Repolarization alternans, defined as period-2 oscillation in the repolarization phase of the action potentials, provides a mechanistic link between cellular dynamics and ventricular fibrillation (VF). Theoretically, higher-order periodicities  are expected but have minimal experimental evidence.We studied explanted human hearts obtained from recipients of heart transplantation at the time of surgery. Optical mapping of the transmembrane potential was performed after staining the hearts with voltage-sensitive fluorescent dyes. Hearts were stimulated at an increasing rate until VF was induced. Signals recorded from the right ventricle endocardial surface prior to induction of VF and in the presence of 1:1 conduction were processed using the Principal Component Analysis and a combinatorial algorithm to detect and quantify higher-order dynamics. Results were correlated to the underlying electrophysiological characteristics as quantified by restitution curves and conduction velocity.A prominent and statistically significant global 1:4 peak (corresponding to period-4 dynamics) was seen in three of the six studied hearts. Local (pixel-wise) analysis revealed the spatially heterogeneous distribution of periods 4, 6, and 8, with the regional presence of periods greater than two in all the hearts. There was no significant correlation between the underlying restitution properties and the period of each pixel.We present evidence of higher-order periodicities and the co-existence of such regions with stable non-chaotic areas in ex-vivo human hearts. We infer from the independence of the period to the underlying restitution properties that the oscillation of the excitation-contraction coupling and calcium cycling mechanisms is the primary mechanism of higher-order dynamics. These higher-order regions may act as niduses of instability that can degenerate into chaotic fibrillation and may provide targets for substrate-based ablation of VF. Therefore, understanding the dynamics of VF and its initiation is of utmost theoretical and practical importance.4 Ablation of monomorphic ventricular tachycardia is the cornerstone therapy for drug-refractory VT or VT storm. Ablation strategies include activation mapping during VT or substrate ablation, which focuses on locating the critical reentry circuit using the local electrophysiological properties, such as the peak-to-peak amplitude and the conduction velocity .7Current management of VF comprises membrane-active antiarrhythmic medications and implantable defibrillators.10 However, due to the lack of detailed mechanistic insight into the initiation of VF, functional substrate ablation of VF is still not possible. For example, the concordant to discordant pathway to VF initiation is well-known and extensively studied but cannot guide ablation because of its spatially dynamic nature.13Conversely, VF ablation is in its infancy. It is performed rarely and only in specific circumstances targeting triggers or fixed anatomical structures, such as the right ventricular outflow tract ablation in Brugada syndrome.the electrical instability starts focally before expanding to the entire ventricles, and ablation of these areas has a stabilizing effect and may reduce the risk of VF.The hope is that by moving beyond alternans to more complex dynamics, we may gain useful localizing information that can be used to find unstable substrates suitable for ablation. Therefore, one of the motivations of the current study is the hypothesis that 14 Most cardiac tissues exhibit APD alternans when stimulated at a sufficiently fast rate. APD alternans can even be detected in clinical settings as the microvolt T-wave alternans (TWA).11 However, classic alternans with period-2 results from the first bifurcation in a cascade of bifurcations with periods of 2, 4, 8... We anticipate that as the cycle length becomes shorter, higher-order periods  appear until the system transitions to chaos and hence VF. The intermediate stages in the period-doubling cascade in cardiac tissue have been experimentally elusive and are reported in only a few animal models,17 but not in human hearts.Before directly testing the feasibility of VF ablation, we need to verify the premise of the hypothesis, namely the existence and detectability of electrically unstable substrates before VF induction. We use complex repolarization dynamics as a surrogate for electrical instability. Action Potential Duration (APD) alternans is the beat-to-beat (period-2) oscillation in the APD, is the simplest quantifier of repolarization dynamics, and is the one used to describe discordant alternans.In this paper, we are particularly interested in these higher-order periodicities. Specifically, using higher-order repolarization dynamics to measure electrical instability, we try to answer the following questions:Do higher-order periods (> period-2) occur in (diseased) human hearts?What is the spatial distribution of the higher-order areas ?Are the baseline electrophysiological characteristics predictive of the local dynamics?The study protocol was approved by the Emory University and Georgia Institute of Technology Institutional Review Boards (IRB). Patients consented to the research protocol before surgery. We obtained hearts from the recipients of orthotopic heart transplantation at Emory University Hospital. At the time of surgery, each patient was fully heparinized and placed on a cardiopulmonary bypass machine after circulatory arrest was induced with the infusion of cold cardioplegia solution. Then, the pericardium was opened, and the recipient’s heart was removed using the bicaval technique.18Within 5 minutes of heart harvesting, the explanted heart was perfused from the arteries with cold cardioplegia solution for 5 minutes and transported to the optical mapping lab. Once in the lab, the left main and right coronary arteries were cannulated, and the heart was perfused with warm (37C) oxygenated Tyrode’s solution until the return of the spontaneous contractions. The heart was placed in an imaging chamber and perfused for at least half an hour to recover from the cardioplegia. Before optical mapping, the cardiac motion was suppressed with the help of the myosin ATPase inhibitor (−)-Blebbistatin, at a concentration of 1.8 uM.19 Staining arterially-perfused explanted whole heart or a segment with voltage-sensitive fluorescent dyes allows for non-contact mapping of the transmembrane potential with high spatial (sub-millimeter) and temporal (milliseconds) resolutions.Optical mapping is one of the main tools to study complex arrhythmias.21 The first voltage measurements were obtained from the epicardial surface as long as there was an acceptable imaging window . Afterward, the right ventricle free wall was removed, and its endocardial surface was imaged. We report on the results of transmembrane potential mapping from the endocardial surface of the right ventricles.In this study, after the heart was prepared as above , it was stained with 1 mg of near-infrared voltage-sensitive dye JPW-6003  dissolved in ethanol.The tissue was excited using a deep red LED coupled with a 660/20 nm bandpass filter, and the emitted fluorescence was directed through a long pass 700 nm filter into an Electron-Multiplying-Charged-Coupled-Camera (EMCCD) camera. Image acquisition was performed at 500 Hz and with a resolution of 128 × 128 pixels.The study protocol was organized as multiple restitution runs. In each run, the right ventricles were paced at progressively