Transmembrane ion channels frequently regulate ion permeation by forming bundle crossing of the pore-lining helices when deactivated. The resulting physical constriction is believed to serve as the de facto gate that imposes the major free energy barrier to ion permeation. Intriguingly, many ion channels also contain highly hydrophobic inner pores enclosed by bundle crossing, which can undergo spontaneous dewetting and give rise to a “vapor barrier” to block ion flow even in the absence of physical constriction. Using atomistic simulations, we show that hydrophobic gating and bundle-crossing mechanisms co-exist and complement one and another in the human TRPV4 channel. In particular, a single hydrophilic mutation in the lower pore can increase pore hydration and reduce the ion permeation free energy barrier by about half without affecting the bundle crossing. We believe that hydrophobic gating may play a key role in other bundle-crossing ion channels with hydrophobic inner pores. Atomistic simulations on TRPV4 reveals that hydrophobic gating and bundle-crossing mechanisms coexist in ion channels, highlighting the significance of understanding these mechanisms and shedding light on the regulation of ion flow in various biological channels. These channels play critical roles in ion homeostasis3, electric potential regulation4, neuronal and neuromuscular transmission and cell division5. Central to the ion channel function is the ability to open and close the pathway in response to various stimuli to regulate ion permeation, which is referred to as gating. Studying how an ion channel gates is critical to understand its function under various physiological conditions. Such efforts have greatly benefited from a huge increase of high-resolution structures of ion channel proteins in recent years thanks to the emergence of cryo-EM technology7. Structural studies reveal that many ion channels contain a physical constriction in their “inner pore” region below the selectivity filter, which is formed by bundle crossing of the pore-lining TM helices9. This bundle-crossing gating mechanism was observed in the very first atomistic ion channel structure, that of KcsA potassium channel in the closed state10, whereas the bundle is splayed open to release the constriction, such as in the Ca2+-bound open state of the homologous MthK channel11. Voltage-gated Kv channels are also believed to use the bundle crossing mechanism to control their opening and closing under various physiological conditions17. Curiously, many ion channels lack bundle crossing and remain physically open in the closed states20. Instead, the nanoscale inner pores of these channels are sufficiently hydrophobic such that they can undergo spontaneous dewetting transition23. The resulting dry vapor region provides a large free energy barrier for ion permeation, effectively closing the pathway. These hydrophobic gating channels include BK25, TWIK-1 channel22, as well as many others that are activated by a wide range of stimuli, including voltage, mechanical force, and/or ligand binding19.Ion channels are integral membrane proteins that provide nanoscale transmembrane (TM) pathways to facilitate ion permeation across the lipid bilayer27, MthK28 and all TRP channels with known structures30. This observation raises an important question on whether bundle crossing and hydrophobic gating mechanisms can co-exist and complement one and another within the same channel. There could be synergistic effects between these two otherwise distinct gating mechanisms, that bundle crossing creates a narrower inner pore to promote hydrophobic dewetting transition and the dewetting transition further drives pore collapse to stabilize the bundle crossing and strengthen the physical constriction. In this work, we investigate this question by focusing on TRPV4, a member of TRPV subfamily that plays important roles in many biological processes, such as thermo-sensation32, osmoregulation37, cell swelling38, bone homeostasis39, nociception41, and cancer cell migration42. TRPV4 has also been implicated in many diseases45 and is considered a promising drug target48. TRPV4 is a calcium-permeable nonselective ion channel that can be activated by several stimuli of strikingly different nature, including heat51, chemical ligands55, mechanical force and osmotic stress57. As shown in Fig. 7 shared by all other TRPV members29. Each monomer contains an ankyrin repeat domain (ARD) that consists of six N-terminal ankyrin repeats on the cytoplasmic side. The pore domain (PD) contains pore-forming TM helices S5-6 and harbors the selectivity filter, and the voltage-sensing-domain-like (VSDL) domain consists of TM S1-4. Note that PDs and VSDLs are domain swapped , forming “the lower gate”. The inner pore region between the lower and upper gates is highly hydrophobic and lined exclusively with hydrophobic residues  simulations in explicit solvent to investigate the hydration property of TRPV4 pore in the closed state, and further determined the free energy profile of K+ to cross. The inner pore region overall resembles an inverted cone with a maximum diameter of ~12 Å near I703, in principle providing a large physical cavity for water molecules and ions. Yet, previous simulations showed that hydrophobic cylindrical nanopores can readily undergo dewetting transition when the pore diameter is reduced below ~10 Å61. Such dewetting transitions have also been observed in biological pores such as those of BK and TWIK-1 K2P channels with diameters of ~10 Å22. Therefore, it is likely that the inner pore of hTRPV4 can undergo the hydrophobic dewetting transition to create a vapor barrier inaccessible to bulk water and ions.In this work, we focus on hTRPV4 due to its biomedical significance. A homology structure model of hTRPV4 in the deactivated state was derived from that of the highly homologous xTRPV47, which has a sequence identity of 86% in the TM region (see Methods). The model reveals that the inner pore of hTRPV4 is lined exclusively with hydrophobic residues Fig. . The HOLα atoms (see Methods). All simulations were initiated with a state where the whole pore region was fully hydrated with water molecules  show that the single ion region spans around −20 to 20 Å, beyond which there are non-negligible probabilities of observing two or more ions . The result reveals a striking decrease of the free energy barrier to less than 10 kcal mol−1 in the I715N mutant channel, which reflects a nearly 50% reduction from that of the WT channel . In this work, we examine if both bundle-crossing and hydrophobic gating mechanisms could both be at play and compliment one and another for ion channels that have been observed to contain both bundle-crossing and a highly hydrophobic pore in the closed state. Using hTRPV4 as an important model system, our atomistic simulations confirm that its lower pore region can spontaneously undergo dewetting transition and form a vapor phase. Free energy analysis reveals that the major free energy barrier for ion permeation locates near the bundle crossing site but is broad and spans the entire dry region in the lower pore. This suggests that both bundle-crossing and vapor barrier contribute to the channel deactivation. We further examined the hydration and free energy profiles of mutant hTRPV4 channel, where a nonpore-facing residue I715 is replaced with the hydrophilic N mutation. The I715N mutant channel has been experimentally shown to display increased channel activity in the resting state30 and can be expected to benefit from both gating mechanisms as illustrated for hTRPV4 in this work. The conformational transitions involved in bundle-crossing formation is often accompanied with changes in the pore environment. For example, in TRPV family proteins65, the S6 helix can either adopt a stable π-helix functioning as a “hinge” in the upper region to allow helix motions to open the pore  or switch between α-helix and π-helix during opening process . Those movements involve the S6 rotation, exposing a different set of side chains to the pore and modulating the pore hydrophobicity and geometry. The synergistic interplay between bundle crossing and hydrophobic gating may enable more versatile regulation of channel activities. Mutations, ligand binding or post-translational modifications could shift water dynamics in the pore region and control the ion permeation-free energy barrier without significantly changing the pore structure. Therefore, hydrophobic gating likely plays an even more prevalent role in ion channel gating than currently thought.Many ion channel structures have now been observed to contain both bundle-crossing and a hydrophobic pore7, which share 78% sequence identity for the whole protein and 86% for the TM region. Multisequence alignment between xTRPV4 (uniprot accession: A0A6I8RQZ3) and hTRPV4 (uniprot accession: Q9HBA0) was performed through Tcoffee web server (https://tcoffee.crg.eu/)  or N-methyl groups (for C-termini). The initial closed conformation of hTRPV4 I715N mutant was constructed directly from the WT homology model by replacing the original sidechain with a suitable rotamer of the new residue that minimizes steric clashes.The closed state structure of hTRPV4 was derived from the cryo-EM structure of the closed state of xTRPV4 (PDB: 6BBJ)74. 