques", comprising CRISPR) demand a strict regulation of any such method, even more severe than rules applied to so-called "Genetically Modified Organisms" (i.e. recombinant DNA processes and products). But their position is unwarranted, since it relies on faulty arguments.While most life scientists have always explained that the trigger for regulation should be the single product and its phenotypic traits, opponents insist that the target should be certain biotech processes.The antagonists maintain that NBTs are inherently risky: this belief is exactly the opposite of a long-standing, overwhelming scientific consensus. NBTs involve unpredictable effects, but it is the same for the results of any other technique. The critics wrongly equate "unintended" with "harmful" and misunderstand two meanings of "risk": the "risk" of not achieving satisfactory results does not automatically translate into health or environment "risks". Generic claims that allergenic or toxic properties are a hidden danger of outcomes from NBTs are unsubstantiated - as they would be for traditional techniques.Among several errors, we criticize the misuse of the Precautionary principle, a misplaced alarm about "uncontrolled spreading" of genetically engineered cultivars and the groundless comparison of (hypothetical) agricultural products from NBTs with known toxic substances.In order to "save" traditional techniques from "GMO"-like regulations, while calling for the enforcement of similar sectarian rules for the NBTs, the dissenters engage in baseless, unscientific distinctions.Important and necessary socio-economic, ethical and legal considerations related to the use of agri-food biotechnologies (older and newer) are outside the scope of this paper, which mostly deals with arguments from genetics, biology, and evolutionary theory that are provided by those who are suspicious of NBTs. Yet, we will provide some hints on two additional facets of the debate: the possible motivations for certain groups to embrace views which are utterly anti-scientific, and the shaky regulatory destiny of NBTs in the European Union.

Measurement of Diaphragmatic Electrical Activity by Surface Electromyography in Intubated Subjects and Its Relationship With Inspiratory Effort.

BACKGROUND: Quantification of patient effort during spontaneous breathing is important to tailor ventilatory assistance. Because a correlation between inspiratory muscle pressure (Pmus) and electrical activity of the diaphragm (EAdi) has been described, we aimed to assess the reliability of surface electromyography (EMG) of the respiratory muscles for monitoring diaphragm electrical activity and subject effort during assisted ventilation. METHODS: At a general ICU of a single university-affiliated hospital, we enrolled subjects who were intubated and on pressure support ventilation (PSV) and were on mechanical ventilation for > 48 h. The subjects were studied at 3 levels of pressure support. Airway flow and pressure; esophageal pressure; EAdi; and surface EMG of the diaphragm (surface EAdi), intercostal, and sternocleidomastoid muscles were recorded. Respiratory cycles were sampled for off-line analysis. The Pmus/EAdi index (PEI) was calculated by relying on EAdi and surface EAdi (surface PEI) from an airway pressure drop during end-expiratory occlusions performed every minute. RESULTS: surface EAdi well correlated with EAdi and Pmus, in particular, after averaging breaths into deciles (R = 0.92 and R = 0.84). When surface PEI was used with surface EAdi, it provided a reliable estimation of Pmus (R = 0.94 in comparison with measured Pmus). CONCLUSIONS: During assisted mechanical ventilation, EAdi can be reliably monitored by both EAdi and surface EMG. The measurement of Pmus based on the calibration of EAdi was also feasible by the use of surface EMG.

A Plea for the Renewal of the ISBR.

The recent meeting of the International Society for Biosafety Research (ISBR) focused on so-called genetically modified organisms. For decades, in most regulatory frameworks, recombinant DNA-modified organisms have been the wrong focus of unbalanced agri-food regulations. The ISBR should instead adopt a scientifically defensible and truly risk-based perspective, abandoning a misleading pseudo-category.

The central dogma, "GMO" and defective epistemology.

The expression "Genetically Modified Organisms" was coined to indicate a group of agricultural products (mostly crops and vegetables), modified through direct DNA recombination in order to obtain useful phenotypic traits or to inhibit undesirable characteristics. But the border between rDNA ("GMO") and other biotech methods is blurred. Moreover, the ill-assorted group is frequently charged with having peculiar, negative characteristics: many activists, part of the public and a few social science scholars think that "GMOs" are all dubious, even inherently dangerous. However, theoretical justifications of this alleged problematic nature which is supposed to be necessarily linked to the "splicing" of DNA, only when applied to agricultural products, are missing: the only text which tries to go in depth on the subject, an article by biologist Barry Commoner, takes aim at the wrong target, misunderstanding the Central Dogma. "GMO" is a term that has no clear reference, let alone in a detrimental sense. The only attempt to give it epistemological dignity fails.

A label-free immunoassay for Flavivirus detection by the Reflective Phantom Interface technology.

Flaviviruses are widespread and cause clinically relevant arboviral diseases that impact locally and as imported travel-related infections. Direct detection of viraemia is limited, being typically undetectable at onset of symptoms. Therefore, diagnosis is primarily based on serology, which is complicated by high cross-reactivity across different species. The overlapping geographical distribution of the vectors in areas with a weak healthcare system, the increase of international travel and the similarity of symptoms highlight the need for rapid and reliable multi-parametric diagnostic tests in point-of-care formats. To this end we developed a bi-parametric serological microarray using recombinant NS1 proteins from Tick-borne encephalitis virus and West Nile virus coupled to a low-cost, label-free detection device based on the Reflective Phantom Interface (RPI) principle. Specific sequential detection of antibodies in solution demonstrates the feasibility of the approach for the surveillance and diagnosis of Flaviviruses.