faster rates, starting at a pacing cycle length of ~2000 ms and down to either the emergence of 2:1 block or induction of a reentrant arrhythmia (VT or VF). Optical signals were recorded from the endocardial surface in 20–40 seconds segments. We compared the signal recorded just before VT/VF induction  to a control recording at 500 ms to detect higher-order periods.The transmembrane potential data for each pixel is low-passed filtered by a cutoff of 50 Hz, and the output is normalized in the 0 to 1 range. Spatial smoothing is performed by applying a variational method (Supplement A).15 Instead, they are localized to a few regions. In response, we have developed two complementary processing pathways, one global to detect low-amplitude higher-order periodic signals from the whole imaging area and the other local to localize period-4 and higher at a pixel level.Period-4 and higher-order dynamics are not distributed uniformly over the recording area.22In addition to global analysis, we apply local analysis to find the dominant periodicity  of each pixel expressed as an integer in the range 1 to 8. The local analysis uses a combinatorial algorithm, which is described in Supplement C.According to the restitution hypothesis, the  lengths . The expPixel-wise conduction velocity is calculated from the smoothed activation map, i.e., the map showing the time of the upstroke of action potentials for each pixel. Specifically, the conduction velocity is inversely proportional to the magnitude of the gradient of the activation time.H1 to H6) removed during heart transplantation surgery. Demographics and clinical history of the hearts are presented in We report on six explanted human hearts  showed a prominent and statistically significant H4). No 1:4 peak was present in two hearts (H5 and H6).Three hearts (H1. The baseline spectrogram at 500 ms shows the expected 1:1 peak (the primary activation) and a small 1:2 peak, signifying repolarization alternans. As the heart was stimulated faster at a cycle length of 310 ms, the 1:2 peak became larger, and a new 1:4 peak emerged. This means that a bifurcation occurred somewhere between 500 ms and 310 ms, and the global dynamics had period-4 periodicity. Pacing this heart faster at 300 ms resulted in VF.H2 follows a similar pattern (panel B). Again, we observed barely discernible alternans at 500 ms with a strong 1:2 peak and a clear 1:4 peak at 270 ms. Similarly, VF was induced while pacing at 260 ms.H5 (panel C) is offset from the 1:4 location and is around ~0.18. This peak is further discussed below.The peak of interest in H6 is a negative example with no 1:4 peak (panel D). There is a prominent 1:2 alternans peak while stimulating at 270 ms, but no significant peak at 1:4. Stimulating this heart faster resulted in a conduction block but no reentrant arrhythmia.The absence of the 1:4 peak at 500 ms in hearts with a prominent 1:4 peak at short cycle lengths significantly reduces the chance that this peak is a processing artifact and points to its dynamic origin. We can probe the dynamics further by looking at the stimulation frequency dependencies of the 1:2 and 1:4 peaks .The distribution of areas with higher-order periodicity is heterogeneous in both space and time and is variable in different hearts. The spatial distribution of the dominant periodicity in four hearts is depicted in H1, which shows a dominant 1:4 peak in global analysis, has areas of stable period-4 periodicity localized to a few discrete islands with roughly circular borders  before transplantation  were on amiodarone, a multi-channel membrane active antiarrhythmic medication  were detected in all six hearts, and they are likely a common occurrence in severely diseased human hearts at sufficiently fast pacing rates. Moreover, we observed the coexistence of higher-period regions with lower-period regions (period-1 and period-2); which suggests there is a period of time when portions of the ventricles are already electrically unstable, but the entire heart is not in VT or VF yet.We observed no significant baseline electrophysiological differences between areas with 1:2 periodicity and areas with period-4, -6, and -8. Specifically, restitution curves remain the same. Therefore, the baseline restitution properties do not predict the ultimate pre-VF dynamics.23 Based on the observed independence of the local dominant period from the restitution properties, we infer that the oscillation of the calcium machinery and excitation-contraction coupling is the primary driver of higher-order dynamics in human hearts. This result is consistent with theoretical works that showed monotonically increasing restitution curves, as is the case for human hearts, cannot generate periods greater than 2.24These results have mechanistic implications. There are two known mechanisms for generating alternans and higher-order periodicities: voltage-driven (dependent on the restitution properties) and calcium-driven.26 Higher-order and chaotic regions may spatially grow to act as niduses of instability that then degenerate regular rhythms into chaotic fibrillation.According to the concordant to discordant alternans pathway, the heart is in a meta-stable but not chaotic condition just before VF initiation, waiting for a trigger. The trigger is usually conduction block or a premature ventricular contraction caused by early after depolarization (EAD) or phase-2 reentry.As mentioned prior, one of the main motivations behind this study was to develop a theoretical framework for possible substrate-directed ablation of VF. Our results, as relevant to VF ablation, are mixed. On the one hand, detecting higher-order periodicities in human hearts, the spatial heterogeneity of these regions, and the co-existence of chaotic pockets and normal rhythm, are promising evidence that target regions exist. On the other hand, we noted that the restitution properties and conduction velocity are not predictors of these areas; therefore, it is unclear how we can locate these targets during ablation. Our results do not preclude the possibility that local differences in calcium cycling properties can predict the site of higher-order dynamics; however, no clinically feasible technique exists to measure these characteristics.We did observe larger non-alternating areas (period-1) in amiodarone-treated hearts relative to higher-order periodicities, which points to the membrane-stabilizing effects of amiodarone. Nevertheless, this effect is partial, and the amiodarone-treated hearts still exhibit higher-order periodicities.27Detecting higher-order periodicities in human hearts may have practical implications beyond its relevance to ablation. If a practical method to detect period-4 from clinical recordings  is developed, it might fix the main shortcoming of T wave/APD alternans in the form of low positive predictive value for malignant ventricular arrhythmias.Human recipient hearts used in this study are very heterogeneous with variable age, sex, underlying disease, pre-transplant ejection fraction, exposure to different antiarrhythmic medications, and the use of mechanical circulatory support before transplantation. In addition, the logistical problems in performing the experiments prevent using a large sample size. The combination of the small sample size and heterogeneous hearts makes it difficult to perform accurate statistical analysis. While the primary finding of the study, i.e., the detection of higher-order periodicities in human hearts, is robust, the secondary findings, primarily as related to the mechanistic differences, are less confident and should be considered mainly as hypothesis generation rather than confirmatory. In addition, we cannot prospectively control for confounding factors, like exposure to amiodarone and inotropes.Transplant recipient hearts used in this study are very diseased and electrophysiologically different from normal human hearts. Therefore, the results obtained here may not apply to normal hearts. Nevertheless, these are precisely the hearts prone to malignant arrhythmias that would benefit from ablation. The results obtained from recipients’ hearts are likely more clinically relevant than studies done on normal hearts.Focal areas of higher-order periods can occur in diseased human hearts under fast pacing. The underlying electrophysiological characteristics, as measured by the restitution properties and conduction velocity, are not predictive of the localization of these areas.