150 mM KCl was added to buffer and neutralize the systems. The final simulation boxes consist of ~57,000 TIP3P water molecules, ~450 lipids and ~220 ions, with a total ~270,000 atoms and a dimension of ~13.6 × 13.6 × 14.3 nm3. The protein was described using the CHARMM36m protein force field75 and lipids using the CHARMM36 lipid force field76. All simulations were performed using GPU-accelerated GROMACS 201978. The non-bonded forces were calculated with a cutoff of 12 Å and a smoothing switching function starting at 10 Å. Electrostatic interactions were calculated using the Particle Mesh Ewald (PME) algorithm79. The SHAKE algorithm was used to constrain all hydrogen-containing bonds80. The system temperature was maintained at 298 K using the Nose-Hoover thermostat82. The system pressure was maintained semi-isotopically at 1 bar at the x-y plane using the Parrinello-Rahman barostat algorithm83.Models of the closed-state conformation were first embedded in independently generated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, and then solvated with TIP3P water using the CHARMM-GUI web serverα atoms of the protein were restrained using a force constant of 2.39 kcal mol−1 Å−2 ) with respect to the reference configuration of the hTRPV4 homology model mentioned above. The restrained simulations aim to evaluate if hydrophobic dewetting transitions depend on the ability of the pore to contract during simulations. Both unrestrained and restrained systems undergo the same equilibration and production procedures. For equilibration, all systems were first subjected to 5000 steps of energy minimization using the steepest descent algorithm and then underwent a series of equilibration steps where the heavy atoms of protein and lipids (and ligands) were harmonically restrained with the restraining force constant gradually decreasing from 10 to 0.1 kcal mol−1 Å−2, as recommended by CHARMM-GUI74. All normal production runs were performed under the NPT condition  with 2 femtoseconds as the MD integration time. To minimize the effect of the missing loop (640-660), Cα atoms of the two ends residues (639 and 661) were harmonically restrained to their initial positions using a force constant of 1.0 kcal mol−1 Å−2 in all simulations. The I715N system was directly created from the WT system through CHARMM-GUI by mutating the 715 site. Then, a similar equilibration, three runs of restrained as well as unrestrained simulations were performed.We performed 3 parallel simulations under unrestrained conditions, where no other extra restraint was added, and 3 parallel simulations under restrained conditions, where all C+ permeation through the pore were calculated using umbrella sampling84 for both WT and I715N hTRPV4. The initial structures of these systems were taken from representative fully equilibrated snapshots from the equilibration simulations described above. The initial conformations for each umbrella sampling window were generated using steered MD simulations, during which Cα atoms of the protein were harmonically restrained using a force constant of 2.39 kcal mol−1 Å−2 ). During the steered MD simulations, a pulling harmonic potential was applied to a pre-selected potassium ion within the pore region with a force constant of 1.91 kcal mol−1 Å−2 ). The initial conformations were then carefully inspected to ensure minimal protein structural distortions. Umbrella sampling windows covered the whole TM region, ranging from −20 Å to 20 Å with respect to the bundle crossing site (COM of the backbone of residue 715 to 718), with an interval of 2 Å  analysis to calculate the both 1D and 2D PMF profiles. Note calculation of 2D PMF along K+ z-position and pore hydration water count does not require biasing in the second dimension, because pore hydration can readily reach equilibrium in each umbrella window along the K+ z-position. Uncertainty of PMF profiles was estimated by calculating standard errors between the first half and the second half of the last 18-ns umbrella sampling. Analysis of potassium occupancy along the Z-axis during the equilibrium simulations (see above) revealed that there is a high probability of double potassium occurrence near the selectivity filter (~30 Å above the bundle-crossing gate)  were calculated; secondly, those counts were converted to probabilities to get the PMFs. We stitched the whole potassium permeation PMF profiles by combining umbrella sampling (range: −20 Å – 20 Å) and normal 500 ns equilibration simulations (range: −80 Å – −20 Å and 20 Å – 54 Å) through linear interpolation  profiles of K85 and averaged to produce the final pore profiles shown. When calculating water density along the Z-axis (Fig. +86.To calculate average pore profile, we extracted the snapshots every 1 ns during the last 100 ns of one of the three parallel 500-ns trajectories. All snapshots were first aligned using all Cα atoms and the pore profiles then calculated using HOLExis Fig. , a cutofxis Fig. . The regAll standard MD simulations were performed in triplicates to quantify the standard errors in all plots. The convergence of the umbrella sampling free energy simulations was assessed using block analysis.Further information on research design is available in the Peer Review FileSupplementary InformationReporting Summary


The aim of this study is to investigate changes in choroidal and optic nerve morphological parameters following MicroShunt PreserFlo implantation. The secondary aim is to investigate how the structural changes relate to the decrease in intraocular pressure (IOP).Prospective observational study on 15 eyes with glaucoma requiring MicroShunt implantation. Optical coherence tomography was used to measure macular choroidal thickness (MCT), peripapillary choroidal thickness (PCT), lamina cribrosa depth (LCD), cup depth and prelaminar tissue thickness (PLT), before and one day after surgery. Results were expressed in median and interquartile range (IQR) and correlated with IOP results.p < 0.001), with a median increase of + 87.7 µm (+ 26.4%). PCT increased from 157.2 (IQR = 109.1) µm before surgery to 206.0 (IQR = 136.1) µm after surgery (p < 0.001). Moreover, we found a significant post-operative decrease in cup depth  and an increase in PLT . On the other side, LCD reduction 24 h after surgery didn’t reach any statistical significance.The IOP decreased from a median of 25 (IQR = 11) mmHg to 8 (IQR = 2) mmHg the day after surgery. Median MCT increased after MicroShunt implantation from 252.1 (IQR = 156.4) µm to a postoperative value of 318.1 (IQR = 166.6) µm (PreserFlo implantation determines retinal structural changes which appear similar to those caused by traditional filtering surgery, confirming the effectiveness of this device, meantime carrying a much smaller complications rate when compared to trabeculectomy. The enhanced depth imaging optical coherence tomography (EDI-OCT) allows a higher image resolution of choroid and anterior surface of lamina cribrosa . Hence, Previous studies have shown an increase in macular and peripapillary choroidal thickness (CT), anteriorization of lamina cribrosa (LC) and an increase in the thickness of the prelaminar tissue following traditional glaucoma surgery. A positive correlation between the magnitude of change and the magnitude of IOP reduction has been found in some reports .PreserFlo MicroShunt is a sub-Tenon filtering device which consists in a 8.5 mm tube composed of an inert polymer that induce minimal tissue fibrosis. The flow inside the 70-micron lumen of the device is laminar and there is no flow when the IOP inside the anterior chamber is less than 5 mmHg . FurtherTo the best of our knowledge, the changes in choroidal thickness and in optic nerve head morphology following PreserFlo MicroShunt implantation have not been investigated yet. Moreover, previous reports of choroidal thickness and optic nerve head morphology changes, reported data from 7 days to 6 months after glaucoma surgery , 7. The This prospective observational study included glaucomatous patients who underwent MicroShunt PreserFlo implantation at Fondazione Policlinico Universitario A. Gemelli—Rome between January 27, 2021 and October 8, 2021.The following inclusion criteria were considered: uncontrolled glaucoma on maximum tolerated medication or drug intolerance with an intraocular pressure (IOP) of 12–45 mmHg; phakic or pseudophakic patients; best-corrected visual acuity (BCVA) of 20/200 or better; and individuals with rapid and significant loss of visual function at visual field analysis [mean deviation (MD), pattern standard deviation (PSD), visual function index (VFI), and glaucoma progression analysis (GPA) and retinal nerve fiber layer (RNFL) analysis using Spectral Domain Optical Coherence Tomography (SD-OCT).The exclusion criteria were: age under 18, history of intraocular inflammation or any retinal abnormalities, history of non-glaucomatous optic neuropathy, optic nerve drusen, low-quality image due to hyper-mature cataract, unstable fixation, refractive error more than – 6.0 or + 6.0 D of sphere and ± 3.0 D of cylinder. In case of concomitant cataracts, indications were given to perform PreserFlo implantation associated with phacoemulsification and IOL implantation. This study was conformed to the Declaration of Helsinki and informed written consent was obtained from all subjects.Fifteen patients were included in this research and we collected parameters for both eyes, in order to compare treated eye with the fellow one.