Intraoperative hypotension is linked to increased incidence of perioperative adverse events such as myocardial and cerebrovascular infarction and acute kidney injury. Hypotension prediction index (HPI) is a novel machine learning guided algorithm which can predict hypotensive events using high fidelity analysis of pulse-wave contour. Goal of this trial is to determine whether use of HPI can reduce the number and duration of hypotensive events in patients undergoing major thoracic procedures.Thirty four patients undergoing esophageal or lung resection were randomized into 2 groups -“machine learning algorithm” (AcumenIQ) and “conventional pulse contour analysis” (Flotrac). Analyzed variables were occurrence, severity and duration of hypotensive events (defined as a period of at least one minute of MAP below 65 mmHg), hemodynamic parameters at 9 different timepoints interesting from a hemodynamics viewpoint and laboratory  and clinical outcomes .Patients in the AcumenIQ group had significantly lower area below the hypotensive threshold  and time-weighted AUT . Also, there were less patients with hypotensive events and cumulative duration of hypotension in the AcumenIQ group. No significant difference between groups was found in terms of laboratory and clinical outcomes.Hemodynamic optimization guided by machine learning algorithm leads to a significant decrease in number and duration of hypotensive events compared to traditional goal directed therapy using pulse-contour analysis hemodynamic monitoring in patients undergoing major thoracic procedures. Further, larger studies are needed to determine true clinical utility of HPI guided hemodynamic monitoring.Date of first registration: 14/11/2022Registration number: 04729481-3a96-4763-a9d5-23fc45fb722d In Europe, approximately 20 million major surgical procedures are performed annually. Up to 5% of these patients will die and 10–15% will develop adverse events that could have been prevented in more than 33% of cases [Intraoperative hypotension (IOH) may increase mortality and morbidity rates in the postoperative period, and is associated with adverse events such as acute kidney injury, cerebral and myocardial infarction . It is sA recent randomized controlled trial reported that preventing intraoperative hypotension reduces the risk of postoperative organ dysfunction by about a quarter , while aAbsolute threshold in defining IOH includes systolic blood pressure (SBP) less than 90 mmHg or mean arterial pressure (MAP) less than 65 mmHg, while a 20% reduction in baseline SBP or a 30% reduction in MAP represents the relative threshold. Most patients in noncardiac surgery experience at least one episode during which MAP decreases to < 65 mmHg, and known causes are anesthetic drugs, uncorrected hypovolemia, preexisting comorbidities, and surgical manipulation .The “Hypotension Prediction Index” (HPI) is a novel hemodynamic monitoring tool that predicts episodes of intraoperative hypotension before they occur. It has recently been shown that applying Acumen  HPI software algorithm on data obtained by invasive blood pressure monitoring sensor during noncardiac surgery enables earlier assessment of causes of impending hypotension and timely response to possible hemodynamic instability . It can Given the significant association between hypotension and postoperative complications and adverse outcomes such as increased incidence of myocardial infarction and cerebrovascular insult, as well as increased hospital stay , it seemThe aim of this study was to examine whether use of machine-learning algorithm guided intraoperative patient hemodynamic optimization reduces duration and severity of hypotension and its complications during and after thoracic surgery compared to conventional pulse contour analysis goal-directed hemodynamic optimization.By design this study is a prospective, randomized, single blinded study.Participants are patients over 18 years of age which were scheduled for elective major thoracic procedure  with planned thoracotomy and intraoperative period of one lung ventilation with planned postoperative admission to the ICU. Exclusion criteria were persistent atrial fibrillation, structural heart defects , preoperative serum hemoglobin levels < 120 g/L and severe heart failure classified as New York Heart Association (NYHA) grade IV. (AcumenIQ) group or “conventional pulse contour analysis” (Flotrac) group. Both groups were hemodynamically monitored using Hemosphere monitoring platform using either AcumenIQ or Flotrac sensors , depending on group allocation.In the anesthesia preparation room, after informed consent was obtained, patients were randomized using coin toss into two groups:“machine learning algorithm”In the operating room and before induction of anesthesia, arterial and central venous lines were placed after sterile skin preparation and local anesthetic infiltration, zeroed to account for hydrostatic pressure differences between the pressure transducer and the heart, and monitoring was initiated.2), stroke volume index , systolic, diastolic and mean arterial pressure (mmHg), heart rate , systemic vascular resistance index , stroke volume variation , hypotension prediction index , systolic pressure increase divided by duration of systole as an indicator of left ventricular contractility  and dynamic arterial elastance .Following hemodynamic parameters were measured: cardiac index , during induction of anesthesia - 3 min after administration of muscle relaxant (T1), 1 min after intubation (T2), 1 min after placing the patient in lateral decubitus position (T3), 1 min after skin incision (T4), 1 min after thoracotomy and initiation of one-lung ventilation (T5), 1 min after removal of tumor (T6), 3 min after skin closure (T7) and 1 min after placing the patient back in the supine position (T8).2 adjusted to achieve peripheral oxygen saturation > 95% during two lung ventilation and > 90% during one lung ventilation. Muscle paralysis was preserved by administration of 8 mcg/kg/min rocuronium bromide.Induction of anesthesia was performed using propofol (1–1.5 mg/kg), sufentanil (0.1–0.5 mcg/kg) and rocuronium bromide (0.6 mg/kg). After intubation, placement of Robert Shaw tube was verified capnographically and the correct position of endobronchial lumen was verified using a flexible fiber bronchoscope. Anesthesia was maintained using oxygen/air/sevoflurane mixture at minimal alveolar concentration (MAC) 0.8–1 and FiOIn patients with thoracic epidural catheter in place, continuous infusion of 1 mg/ml levobupivacaine and 1 mcg/ml sufentanil was administered at rate of 1–2 ml/h, according to attending anesthesiologist’s assessment of patient’s nociceptive response, and in those without an epidural catheter, 5—10 mcg sufentanil i.v. boluses were administered as needed.PBW during two-lung ventilation and 4–6 ml/kgPBW during one lung ventilation, with frequency set to maintain etCO2 3.5 - 4.5 kPa and I:E ratio was set to allow expiration duration of at least 3 RC.Patients were ventilated  using a tidal volume of 8 ml/kgAcumenIQ group HPI values ≥ 90 triggered an alert after which the attending anesthesiologist analyzed the “clinical decision tree” screen with hemodynamic variables displayed in a structured manner  after which a goal directed therapeutic intervention was performed .In the Flotrac group, hemodynamic optimization was guided by measured values displayed on the monitor and interventions were aiming for hemodynamic targets: MAP > 65 mmHg, CI ≥ 2.4 l/min/m2, SVI ≥ 30 ml/beat/m2 and SVRI 1700–2400 dyn·s·cm−5/m2.In the 2 was not met after adequate volume resuscitation dobutamine infusion was started until the threshold was reached, and if MAP > 65 mmHg was not achieved with adequate CI, norepinephrine was used as a predefined pressor of choice and was administered either as 2–10 mcg bolus or in a continuous infusion until target was achieved.In supine patients SVV ≥ 13% defined volume responsiveness , while iPrimary endpoint is time weighted average of area spent under 65 mmHg for MAP per patient (AUT - depth of hypotension below a MAP of 65 mmHg × time spent below a MAP of 65 mm Hg divided by procedure duration in minutes) of AUT (TWA-AUT).Secondary endpoints are AUT, number of hypotensive episodes (defined as MAP < 65 mmHg for at least 1 min) during the procedure and cumulative duration of hypotension during the procedure.2 gap (kPa) [2, %) and serum lactate levels (mmol/L); duration of postoperative mechanical ventilation (hours), ICU stay (hours), hospital stay (days from day of surgery to discharge) and in-hospital and 28-day mortality, as well as occurrence of postoperative complications: acute kidney injury (defined according to RIFLE criteria [Volume of i.v. fluids (ml/kg/h) and norepinephrine (mcg/kg/min) administered in the operating theater, urine output (ml/kg/h), postoperative arterial blood pH, arterio-venous COap (kPa) , base excriteria ), acute Hemosphere monitor via USB as Microsoft Excel .xls files and were analyzed with the Acumen Analytics  software package which generated reports from which derived data about intraoperative hypotension events was collected.Hemodynamic measurements for each patient were exported from Continuous variables are displayed as either mean and standard deviation (SD) for values with Gaussian distribution, or median and interquartile range or 95% confidence interval (CI) for data that does not follow normal distribution. Normality of distribution was assessed using the Shapiro–Wilk test. Categorical variables are displayed as counts and percentages.2 or Fisher’s exact test for 2 × 2 tables.Differences in independent continuous variables between 2 groups were tested for statistical significance using Student’s t test for independent samples or Mann–Whitney U test, depending on distribution of data. For measurements at various time points, repeated measurements two-way analysis of variance (RM-ANOVA) with post-hoc Holm-Bonferroni correction was performed, and values are expressed as marginal mean and 95% confidence interval (CI). Differences between groups in categorical variables were tested for statistical significance using χPilot trial with 5 participants per group was conducted for difference in means of TWA-AUT and effect size (d) of 1.08 was obtained. For ɑ error probability of 0.05 and statistical power (1-ꞵ) of 0.8 sample size of minimally 16 patients per group was calculated. Software package G*Power v 3.1.9.7 , 16 was P values < 0.05 were considered statistically significant. Software package jamovi v2.3.2 [i v2.3.2  was usedAcumenIQ sensor which uses machine learning guided HPI algorithm, and 17 were monitored with the Flotrac sensor and conventional goal directed therapy was utilized.There were 36 patients screened for inclusion, of which 1 was not randomized due to presence of an exclusion criterion, and 1 declined to participate. 