Macrocyclic hosts as prototypical receptors to gaseous and drug-like guests are crucial components in pharmaceutical research. The external guests are often coordinated at the center of these macromolecular containers. The formation of host–guest coordination is accompanied by the broken of host–water and host–ion interactions and sometimes also involves some conformational rearrangements of the host. A balanced description of various components of interacting terms is indispensable. However, up to now, the modeling community still lacks a general yet detailed understanding of commonly employed general-purpose force fields and the host dynamics produced by these popular selections. To fill this critical gap, in this paper, we profile the energetics and dynamics of four types of popular macrocycles, including cucurbiturils, pillararenes, cyclodextrins, and octa acids. The presented investigations of force field definitions, refitting, and evaluations are unprecedently detailed. Based on the valuable observations and insightful explanations, we finally summarize some general guidelines on force field parametrization and selection in host–guest modeling. Macrocyclic hosts are employed as drug carriers and reservoirs in pharmaceutical applications ,2. The en]urils (CB[n] or CBn), pillar[n]arenes, and cyclodextrins (CD) are symmetrical two-rim macrocycles with different compositions , while the other host family octa acid (OA) features a basket-like single-entrance pocket. For macrocyclic hosts, the number of repeating units as a tunable parameter is used to tune the cavity volume and entrance size. As the prototypical hosts often exhibit non-satisfactory physiochemical behaviors, chemical modifications are often imposed on the host structure in order to fine-tune the host properties, e.g., solubility, guest-binding strength, and entrance size. To cover the commonly employed species from the four host families, we carefully select molecules with representative features as investigation targets, which are summarized in Four popular host families are considered in this work. Cucurbit. This is a relatively well-studied host family with extensive experience in host–guest binding features and thermodynamics  family.The first step in force field comparison is investigating the detailed definitions of force field terms. Using the CB7 host as an example, we identify two torsional terms shown in After obtaining a term-specific understanding of the similarities and differences of GAFF derivatives, we then perform 500 ns unbiased simulations in explicit water to probe the dynamics of the host molecules. For this simulation, we use GROMACS 2020.6 , the velThe relatively flexible behavior under GAFF and the stiffer GAFF2 host are expected and coincide with the analysis of torsional potentials, but the similar host dynamics under GAFF and GAFF2 for CB6 and CB7 are rather unexpected. Thus, differences in torsional terms do not necessarily lead to differences in the dynamic behaviors of macrocyclic hosts, and we cannot simply infer the host dynamics from force field definitions. From CB6 to CB8, the number of repeating units is increased, which is accompanied by an increase in the ring flexibility . Note that the flexibility discussed here refers to the intrinsic flexibility of the host ring rather than that defined by torsional potentials. The real dynamics are determined by both the intrinsic flexibility of the host ring and the force field definitions. Following this flexibility-increasing trend, we could further infer that species in this family with even larger numbers of repeating units  could exhibit more complex dynamic behaviors under general-purpose force fields.The above-detailed comparison of force field definitions confirms the noticeable differences between the two commonly employed GAFF derivatives for cucurbiturils, but whether the differences in torsional potentials would trigger alterations of dynamic behaviors is related to the intrinsic flexibility of the host ring. Only when the number of repeating units hits eight does the torsional potentials begin to incur noticeable deviations. For CB7, the dynamic properties under GAFF and GAFF2 are very similar, but it would be interesting to evaluate their closeness to higher-level Hamiltonians and identify problematic parameters in order to provide a direct energetic perspective of the force field quality. Also, we refit the transferable parameter set, expecting to reach the accuracy limit of the functional form of the force field and secure a high-accuracy close-to-ab initio description. The details of the force field refitting and evaluation have been provided at the beginning of n] family is ~0.53 kcal/mol relative to high-level ab initio calculations.We first present the quality of energetics produced by the general-purpose GAFF(2) and the refitted force fields in As this energy scalar does not provide detailed information on the error distribution inside each molecule, we then check the time series of the atom-specific force errors in The above investigation of energetics and atomic forces confirms the room for improvement in the transferable parameter set. We then perform unbiased sampling in explicit solvent to probe the dynamic behavior produced by the refitted parameter sets in order to investigate whether the parameter adjustment would incur noticeable variations of the host dynamics. Similar to the GAFF(2) results in n  investigations. As the refitted molecule-specific FM-B97-3c parameter sets are expected to be more accurate than transferable selections, and the two FM-B97-3c parameter sets produce consistent dynamic behaviors of the CB10 host, we reach the conclusion that even GAFF2 is not satisfactory enough. As none of the existing general-purpose force fields is able to produce the correct host dynamics for enlarged cucurbiturils, it seems necessary to refit existing force fields in the molecular modeling of large rings.Based on the above-detailed investigation of commonly encountered cucurbiturils, including CB6, CB7, and CB8, the above conclusion on the superiority of GAFF2 over GAFF and the closer-to-higher-level behavior of GAFF2 seems solid enough. However, as inferred in the above discussion, more complex dynamic behaviors could be expected for further enlarged rings. The most popular specie with such a property should be CB10, which features 10 repeating units forming its large macrocycle. We similarly perform 500 ns explicit-solvent sampling of this macrocycle under