® SD-OCT Heidelberg Engineering GmbH, Heidelberg, Germany. In the case of concomitant cataracts, they underwent ocular biometry .Patients were clinically evaluated at baseline and 1 day after surgery. At baseline, all patients underwent a comprehensive ophthalmic examination including best-corrected visual acuity (BCVA), slit lamp examination, Tonopen  and Goldmann applanation tonometry, gonioscopy, accurate fundus examination fundus with indirect ophthalmoscopy, i.e., an examination conducted with a slit lamp with a 90 D lens by Volk, corneal pachymetry , computerized or manual visual field examination with SITA standard 30–2, Humphrey Field Analyzer—HFA II; Carl Zeiss Meditec, EDI-OCT of optic nerve head (ONH) and macula SpectralisFollow-up examinations included: IOP, choroidal thickness in macula with a mean of 7 measurements at 500 μm distance, choroidal thickness in peripapillary area with a mean of 6 points at 500 μm distance, lamina cribrosa depth, CUP depth and prelaminar tissue thickness.Morphological parameters of the optic nerve head and choroidal thickness of the treated eye were compared with the adelph eye, as the control, and related to IOP.The choroidal layer and optic nerve head were imaged by SD-OCT EDI mode . All measurements were taken within a limited time frame from 8:00 am to 11:00 am to reduce the influence of diurnal variation of choroidal thickness. Images obtained were assessed by two ophthalmologists that were masked for patient status using caliper tool of Eye Explorer software version 5.4.In each scan of ONH, a horizontal reference line was drawn connecting the two terminations of Bruch's membrane. Taking as a reference the deepest point of the cup, a vertical reference line was drawn. Two other vertical measurements were made at 100 μm and 200 μm from the vertical reference line. The distances from the horizontal reference line to the anterior surface of the prelaminar tissue were measured at the above three points. The average of the three measurements was defined as “CUP”. Distances from the same 3 points of the reference line to the anterior surface of the lamina cribrosa were also measured. The average of these measurements was defined as “lamina cribrosa depth” (LCD). The thickness of the prelaminar tissue (PLT) was considered as the difference between the cup and the LCD.On peripapillary scans, the choroid was manually segmented and measured between the Bruch membrane hyperreflective line and the hyperreflective line of the inner surface of the sclera. The CT was assessed in the temporal and nasal sectors by 3 measurements spaced 500 μm for each sector Fig. .Fig. 1OpIn horizontal macular scans, CT was measured using the same anatomical boundaries. First, the subfoveolar choroidal thickness (SFCT) was measured and 3 measurements were then carried out equidistant 500 μm from the fovea in the nasal and temporal sectors, respectively.Mean macular choroidal thickness (MCT) was defined as the average of these seven measurements Fig. .p value < 0.05 was considered as statistically significant.The sample size calculation was performed using G*power (3.1.9.7 software) by setting the desired power of the study to 80%, the alpha error to 5% and a clinically significant difference of 7 µm in cup depth. Statistical analysis was conducted using SPSS software (IBM SPSS Statistics 26.0). Qualitative variables were described as number of cases over total and percentage. Since the distribution of quantitative variables resulted to differ from normality according to Shapiro–Wilk test, the analysis was performed with non-parametric statistics. Quantitative measures were thus described using median and interquartile range (IQR). Wilcoxon test was used to compare paired data for each group. Mann–Whitney test was performed to detect differences in the variation of the parameters  between the two groups (unpaired data). Linear correlation of quantitative values was assessed with Spearman test. The agreement between the two graders in manual measurements was determined through Bland Altman plot analysis. For all the aforementioned tests, a Thirty eyes of 15 patients were included in the study. There were 7 men (46.7%) and 8 (53.3%) women. The average age of the patients treated at diagnosis was 67.3 years (range 45–85 years). All enrolled patients were Caucasian. In the study group, 14 eyes (93.2%) of 15 patients had a diagnosis of primary open angle glaucoma, 1 patient (6.7%) had a diagnosis of closed-angle glaucoma. Among patients diagnosed with open-angle glaucoma, 6 patients had a diagnosis of pseudoexfoliative glaucoma, 1 patient had a diagnosis of pigmentary glaucoma, 1 patient had a diagnosis of juvenile glaucoma and 7 patients had a diagnosis of primary open-angle glaucoma (POAG). At baseline, all patients were treated with at least 2 active principles. The mean of active topical drugs in use at the time of diagnosis was 3.7 ± 1.1. None of the eyes enrolled in the study had already undergone previous surgery involving the conjunctiva. Five eyes were pseudophakic at the time of Preseflo implantation. Out of the 10 phakic subjects who underwent PreserFlo implantation, 4 were simultaneously subjected to combined phacoemulsification and IOL implantation in association with PreserFlo. Manual measurements revealed sufficient closeness of agreement between graders for all the analyzed parameters. A review of demographic characteristics of the study population is available in Table p < 0.001). Moreover, median decrease in IOP of − 13 (IQR = 10.5) mmHg in the treated eye differed significantly from median decrease of 0.0 (IQR = 2) mmHg detected in the fellow eye (p < 0.001). A review of all pre and post-operative morphological changes is available in Table In the study group, median preoperative IOP was 25 (IQR = 11) mmHg while median IOP at 1 day after surgery was 8 (IQR = 2) mmHg, indicating both a clinically and statistically meaningful variation induced by the surgery as a proof of its effectiveness (p < 0.001) while it didn’t change significantly in the fellow eye (see Table p < 0.001). PCT also increased in the treated eye as a result of the surgery, passing from 157.2 (IQR = 109.1) µm before surgery to 206.0 (IQR = 136.1) µm after surgery (p < 0.001). Once again, this finding wasn’t present in the control group (p = 0.306). Table As concerns macular morphological parameters, MCT changed from a preoperative median value of 252.1 (IQR = 156.4) µm to a postoperative value of 318.1 (IQR = 166.6) µm in the treated eye (p = 0.12). No significant correlation was found between the magnitude of increase in macular and peripapillary CT and the magnitude of reduction of the IOP after surgery. Also no correlation was found between IOP at baseline and changes in choroidal structural parameters after surgery.Comparing the nasal sector with the temporal sector, PCT increase was, respectively, + 25.17% and + 36.6%. However, this difference was not statistically significant (Median preoperative and postoperative LCD in the study group were 456.7 (IQR = 186.5) µm and 447.0 (IQR = 157.6) µm, respectively, a reduction that didn’t reach statistical significance. Despite the detection of a median decrease in LCD in the treated eye of − 16.3 IQR = 71.2) µm compared to an increase of + 3.7 (IQR = 9.2) µm in the control eye, also this difference wasn’t found to be significant. Nevertheless, we detected a liner correlation between the age of the patient and LCD variation in the treated eye following surgery   and median surgical induced variation differed significantly from the control eye (respectively − 29.3 (IQR = 48.9) µm and − 5.0 (IQR = 18.3) µm, p < 0.001). In addition, PLT measurements revealed a significant increase after surgery in the study eye (from 132.7 (IQR = 174.5) µm preoperatively to 183.0 (IQR = 177.5) µm postoperatively, p = 0.028) while the contralateral eye’s increase didn’t reach statistical or clinical significance. Nevertheless, comparison between the groups as concerns pre and postoperative PLT variation didn’t detect any statistically significant difference between the two (p = 0.141). In order to evaluate the secondary objective, the IOP and the structural parameters taken into consideration were correlated. No particular correlations emerged from this analysis.By contrast, cup depth in the study eye varied significantly comparing pre and postsurgical measurements (respectively 355.0 (IQR = 187.2) µm and 274.7 (IQR = 203.8) µm, In accordance with previous studies performed on trabeculectomy, we found a significant increase in CT both in macula and in peripapillary region after the decline of the IOP, already 24 h after surgery , 7–10.In literature, the increase of CT after filtering surgery has been attributed to different factors including the increase in the size and number of fenestrations of the capillaries at the level of choriocapillary and the active synthesis of osmotically active proteoglycans that attract fluid at the level of the choroid. This mechanism finds confirmation in the study carried out by Zhang et al.  in whichAt time T1, we also found a significant increase in the thickness of the prelaminar tissue with a consequent reduction of the cup. This data, even with a shorter follow up, agrees with the findings published by Bouillot et al., who observed a significant increase in the thickness of the prelaminar tissue at 1 week after surgery both in the trabeculectomy and in the deep sclerectomy group. The same data have been reported by Lee et al., up to 6 months after filtering surgery and by Agoumi et al. and Fazio