34 participants were included, of which 17 were randomized to be monitored with the No statistically significant differences between groups were observed in regard to their baseline demographic, clinical and anthropometric data, severity of comorbidities (Charlson comorbidity index—CCI) as well as type of surgery performed, and most hemodynamic measurements. Only baseline heart rate was higher in the AcumenIQ group Table .Table 1BFlotrac group there were a total of 50 hypotensive events (13/17 patients) with a total duration of hypotension of 105 min, compared to 13 events in the AcumenIQ group (7/17 patients) with total duration of hypotension of 22 min. Patients in the AcumenIQ group had significantly lower number of hypotensive events, duration of hypotensive event per patient and area under 65 mmHg for MAP per patient (AUT) and weighted average (TWA) of AUT. There was no significant difference in the number of patients that were hypotensive under 50 mmHg and MAP during hypotensive episodes  outcomes (Table 2).There was no significant difference between groups in the amount of administered intraoperative i.v. fluids, packed red blood cells or norepinephrine, clinical  or laboratory  and an increase after intubation in the AcumenIQ group (P < 0.001), but without difference between groups  and an increase after intubation in the AcumenIQ group (P = 0.002), but without difference between groups  but without other significant differences between groups and measurements  and drop after intubation , followed by an increase after intubation  significantly reduced the number and duration of intraoperative hypotensive episodes, as well as AUT and the primary outcome—TWA of AUT.These results are in agreement with results obtained by Wijnberge et al.  and GrunA recent study by Enevoldsen et al.  discusseFlotrac group was 3.7 min and 0.0 min in the AcumenIQ group. These results show that both reactive or proactive approaches to hemodynamic optimization will achieve similar clinical outcomes, as long as the anesthesiologist's reaction to occurrence of intraoperative hypotension is rapid and goal directed.Our results show that while incidence and duration of hypotension were significantly reduced in patients that used HPI algorithm, there were no significant differences in clinical outcomes, however these results should be interpreted with caution due to sample size and inadequate statistical power. Results of a large retrospective study by Gregory et al.  (over 35AcumenIQ group (who had zero hypotensive events) that died in the hospital due to surgical complications which required subsequent re-do procedures and resulted in a fatal outcome on 27th post-op day.There was one patient in the AcumenIQ group, there was no significant difference in the amount of i.v. fluid or vasopressors administered between groups. In a study by Wu et al. [While hemodynamic stability was much better preserved in the u et al.  both oveu et al. . PatientHypotension prediction index has proven to be reliable enough in prediction of intraoperative hypotensive events even when used with non-invasive volume clamp method, with similar sensitivity and specificity levels as when used with arterial lines , 24.Since most elective thoracic procedures (lung and esophagus resection) are generally not associated with major blood loss and fluid shift, most hemodynamic changes are due to surgical manipulation, the patient’s position and changes in cardiac output during OLV, which rises after pleural opening . In our AcumenIQ group is the fact that dynamic parameters such as stroke volume variation are not a viable method to determine fluid responsiveness during OLV, and once hypotension occurs, valuable time is lost during a fluid responsiveness trial, while once the HPI alarm goes off, there is enough time to react before the hypotension itself and it can be either avoided, or its duration can be reduced, as our results show.One probable reason why both incidence and duration of hypotensive events were significantly lower in the Further extension of machine learning algorithms in order to preserve intraoperative hemodynamic stability is application of closed loop systems that automatically administer fluids and vasopressors according to measured hemodynamic variables, which have reduced duration of intraoperative hypotension (< 65 mmHg) by 21.1%, as well as a reduction of intraoperative fluid balance (+ 1600 vs + 2050 ml) and lower serum lactate levels (1.2 vs 2.7 mmol/L) in patients undergoing moderate to high-risk surgery as demonstrated by Joosten et al. .It must be noted that most patients in both groups were graded as ASA 3, and although significant comorbidities are present (most of which are associated with smoking), careful multidisciplinary approach and preoperative assessment of cardiac and lung reserve are essential in decreasing the duration of mechanical ventilation, ICU and hospital stay, and improving patient outcomes in general .Since surgical manipulation  in thoracic procedures will result in rapid hemodynamic changes that cannot be predicted by an algorithm, there were concerns that use of HPI to guide hemodynamic optimization in such procedures is futile. However, obtained results show that even with that shortcoming, there were significant differences between groups in hypotension related outcomes.One other question that is usually raised in studies which rely on arterial waveform pulse contour analysis is their utility in patients with an open thorax and/or reduced left ventricular ejection fraction, as studied by Vetrugno et al. in patients that were monitored using second generation algorithm . In our P = 0.42), statistical power is 0.60 and to achieve 0.8 at least 45 participants per group are needed. Due to the fact that no external funding was received and considering the cost of a single-use pressure transducer set needed for hemodynamic monitoring, we were unable to conduct a larger study.In terms of the number of participants included in this study, sample size was determined for primary outcome measure, for which the null hypothesis was rejected. However, in terms of secondary clinical outcomes, the study is underpowered, and a much larger sample is needed to draw relevant conclusions. For example, while there was no significant difference in lactate levels between groups   but opteAlso, during statistical analysis, some concerns were raised about the validity of the coin-toss method of randomization. However, since there were no significant differences in relevant baseline characteristics between groups, choice of randomization method did not result in sampling bias, as can be seen in Table Results of this study suggest that use of machine learning algorithm guided hemodynamic optimization can significantly decrease both number and duration of hypotensive events compared to conventional pulse contour analysis guided monitoring in patients undergoing major thoracic procedures. There were no significant differences between groups regarding secondary clinical outcomes and larger multi-center studies are needed to determine true clinical utility and feasibility of predictive hemodynamic monitoring in thoracic surgery.


Filming atomic motion within molecules is an active pursuit of molecular physics and quantum chemistry. A promising method is laser-induced Coulomb Explosion Imaging (CEI) where a laser pulse rapidly ionizes many electrons from a molecule, causing the remaining ions to undergo Coulomb repulsion. The ion momenta are used to reconstruct the molecular geometry which is tracked over time  by ionizing at an adjustable delay with respect to the start of interatomic motion. Results are distorted, however, by ultrafast motion during the ionizing pulse. We studied this effect in water and filmed the rapid “slingshot” motion that enhances ionization and distorts CEI results. Our investigation uncovered both the geometry and mechanism of the enhancement which may inform CEI experiments in many other polyatomic molecules. 2O that induces this distortion and elucidate the underlying mechanism of enhanced ionization.Laser-induced Coulomb explosion imaging allows the study of molecular geometries over time, but the results are often distorted by ultrafast motion during the ionizing laser pulse. Here, the authors film the rapid slingshot motion in D Resolving the femtosecond-scale motion of these light atomic species is therefore central to the fields of molecular physics and quantum chemistry, and motivates continued efforts to film atomic-scale “molecular movies”6.Hydrogen atoms within molecules can move extremely rapidly in response to the sudden intramolecular forces introduced by ionization or photoexcitation. These atoms can accelerate to traverse Angstrom-scale distances in just tens of femtoseconds. Such ultrafast motion may mediate many biologically important light-matter interactions, including photosynthesis, photochemical damage mitigation in DNA, and vision9, laser-induced electron diffraction11, and hard x-ray diffraction14; spectroscopic techniques such as high harmonic generation17; and momentum imaging techniques such as Coulomb Explosion Imaging (CEI)33. Diffractive techniques, however, lose sensitivity with decreasing atomic mass and spectroscopic techniques typically rely on indirect observables of atomic motion such as electronic structure. Among the methods listed, only CEI probes the direct momenta of all atoms, irrespective of mass, within a molecule34.Many methods exist to record ultrafast motion in molecules including diffractive imaging techniques, such as ultrafast electron diffraction35, intense infrared (IR) lasers36 or ultrafast x-ray pulses37. Intense IR lasers are especially attractive for time-resolved CEI; intramolecular dynamics can be initiated with a few-femtosecond table-top IR laser pulse and probed with sub-femtosecond temporal jitter using another pulse derived from the same source39.CEI deduces the positions of atoms within molecules by stripping away binding electrons and measuring the momenta of fragments produced in the resulting Coulomb repulsion of the ions. The binding electrons can be removed using thin foils41. The ultimate dissociation pathway of the fragments is therefore rarely determined by idealized Coulomb repulsion