GAFF and GAFF2 and further refit a force field based on the B97-3c statistics (FM-B97-3c from GAFF and GAFF2). The Rg time series is presented in n]arene. Unlike the previous CB[n] family that is better studied and applied in much pharmaceutical research, pillararenes are relatively unpopular and seldom studied in a detailed manner computationally. The modeling reports on host–guest binding affinities in the recent SAMPL9 challenge serve as a good addition to the literature  case, we start with a detailed comparison of force field definitions under the two GAFF versions. The most critical parts for intra-molecular conformational preference, the torsional potentials, are still compared in the first place. The small -SO3− tails do not involve complex torsional interactions, and all torsional potentials defined for this SPn family are presented in Similar to the first CB[Similar to the cucurbiturils case, we conduct 500 ns explicit-solvent sampling for the three hosts in this sulfur-substituted pillararene family with explicit sodium counterions neutralizing the net charges. Thus, the host cavity could be occupied by water or counterions. The structural overlay under the two GAFF derivatives is presented in 32− rims is limited to a small region, and the host backbone fluctuates minimally. When an additional repeating unit is added (SP6), the host dynamics become more flexible. Both the pillararene backbone and the -SO32− rims become noisier on the n family is similar to the cucurbiturils, i.e., larger rings with nosier dynamics. This phenomenon is also expected to be generalizable to other host families, such as cyclodextrins .Aside from the GAFF-vs-GAFF2 comparison, another interesting variation is the size of the host ring (the number of repeating units). For the smallest ring SP5, the host dynamics is rather regular/stiff. The rotation of the -SOn, there is no multi-state behavior, and only a single conformational state is observed. The host cavity is not collapsed like the GAFF CB8 in 32− rims could occupy the entrance of the host cavity or fluctuate away from the cavity and become fully solvent-exposed. The aromatic rings could stay in their well-shaped conformation  in general-purpose force fields. As for the GAFF-vs-GAFF2 comparison, the energetic and force data (RMSE) suggest that GAFF is slightly closer to the high-level reference than GAFF2. As both GAFF and GAFF2 provide similar dynamic behaviors, energetically speaking, GAFF is superior to GAFF2.The above-detailed GAFF-vs-GAFF2 comparison suggests that the two general-purpose force fields, although having different bond stretching, angle bending, and vdW parameters, produce almost the same dynamic behavior for the SPThe above numerical data of energetics and atomic forces provide valuable information about the closeness of transferable parameter sets to high-level reference and successfully locate the problematic terms. We then perform 500 ns unbiased explicit-solvent sampling of the solvated host under the refitted parameter sets in order to probe whether the pronounced energetic and force betterments would lead to noticeable differences in host dynamics. The host structures are superposed in Cyclodextrins are probably the most commonly applied macrocycles due to their readily availability, low cost, and tunable solubility ,63,64. T2-OMe at the primary surface and -OMe at the secondary surface (see more detailed discussions about this surface difference in our previous work) [In existing studies involving a comparison between GAFF derivatives for the CD family, the focused investigation targets are mostly binding thermodynamics between host–guest complexes and sometimes the dynamic behaviors produced by different parameter sets. To our best knowledge, a detailed comparison at a term-specific level was never presented up to now. Thus, the first step of our force field investigation fills this critical gap. As the differences in bond stretching, angle bending, and vdW terms have been shown to have small impacts on host dynamics in the pillararene investigation presented in the previous section, we thus focus on only the torsional terms. Unlike pillararenes and cucurbiturils, having only several torsional terms regulating the dynamics of the macrocyclic cavity, we identify a larger number (~20) of torsional terms in the CD hosts. According to the presence/absence and types of variations between the two GAFF derivatives, the torsional potentials in Hβ-CD could be divided into five groups, all of which are depicted in us work) , there in is observed. Concerning the relative accuracy of GAFF and GAFF2, it is observed that the GAFF2 errors (either RMSE or MAE) are systematically smaller than GAFF. A similar phenomenon is observed for the refitted FM-B97-3c force field that is optimized in the presence of L2 restraints restraining the parameter space in the neighborhood of the initial guess. These phenomena suggest that energetically GAFF2 should be closer to the high-level reference B97-3c than GAFF.We then turn to the force field refitting and evaluation part to grab perspectives from energetics and about the closeness to high-level descriptions. The computational protocol is still similar to the previous cases and would not be repeatedly discussed. The energy deviations of the transferable force fields and the refitted parameter sets are presented in To grab further insights into the sources of errors, we turn to the atom-specific heatmaps of force errors shown in Finally, with a detailed understanding of the similarities and differences of transferable GAFF derivatives and the verified higher accuracy and reliability of the refitted FM-B97-3c parameter sets, we compare the host dynamics under pre-fitted general-purpose and refitted molecule-specific force fields. The superpositions of the 500 ns explicit-solvent simulations under the four parameter sets are depicted in According to the structural overlay in Overall, the detailed investigation of the methylated β-CD ring provides insights into the similarities and differences of transferable GAFF derivatives, their closeness to high-level descriptions, their room for improvements, and the dynamic behaviors of pre-fitted and refitted force fields. When accurate descriptions of the host dynamics are pursued, force field refitting seems necessary. However, as this requires costly ab initio calculations if the practitioners insist on employing transferable force fields, the GAFF2 option could be recommended, considering its proven successes in host dynamics and binding thermodynamics for the prototype β-CD reported in our previous work  and its The last host family under investigation is the basket-like OA. Unlike other macrocycles investigated in this work, the hosts from the OA family have a single entrance to a deep hydrophobic cavity. Consequently, OA hosts are often recognized as deep-cavity cavitands . The car− region. However, as this height of torsional potential is still as high as the thermal energy , the -COO− region is still expected to be rather regular and does not exhibit flexible rotation like the -CH2-OMe and -OMe tails in the previous methylated β-CD. The latter strengthens the cavity entrance, but its low barrier height of ~0.9 kcal/mol may not perturb the existing dynamic behavior significantly. The other newly added and differing terms are mainly imposed on the bottom of the host and make this region more regular under GAFF2. Aside from the torsional potentials, the other