et al., who observed the inverse phenomenon, i.e., a reduction of prelaminar tissue thickness after sudden increase in IOP [Many explanations are reported in the literature about the mechanism underlying this phenomenon. Previous studies hypothesized that the increase in thickness is mainly related to an increase in the size of the vessels due to the increase in perfusion after the reduction of the IOP . Reis etWith regard to the LCD, we found an anteriorization of the lamina cribrosa which was not statistically significant 24 h after MicroShunt implantation. Our data analyzed the immediate morphological alterations of the LC after the filter surgery, differently from other studies in literature, which evaluated this parameter at 1 week, 1 month, 3 months and 6 months after surgery . CompareIn our study, the magnitude of morphological changes in the choroid and laminar region did not appear to be correlated with the IOP reduction magnitude or the baseline IOP value. Conversely, Buillot et al. found a correlation between IOP variation linked to the MicroShunt and changes in macular choroidal thickness one week after surgery .With the exception of few researches in which a relationship between the IOP and structural changes was not found, since the measurements were influenced by circadian variations, the majority of data published in literature show a significant correlation between the percentage of IOP reduction and the percentage of modification of structural parameters following traditional filtering surgery . We hypoFurthermore, in patients with advanced glaucomatous damage treated with traditional filtering surgery, the loss of central vision known as "wipe-out" syndrome has been described –19. ThisOne of the main limitations of the study presented is that the series includes a high percentage of patients with pseudoexfoliative glaucoma. This condition affects the biomechanical properties of elastic tissues, including the lamina cribrosa. Vazquez et al. have shown that patients with this condition have greater weakness of lamina cribrosa, and consequently are more likely to undergo structural alterations following IOP changes after surgical procedures .Further limitations of the present study are: the sample size, the lack of an arm undergoing late filtering surgery to be able to directly compare the parameters evaluated, the lack of complete data of the long-term follow-up, currently in progress, which allows for the evaluation about how these parameters may change over time.This study is the first to investigate early structural changes after MicroShunt PreserFlo implantation. Thanks to the minimally invasive nature of the procedure, it was possible to perform high quality EDI-OCT scans already 24 h after surgery. In agreement with the data presented in literature focusing on retinal structural changes after filtering surgeries, we found a statistically significant increase in macular and peripapillary choroidal thickness and prelaminary tissue thickness. The magnitude of modification of structural parameters was not found to be related to the magnitude of reduction of the IOP. This finding, in association with the possibility of early evaluation of these parameters, represents a further index of safety and minimally invasive procedure.We speculate, therefore, that the PreserFlo MicroShunt implantation is a minimally invasive technique compared to traditional filtering surgery, not only at the surgical site level, but also at the level of the structures functionally involved in the progression of glaucomatous opticopathy.


The entire rotational ladder from J = 24 to J = 364 was resolved simultaneously which enabled a more accurate measurement of the centrifugal distortion constants for CO2. Remarkably, coherence transfer was directly observed, and time-resolved, during the field-free relaxation of the trap as rotational energy flowed into bending-mode vibrational excitation. Vibrationally excited CO2 (ν2 > 3) was observed in the time-resolved spectra to populate after 3 mean collision times as a result of rotational-to-vibrational (R-V) energy transfer. Trajectory simulations show an optimal range of J for R-V energy transfer. Dephasing rates for molecules rotating up to 5.5 times during one collision were quantified. Very slow decays of the vibrational hot band rotational coherences suggest that they are sustained by coherence transfer and line mixing.Optical centrifuges are laser-based molecular traps that can rotationally accelerate molecules to energies rivalling or exceeding molecular bond energies. Here we report time and frequency-resolved ultrafast coherent Raman measurements of optically centrifuged CO In this work the authors use coherent anti-Stokes Raman scattering to study collisional vibrational excitation in highly rotationally excited CO2 molecules prepared in an optical centrifuge. One method is the optical centrifuge1, which traps and uni-directionally accelerates molecules to extreme rotational states  and in some cases, dissociation7. As a consequence of being able to induce such high rotational excitation, optical centrifuges have been used to investigate these so-called “super-rotors” and their collisional dynamics and energy transfer14. In gas-phase molecular systems, energy transfer between molecules is primarily mediated by molecule-molecule collisions. For collisions involving super-rotors, the super-rotors can be thought of as molecular gyroscopes, where high angular momentum stabilizes the super-rotor orientation13, promotes rotationally adiabatic collisions, and prolongs synchronized rotation10. In particular, entry into the rotationally adiabatic regime requires a molecule to rotate more than once during a collision. This violates traditional notions of molecular collisions as “sudden” and rotationally frozen16. Therefore, super-rotors are typically defined by the requirement that the molecules make at least one rotation within the duration of a collision or equivalently have an adiabaticity parameter greater than π 10. The eventual thermalization of super-rotors can produce macroscopic phenomena such as vortex flows12 as well as acoustic waves12. Optical centrifuges have also been used to study molecular potentials7, remote magnetization of gases17, control of chiral molecules18, three-dimensional alignment of molecules20, chemical reactivity21, and new spectroscopic transitions22.Over the past few decades, there has been significant development in quantum state control of molecules and their degrees of freedom using lasers for manipulating chemical reactions and quantum information2 is of primary importance due to the role of CO2 as a greenhouse gas. It is also important for analyzing astronomical observations of CO2 on hot exo-planets such as those recently made by the James Webb Space Telescope23. For space travel, shock heating of spacecraft entering extra-terrestrial atmospheres of planets such as Mars motivates the need to understand energy transfer of CO2 rotational states that are populated well above 10,000 K24. On Earth, it was predicted that collisional inter-molecular rotational-to-vibrational (R-V) energy transfer plays a significant role in strong shock waves surrounding spacecraft during atmospheric re-entry27. Quantitative understanding of R-V energy transfer could have significant impacts on modelling of hypersonic planetary entry flows and spacecraft heat shield design. However, there has been relatively little attention paid to R-V energy transfer in comparison to vibrational-to-rotational (V-R) energy transfer28. One of the few state-to-state resolved observations of R-V gas-phase energy transfer was in vibrationally excited Li2 (v = 5 and 9) vapor as a result of quasi-resonance between rotations and vibrations30. A later theoretical study showed that Li2 super-rotors may induce quasi-resonant Δv = 1 transitions from the vibrational ground state31.Understanding gas phase energy transfer and the rotational structure of CO2 super-rotors prepared by an optical centrifuge produced vibrationally excited molecules with 3 quanta in the bending mode within 50 molecular collisions8. Following work explored anisotropy of energy release and rotational adiabaticity in CO2-CO213 and CO2-Ar/He14 collisions. However, the question of whether the observed vibrational excitation in CO2 arises from the optical centrifuge trap itself or collisional energy transfer has not been fully answered. If the latter was responsible, then it is still unknown whether rotations directly populate vibrational states and on what time scale this occurs due to the limited temporal resolution of previous experiments. Measurements of centrifuged N2O have shown that 1 quanta of bending vibrations in the J = 68 state was populated after 6 collisions22. However, this vibrational state is populated at ambient temperature, so it is unclear whether this was the result of R-V energy transfer or pure rotational energy transfer. Furthermore, the full rotational distribution of the centrifuged molecules was not measured in these studies due to the limited scanning range of the infrared diode laser. Therefore, it is still unknown what ultimate rotational frequency of optically centrifuged CO2 can reach and whether known spectroscopic constants of CO2 hold at super-rotor rotational frequencies.It was previously reported that an ensemble of CO34. We use both the time-domain coherence decays and the CARS frequency-domain