between point-like ions. This problem is greatly exacerbated by a strong-field phenomenon known as Enhanced Ionization (EI), where a “critical” spacing among the constituent atoms of a molecule increases the ionization yield47, distorting the momentum distribution observed via CEI to favor the critical geometries that undergo EI.One of the greatest limitations of laser-induced CEI is that multiple ionization typically occurs sequentially; as a result, intermediate charge states can drive nuclear dynamics prior to Coulomb explosion50 and have been more recently studied in triatomic molecules53 such as water59. Diatomics have long been known to undergo EI en route to dication formation: strong-field ionization of the first electron drives nuclear motion toward a geometry that facilitates ionization of the second electron. In this geometry the two atoms are always stretched to a critical distance and aligned with the polarization axis of the laser64. If both electrons are removed within the same pulse, it has been found that decreasing the pulse duration generally reduces the fraction of molecules that undergo EI65. A similar phenomenon exists in multiply-charged linear triatomics66, and has recently been studied both experimentally46 and theoretically67 in triply charged water, the bent triatomic considered in this work.Strong-field distortions of molecular dissociation dynamics were first studied extensively in diatomic molecules2O as a model molecular system to film an EI process that proceeds within just 20 fs. To do so, we first verified that the formation of D2O3+ via strong-field multiple ionization yields CEI results with clear distortions indicative of EI. We characterized how the severity of these distortions greatly depends on the ionizing pulse duration. We then demonstrated that the conditions for EI can be reproduced by launching rapid “slingshot” motion in D2O2+. We modeled this motion using ab initio theory, and the correspondence between theory and experiment allowed the direct retrieval of the time-resolved molecular geometry. The resulting molecular movie revealed the critical geometry at which EI occurs and unveiled the underlying mechanism that induces the enhancement. This improved understanding of EI can not only aid the analysis of future CEI data, but can also be employed to highlight particular features of polyatomic motion in future atomic-scale molecular movies.Here, we used Dτ = 6, 10, 19, and 40 fs) but constant peak intensity (I0 = 2 × 1015 W/cm2) ionize neutral D2O to form D2O3+. Here, the intermediate charge states, D2O+ and D2O2+, undergo field-assisted dynamics within the envelope of a single pulse. In the second scheme, a 6-fs 750 nm pulse (I0 = 1 × 1015 W/cm2) doubly ionizes the neutral molecule to create the dication D2O2+ before an identical cross-polarized pulse follows at an adjustable delay (Δt = 10–110 fs) to form the trication D2O3+. Here, the dication undergoes field-free dynamics in the time between pulses. Following trication formation, the molecule rapidly Coulomb exploded into three bodies (D+/D+/O+), each of whose three-dimensional momenta were captured in coincidence by a high-resolution position- and time-sensitive detector68. Two experimental schemes were used to induce EI in water, both of which are depicted in Fig. 2O than single short pulses at the same intensity  of triply- (D+/D+/O+) to doubly-charged (D+/D+/O) three-body dissociations undergoes a 27-fold increase as the pulse duration lengthens from 6 to 19 fs  in the lower left corner of Fig. +/D+ coincidence following ionization at each of the four pulse durations studied. In Fig. + cluster results from triple ionization (D+/D+/O+), and the lower-momentum D+ cluster results from double ionization (D+/D+/O)59. The ratio of these two fragmentation pathways for the four pulse durations is shown in Fig. +/D+/O feature appears when τ = 40 fs which we attribute to the relatively slow unbending of D2O+58 facilitating second ionization and subsequent three-body dissociation). As this ratio changes, the momentum distributions within the D+/D+/O+ channel also change due to particular geometrical distortions: stretching of the OD bond lengths (rOD) reduces the momenta of all fragments, unbending of the DOD bond angle (θDOD) increases the angle between the two D+ momenta (β), and alignment sees the angle between the D-D axis and the laser polarization axis (θ) tend toward 0 and 180°.The fragment momenta captured following ionization with single pulses are strongly suggestive of ultrafast nuclear motion. These momenta were transformed to the molecular frame defined  pulses.Next we compare the single-pulse momentum distributions to the double-pulse momentum distributions at an interpulse delay of 18 fs, the delay at which 2O to the accurately computed potential energy surface of any one of the nine states of D2O2+ with two vacancies in the valence orbitals70. These nine states are detailed in Table t, before arrival of the second pulse and formation of the trication. Upon formation of the trication, the trajectories are continued under simple Coulomb repulsion of three singly-charged fragments, allowing the straightforward extraction of the asymptotic fragment momenta for each delay (see Methods for further detail on this procedure).Ionizing with pulse pairs and tracking the CEI observables as a function of interpulse delay offers a wealth of information on the intramolecular dynamics leading to enhanced trication formation. When paired with detailed ab initio calculations, these observables serve as an unambiguous probe of state-selective interatomic motion. In our theoretical treatment of the nuclear dynamics, the leading pulse in the pair promotes the Wigner phase space distribution of the ground vibrational state of neutral D2O2+ ultimately contribute to the formation of D2O3+. In Fig. +/D+/O+ coincidences over interpulse delay: the magnitude of the deuteron momentum (β). In Fig. Comparing the experimental observables to their simulated counterparts allows us to identify which nuclear dynamics in Dt. The first trajectory, labeled “slingshot”, is an example of symmetric breakup into three bodies. Here, both deuterons move symmetrically following double ionization and the molecule undergoes a rapid slingshot motion in which the bend-angle is inverted about the zm axis. As the molecule unbends and stretches, it briefly becomes linear at Δt = 20 fs before re-bending the other way. This kind of slingshot trajectory only occurs on three of the nine states of D2O2+, and it is most common in the relatively high-lying 2 1A1 state70 70.In order to emphasize the distinct features within Fig. β is centered at 180° after only 10 fs. Figure θDOD of only 146° and 158°, respectively. This discrepancy between β and θDOD represents a clear example of what is known as nonaxial recoil or a breakdown of the axial recoil approximation70. In the slingshot trajectory, the molecule becomes linear at Δt = 20 fs, pictured in Fig. β has already bent backwards, to 148°. The backwards bend also reverses the momentum of the oxygen ion, as seen in Fig. Atomic motion is revealed in detail by comparing the data to the two trajectories highlighted in Fig. β ~ 0° and then unbends after about 60 fs. The two-body trajectory in Fig. β = 0°. Likewise, in Fig. ℏ/a0 at Δt ~ 60 fs can also be attributed to this same motion in the two-body trajectory. Here, both deuterons are oriented to act together in repulsing the oxygen ion, giving it maximal momentum , the slingshot trajectory, for example, traverses through the geometry in which the molecule is briefly linear  involves forming at least one vacancy in the 3a1 molecular orbital while the 1b2 orbital remains doubly occupied .The inset of Fig. 2O2+. Models of EI in diatomic cations such as H64. In this picture, the presence of the downhill hydrogen suppresses the tunneling barrier for electrons localized on the uphill hydrogen. The critical geometry is determined by balancing two competing factors: smaller bond lengths cause greater barrier suppression but larger bond lengths trap electronic population more effectively on the uphill hydrogen. Here, we will invoke a similar tunneling picture to explain EI in D2O2+ while noting the ways in which this case differs from the prototypical case of HWe now attempt to model the enhancement mechanism that facilitates ionization of the third electron in D+/D+/O+ coincidences peaked at θ = 0 and 180°. Utilizing the pulse pair data, the distribution in θ can be plotted as a function of delay, generating Fig. t = 18 fs. The distribution in θ becomes increasingly uniform far from this delay. A tunneling picture of ionization suggests that, for a brief window of time around Δt = 18 fs, the barrier to ionize is suppressed along θ = 0°.The observed enhancement has a preferred polarization Fig. h. All tr2O3+ at the approximate critical geometry (rOD = 2.2Å and θDOD = 180°), subjecting it to our peak field strength (ε0 = 0.17 Eh/ea0), and making two 1-dimensional cuts in θ (θ = 0° and θ = 90°), yields Fig. 2O2+ ( ~ 40.7 eV) in Fig. θ dependence of the ionization barrier becomes apparent . When θ = 90°, an electron localized near the oxygen atom must tunnel through a substantial barrier to ionize; however, when θ = 0°, the barrier is nearly suppressed below the binding energy of the electron by the charge of the downhill deuteron, facilitating tunnelling (if not over-the-barrier ionization). This mechanism of barrier suppression is typical of EI phenomena previously observed in both diatomics64 and linear triatomics66. Because the molecule is linear and stretched symmetrically, this same process may also occur on the opposite side of the molecule during the next half-cycle of the field. The third ionization most likely creates a vacancy in the σ orbital of the linear molecule , pictured in Fig. rOD = 2.2 Å, but unlike the other two valence orbitals, has the largest value of electron density at the tunnelling barrier, and is doubly occupied in all three rapid slingshot states. The additional degeneracy introduced by unbending and symmetric stretching70 may also supplement the tunneling current due to field-assisted couplings between states.To construct our model of EI, we generated the molecular electrostatic potential (MEP) during the enhancement using the critical geometry discussed in the previous section. Modelling the MEP for the ground state of D64. Another contributing phenomenon is electronic collision with the internal barrier of the double-well potential. Here, electronic population becomes trapped at a