terms  also exhibit differences between GAFF derivatives. However, according to the analysis in the sulfated pillararene, these terms would not incur noticeable alterations in host dynamics. Therefore, according to the similarities and differences identified in the detailed force field analysis, the stiffness of the host entrance and the rotational dynamics in the -COO− region could be altered when shifting from GAFF to GAFF2, but the magnitude of variations could be insignificant.We still first compare the detailed definitions of torsional terms in GAFF and GAFF2 to grab some preliminary insights into host dynamics without dynamics simulations. Similar to CDs, the OA host has a large number of torsional potentials regulating its dynamic behavior. The GAFF2 definitions of the four terms in We then proceed to the refitting and evaluation stage for the OA host. The energy deviations of the transferable GAFF derivatives and the refitted force fields are depicted in Although the by-term force field comparison identifies differences between GAFF derivatives, the two transferable force fields do not have noticeable distinctions for energetic and force metrics. Thus, we expect to grab more insights from the dynamic viewpoint presented here. Still, 500 ns unbiased sampling is performed under each parameter set, and the structural overlap and the Rg time series are presented in Accurate modeling of host–guest systems remains rather challenging in the modern computational community. One of the main influencing and limiting factors is the force field accuracy. Despite the massive application of general-purpose force fields in the molecular modeling of host–guest complexes, the accuracy levels and dynamic behaviors under these transferable parameter sets are not well-understood. To answer this long-standing question and consolidate modeling protocols of host–guest systems, we devote the current paper to an unprecedently detailed investigation of commonly employed fixed-charge force fields.The first force field component that is believed to exhibit significant system dependencies in host–guest modeling is the atomic charge. Common practices are semi-empirical models  and the rigorous regularized least-squares RESP regime with different levels for ESP scans. Comparison between ESP RRMSE suggests that AM1-BCC models produce acceptable results in some cases, but the RESP regime serves as a fail-safe option that is recommended in general, as long as sufficient computational resources are available. It is often argued that the traditional HF/6-31G* selection is too old and crude to be applied in modern modeling studies. However, our detailed validation based on a huge basis set def2-QZVPP  and a scan of modern DFT selections supports the opposite conclusion. There is no solid numerical evidence supporting the shift to larger basis sets and higher and costlier ab initio levels. Although we would not fully discommend the use of DFT levels, it should be emphasized that the traditional HF/6-31G* still serves as an acceptable option in charge generation and should not be criticized in practical applications.The other force field components, including bond stretching, angle bending, torsional, vdW, and many other related parameters, are predominantly extracted from transferable force fields, especially GAFF derivatives. These parameters are commonly believed to exhibit small system-dependent behaviors. However, applications without justification are rather dangerous, and detailed force field validations should be properly conducted. Lucky shots due to fortuitous and possibly erroneous error cancellations are poisonous to this field. The obtained structural and thermodynamic insights could be biased, and the reliability of the host–guest interaction picture would be questionable. Therefore, the main results of the current paper focus on detailed comparison, validation, and refitting of transferable force fields, in order to identify their similarities and differences, their deviations from ab initio references, and their room for improvement.n is formed by n glycoluril monomers linked by 2n methylene bridges. The hydrophobic cavity encapsulates external species, while the hydrophilic carbonyl portals act as hydrogen-bond acceptors. The 6-, 7-, and 8-unit species  are popular in practical applications due to the similarity of their portal sizes and pharmaceutically active molecules. By comparing the detailed force field terms of GAFF and GAFF2, we identify two torsional potentials with varied definitions under GAFF derivatives. As these terms are directly acting on the host backbone, the stiffness of the host ring is expected to exhibit a force field-dependent behavior. However, practical simulations in explicit solvent reveal that only the CB8 dynamics exhibit noticeable differences between the two GAFF derivatives, while the structural overlays under GAFF and GAFF2 are virtually the same for the other two species. This phenomenon suggests that the host dynamics are not only determined by the force field definitions and are also related to the intrinsic flexibility of the host ring. Only when the ring size is increased to a sufficient level the force field differences could finally impact the dynamic behavior of the host cavity. In energetics evaluation and parameter refitting, the 7-unit member is selected as the investigation target, as the other  has been studied thoroughly in our previous work [n family. More complex dynamics are observed for further enlarged rings; for enlarged cucurbiturils such as CB10, the flaw/inaccuracy of the GAFF2 parameter set begins to produce noticeable deviations from the FM-B97-3c parameter sets. The host dynamics under all existing general-purpose force fields do not agree well with the higher-level description. Thus, the refitted force field as the ultimate fail-safe treatment is necessary for accurate host dynamics in enlarged cucurbiturils.The first family under investigation, cucurbiturils, is relatively well-studied, with many publications from both experimental and computational sides. The pumpkin-like family CBous work . It is on shares the same torsional description under GAFF and GAFF2. Thus, the investigation of the SP6 host distills the impact of variations of bond stretching, angle bending, and vdW parameters. Despite the differences in these terms, in practical explicit-solvent simulations, the host dynamics are not altered noticeably. The ring-size-dependent behavior in this pillararene family is similar to cucurbiturils, i.e., with the increase in the size of the host ring, the intrinsic flexibility is increased, and the host dynamics become more complex. A difference between pillararenes and cucurbiturils is that in SPn, the host dynamics do not exhibit a multi-state behavior for the largest component of the host family. The host conformation becomes twisted in enlarged rings, but there is no explicit barrier between different conformations. The stabilizing factors for the twisted conformations have two origins. The intra-molecular components include the intrinsic flexibility of the host cavity (ring size) and the force field-defined behavior, while the inter-molecular component is the coordination between the host and the sodium cations. As for the energetics perspective of the force field quality, we observe unusually betterments upon force field refitting. Detailed analyses reveal that the significant improvements are closely related to the problematic parameters in