spectra to probe the molecular energy transfer dynamics. We simultaneously observe the entire rotational energy ladder of CO2 from J = 24 to J = 364 with rotational energies up to 6.14 eV . We present state-resolved observations of CO2 spinning with energies beyond the ground-state bond dissociation energy as well as coherence decay measurements of molecules rotating up to 5.5 times during a single collision. This is more than three times as many rotations during a single collision as compared to past studies35. Using high frequency resolution nanosecond (ns) probing and detection from a highly-dispersive virtually imaged phased array (VIPA) spectrometer36, the centrifugal distortion constants  for CO2 are revised. We use this data to resolve an ongoing discrepancy in the literature concerning the ground state spectroscopic constants determined from high-resolution ro-vibrational infrared measurements39. Time-resolved coherence decay measurements were conducted with a 65 ps probe laser. After ~500 ps or 3 mean collision times at 380 Torr, odd rotational energy level peaks corresponding to vibrationally excited CO2 emerge in the pure rotational CARS spectra. We use the VIPA spectrometer to analyze the hot band structure, limited only by the spectral width of the probe pulse (0.3 cm−1). We find that 3–6 vibrational quanta in the ν2 bending mode are necessary to describe the width and positions of these peaks. Thus, we directly observe that molecular super-rotors can transfer significant amounts of rotational energy to vibrational energy within a few collisions. Quasi-classical trajectory simulations42 show that moderately excited CO2 super-rotors are the primary drivers of R-V energy transfer with average per-collision probabilities of R-V energy transfer of up to 25%.In this work, we utilize ultrafast pure rotational coherent anti-Stokes Raman scattering (CARS) to monitor the entire rotational coherence established by the optical centrifuge pulse with tens of picoseconds (ps) of temporal resolution2 at a probe delay of 100 ps is shown in Fig. J = 364 were detected. The presence of a centrifugal barrier prevents the CO2 molecules from dissociating at these rotational energies like in Cl2 and other molecules7.An example spectrum taken in 380 Torr of COJ = 2:Bv, Dv, Hv, and Lv are the vibrational level dependent rotational constant and centrifugal distortion constants, respectively. Note that sensitivity to the higher order centrifugal constants Hv and Lv requires high spectral resolution or large J. Details on the spectral fitting can be found in the J = 145 in their fitting algorithm43 and transitions up to J = 100 were measured in past CO2 optical centrifuge work13. This motivates the question of whether spectroscopic constants determined at lower J can extend far into the super-rotor regime.The observed Raman shifts can be derived from following expression for the rotational energy and the S-branch selection rule of Δ2 using a centrifugal distortion corrected rotational constant44 were plotted alongside the experimental spectrum. Using a grating spectrometer, the existing centrifugal coefficients were accurate to within the spectrometer resolution (0.4 cm−1/pixel). Errors in the Raman shift from a rigid rotor assumption were as large as 61.5 cm−1  VIPA and had a 0.0025 cm−1/pixel resolution and a seeded nanosecond Nd:YAG laser was used for the probe (0.003 cm−1 linewidth). Every peak between J = 26–364 was well-resolved  with the value of B taken from37. Using the fitted centrifugal distortion constants eliminates the observed error for all observed rotational states (Jmax = 364) to within 0.01 cm−1 and within 0.0065 cm−1 for the highest rotational states.From Fig. 38 agreed well with our measured Raman shifts and fitted constants. The constants determined by Long et al.37 do not include L, which explains the deviation at high J. Unexpectedly, the spectroscopic constants with the lowest reported uncertainty39 had the highest Raman shift error at high J. The H and L constants determined by Reed et al.39 are significantly larger than those determined by our fits and by Wu et al.38. Agreement with our data is uniquely sensitive to the values of H and L as a result of having resolved over three times more rotational states than in previous studies. The Cs-stabilized frequency comb measurements39 are presumed to be more accurate than stabilized by Rb38 due to Cs being a SI primary frequency standard. Therefore, we attribute this error at high J to the differences in the multi-band fitting routines and data inputs which led to divergent solutions, particularly in H and L.The values of our fitted constants and their comparisons with those found in the literature are summarized in the −1 that extends up to a probe delay of 1 ns. A close up evaluation of the spectra at 200 and 500 ps is shown in Fig. 2 only contains even rotational energy levels at room temperature and requires high temperatures to populate bending vibrational modes for odd rotational energy levels to appear45. With no vibrational excitation, odd rotational energy levels lie in between the even rotational energy levels with a spacing of 1.5 cm−1. However, additional vibrational quanta modifies the rotational constant through the molecule’s moment of inertia44. Here, the observed shifts are ~1.1 cm−1, which suggest significant vibrational excitation in these so-called “hot bands.”Shown in Fig. By a probe delay of 200 ps, the rotational coherences evolved in a field-free environment. The appearance of hot bands after 500 ps suggests that they are the result of collisional rotation-to-vibration energy transfer. To investigate this, the pressure was varied and the onset times of the hot bands in the rotational spectrum are shown in Fig. −1 of the ground state peaks, a VIPA with a free spectral range of 2 cm−1  was used to resolve their structure. As a result, these spectra are probe pulse bandwidth limited (0.3 cm−1). At a probe delay of 100 ps, only even ground state rotational lines are visible and the influence of centrifugal distortions can be seen in the fine dispersion axis as an upward curve at high J. At a 500 ps probe delay  are a pre-requisite for observing the hot bands. On one hand, energetic rotational states provide a reservoir of energy necessary for vibrational excitation. On the other hand, energy gap laws dictate that energy transfer with large energy gaps are more improbable than those with smaller ones. Therefore, the fastest super-rotors may not play much of a role here. In Fig. J = 114. Under such conditions, the hot bands still appear in the rotational spectrum. Furthermore, at a probe delay of 1 ns . This suggests that the hot band is composed of a distribution of vibrational states. Also shown are possible vibrational state assignments to the observed spectrum using known rotational constants of CO244. Surprisingly, 3–6 vibrational quanta in the bending modes are required to describe the hot band distribution. Lower energy states likely play a role in the lower frequency wing, but the peak is well-described by large excitations in the bending mode. This suggests that excitations of bending modes are preferred. This could proceed from in-plane collisions where imparted angular momentum can be taken up by a bending vibration rather than additional angular velocity. The ratios between the hot bands and ground state peaks are also not dramatically affected by whether the centrifuge pulse is cut short to Jmax = 114 or not  results in hot bands shifting to lower rotational quantum numbers  do not contribute much to R-V energy transfer. This explains why experimentally cutting the centrifuge pulse to Jmax = 114 does not eliminate the hot bands as these rotational states still have high probability of R-V energy transfer. The fact that ΔJ = − 30 at J = 80 also clarifies why further cuts to Jmax = 80 limits the maximum J of the hot band lines to <50. On average, molecules with initial rotational states from J = 60 to J = 80 will lose 30 quanta of rotational energy and may simultaneously gain vibrational energy. This makes observations of hot bands at J > 50 unlikely when Jmax = 80.To provide additional insight into the collisional energy transfer, quasi-classical trajectory simulations were performed for COJ agree well with those measured by ps CARS coherence decays34 as well as those from high-resolution infrared absorption spectroscopy44. However, the fits derived from infrared measurements only extend to J = 121. Here, we have extended the available linewidth data by nearly three times to J = 362, with CO2 molecules transitioning into super-rotors at J = 66 as shown in Fig. J = 8635. As a consequence, the measured linewidths involve molecules spinning more than five times during a single collision, far above the super-rotor threshold of one rotation during a collision. Previous frequency-resolved linewidth measurements using optical centrifuges reached at most 1.8 rotations during a collision10. The super-rotors dephase an order of magnitude slower than slower rotors and the shape of the overall distribution appears to be exponential.The rotational transition linewidths extracted from exponential fits to the coherence decays are shown in Fig. 47, the hot bands also dephased more than order of magnitude slower than those in the ground state. We were able to observe these hot bands persist even at a 1.6 ns probe delay after the centrifuge pulse were found to not