particular atomic site as the internal barrier of the double-well potential grows in height with increasing internuclear distance. We have evidence to suggest that these two phenomena do not play such prominent roles during EI in D2O2+.A distinct feature of EI in H+/H and H/H+, two “charge resonant” states that each localize electron density preferentially on a particular atomic site72. By contrast, when symmetrically stretching D2O2+, none of the nine field-free electronic states considered here continue to distribute electron density appreciably on either deuterium past an OD bond distance of ~ 2 Å. As a result, the charge distribution of D2O2+ as a function of symmetric stretch in all 9 states becomes D+/D+/O and notably not D+/D/O+70. This behavior not only precludes charge resonance at large internuclear distances, but additionally suggests that enhanced ionization in D2O2+ will occur at much smaller internuclear distances than in HAs the ground electronic state of H2O2+ → D2O3+, the global minimum of the tunneling barrier occurs at rOD ~ 1.8 Å. This distance is smaller still than the critical OD bond distance recovered in Fig. rOD = 2.2 Å), suggesting that the critical geometries extracted from our analysis may be more the result of the particular trajectories launched by double ionization, rather than representative of the global optimum in bond length for EI. This disparity does not occur in diatomic molecules because motion is only along one dimension; it is a feature in polyatomics due to the increased degrees of freedom: constraints exist for EI in both bend angle and bond length.In the proposed model of EI for D63: one of the field-dressed states of the water dication, monocation, or both, could drive motion that unbends the molecule with less rapid stretching73. Evidence for propagation on field-dressed states is already visible in the alignment preference for longer pulse durations  pulses57.Figure ons Fig. h where t2O that induces EI within 20 fs. This motion is initiated by double ionization and creates, for a brief window in time, a linear DOD molecule with each OD bond distance stretched to about 2.2 Å. Within this window, a sufficiently strong and linearly polarized laser field oriented along the molecular axis can distort the molecular potential to facilitate ionization of a third electron and form D2O3+. Electrons localized near the oxygen atom see a suppressed tunneling barrier due to the charge of the downhill deuteron, and are thus more easily ionized. We see maximum EI when ionizing with 6 fs cross-polarized pulse pairs at pulse separations of 18 fs, but we also observe substantial EI when ionizing with single pulses of 19 fs duration or longer. In both cases EI appears to be associated with unbending and symmetric stretching of the molecular bonds, but the KER are different, suggesting additional field-dressed dynamics in the case of single-pulse EI.We have spatially and temporally resolved the rapid slingshot motion in D2O2+ represent a new understanding of EI in polyatomics that can drastically affect the interpretation of CEI experiments. The largest limitation of CEI is often ultrafast motion of light atomic species in the intermediate sequence of charge states that are formed en route to Coulomb explosion. The distorting effects of EI are particularly egregious examples of this limitation. Understanding this motion is therefore required before CEI can be applied to more complex molecular systems.Our measurements of the rapid intramolecular dynamics that produce EI in D21. Control of EI in polyatomic molecules could therefore extend the applicability of laser-induced CEI, and may spark a renewed interest in its use to film ultrafast motion within polyatomic molecules.This study shows not only how this motion can be measured in polyatomic molecules, but how the phenomenon of EI can be employed to do so. We demonstrated how EI can act as a structural filter to highlight a single kind of interatomic motion in water. It may be possible to tailor this EI process and select a subset of dynamics to highlight by changing the shape, intensity, or polarization of the ionizing laser field. This capability may have already been seen in recent experiments examining an elusive roaming reaction in formaldehydeτ = 6, 10, 19, and 40 fs) and constant peak intensity (I0 = 2 × 1015 W/cm2) were used to triply ionize neutral D2O. Here, τ is a measure of the full width at half maximum (FWHM) of the temporal intensity profile for each pulse. To create the 6 fs pulses, a 40 fs 800 nm 1 kHz Ti:sapphire laser pulse was spectrally broadened and chirped in a 1 m neon-filled hollow-core fiber (at 45 psi Ne) with a 250 μm diameter. The pulse  was then recompressed using a series of bounces from two chirped mirror blocks. The 10 fs pulses were created similarly but using a lower gas pressure (35 psi Ne) to elicit less spectral broadening. The 19 fs pulses were created by positively chirping the 10 fs pulses using two fused silica wedges. Negatively chirping the 10 fs pulse to a duration of 19 fs yielded similar results. The 40 fs pulse was supplied directly by the output of a Ti:sapphire laser oscillator and amplifier. All peak intensities were kept constant by altering the energy per pulse using a series of neutral density pellicle filters with negligible dispersive effects. For the 6 and 10 fs pulses, temporal characterization was performed via the dispersion scan method utilizing the aforementioned fused silica wedges to apply variable dispersion74. For the longer pulse durations (19 and 40 fs), temporal characterization was performed via intensity autocorrelation75.In the first experimental scheme, single pulses of variable duration (I0 = 1 × 1015 W/cm2) were used to triply ionize D2O. These pulses were generated by first creating a single 6-fs pulse (as described in the section above) before splitting this pulse using a Mach-Zehnder interferometer. The interferometer splits the pulse into two pulses of equal intensity and variable delay. Each arm contained an additional polarizer at ± 45° to create a cross-polarized pair at the output directed along a common beam path. Pulse characterization was performed via dispersion scan utilizing two BK7 wedges to apply variable amounts of dispersion74. The interpulse delay was extracted with high precision from the spectral interference between the two beams in the unused output port of the interferometer.In the second experimental scheme, a pair of 6 fs 750 nm 1 kHz pulses with equal peak intensity , the laser was ultimately directed into a vacuum chamber with a pressure of 6 × 10xm, ym and zm. Here, + momenta, + momenta, and zm and − zm. Because the bisector of the two deuteron momenta is always defined as + zm, there is nothing distinguishing a molecule that has been inverted about zm from one that has not. This inversion is only apparent after considering the evolution of certain time-resolved observables with pulse-pair separation. We see β reach 180° before returning to more acute angles, indicating backward bending. We also see the magnitude of the oxygen-ion momentum  were then easily distinguishable by the magnitudes of the deuteron momenta.By focusing our analysis primarily on D2O following double ionization, we simulated the motion of a nuclear wavepacket propagating on the dication potential energy surfaces semi-classically in the same way as was done in references69 and70, by using classical trajectories whose initial conditions are given by the Wigner phase space distribution of the initial vibrational state. The Wigner distribution in the harmonic approximation used here isQj and Pj are the coordinates and momenta respectively of the three normal modes, and ωj are the associated frequencies. The normal modes were calculated in a complete active space self-consistent field (CASSCF) calculation with the same active space used in the calculations of the dication potential surfaces described below. This semiclassical phase space distribution was propagated by sampling it to initiate a set of 2048 classical trajectories on each of the nine states of D2O2+. The vertical transition energies, symmetries, orbital vacancies in the dominant configuration at the equilibrium geometry, and branching ratios for all of these states are displayed in Table To model the interatomic dynamics of Dmentclass2pt{minim69 and the ninth, the 31A1 state, was calculated by Streeter et al.70. Briefly, the potentials were produced by internally contracted multi-reference configuration interaction (icMRCI) calculations at the configuration interaction singles and doubles (CISD) level including the Davidson correction for quadruple excitations. The calculations, which were performed on extensive grids of geometries, employed the cc-pVTZ Dunning correlation consistent basis76 and were based on a CASSCF reference space. For example in the case of the 31A1 state, the active space used orbitals from calculations on the lowest 3B1 state in Cs symmetry with one a78 were fit to a linear combination of 100 basis functions that represent the Coulomb and polarization interactions at intermediate and long interatomic distances together with screened Coulomb and multipole interactions at short distances as described in reference69.Eight of the three-dimensional potential energy surfaces used for this simulation were calculated by Gervais et al.2O and D2O70. This treatment of the nuclear dynamics closely reproduces experimental final momentum distributions for the three-body breakup channels as well as internal energy distributions in the two-body channels in those studies. Those benchmark comparisons underlie our confidence in the present investigation in using these detailed dynamics to interpret the experimental trajectories as described in this text.The reliability of these potential surfaces and the Wigner phase space propagation on them for dissociative dynamics has been established in detailed comparisons with experimental momentum imaging experiments on one-photon double photoionization of H2O2+ for a time commensurate with the interpulse delay, Δt, the formation of the D2O3+ and the resulting Coulomb explosion were modelled by an instantaneous transition (preserving the positions and momenta of the particles on the dication states) to a purely repulsive potential, V. This potential simply represents mutual Coulomb repulsion between three single charges:qi is the net charge on each fragment (qi = 1 in atomic units), i = 1, 2, 3 correspond to D+ respectively. The inherent timing ambiguity due to the finite width of the two pulses