the -OSO32- rims, especially the bond-stretching potentials of the O-S linkage. However, despite the huge energetic biases due to the bond-stretching errors, the host dynamics produced by transferable force fields and the accurately refitted FM-B97-3c parameter sets are virtually identical. Thus, applying either GAFF or GAFF2 would not trigger huge problems in practical applications. However, in cases where bond-stretching motions are crucial, or the reproduction of these energetics is desirable, refitting to secure an accurate description is still preferable.The pillararene hosts, as another crucial family of macrocycles, are relatively unpopular, and their dynamic behaviors are not well-understood. Due to the unsatisfactory physiochemical properties of the prototype pillararenes  and the size comparability of the 6-unit species, we pick the 6-unit pillararene backbone and consider the sulfur-substituted form SP6 as the investigation target. By-term force field investigation reveals that the sulfur-substituted pillararene family SPThe third host family studied is CD. This is probably the most popular family with wide applications due to their readily availability and tunability. The prototype 7-unit β-CD has been studied extensively in our previous work, where a complex multi-modal binding behavior is observed for CD-guest complexes, and the GAFF2 parameter set prevails among GAFF derivatives. However, as the prototype CDs do not often satisfy the intended use, chemical modifications are often introduced. Thus, it is preferable to have some detailed yet general insights into the dynamics of these chemically modified rings. The methylated Hβ-CD featuring the drug-compatible 7-unit portal size and chemical modification (methyl substitutions) serves as a good example illustrating the basic features of commonly applied CD derivatives. The unprecedently detailed by-term force field investigation identifies a large number of torsional potentials, most of which differ between GAFF derivatives. These differing terms are all acting on the CD backbone, while no torsional potential is imposed on substituting methyl groups. Thus, the backbone motions under GAFF derivatives are expected to exhibit differences, while the rotational dynamics of the methyl rims should be similar. Regularized force field refitting leads to closer-to-ab initio descriptions. In terms of both system energy and atomic force, the GAFF2 error is smaller than GAFF, and a similar phenomenon is observed for the refitted force fields. Thus, generally, GAFF2 is indeed better than GAFF. As similar conclusions have been reached in our previous investigation of the prototype β-CD, we expect the superiority of GAFF2 would be rather general for the CD family. However, when monitoring the host dynamics produced by the pre-fitted GAFF derivatives and the refitted FM-B97-3c parameter sets, we observe that neither GAFF nor GAFF2 provides an accurate description of the host dynamics. The GAFF cavity remains squashed in unbiased explicit-solvent sampling. The GAFF2 ring seems stiffer, but the cavity entrance is sometimes occupied by the methyl rims. In stark contrast, the host dynamics under FM-B97-3c exhibit an initial-guess-independent behavior, and the host cavity remains fully open with few penetrations by host rims. Therefore, when accurate descriptions of host dynamics are expected, the refitting of CD force fields seems necessary. For practitioners who lack resources to support costly ab initio calculations needed in force field refitting, we believe the GAFF2 option to be a workable solution, considering the proven success in binding thermodynamics and host dynamics for the prototype β-CD reported in our previous work [The last host family under investigation, the OA family, is structurally dissimilar to the previous three. The bowl-like OA host has only a single entrance to a deep hydrophobic cavity. Different chemical modifications could be introduced to regulate the entrance size. Similar to CD hosts, still a large number of torsional potentials could be identified under GAFF derivatives. For some torsional terms, the GAFF and GAFF2 definitions are exactly the same, while for others, the force field definitions are varied  between GAFF derivatives. However, the magnitudes of variations are often small, and thus no significant alteration of host dynamics is expected. When comparing the GAFF(2) energetics and atomic forces with ab initio calculations, the deviations from high-level references are similar for the two GAFF versions. A similar phenomenon is observed for refitted force fields. Thus, GAFF and GAFF2 are comparable in accuracy. Practical simulations under pre-fitted transferable and refitted molecule-specific force fields reveal minor differences between host dynamics produced by GAFF derivatives and FM-B97-3c force fields. Specifically, GAFF derivatives are similarly describing a stiff host backbone with occasional up–down flipping of benzoic acid groups at the cavity entrance, which involves the interplay of host–ion coordination and intra-molecular torsional potentials. The stiffness of the host backbone under the high-accuracy FM-B97-3c parameter sets is similar to the GAFF(2) description, but the dynamics of benzoic acid groups become more regular with negligible visits of the unexpected up conformation. Overall, when force field refitting is impractical , both GAFF and GAFF2 could be applied for the OA family. When high-accuracy energetics and host dynamics are pursued, still, refitted force fields are preferred.


India implemented the largest COVID-19 vaccination drive in the world, through which it vaccinated the majority of its population. Lessons from the Indian COVID-19 vaccination experience can be invaluable for other LMICs as well as for preparedness for future outbreaks. Our study is designed to explore the factors associated with COVID-19 vaccination coverage in India at the district level. We used data from COVID-19 vaccination in India combined with several other administrative data to create a unique data set that facilitated a spatio–temporal exploratory analysis by uncovering the factors associated with vaccination rates across different vaccination phases and districts. We found evidence that past reported infection rates were positively correlated with COVID-19 vaccination outcomes. Past cumulative COVID-19 deaths as a proportion of district populations were associated with lower COVID-19 vaccination, but the percentage of past reported infection was positively correlated with first-dose COVID-19 vaccination, which might indicate a positive role of higher awareness created by a higher reported infection rate. Districts that on average had a higher population burden per health centre were likely to have lower COVID-19 vaccination rates. Vaccination rates were lower in rural areas relative to urban areas, whereas the association with literacy rate was positive. Districts with a higher percentage of children with complete immunisation were associated with higher COVID-19 vaccination, whereas low vaccination was observed in districts that had higher percentages of wasted children. COVID-19 vaccination was lower among pregnant and lactating women. Higher vaccination was observed among populations with higher blood pressure and hypertension (which were a few of the co–morbidities associated with COVID-19 infection). The adverse impacts of the COVID-19 pandemic have been felt in all spheres of human life, with