Product, not process! Explaining a basic concept in agricultural biotechnologies and food safety.

Most life scientists have relentlessly recommended any evaluative approach of agri-food products to be based on examination of the phenotype, i.e. the actual characteristics of the food, feed and fiber varieties: the effects of any new cultivar (or micro-organism, animal) on our health are not dependent on the process(es), the techniques used to obtain it.The so-called "genetically modified organisms" ("GMOs"), on the other hand, are commonly framed as a group with special properties - most frequently seen as dubious, or even harmful.Some social scientists still believe that considering the process is a correct background for science-based understanding and regulation. To show that such an approach is utterly wrong, and to invite scientists, teachers and science communicators to explain this mistake to students, policy-makers and the public at large, we imagined a dialogue between a social scientist, who has a positive opinion about a certain weight that a process-based orientation should have in the risk assessment, and a few experts who offer plenty of arguments against that view. The discussion focuses on new food safety.

The EU legislation on "GMOs" between nonsense and protectionism: An ongoing Schumpeterian chain of public choices.

The EU regulation of agricultural biotechnology is botched and convoluted: the pseudo-concept of "Genetically Modified Organisms" has no coherent semantic or scientific content. The reasons of the paradox by which the cultivation of "GMOs" is substantially banned in Europe, while enormous quantities of recombinant-DNA cereals and legumes are imported to be used as feedstuff, are explained. The Directive 2015/412, giving Member states the choice to refuse the cultivation of genetically engineered crops at a national or local level, paves the way for a mosaic-like, Harlequinesque form of protectionism: nothing resembling a well-regulated free market. In the meantime, importation of "GMO" feed goes on at full speed all over Europe. A proposal by the Commission to adjust the rules on importation according to those for cultivation has been rejected by the Parliament.This dynamics may be seen as an ongoing "Schumpeterian" chain of public choices: the calculus of consent drives politicians more than a science-based approach to law-making. The EU should restart from scratch with the right concept, i.e. the careful examination of the pros and cons, the costs and benefits of each new agricultural product ("GMO" or otherwise), freely cultivated and/or imported, assessed case by case, at last acknowledging that the biotech processes used to create new varieties are of no practical or legal relevance. In doing so, the EU would pursue its stated "better regulation" approach, cancelling any sectoral and sectarian regulation.

The Precautionary principle: its misunderstandings and misuses in relation to "GMOs".

References to the Precautionary principle (PP) in relation to "GMOs" are commonplace. Those who oppose the DNA recombinant approach to create new agricultural products either have not read the PP (misunderstanding), or they want to exploit the PP for their propaganda while forcing it (misuse). Proponents of a stricter approach to the regulation of biotechnologies must forge a new expression, since the PP is something else - historically and theoretically. In any case, a legitimate very circumspect attitude, to be coherent, must be applied to each and every biotechnology, not only to "GMOs".

Systematic comparison of single-chain Fv antibody-fusion toxin constructs containing Pseudomonas Exotoxin A or saporin produced in different microbial expression systems.

BACKGROUND: Antibodies raised against selected antigens over-expressed at the cell surface of malignant cells have been chemically conjugated to protein toxin domains to obtain immunotoxins (ITs) able to selectively kill cancer cells. Since latest generation immunotoxins are composed of a toxic domain genetically fused to antibody fragment(s) which confer on the IT target selective specificity, we rescued from the hydridoma 4KB128, a recombinant single-chain variable fragment (scFv) targeting CD22, a marker antigen expressed by B-lineage leukaemias and lymphomas. We constructed several ITs using two enzymatic toxins both able to block protein translation, one of bacterial origin (a truncated version of Pseudomonas exotoxin A, PE40) endowed with EF-2 ADP-ribosylation activity, the other being the plant ribosome-inactivating protein saporin, able to specifically depurinate 23/26/28S ribosomal RNA. PE40 was selected because it has been widely used for the construction of recombinant ITs that have already undergone evaluation in clinical trials. Saporin has also been evaluated clinically and has recently been expressed successfully at high levels in a Pichia pastoris expression system. The aim of the present study was to evaluate optimal microbial expression of various IT formats. RESULTS: An anti-CD22 scFv termed 4KB was obtained which showed the expected binding activity which was also internalized by CD22+ target cells and was also competed for by the parental monoclonal CD22 antibody. Several fusion constructs were designed and expressed either in E. coli or in Pichia pastoris and the resulting fusion proteins affinity-purified. Protein synthesis inhibition assays were performed on CD22+ human Daudi cells and showed that the selected ITs were active, having IC50 values (concentration inhibiting protein synthesis by 50% relative to controls) in the nanomolar range. CONCLUSIONS: We undertook a systematic comparison between the performance of the different fusion constructs, with respect to yields in E. coli or P. pastoris expression systems and also with regard to each constructs specific killing efficacy. Our results confirm that E. coli is the system of choice for the expression of recombinant fusion toxins of bacterial origin whereas we further demonstrate that saporin-based ITs are best expressed and recovered from P. pastoris cultures after yeast codon-usage optimization.