in the pair (τ = 6 fs) was accounted for by “blurring” the dication dynamics by ± 3 fs: that is, randomly shifting the timing of all 2048 trajectories forward and back by an amount between 0 and 3 fs and averaging over the results.After propagating on a given state of D2O3+ displayed in Fig. 79 with a 6-31G Gaussian basis set. A DC electric field of strength ε0 = 0.17 Eh/ea0 (I0 = 1 × 1015 W/cm2) was then applied to this MEP along the ym and zm axes in order to yield the tunneling pictures in Fig. θ = 0° and 90° respectively.The molecular electrostatic potential (MEP) for the ground quartet state of Dψ(r)∣2 of the field-free (ε0 = 0) 1b2 molecular orbital for the triplet ground state of D2O2+ displayed in Fig. The probability distribution ∣3Σ−) of H2O2+ and that of H2O3+ (4Σ−) under the influence of an applied field of ε0 = 0.17 Eh/ea0. In these calculations, performed with the Psi480 suite of codes, the Gaussian basis was again the cc-pVTZ Dunning correlation consistent basis76, and the OD distance was either 2 or 2.5 Å. Results were interpolated to yield the IP at 2.2 Å. The two tightest a1 orbitals were doubly occupied in these calculations and the active space for calculations on both systems consisted of 2 a1 2 b1 and 2 b2 orbitals. Correlated calculations like this give ionization energies in the presence of an electric field several eV larger than ROHF descriptions. The difference evidently arises because CASSCF calculations can allow orbitals in correlating configurations to occupy the vicinity of the down-field deuteron, while single configuration-calculations spread the highest occupied orbital over both the oxygen and deuteron. As noted in the main text, these estimates of the ionization energy, and therefore the energy in a one-dimensional model at which tunneling or over-barrier ionization takes place, are very approximate. The calculations are complicated in particular by the difference in the electron correlation energies of the two systems, because the trication is dramatically less correlated than the dication. In addition, electronic states in intense fields have finite lifetimes and therefore energy widths, which are neglected in these calculations. Even if these computational estimates of the ionization energy and barrier height are off by one or two eV, the proposed mechanism for EI remains reasonable.The ionization potential (IP) plotted in Fig. Peer review fileSupplemental Information (PDF)


The existenceof moiré magnons is further confirmed via inelastic quasiparticleinterference, showing the appearance of a dispersion pattern correlatedwith the moiré length scale. Our results provide a direct visualizationin real-space of the dispersion of moiré magnons, demonstratingthe versatility of moiré patterns in creating emergent many-bodyexcitations.Two-dimensional magnetic materials provide an ideal platformtoexplore collective many-body excitations associated with spin fluctuations.In particular, it should be feasible to explore, manipulate, and ultimatelydesign magnonic excitations in two-dimensional van der Waals magnetsin a controllable way. Here we demonstrate the emergence of moirémagnon excitations, stemming from the interplay of spin-excitationsin monolayer CrBr Using the twist degree of freedomhas emerged as a powerful strategy to design new quantum states.23 Paradigmatic examples are the emergent correlated and topologicalstates in graphene moiré multilayers,25 ferroelectricity in hexagonal boron nitride moiré bilayers,26 moiré excitons in twisted MoSe2/WSe2,27 and moiré magnetismin CrI3 moiré bilayers.28 The emergence of moiré phenomena in magnetic van der Waalsmaterials is a newly explored field, and in particular, the possibilityof creating moiré magnon excitations remains an open problemin twistronics.The recent discovery of two-dimensionalvan der Waals (vdW) monolayer magnetic materials has opened new avenuesfor scalable, defect-free samples for spintronic applications andartificial designer materials.3, X = Cl,Br, and I, 29 with all three showing ferromagnetic order, where the easyaxis is out-of-plane for CrBr37 andCrI3,2 and in-plane for CrCl3.30 We have carried out low-temperaturescanning tunneling microscopy (STM) and spectroscopy (STS) to probethe magnon excitations in monolayer CrBr3. We show thatthe results can be understood in terms of moiré magnons arisingfrom a reconstruction of the magnon dispersion by the moirépattern formed by the lattice mismatch between CrBr3 andthe substrate. This leads to new van Hove singularities in the magnonspectral function that are correlated with the moiré lengthscale. Furthermore, by employing quasiparticle interference with inelasticspectroscopy, we directly probe the magnon dispersion in reciprocalspace, allowing us to map the moiré magnon spectra. Our resultsdemonstrate the emergence of moiré magnons and the impact ofmoiré patterns on the magnetic excitations of 2D materials.Chromium trihalides (CrX3 monolayerson a highly oriented pyrolytic graphite (HOPG) substrate at a T = 350 mK (see Supporting Information (SI) for more experimental details). Typical STM topographyimage  and they should result in bias-symmetric steps in the dI/dV signal.33 We observe clear inelastic excitations experimentally as demonstratedin I/dV (symmetrized)and numerically differentiated and smoothened d2I/dV2 signals (see SI for details). As schematically illustratedin I/dV to correspond to the integrated magnon density of states(DOS) while the d2I/dV2 signal directly corresponds to the local magnon spectralfunction. It is immediately obvious that our experimental d2I/dV2 contains manymore peaks than expected for a typical magnon spectrum. We explainthis discrepancy below as arising from the moiré-induced modificationof the magnon spectrum.We have carried out experiments on CrBrhyimage 1b shows35 describing the spin excitations in a magnetic two-dimensional system(see SI for details)Jij the spatially modulated isotropic exchange coupling, Kij the anisotropic exchange,and Snα the local S =3/2 operators in the Cr atoms forming a honeycomb lattice  over arange of CrBr3 islands with different moiré periodicitiesallowing us to address the impact of the underlying moiré patternon the magnetic excitations. 2I/dV2 (details in the SI) shows strong peaks that are spatially quite uniform as shown in 46 Therefore, we focuson the lower energy features and comparing SI for the statistics of inelastic peak energies).We have carried out inelastic tunneling spectroscopy experiments; however, the HOPG substrate shows very clear andstrong signatures of Landau levels at high magnetic fields that completelyoverwhelm the signal from the magnetic excitations in the CrBr3 layer. It is also not possible to subtract the signal fromthe Landau levels as the Landau level spectra of bare HOPG and HOPGcovered by CrBr3 are different arising from the sensitivityof the Landau levels to the local potential.48 At low-magnetic fields (<0.5 T), the signal due to the magneticexcitations is clearly visible, but the shifts due to the Zeeman energyare too small to be reliably detected.While our experiments are consistent with the expectationthatthe observed inelastic features correspond to magnetic excitations,they could also correspond to inelastic excitations of phonons. However,earlier experiments on tunneling devices have shown that the modeswith sufficient electron–phonon coupling are at higher energies(above 25 meV)2.49 The differential conductancedI/dV is proportional to the totalnumber of magnons that can be excited with that energy. In the presenceof weak scattering, the total number of magnons will be spatiallymodulated. The dispersion of the moiré magnons can be directlyprobed by visualizing the Fourier transform of the spatially resolveddI/dV, shown in I/dV, in the following denoted as Ξ stemsfrom inelastic magnon tunneling processes as Ξ ∼ ∫AAd2k, where A is the magnon spectral function. As a result, the magnon QPI reflectsa self-convolution of the magnon dispersion, directly reflecting magnonreconstructions in reciprocal space.The presence of moirémagnons can be demonstrated even moreconvincingly through inelastic quasiparticle interference spectroscopythat allows a direct visualization of the length scale of the moirémagnons. We note that this technique has been used to demonstratethe emergence of quantum spin liquid signatures in monolayer 1T-TaSeI/dV maps at various energies(parameters mentioned in the SI). The typicalFFT of the dI/dV maps has strongpeaks at characteristic reciprocal space points, indicating differenttopographic periodicities present. The green, magenta, red, and yellowdotted circles in K- points have features around them.To explore the dispersion of the magnonic bands,we performed constantcurrent dk) ∼ |k|2 as shown in Theoretically,in the absence in the moiré pattern, themagnon spectral function at energies below 8 meV should feature asimple circular shape coming from the magnon dispersion ϵ(3.These theoretical moiré QPI predictions canbe directlycompared with our experimental data. In order to factor out the impactof the topographic moiré modulation in the QPI, we first removethe peaks associated with the moiré length, whose origin ispurely structural. Around the Γ-point, after removing the intensitydue to the moiré, we see an internal interference pattern stronglydependent on the energy and ultimately vanishing above ∼25mV 3d. Itmu3 is an insulator and has no electronicstates close to the Fermi level. We could of course still in principleobserve QPI from the electronic states of the HOPG substrate; however,in that case one would expect QPI signal over a large bias range sinceHOPG has states at all energies. This is in contrast to our experimentalresults and, hence, the QPI features most likely correspond to themagnon excitations.While this kind of QPI featurescould also arise from elastic scatteringbetween electronic states, it is very unlikely in the present case.First of all, CrBrTo summarize, we have demonstrated the emergenceof moirémagnon excitations in 2D monolayer ferromagnet. By using inelasticspectroscopy, we showed that the existence of moiré patternswith different moiré lengths leads to different reconstructionsof the moiré spectra. The existence of moiré magnonsis further confirmed via inelastic quasiparticle interference, showingthe appearance of a dispersion pattern correlated with the moirélength scale. Our results provide a direct visualization in real spaceof the dispersion of moiré magnons, demonstrating the versatilityof moiré patterns in creating emergent many-body excitations.