unprecedented challenges to public health and the economy. Herd immunity has been the key to minimise disruptions caused by the pandemic. The sooner the population acquires herd immunity, the sooner human life and activity can be restored to pre-pandemic levels . HoweverWhile scientists have developed vaccines for COVID-19 at unprecedented speed, the process of translating them into manufactured products on a large scale and making them further available to the masses through an uninterrupted and efficient supply chain is an inevitable component of mass inoculation ,9. VacciCOVID-19 vaccination rates have been the lowest among low- and middle-income countries (LMICs) ,15,16. AIn this article, we analyse COVID-19 vaccination rates in India. Our research is built upon a novel data set that has been created by combining several administrative data sets and COVID-19 data sets for India. While researchers have analysed the district-level COVID-19 infections and fatalities ,36, as fIndia has a history of public immunization programs since its colonial times . India sThe COVID-19 vaccination drive in India began on 16 January 2021 with two approved vaccines. The COVID-19 vaccination being voluntary in nature left ample scope for vaccine hesitancy. The COVID-19 vaccination drive in India prioritised the section of the population at the highest risk of infection, which was the rationale for the phased rollout of the vaccines . The timPhase-1 COVID-19 vaccination started on 16 January 2021 and went on until 28 February 2021 and allowed first-dose vaccination of frontline workers who were actively involved in containing the spread of the pandemic. The frontline workers included health workers, security staff and people who provided essential services to the general public. We earmarked the period before Phase 1—from the beginning of COVID-19 in India in March 2020 up to 15 January 2021—as the pre-vaccination phase or Phase 0, though technically there was no Phase 0 in the vaccination drive. During Phase-2, which was implemented between 1 March to 31 March 2021, population aged 60 years and above was eligible for fist vaccine dose. In addition, individuals aged 45 years and above with comorbidities such as hypertension, diabetes, HIV infection, etc., were also eligible. Phase 3, which ran between April 1 and 30, made all individuals aged 45 years and above eligible for first dose of COVID-19 vaccination. All adults aged 18 years and above were eligible for the first dose of vaccine from 1 May 2021 onwards, which was the last and final phase of COVID-19 vaccination in India. Any individual eligible for vaccination during a particular phase remained eligible for vaccination in all subsequent phases.We used data from various secondary sources to create a unique data set at the district level that allowed us to investigate the heterogeneity of vaccination rates across different districts and vaccination phases. We obtained district-level data for daily cumulative COVID-19 first-dose vaccination numbers, daily cumulative COVID-19 deaths and daily cumulative reported caseload numbers from the Data Development Lab . From thWe used the data from the Rural Health Statistics (RHS) for the year 2020–2021 from the Health Information Management System (HMIS) portal of India to construct a measure of district-level health infrastructure. RHS data provided us with the number of sub-health centres, primary health centres and community health centres in each district, which were converted into an equivalent number of sub-health centres by using the equivalence ratio provided in the RHS report  . Two meaWe also included district-level data from the fifth round of the National Family and Health Survey (NFHS) conducted between 2019–21 in order to take into account district-specific heterogeneity in the health status and its association with COVID-19 vaccination outcomes. NFHS is an important database that tracks health status over time and has had administrative units in India collecting data every four to five years since 1992–1993. Some of the notable variables from NFHS-5 that we included in our analysis include child and maternal health (including immunization and nutrition), years of schooling, family planning, etc.District-level demographic and socio–economic variables, including percentage of rural population, literacy, gender, social and religious minorities and population density from the 2011 census of India were also included. In addition, we also included an indicator for aspirational districts. The government of India introduced the aspirational districts programme in early 2018 to reduce inter-district and inter-state disparity by bringing the state and the central governments together on various developmental schemes through healthy competition among the districts to achieve improved targeted health, education and infrastructure outcomes. The programme identified the 117 most-underdeveloped districts as aspirational districts based on a composite index that captured deprivation in health, education and infrastructure domains .We employed ordinary least squares (OLS) regression to find the associations between COVID-19 vaccination and other relevant variables from our data set. Our primary dependent variable was the percentage of the population vaccinated with a first dose of COVID-19 vaccine during every time period (denoted by phases and Months 1 and 2 for Phase 4). The independent variables that we used in our regression included phase-wise vaccinations, cumulative reported infections and cumulative deaths as a percentage of the population. In addition, we also used time-invariant (vaccination phase), district-specific demographic, socio–economic and health variables as independent variables in our regressions. We estimated a separate OLS regression for each time period (or phase). For all regressions, we used state-fixed effects to control for unobserved state effects. We also used clustered robust standard errors, which were clustered at the state level.The results of phase-wise OLS regression are shown in Columns 1 to 5 of the regression All regression models included four COVID-19-specific explanatory variables that varied across districts and phases—PIR, PDR, (PIR/PDR) and IR, along with demographic and socio–economic variables and measures of health infrastructure. The results have been tabulated in In COVID-19 vaccination in India was provided free of charge to all. Despite this, people might not have perceived it to be costless due to several opportunity costs and unobserved psychological costs in addition to lack of trust in the vaccine that might have led to vaccine hesitancy. Vaccine hesitancy could have been a major driver of low vaccine demand and subsequently low vaccination rates despite the availability of the vaccine. Availability of a vaccine does not mean that people will rush to become inoculated. The observed vaccination rate is an outcome of the complex interaction between the supply as well as the demand for the vaccine rather than one single component alone.The reported infection rate in a district, while likely to be indicative of the actual infection rate, was also confounded with COVID-19 testing efforts. Districts that implemented more rigorous COVID-19 testing were also likely to report a higher number of COVID-19 cases compared to districts that had lower COVID-19 testing rates. On the contrary, unlike reported infection rates, COVID-19 fatalities were more closely associated with