have a significant role in R-V energy transfer. Lastly, the hot band transitions were found to persist unusually long, and these transitions were hypothesized to be sustained by coherence transfer. The presented measurements and simulations open the door to fully resolved pure rotational molecular structure and centrifugal distortions beyond bond-dissociation energies as well as studies of super-rotor R-V energy transfer and coherence transfer occurring on the time scale of molecular collisions.In summary, R-V energy exchange and coherence transfer in optically centrifuged CO9. The schematic diagrams can be found in Figs. 9. The two pulses were recombined in a polarizing beam splitter cube with orthogonal linear polarizations. The overall transmission efficiency was approximately 40%. The combined pulse had an energy of either 4 or 7 mJ and was passed through a λ/4 waveplate to circularly polarize the light. The resultant optical centrifuge pulse was then focused into a gas cell filled with CO2 at pressures ranging from 50 Torr to 380 Torr. The purity of the CO2 gas cylinder was 99.99% . The CARS spectra were generated by a 65 ps pulse from a ps Nd:YAG regenerative amplifier and was collinearly phase-matched with the centrifuge pulse. Probe energy up to 1 mJ was used. For high resolution spectra, a seeded Nd:YAG  was used which provided 6 ns pulses with energies of 10 mJ. The linewidth of the seeded Nd:YAG laser was specified by the manufacturer to be 0.003 cm−1. Both probes had a horizontal linear polarization. A dichroic mirror was used to separate the probe and CARS signal from the centrifuge pulse. The oscillators of the ps Nd:YAG and Ti:Sapphire regenerative amplifiers were phase locked to within 1 ps, and the relative probe pulse delays were controlled electronically . The seeded Nd:YAG was triggered by a delay generator  synchronized to the Ti:Sapphire regenerative amplifier. The CARS signal was measured by a Czerny Turner spectrometer  and charge coupled device (CCD) camera . For all measurements presented here, 500–4000 laser shots were averaged per spectrum. The linewidths reported in Fig. The optical centrifuge pulse was generated from the output of a chirped pulse Ti:Sapphire regenerative amplifier , similar to previous implementations36. The CARS signal was spatially filtered with a 200 micron pinhole and imaged into the VIPA (LightMachinery) using an achromatic lens and a cylindrical lens with focal lengths of 100 mm and 300 mm, respectively. The VIPA dispersed signal was then focused into a Czerny-Turner grating spectrometer  with two orthogonally oriented cylindrical lenses. The focal lengths of theses lenses were 75 mm and 500 mm. The 500 mm lens imaged the VIPA dispersion onto the slit in the vertical direction, while the 75 mm lens focused the light onto the slit in the horizontal direction. The spectrometer slit length was oriented vertically and the grating dispersion direction was horizontal. The cross-dispersed CARS signal was detected by an intensified CCD camera  with a gate width of 100 ns. For nanosecond probing, a VIPA with a 0.5 cm−1 free-spectral range was used, while for picosecond probing, a 2.0 cm−1 free spectral range was used. The VIPA free spectral ranges were specified by the manufacturer to be 0.5 ± 0.005 and 2 ± 0.02 cm−1 and were measured to be 0.4965 cm−1 and 1.9954 cm−1 using CO and N2 rotational Raman spectrum as calibrations, respectively. Rotational constants of CO have been fit using high frequency resolution data with uncertainties ranging from 10−3 cm−1 to 10−8 cm−1 and rotational states up to J = 13253. For reference, we observed optically centrifuged CO up to J = 67, which spanned across the entire CCD chip. Therefore, CO was used as the calibration gas for the 0.5 cm−1 FSR measurements to ensure the highest level of accuracy for determining new centrifugal distortion constants. The entire rotational expansion (up to O0 J7(J+1)7) for vibrational ground state 12C16O from53 was used in the calculation of rotational energies. For the 2 cm−1 FSR VIPA, N2 was sufficiently accurate for frequency calibration, particularly in the low J regions where vibrational hot bands were observed. The N2 rotational constants used were B = 1.99824 cm−1 and D = 5.7610 × 10−6 cm−1 54. For observations of the highest rotational states in this work, 1800 gr/mm gratings were used in order to detect the entire spectrum on the CCD chip. Otherwise, the spectra were dispersed with a 2400 gr/mm grating to maximize the spectral resolution. Frequency axes were determined by a second order polynomial fit to the observed rotational Raman line positions for each calibration gas.For high spectral resolution images, a virtually imaged phased array (VIPA) spectrometer was used to disperse the CARS signale time constant can be related to the Lorentzian linewidth using the following relation:c is the speed of light, and τ is the 1/e time constant of the coherence decay.Linewidths were obtained by fitting an exponential function to each coherence decay. The 1/2 + CO2 potential energy surface was generated using strategies described and validated previously55. The interaction potential was represented using permutationally invariant polynomial (PIP) expansions56 and the computer code PIPPy57. The training data set consisted of 72,000 counterpose corrected SAC MP2/CBS energies, where the complete basis set (CBS) limit was estimated from aug-cc-pVDZ and aug-cc-pVTZ energies and a scaling all correlation  correction factor of 1.08 was employed to improve the binding energy. At this level of theory, the CO2 dimer binding energy is 513 cm−1, in good agreement with a recent CCSD(T)/CBS calculation . Convergence with respect to the order of the PIP expansion was demonstrated by comparing results for 3rd and 4th order expansions; the 4th order expansion is four times larger than the 3rd order one, and both expansions gave collisional outcomes that agreed within the present statistical uncertainties; attention is limited to results for the 4th order expansion below. The fitted CO2 monomer potential was adjusted to give harmonic frequencies  and an equilibrium rotational constant (Be = 0.390 cm−1) in good agreement with experiment .Collisional energy and angular momentum transfer was theoretically calculated as follows. The required full-dimensional CO60) and the dynamics code DiNT42 were used to prepare state-specific initial conditions with zero-point energy in each vibrational mode. We confirmed that the highly averaged collision outcomes of interest here were similar when vibrational states were instead selected from a thermal distribution at 300 K. One of the pair of CO2 colliders (the target) was prepared with a well-defined initial rotational state J, while the initial rotational state of the other CO2 collider (the bath) was sampled from either a thermal distribution at 300 K (J < 50) or from the experimentally determined high-J distribution (J ≈ 60–360). Numerical parameters controlling integration and the initial and final collider separation were chosen such that the total energy was conserved to better than 2 cm−1, well below the finite-ensemble statistical error of 10 cm−1 (7200 trajectories). The final total energy and angular momentum J’ of the target CO2 were computed, and the change in the total energy ΔE, the change in the rotational energy ΔEr = B0(J’(J’+1)−J(J+1)), and the change in the vibrational energy ΔEv = ΔE − ΔEr were computed; these averages were scaled to per-collision averages using a mean collision time of 168 ps, consistent with the present experimental analysis. Using the semiclassical assumption that ΔEvib/ωbend is the per-collision probability of vibrational excitation allows for the interpretation of Fig. J states. Here, we used ωbend = 668 cm(−1) to calculate example probabilities for J = 40 and J = 100 from Fig. Quasi-classical trajectories ωi. Because of the strong coupling of the linear bending mode energy and rotational energy, this approach poorly describes the bending energy. Instead, we used this approach to confirm negligible excitation to the symmetric and antisymmetric stretches. Changes to the total energy were therefore attributed to changes in the rotational state and to excitation of the bending mode.Vibrational mode energies were monitored along each trajectory by projecting the target collider’s instantaneous momenta onto equilibrium normal modes after aligning rotational axes. To convert projected vibrational energies to quantum numbers Supplementary InformationPeer Review File


The correct sample size is 3 544 636 admissions. This article has been corrected.1In the Original Investigation titled “Trends in Racial and Ethnic Disparities in the Receipt of Lifesaving Procedures for Hospitalized Patients With Decompensated Cirrhosis in the US, 2009-2018,”