The precise molecular events initiating human lung disease are often poorly characterized. Investigating prenatal events that may underlie lung disease in later life is challenging in man, but insights from the well-characterized sheep model of lung development are valuable. Here, we determine the transcriptomic signature of lung development in wild-type sheep (WT) and use a sheep model of cystic fibrosis (CF) to characterize disease associated changes in gene expression through the pseudoglandular, canalicular, saccular, and alveolar stages of lung growth and differentiation. Using gene ontology process enrichment analysis of differentially expressed genes at each developmental time point, we define changes in biological processes (BP) in proximal and distal lung from WT or CF animals. We also compare divergent BP in WT and CF animals at each time point. Next, we establish the developmental profile of key genes encoding components of ion transport and innate immunity that are pivotal in CF lung disease and validate transcriptomic data by RT-qPCR. Consistent with the known pro-inflammatory phenotype of the CF lung after birth, we observe upregulation of inflammatory response processes in the CF sheep distal lung during the saccular stage of prenatal development. These data suggest early commencement of therapeutic regimens may be beneficial.The online version contains supplementary material available at 10.1007/s10142-023-01050-y. FGF10) gene, are a susceptibility factor for chronic obstructive pulmonary disease   were used in this study. All animal studies were approved and monitored by the Institutional Animal Care and Use Committee (IACUC) at Utah State University (IACUC protocol # 10089) and conformed to the National Institute of Health guidelines. WT sheep were bred according to standard protocols. Briefly, ewes were synchronized at estrus using an intramuscular (IM) injection of 2 mL EstruMate containing 250 μg/ml of cloprostenol, a synthetic analogue of Prostaglandin F2α  and introduced to the ram on the same day. Ultrasonography around 40 days confirmed pregnancy status and day 1 of gestation was defined as ~ 48 h post injection. CFTR−/− sheep were generated by somatic cell nuclear transfer (SCNT) using genetically modified sheep fetal fibroblast (SFFs)  derived from American Romney fetuses as described previously  in a dry ice-cooled MP Biomedicals FastPrep-24 Classic homogenizer using Lysing Matrix D tubes  for 1 or 2 × 20 s cycles at 4.0 m/s. RNA was isolated using the TRIzol manufacturer’s protocol. RNA quality was assessed by NanoDrop, formaldehyde gel electrophoresis, and Tapestation prior to choosing high quality samples for RNA-seq.https://github.com/alexdobin/STAR)   and an adjusted p-value ≤ 0.001 . Data are deposited at GEO:GSE202019.RNA samples passing strict QC criteria were used as templates for cDNA library synthesis with oligo dT priming, and amplified libraries were pooled and sequenced (100 bp paired ends) on a NovaSeq 6000 machine. Raw sequence reads were aligned to the Oar_v4.0 (oviAri4) genome of the Texel sheep breed with STAR version 2.7.0e_0320    and glucose 6 phosphate dehydrogenase (G6PD)  with random hexamers, or oligo dT for CFTR−/− animals did not show gross histological changes from WT animals through gestation  at 50-, 65-, 80-, 100-, and 120-day animals during gestation and 147-day (term)  Fig. B.Principal component analysis (PCA) of RNA-seq data is shown in Fig. p-value filter of ≤ 0.001 and log2fold change of ± 0.58 (± 1.5 fold change). We compared 65 days to 80 days, 80 days to 100 days, 100 days to 120 days, and 120 days to term. Proximal and distal lung samples were evaluated separately. For each sample set gene ontology process enrichment analysis was performed on the DEGs to identify upregulated and downregulated processes across gestation.First, we conducted pairwise comparisons of differentially expressed genes (DEGs) between each time point of gestation in WT sheep lungs Table . DEGs weAt the earlier stages of development before 100 days, few gene ontology terms were annotated between time points. Comparing 100 and 80 days, upregulation of multiple genes involved in response to external biological stimuli are annotated in gProfiler Fig. A. Comparp-value   Table . Again, SLC1A1, the upregulated solute carrier genes are different in WT and CF animals. Contrasting 120- to 100-day gestation, gene ontology molecular functions annotated from upregulated genes were divergent in CFTR−/− animals from those seen in WT animals. In the CFTR−/− animals, oxidoreductase and aldehyde oxidase molecular functions were identified, albeit driven by very few genes . Also of interest was the upregulation of genes involved in the innate immune response and anti-inflammatory response, which are included in the cellular compartment defined as extracellular space. These genes include surfactant proteins , secretoglobins (SCGB1A1), and the PLUNC/BPI fold containing family B member 1 (BPIFB1). However, prior to 120 days, no significant gene ontology biological processes are called, only molecular functions. Lastly, comparing term to 120 days in CFTR−/− animals, the most upregulated gene (log2fold change > 9) is the macrophage receptor with collagenous structure (MARCO), a key component in innate antimicrobial immunity. However, there are many fewer DEGs between these two time points in the CF animals (~ 150) compared to WT (~ 1000). Regulation of immune system is called as a significant process by gProfiler when comparing these timepoints  and an adjusted p-value ≤ 0.001 and so gene ontology analysis was not fruitful. However, robust upregulation of both SFTPB and SFTPD was of significant interest (Table CXCL8 (encoding the chemokine IL-8), kininogen (KNG1), chitinase 3-like 1 (CHI3L1), complement C4A (LOC101123672), and WAP four-disulphide core domain-1 (WFDC1). Of note, WFDC2 is also robustly upregulated (Table WFDCs)  Fig. A. These Cs) Fig. B.CFTR−/− animals to WT animals at each time point  and myocardin (MYOCD) both of which regulate aspects of smooth muscle cell replication, differentiation, and migration, in addition to other functions. At 80 days, higher expression in CFTR−/− lungs than WT of scavenger receptor cysteine-rich family member with 5 domains (SSC5D), extracellular matrix protein 1 (ECM1), and integrin subunit alpha 7 (ITGA7) predicts extracellular matrix (ECM) binding as a molecular function in gProfiler. SSC5D is active in the ECM where it has multiple functions in laminin and fibronectin binding in addition to scavenger receptor activity  was among the most differentially upregulated genes in CFTR−/− compared to WT proximal lungs at 120 days. At term, the ~ 400 DEGs between CFTR−/− and WT predict multiple highly significant biological processes, with anatomical structure development/ morphogenesis having the highest p-values, but response to hypoxia and related processes also reaching statistical significance. Among upregulated genes driving the hypoxia signature are Egl-9 family hypoxia inducible factor 3 (EGLN3), a cellular oxygen sensor that hydroxylates proline residues in hypoxia inducible factor 1 subunit alpha (HIF1A) and other targets  changes in expression of a few genes including potassium voltage-gated channel subfamily members (KCNH2 and KCND3). In contrast by 100 days gestation, the ~ 230 DEGs between CFTR−/− and WT animals resulted in annotation of ion transport processes by gProfiler based on the significantly upregulated genes in the CFTR−/− animals. Multiple genes contributing to these processes include the voltage-gated calcium channel cation channel sperm associated 4 (CATSPER4), anoctamin 9 (ANO9) a member of the calcium-activated chloride channel which may not transport chloride, the ATP binding cassette transporter family members ABCC5 and ABCC10, and polycystin 1 (PKD1). At 120-day gestation, the ~ 210 DEGs did not identify any robust signatures for biological processes between the genotypes. As for the proximal lung, at term, the ~ 640 DEGs predict anatomical structure development, angiogenesis, and response to hypoxia among the significantly upregulated genes annotated by gProfiler and downregulated genes are associated with cell cycle-related processes in the CFTR−/− animals compared to WT. Many genes contribute to all these processes and indicate a term lung that is still undergoing development but shows reduced cell division in the term CFTR−/− animals.The ~ 250 DEGs between CFTR−/− and WT animals and then validated by RT-qPCR for both proximal and distal lungs ,  , Fig. A–D, the O1) Fig. E, F, anderm Fig. A–D, conserm Fig. A, B. At als Fig. C, D. ANOals Fig. E, F. In ung Fig. , consistung Fig. .Fig. 4DeWFDC2), the lung epithelial cell protease inhibitor that has a predicted role in lung surface liquid and innate immunity  .Also, highly relevant to lung functions are the components of the innate immune response, which are challenging to study in the fetus prior to birth  . For comparison in these studies, we assayed the expression of a lung epithelial integrity protein , which as expected did not alter significantly through gestation in proximal or distal lung irrespective of genotype  Fig. C–F. SFTPls Table . In the CFTR), C3, and CXCL8 (encoding IL-8) at 120 days are marked in Fig. KNG1, CHI3L1, and C4A transcripts  and the detailed information on the differentiation and migration of individual cell types that it provides, it can still yield important insights into organ development and disease-associated deficits.Human lung development is separated into 5 overlapping stages, largely based upon embryological transitions (reviewed in Burri  Nikolic), and byCFTR−/− animals. For WT proximal lung, our data show that upregulated genes are significantly involved in biological processes relating to response to external stimuli, both chemical and biological between 80 and 100 days, extracellular matrix organization and preparation for gas transport between 100 and 120 days, and most notably, the innate and adaptive immune responses at term, which is accompanied by marked downregulation of genes involved in developmental processes. In WT distal lung, upregulation of genes involved in cation homeostasis and blood vessel development is seen between 80 and 100 days, with the latter processes also annotated for genes upregulated between 100 and 120 days. The changes in biological processes between 120 days and term are the same as observed in proximal lung. For CFTR−/− proximal lung, when using the stringent filters for statistical significance applied here few biological processes were identified until term when immune system processes were annotated from genes upregulated compared to 120 days of gestation. However, in the CFTR−/− distal lung, gProfiler annotates wound healing, coagulation, and defense response from genes upregulated between 100 and 120 days and using the same gene list DAVID identifies the inflammatory response and neutrophil activation over this time period. As in WT lungs, there is a marked upregulation of genes involved in both innate and adaptive immune system processes between 120 days and term in the CFTR−/− distal lung, which is accompanied by downregulation of cell cycle-associated genes rather than the developmental processes seen in WT. The mechanisms underlying this difference in downregulated genes at term in the CFTR−/− and WT lung are unclear. Although it is possible that earlier changes in the CFTR−/− lung have a differential effect on lung development, for example through inflammation and associated tissue remodeling  ewes and rams will enable us to address these processes in detail.There are two major aspects of interest in the work presented here, the first being the detailed transcriptomic analysis of WT lung development, which has not been done previously in a large animal, and the second is the differences observed in the CFTR−/− animals when compared to WT, particularly in the proximal lung, is somewhat surprising since we did not detect any consistent abnormalities in lung structure or development through gestation by histological analyses . Another factor affecting the transcriptome of the term lungs that warrants consideration is the precise time of euthanasia and tissue collection after birth. As part of the alveolar stage of development, the lung epithelium undergoes a period of rapid change immediately after birth. Pathways of ion transport become modified to facilitate respiration in air and the alveolar surface is coated with the necessary balance of surfactant proteins and antimicrobial agents. Indeed, we confirmed the dramatic upregulation of transcripts encoding these compounds between 100-day gestation and term in both regions of the lung and in WT and CFTR−/− animals  are members of the glycoside hydrolase family 18 proteins. Their role in innate immune pathways and tissue injury/repair are well characterized (reviewed in (Sutherland KNG1) may have several functions in inflammation since it encodes two different proteins by alternative splicing. One of these, high molecular weight kininogen (HMWK) is required for blood coagulation, but bradykinin, which is released from it also has antimicrobial peptide properties and is associated with lung inflammation  from which gProfiler and DAVID retrieve data. For example, C3, the most differentially upregulated gene in the 120 day CFTR−/− distal lung  among the most significantly upregulated genes between 100 and 120 days only in the CFTR−/− distal lung warrants further discussion. In fact, C3 is also upregulated in the CFTR−/− proximal lung at this time point, although not in WT proximal or distal lung. Complement has a key role in the innate immune response as its activation causes local inflammation, the clearing and direct killing of pathogens and the initiation of the adaptive immune response Supplementary file2 (XLSX 8206 KB)Supplementary file3 (XLSX 8754 KB)Supplementary file4 (XLSX 355 KB)Supplementary file5 (XLSX 10 KB)Below is the link to the electronic supplementary material.