As a result, the measurement sensitivities for thermal conductivity and specific heat capacity are increased by 4.1 and 1.6 times, respectively. When differentiating between deionized water (H2O) and heavy water (D2O) with similar thermophysical properties and ~10% different mass densities, the signal-to-noise ratio (property differences over standard error) for H2O and D2O is increased by 9 and 5 times for thermal conductivity and specific heat capacity, respectively.For simultaneous and quantitative thermophysical measurements of ultrasmall liquid volumes, we have recently developed and reported heated fluidic resonators (HFRs). In this paper, we improve the precision of HFRs in a vacuum by significantly reducing the thermal loss around the sensing element. A vacuum chamber with optical, electrical, and microfluidic access is custom-built to decrease the convection loss by two orders of magnitude under 10 There have been continuous efforts to measure thermophysical properties with microfluidics-integrated sensors6 that offer the advantages of fast response time and low sample consumption. Since most microfluidic platforms for thermophysical property measurements are not equipped with direct volume or mass measurements, their measurement results tend to be extensive and thus proportional to sample quantity. To integrate thermal sensing with gravimetric sensing metrology10, we recently introduced simultaneous, fast, and precise thermophysical property measurements using heated fluidic resonators (HFRs)11. With HFR, three intensive properties—thermal conductivity, specific heat capacity, and density—are simultaneously obtained by resistive heating/thermometry and resonant densitometry.Thermophysical properties  of liquids are relevant to numerous applications, including thermal management of high-power electronic devices13, thermophysical property measurement15, or single-cell analysis16, there have been various studies that reduce convection loss by creating a vacuum environment around the microfluidic channel. The effect of minimizing convection loss is more significant in micro/nanostructures due to the scaling effect of heat transfer17, which indicates that convection coefficients tend to increase with decreasing characteristic dimensions. Since our previous report11 is based on the atmospheric pressure operation of HFRs, the performance of these devices can be improved by decreasing convection loss in a vacuum.To increase sensitivity in thermal sensing including calorimetryIn this paper, we propose such advanced operation of HFRs in a vacuum to improve their performance during thermophysical property measurements. Minimizing convection loss in a vacuum causes more heat generation within the integrated heater to be dissipated by conduction through the liquid sample inside the integrated channel. Therefore, the temperature responses of HFRs during pulse heating with fixed power levels show higher contrast for different liquids and enhance the sensitivity of thermophysical property measurements. With a custom-built vacuum chamber, we thoroughly characterize and compare measurement variables–resistance, and resonance frequency–in atmospheric and vacuum pressures. We confirm that the vacuum operation of HFRs improves the sensitivity as well as the signal-to-noise ratio for thermophysical property measurements.20. As shown in Fig. 21. Steady-state resistance differences between the on and off states of the heating pulse and time constants of resistance at the rising edge are extracted to characterize the temperature response. Considering 30 ms of a pulse heating time as used previously11 and the increased time constant (~1 ms in atmospheric pressure) of HFR due to slowed thermal diffusion in a vacuum, pulse heating with 80-ms width and 160-ms period (50% duty) is used. A 3-mW amplitude and 3-mW DC offset are applied, which corresponds to 3 °C modulation at an average temperature of 27 °C in vacuum. Heat generation by Joule heating along the bottom heater surface is balanced with the sum of convection and conduction loss through the solid structure and liquid channel, as shown in the heat transfer diagram of the HFR , and 4-µm thick (3-µm channel height) suspended structures . The approach for mass sensing follows the procedure of previous liquid density measurements using a channel resonatorHFR Fig. . Differe11.Thermophysical property measurement in a vacuum is performed with a custom vacuum chamber Fig.  that canR is the electrical resistance (R) difference between the highest and lowest levels at the steady state during square pulse heating with a 50% duty cycle. τ is the characteristic time response extracted by fitting resistance changes with the exponential decay function  under forced oscillation between the air-filled (f0) and specific liquid-filled HFR, simultaneously obtained with resistance monitoring.ΔR) of HFR filled by an ethanol–water 50:50 binary mixture under atmospheric (~103 mbar) and vacuum pressures (10−4 mbar) is shown in Fig. R in a vacuum environment, the resistance difference (ΔR) with the same heating power is higher in a vacuum due to the reduced convective heat loss. Another feature of operation in the vacuum environment is a long time constant (τ) due to slowed thermal diffusion. The distinct thermal behavior differences between the two environments occur due to the significant difference in the convective heat transfer coefficient for the HFR, which is expected to be ~1000 W/m2 K based on a comparative analysis of spatial temperature mapping between Raman thermometry and finite element analysis  simultaneously measured with Fig. f in vacuum. Resonance spectra of HFR filled with a 50% ethanol-water binary mixture in atmospheric and vacuum pressure environments are illustrated in Fig. f is upshifted (~6.4 kHz) and the quality factor (Q-factor)27 is increased  of the filled liquids. For measuring deionized water and ethanol binary mixtures, increasing the concentration of ethanol (which has a lower k than water) increases ΔR. In addition, the increase in τ depends on the total thermal capacitance of the HFR, which is related to the thermal mass or volumetric heat capacity (ρcp) of the filled liquids. Thus, increasing the concentration of ethanol reduces τ because ethanol has a lower ρcp than water. Considering the equivalent electrical circuit of a heat transfer system within the HFR  ΔR = A/(k‒B) + C for k, (2) τ/ΔR = aρcp + b for volumetric heat capacity (ρcp), and (3) τ/ΔR/|Δf| = dcp + e for specific heat capacity (cp), where A, B, C, a, b, d, and e are calibration coefficients  with density (ρ) reference from the literature32 are used. To enhance the precision of measurement, the average value and standard error from 800-pulse repetitions are obtained for both calibration and subsequent measurements.To ensure measurement reliability, physical relationships between the measurement variables and thermophysical properties are obtained by dimensional analysisk is defined as the 1st derivative of a fitting function for ΔR  is retained in a vacuum. Moreover, the sensitivity of ρ, ρcp, and cp can be represented by their slope from linear function fits, where the sensitivity of ρ with f  divided by the effective mass33 (meff). In a vacuum, the pressure difference between the surroundings and inside the channel can induce bulging34 in the direction of channel thickness, leading to an increase in km and meff. Among those factors, an increase in km is more dominant in the characteristics of the system and leads to an improvement in ρ sensitivity. On the other hand, frequency noise is related to f, km, and the Q-factor. In a vacuum, a decrease in the density of the surrounding air reduces viscous damping, which increases the Q-factor and reduces inertial mass, which increases f. Since normalized frequency noise is reduced by the increase in km, Q-factor, and f, theoretical ρ resolution is improved by considering both ρ sensitivity and normalized frequency noise. The cp sensitivity is proportional to τ/ΔR/|Δf|. All of the variables τ, ΔR, and |Δf|, are increased in a vacuum, and the dominant increase in τ enhances cp sensitivity. Since the temperature response of the HFR is affected by both thermophysical properties and the convective heat transfer coefficient, the convective heat transfer coefficient cannot be extracted solely by monitoring temperature changes over time. By comparison of ρcp sensitivity, where the heat transfer coefficient is the sole factor to change, with FEM  was chosen as a test material  exhibit comparable k, falling within a range where the sensitivity to k is low in ambient measurements. The higher value of ΔR in D2O occurs due to its lower k than H2O, while the higher τ of D2O occurs due to its larger ρcp, and the lower f occurs due to its larger ρ than H2O. Thermophysical properties are extracted using the calibration curve in Fig. 2O) and dashed lines (H2O) in Fig. k and cp of H2O). The difference between the measurement results and the local standards is mainly attributed to the fact that the properties of H2O are near the upper limit of the liquid properties used for calibration. If SNR is defined as the difference between H2O and D2O divided by the standard error, then the vacuum shows enhanced SNRs of those three variables  compared to the air environment, as shown in Fig. 2O. Figure To compare the signal-to-noise ratio (SNR) or sensitivity of ial Fig. . D2O and2 K  to 25 W/m2 K . The reduced thermal loss and slowed thermal diffusion in vacuum increase the differences in resistance and time constants recorded for the various liquid samples. As a result, the measurement sensitivities for all three intensive thermophysical properties reported here are increased by 4.1 times for thermal conductivity, 1.8 times for volumetric heat capacity, and 1.6 times for specific heat capacity. Based on their similar thermophysical properties, using H2O and D2O allowed us to show that the signal-to-noise ratios of thermal conductivity and specific heat capacity are increased by 9 and 5 times, respectively. Thus, we highlight the sensing performance of HFR with the lowest measurement error among the real-time thermophysical property measurement methods inside microfluidics and the importance of thermal loss management in micro/nanoscale sensors.In this paper, we improve the performance of thermophysical property measurements using our recently reported heated fluidic resonators (HFRs) by operating them in a vacuum. The vacuum chamber is constructed to decrease convection loss around the HFR, resulting in a two-orders-of-magnitude reduction in the convective heat transfer coefficient from ~1000 W/mSupporting Information