P-value of 0⋅05. The magnitude of minimum acceptable diet in the study area was 16⋅7 % . Sex of child, getting infant and young child feeding counselling at antenatal care, infant feeding practice-related knowledge and childhood illness are the variables that were found to be an independent predictor of minimum acceptable diet. Health facilities should strengthen infant feeding counselling starting from antenatal care visits during pregnancy for the recommended minimum acceptable diet is crucial.The first 2 years of life are a critical window of opportunity for ensuring optimal child growth and development. In Ethiopia, the magnitude of the minimum acceptable diet ranges from 7 to 74⋅6 %. The evidence revealed the variation and unrelated data on the prevalence of minimum acceptable diet. Therefore, the present study aimed to assess the minimum acceptable diet and its associated factors among children aged 6–23 months in Lalibela town administration, northeast Ethiopia. A community-based cross-sectional study was conducted in Lalibela town administration, northeast Ethiopia among 387 mothers/caregivers with children aged 6–23 months from May 1 to 30, 2022. The data were entered by Epidata version 3.1 and analysed by SPSS version 25.0. A multivariable binary logistic regression model was fitted to identify factors associated with minimum acceptable diet. The degrees of association were assessed using an adjusted odds ratio with a 95 % confidence interval and  Infants and young children (IYC) are vulnerable to malnutrition because of their high nutritional requirements for growth and development and they are particularly vulnerable during the transition period when complementary feeding begins, at 6 months. Despite some improvements in selected nutrition indicators, progress is insufficient to meet the 2025 global nutrition targets.Minimum acceptable diet (MAD) is the proportion of children aged 6–23 months who had at least minimum meal frequency and minimum diversified diet during the previous day.Adequate nutrition during infancy and early childhood is fundamental to the development of each child's full human potential. It is well recognised that the period from birth to 2 years of age is a ‘critical window’ for the promotion of optimal growth, health and behavioural development(4). In African countries, Ghana, Uganda and Kenya in which 29⋅9, 23⋅9 and 48⋅5 % of children received recommended MAD, respectively–8 and Malawi (8⋅36 %), Nigeria (7⋅3 %) and Philippines (6⋅7 %) of children aged 6–23 months received the recommended MAD–13. In Ethiopia, the magnitude of MAD ranges from 7 to 74 %,11,14–16.Globally, the 2020 global nutrition report: ‘action on equity to end malnutrition’ revealed that 18⋅9 % of children aged 6–23 months received a MAD. Globally, ensuring optimal complementary feeding can avert a substantial proportion of childhood deaths. An undernourished child has a nine times higher risk of mortality as compared with an optimally nourished child. Almost half (45 %) of all child death resulted from the effect of malnutrition, and the highest figure was in Africa.More than two-thirds of malnutrition-related child deaths are associated with inappropriate feeding practices during the first 2 years of life–28.Previous studies conducted elsewhere on factors associated with inappropriate complementary feeding practices of children aged 6–23 months show higher maternal and paternal education, better household wealth, exposure to media, adequate antenatal and post-natal contacts, child's sex and age, institutional delivery, low parity, maternal occupation, urban residence, knowledge and frequency of complementary feeding and receiving feeding advice in immunisation as determinant factors for appropriate complementary feeding, Health Sector Transformation Plan (HSTP), Health Extension Program (HEP), Sustainable Undernutrition Reduction in Ethiopia (SURE) and Seqota declaration. Despite efforts done by the Ethiopian Government and other stakeholders, only 7 % of children aged 6–23 months have met the MAD.The Ethiopian government also set targets to improve the nutritional status of children and to end child malnutrition by 2030 through implementing different programmes and strategies such as the National Nutrition Program (NNP),14–16. Moreover, as far as the researcher's knowledge is concerned, no documented data were accessible specifically in the study area. Therefore, the present study aimed to assess MAD practice and its associated factors among children aged 6–23 months in Lalibela, northeast Ethiopia.Even though studies were conducted about the determinants of the optimal complementary feeding practices in Ethiopia, inadequate efficient information was documented about MAD practice and its associated factors independently and most of the studies were not representative especially for rural communitiesA community-based cross-sectional study was conducted in Lalibela town administration, northeast Ethiopia from May 1 to 30, 2022. Lalibela is found in the North Wollo zone 300 km away from Bahir Dar town which is the capital city of the region and 701 km from Addis Ababa. The town has a total population size of 42 975 people of which 21 057 are males, 21 918 are females and about 1528 estimated numbers of children 6–23 months age. There are 5 Kebeles in the town based on the Lalibela town health office 2021 report. The town has one general hospital, one health centre and six health posts..Lalibela and its surrounding called Lasta remains in use by the Ethiopian Orthodox Christian Church to this day, and it remains an important place of pilgrimage for Ethiopian Orthodox worshipers, as a home to clergy, which increasingly brings together religious adherents. In the study area, there is household's food insecurity and their coping strategies were temporal and might not systematically link to the ever-increasing climate change and other related hazards, which were more vulnerable to food insecurity. And also, up to today, it is one site of long-term development interventions through the Government of Ethiopia-led Productive Safety Net Program (PSNP), which aims to reduce chronic food insecurityAll mothers/caregivers with children aged 6–23 months in Lalibela town administration were the source population. Mothers/caregivers with children aged 6–23 months who lived in the randomly selected Kebeles were the study population. Individual mothers/caregivers having children aged 6–23 months lived in the randomly selected Kebeles and participated in the actual data collection interviewee was the study unit. Mothers/caregivers with children aged 6–23 months on therapeutic feeding were excluded from the study., a confidence level of 95 % 1⋅96, and a 0⋅05 margin of error the sample size became 352. By adding a 10 % non-response rate, finally became 387.The sample size was calculated for the first objective using single population proportion formula; by considering the proportion of MAD in Mareka District, southern Ethiopia 35⋅5 % of the children aged 6–23 months met the recommended MADThere are 5 Kebeles in the Lalibela city administration, out of them 2 Kebeles (with estimated 720 study participants) were randomly selected by a lottery method. To give equal chance in the selection of mother–child pairs, a proportional allocation technique was employed across each selected Kebeles. Finally, systematic sampling techniques were applied every other lactating mother to select the study participants. If there has been more than one mother–child pair in one household unit, one mother with the youngest children is selected. In the case of twin newborns, the lottery method was used to select the study participants.Dependent variables: Minimum acceptable diet.Independent variables: Socio-demographic and economic characteristics, obstetrics and health service utilisation and maternal knowledge.Complementary feeding is described as the introduction of safe and nutritionally balanced solid, semi-solid or soft foods in addition to breast milk for children aged 6–23 months.Minimum acceptable diet: Proportion of children aged 6–23 months who had at least minimum meal frequency and minimum diversified diet during the previous day,2.Minimum meal frequency: Proportion of breast-feeding and non-breast-feeding children aged 6–23 months who receive soft, solid and semi-solid foods  in the last 24 h. These food groups used for this indicator are breast milk, grains, roots and tubers; legumes and nuts; dairy products ; Flesh foods ; eggs; vitamin A-rich fruits and vegetables; and other fruits and vegetables. Quality and quantity of any amount from those groups were considered sufficient to count. Breast-feed infants aged 6–8 months two times in the last 24 h; and breast-feed infants and young children aged 9–23 months three times in the last 24 h. For non-breast-feeding infants and young children aged 6–23 months at least four times in the last 24 h,36.Minimum dietary diversity: Proportion of children aged 6–23 months who receive five or more food groups out of the eight food groups in the last 24 h.Maternal knowledge on IYCF practice: Mothers who score above the mean of ten knowledge questions related to infant and child feeding practice were categorised as knowledgeable and those who score below the mean were categorised as not knowledgeable.,37,38–41.Data were collected by structured questionnaires using a face-to-face interview which consist the socio-demographic characteristics of mothers, history of antenatal care (ANC) visit and other variables that use to assess child-feeding practice. The questionnaire was developed by adapting from different literatureThe data collectors were two nurses and two midwives who trained for 2 days regarding the purpose of the study and the procedures to be followed for data collection.The structured questionnaire was translated and prepared in Amharic and back to English by other persons to check its consistency. Two days of rigorous and extensive training were given for data collectors and supervisors on methods of obtaining consent, study objectives, contents of the questionnaire and interviewing technique prior to pre-test. A pre-test was conducted on 5 % of the total sample size on lactating women living in an area other than the study site. Overall, data collection was monitored daily and the questionnaire was checked for completeness and consistency at the end of the data collection date.Data consistency and completeness were checked throughout the data collection. Data were entered using Epidata version 3.1 and exported to SPSS version 25.0 for analysis. Descriptive statistics was utilised to summarise data on respondent characteristics and presented in narrative and graphs, charts and tables. For numerical variables such as age, number of children, number of ANC mean and standard deviation were calculated for normally distributed data.P < 0⋅05 as significant. Sampling adequacy for principal component analysis was checked with Kaiser-Meyer-Olkin (KMO) and the measurement accepted if it was >0⋅5. Varimax rotation is employed during factor extraction to minimise cross-loading of items on many factors.Principal component analysis (PCA) was conducted to identify variables that explained high variability among household wealth response ranked into tertile. Bartilett's test of sphericity was checked and taken P-value <0⋅25 in the bivariable analysis were selected as a candidate for multivariable logistic regression. The multivariable binary logistic regression model was fitted to identify factors affecting the MAD. Adjusted odds ratio with 95 % confidence interval and P-value less than 0⋅05 were considered statistically significant.Binary logistic regression analysis was done to check the association between dependent and independent variables. All variables that had a significant association with P-value 0⋅196 which is greater than 0⋅05 considered as the model good fit to the data. And also, multicollinearity was checked by using a variance inflation factor: a maximum variance inflation factor (VIF) of 1⋅43 which was less than ten considered as there were no threat of multicollinearity.The results of multivariable logistic regression with the backward method after checking of model fitness test by the Hosmer and Lemeshow test were tested and This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects/patients were approved by the ethical committee of the Zemen Postgraduate College, Department of Public Health with a reference number of ZPC/000703/2014. Written permission letter was also obtained from the Lalibela town administrative and health office. The participants enrolled in the study were informed about the study objectives, expected outcomes, benefits and the risks associated with it. Finally, verbal and written informed consent was taken and formally recorded from the participants before the interview. Furthermore, the confidentiality of participant's information was assured and information recorded anonymously. Those who are practicing inappropriate complementary feeding were advised to correct their complementary feeding practice.sd) months and 181 (50⋅4 %) were females. More than half (54⋅3 %) of the mothers were aged between 25–34 years old and more than three-quarters (85⋅5 %) were married. More than one-thirds (39⋅6 %) mothers and 37⋅9 % of fathers attended college degree. More than two-thirds (69⋅4 %) of the respondents had a family size of ≤4 and 33⋅4 % had a poor wealth index  participants were interviewed in the study period. The mean age of the infants was 13⋅31 ± 2⋅07  of the mothers had been birth ≤2, (93⋅9 %) attended ANC during pregnancy of the index child and (59⋅6 %) received IYCF counselling during the ANC visits. Almost all mothers (90 %) attended PNC and 62⋅4 % had received IYCF counselling during their PNC visits. Almost all (92⋅2 %) were delivered in health facility and the majority (59⋅4 %) had no media access. With respect to comorbidity, 52⋅4 % had history of mother and the childhood illness .Table 2Nearly three-fourths 74⋅3 %) had good knowledge towards complementary feeding and half (50⋅5) of study participants ever breast-feed . Grains,⋅3 % had P-values <0⋅25), the variables selected for inclusion in the multiple regression model were child age, sex, maternal education level, getting IYCF counselling at ANC, place of delivery, IYCF practice counselling at PNC, infant feeding practice-related knowledge and childhood illness ]. Mothers who received IYCF at ANC were 3⋅5 higher odds of minimum acceptable diet compared with mothers who had not received counselling [AOR 3⋅43 (95 % CI 1⋅32–8⋅86)]. Mothers who had good knowledge towards complementary feeding practice had three times higher odds of feeding MAD than those who have poor knowledge [AOR 3⋅07 (95 % CI 1⋅41–6⋅67)]. The odds of achieving MAD among children who were ill were six times higher than their counterparts [AOR 5⋅87 (95 % CI 2⋅56–13⋅45)].To reduce malnutrition in a developing country like Ethiopia, adequate and safe infant, and young child feeding practice is crucial. The present study demonstrated that the magnitude of MAD was 16⋅7 %. Sex of child, getting IYCF counselling at ANC, infant feeding practice-related knowledge and childhood illness are the variables that were found to be an independent predictor of MAD., Addis Ababa, 74⋅6 % and other countries like Ghana, Uganda and Kenya in which 29⋅9, 23⋅9 and 48⋅5 % of children received recommended MAD, respectively–8 and the 2020 global nutrition report (18⋅9 %). The variation might be due to the present study was conducted in the dry season what we call ‘winter’ in which the nutritional availability of most fruits and vegetables might be low compared to seasons especially ‘summer’ a period in which this study was conducted. Moreover, the countries’ level differences might be due to differences in study design, sample size, study period and difference in socio-demographic characteristics.This magnitude of MAD was lower compared with a study in Mareka District, southern Ethiopia 35⋅5 %, northwest Ethiopia, Dembecha, 8⋅6 %, rural community of Goncha District, Amhara Region, Ethiopia, 12⋅6 %, Ethiopia multilevel analysis report of EDHS 2016 (6⋅1 %) and other countries like Malawi (8⋅36 %), Nigeria (7⋅3 %) and Philippines (6⋅7 %) of children aged 6–23 months received the recommended MAD–13. The difference might be due to EDHS was conducted on a culturally different population, which may underrate child feeding practices while this study was conducted on an almost culturally homogeneous population with similar feeding practices. Furthermore, the reason for a high percentage of feeding practice compared to other countries might be due to variations in study design and data collection period.In contrary, the result was higher compared with the EDHS report of 2016, only 7 % of children aged 6–23 months received a MAD, West Guji Zone, Oromia, Ethiopia and Sodo Zuria District. This might be due to the cultural and/or traditional perceptions in Ethiopia mostly giving high priority to the male baby than females. This implies that female children should get attention to their requirement of dietary intake like male.The odds of achieving MAD among male children were two times higher than females. The finding was supported with the study done in Addis Ababa and Gondar, northwest Ethiopia. This might be due to the fact that counselling and health education provides better health knowledge, attitude and practice towards the timely introduction of complementary feeding. This implied that ANC check-up is an appropriate time to provide essential messages about proper infant feeding practices.Children from mother/care who received IYCF counselling at ANC were four times more likely to achieve the MAD as compared with those who received counselling at ANC. The result was consistent with another study done in Semera logia, northern Ethiopia. This might be due to the fact that improving caregivers’ knowledge of general nutrition had a positive impact on IYCF and child health.Mothers who had good knowledge towards complementary feeding practices had three times higher odds of feeding MAD than those who have poor. The supporting systematic review study was found in East Africa and Sodo town, southern Ethiopia. This might be due to the exposure of parents to health facilities and getting health education and counselling, this leads the parent best take care of their children.The achievement of MAD among those who got sick was six times higher than the counterpart. The supporting study was found in Addis AbabaDue to the retrospective nature of the study, there might be a recall bias, especially for pregnancy-related responses like ANC visit frequency, and getting IYCF counselling at ANC. In addition, a self-reported study might not give the exact figure of the minimum dietary diversity practice .The magnitude of MAD among children in the study area was unacceptably low, almost one of six children meet the recommended minimum criteria. Sex of child, getting IYCF counselling at ANC, infant feeding practice-related knowledge and childhood illness are the variables that were found to be factors associated with MAD. Health administrative should strengthen infant feeding counselling specifically about the benefit of appropriate complementary feeding and recommended MAD are important to improve the IYCF in the study area. It is important to encourage mothers for ANC and PNC check-ups to take appropriate complementary practice counselling.