Currently available antiretroviral 2-drug regimen (2DR) fixed dose combinations may not be suitable for specific situations including the presence of resistance associated mutations (RAM) or drug − drug interactions (DDI). The data on the use of the non-nucleoside reverse transcriptase inhibitor doravirine (DOR) and the integrase inhibitor dolutegravir (DTG) as an alternative 2DR remain scarce.People living with HIV with DOR + DTG as a 2DR are being followed in a prospective observational study.This analysis describes 85 participants with a median age of 57 years. Median CD4-nadir was 173/µl and a majority (66%) had a history of HIV-associated or AIDS-defining conditions. Antiretroviral history was mostly extensive, and documentation of RAM was frequent. The main reasons for choosing DOR + DTG were DDI (29%), tolerability (25%), and cardiovascular risk reduction (21%). Plasma viral load at switch was < 50 copies/ml in all but 3 instances, median CD4 count was 600/µl. DOR + DTG was later changed to another regimen in 10 participants after a median of 265 days, the other 75 participants have remained on DOR + DTG for a median of 947 days.DOR + DTG as a 2DR proved to be a durable treatment option even in extensively pretreated individuals. People living with HIV/AIDS (PLWH) with access to antiretroviral therapy (ART) suppressing plasma viral load (VL) have an almost normal life expectancy . TherefoA fixed-dose combination (FDC) 2DR of the nucleoside reverse transcriptase-inhibitor (NRTI) lamivudine (3TC) and the integrase-inhibitor (INI) dolutegravir (DTG) is considered a first-line recommendation for PLWH starting ART . HoweverAnother 2DR combining the non-NRTI (NNRTI) rilpivirine (RPV) again with DTG can be an attractive option even for PLWH with extensive treatment history. Although RAM limiting the use of RPV/DTG are far less frequent, drug − drug interactions (DDI), e.g. with gastric acid reducing medication including proton pump inhibitors (PPI)  frequentThe next-generation NNRTI doravirine (DOR) was introduced in 2018 and is available both as single drug for combination with other antiretrovirals and as 3-drug FDC with tenofovir-DF (TDF) and 3TC. Solid evidence for its safe and effective use in both contexts is available from several randomized trials –11, and However, despite being a theoretically attractive and both FDA- and EMA-approved combination, DOR + DTG has not been specifically studied in any randomized trial. Even observational data remain scarce , 16. TheAll adult PLWH whose ART was switched to a 2DR consisting exclusively of DOR + DTG at 4 university outpatient clinics and 3 private practices in Germany and Austria were asked to participate in this ongoing observational study. Written informed consent was obtained from all participants, the respective ethics committees approved the study (project nr. 21–0044 at LMU Klinikum Munich). Demographic and medical data were abstracted from charts and electronic records with a planned annual follow-up of a maximum of 5 years after the switch to DOR + DTG. This brief report provides the descriptive analysis of patient characteristics at the time of switch to DOR + DTG as well as a preliminary assessment of durability of this 2DR.Through April 2023, 85 PLWH with a previous ART-switch to DOR + DTG have been included in the study Table . The swiFor 66 participants (77.6%) (cumulative) genotypic resistance testing data were available, in 9 instances (10.6%) resistance testing had never been performed, and for 10 others (11.8%) information on prior resistance testing was unavailable. Protease-RAM were documented in 32 cases (38%), NRTI-RAM in 43 (51%). For 30 participants (35%) RT-RAM were considered to affect NNRTI. For 21 participants (25%) the German GRADE 01/2023 algorithm  identifiIn 2 participants (2%) integrase RAM scored by prediction algorithms had been documented. The presence of N155H resulted in the use of DTG 50 mg twice daily for one patient, in the other the T74I and T97A mutations were not considered to significantly impair the effect of DTG . An overn = 21, 25%), DDI  and specifically gastric acid inhibiting agents , as well as reduction of cardiovascular risk ; 44 participants (52%) received gastric acid reducing agents at the time of switch. Plasma viral load (VL) approximately one year before the switch was < 50 copies/ml in all but one participants and at the time of switch was < 50 copies/ml in all but 3 participants, median CD4 count at the time of switch was 600/µl (range 110–1873). In all 3 instances where the switch to DOR + DTG was made in the presence of detectable viremia of 80, 750, and 160,000 copies/ml, respectively, VL was resuppressed at the next available follow-up while on DOR + DTG.The main reasons for the switch to DOR + DTG were tolerability of the preceding regimen  after a median of 265 days (range 20–1390): 5 patients continued to experience potentially ART-associated side effects despite the switch to DOR + DTG, one wished to return to a single tablet regimen of RPV/DTG after stopping pantoprazole, and one wished to switch to long acting injectable ART. For one participant the identification of the DOR-RAM V106A in an historic genotypic resistance test led to the recommendation to switch off DOR + DTG despite a continuously undetectable VL. In 2 participants persisting low-level viremia of < 100 copies/ml led to the add-on of lamivudine. The other 75 participants (88%) have remained on DOR + DTG for a median of 947 days (range 43–1543) with no occurrence of virologic failure. While on DOR + DTG, 4 male participants died from non-AIDS comorbidities at age 62, 63, 66, and 82, respectively.PLWH whose ART was switched to a 2DR of DOR + DTG had a median age of 57 years and thus were significantly older than the median of about 50 years among PLWH in Germany . The majDespite this, the switch to DOR + DTG mostly occurred in an immunologically stable situation with only 9 individuals (11%) having a CD4-count of < 350/µl at the time of switch, which never occurred due to virological failure of the previous regimen. The main reasons for the switch were rather the attempt to optimize ART with respect to current treatment-associated adverse effects and DDI, specifically compatibility with a PPI, and the intent to reduce the risk for potential future complications of ART. To achieve this, physicians resorted to the off-label prescription of DOR in 31 cases (36%) due to the presence of NNRTI-RAM.Although the majority of participants still continue on DOR + DTG, 10 individuals (12%) have switched to other regimens. So far, only 2 instances of low level viremia  have been observed among this group of individuals living with HIV, confirming the high barrier to resistance expected for this combination. In all instances tolerability issues resulting in switching off DOR + DTG represented persistence of symptoms already present during previous ART. While for 10 participants the convenience of 2 pills without food requirements was the main reason to choose DOR + DTG, 2 others preferred more convenient options (single tablet regimen and injectables) as soon as these became available to them.The characteristics of PLWH on DOR + DTG observed in this study indicating older ager and extensive previous ART experience, the main reasons for switching, as well as the so far favorable course of treatment are in accordance with anecdotal reports and also two smaller observational studies of 18 PLWH from Italy  and 12 PThe limitations of our study include its observational nature and obvious potential for selection bias. We believe; however, that the inclusion of PLWH from university hospital clinics as well as private practices allows for the documentation of representative real-life treatment situations in Austria and Germany. The predominance of patients from university hospital clinics may also reflect the complex treatment situation of patients on DOR + DTG. The still limited number of participants and the absence of a control group both preclude a comparative analysis, e.g., with other ART regimens. Given the extensive and diverse history of ART and HIV-associated complications among most participants, any such analysis will require a larger number of participants to allow adjustment for confounding variables. Therefore, we focused on the description of patient characteristics and their respective reasons for switching to DOR + DTG.To our knowledge, this is the largest published report on the use of DOR + DTG as a 2DR so far. Both inclusion of additional participants from other institutions and further analysis of follow-up data are planned.A 2DR of DOR + DTG (“DoDo”) can be a valuable option for optimizing ART also in PLWH with extensive treatment history. Further study of this combination with a high barrier to resistance and low potential for DDI is clearly warranted.