the actual infection rate and were unlikely to be influenced by COVID-19 testing efforts. Both COVID-19 reported infection rates and death rates were likely to be correlated with vaccination rates, and hence we included these variables in all regression models in For all phases, we observed vaccination rates to be positively correlated with past cumulative reported infection rates, PIR, as a percentage of the total district population. The above association might have been driven by higher COVID-19 awareness in districts with higher reported infection rates that could be either due to higher testing efforts or the prevalence of an actual higher infection rate. We also included reported infection rates for the current phase IR%, which stood out to be positive and significant only during the first month of Phase 4. The initial first month of Phase 4 coincided with the deadly second wave of COVID-19 infections in India, which might have pushed up the demand for vaccination. There was a negative association between past COVID-19 deaths (PDR) and vaccination in any district, though this was significant for Phases 1 and 4 only. PDR% was defined as the past cumulative death rate out of the total district population. On the contrary, the association between vaccination rate and past COVID-19 deaths as the proportion of the past infection rate (PDR/PIR) was positive and statistically significant for the first and fourth phases. The opposite signs of COVID-19 death variables when measured as a proportion of the population and when measured as a proportion of the infection rate might be capturing two different phenomena. At unchanged reported infection rates, while a higher PDR in a district indicated higher fatalities among the population, a higher (PDR/PIR) in a district indicated higher COVID-19 deaths for unit infection in that district. A higher number of COVID-19-linked deaths might have pushed up the demand for COVID-19 vaccination.In the above discussion, we attempted to think of possible reasons for the observed relationship between past and current reported infections and deaths, which tell a demand-side story. However, it cannot be undermined that COVID-19 infections and deaths were also used as critical inputs in vaccine allocation to different districts. Vaccination rates were likely to be lower in districts that were constrained due to a limited supply of vaccines. However, vaccination rates during an entire phase are likely to be less volatile than during a particular week or day due to volatility in the vaccine supply. Therefore, while not impossible, it is less likely that a district would have faced severe supply constraints due to a short supply of vaccine during the entire vaccination phase. Nonetheless, we would not attribute lower vaccination rates to low demand only, but rather a combination of both demand- and supply-side factors ,50.The health infrastructure burden was captured by the average population served by each sub-health centre in a district. A lower health burden is indicative of better health infrastructure and is negatively associated with vaccination rates across all vaccination phases, and the coefficient was significant except during Phase 1. Since Phase 1 targeted vaccination of health workers and frontline workers, it is not surprising to not find a significant correlation between health infrastructure and vaccination rates across districts. Health infrastructure density measured the average number of sub-health centres per square kilometre in a district. During the vaccination phases used in our analysis, COVID-19 vaccines were available on demand at vaccination sites only, which implied that individuals had to travel to the vaccination centres to become vaccinated. While a lower density is likely to be an indicator of a greater distance from the health centre for an average person in a district, we found an inverse and significant correlation between health infrastructure density and COVID-19 vaccination rates in early Phase 4. The beginning of Phase 4 COVID-19 vaccination in India coincided with the deadly second wave  in the literature ,57. As sVaccination was positively correlated with higher literacy rates. COVID-19 vaccination campaigns in India required an online registration to become vaccinated during the period of our analysis. In addition, during the period of our analysis, the registration platform was only accessible in English. Therefore, appointment registration for COVID-19 vaccination required access to a smart phone and internet along with English literacy. These factors could have partially driven the relationship between literacy and vaccination in addition to differences in attitude and perception towards the vaccine shaped by literacy.Districts with higher concentrations of marginalized communities—Scheduled Caste (SC) and Scheduled Tribe (ST)—also had lower vaccination rates. Lower vaccination rates were observed among concentrations of Muslim populations during Phase 2. However, during the second month of Phase 4, vaccination rates were relatively higher in districts with higher Muslim populations. For Muslims, Ramadan, the holy month of fasting, coincided with the first month of Phase 4. Therefore, it might have been that vaccination uptake was higher among Muslims once Ramadan concluded ,63,64.We found vaccination rates to be associated with selected maternal health indicators. Higher maternal protection against neonatal tetanus was positively correlated with COVID-19 vaccination. We found negative significant correlation between vaccination rates and pregnancy rates (between the ages of 15 and 19 years) and percentages of children exclusively breastfed. Women of childbearing age were eligible for vaccination during the fourth phase of vaccination, but a lack of scientific evidence and clear communication surrounding potential side-effects of the vaccine on a pregnant or a breastfeeding woman might have driven this finding ,66,67.Similarly, we also found COVID-19 vaccination to be associated with child health indicators. We found positive and significant correlation between complete immunisation of children and COVID-19 vaccination rates during Phases 3 and 4 of the vaccination ,69. HoweA positive and significant correlation between COVID-19 vaccination rate and the proportion of the population on medication for blood pressure during Phase 2 and Phase 3 was observed. Since high blood pressure and hypertension were some of the identified comorbidities associated with COVID-19 infection, it may have driven the COVID-19 vaccination rate higher. We also found a positive correlation between health insurance coverage and vaccination rates. Vaccination rates were also higher in aspirational districts during the initial first two phases of vaccination.We compiled district-level data from various secondary sources on COVID-19 vaccination, COVID-19 infections and COVID-19 deaths, which we combined with demographic and health data in order to facilitate heterogeneity analysis across districts and phases. We would like to state that the observed relationships in our analysis should not be interpreted as causal relationships between vaccination rates and other explanatory variables. There might be some element of causality in our analysis, which would be invaluable if it could be identified; a causal analysis is limited due to the unavailability of requisite data. We also understand that there might have been measurement errors, particularly with respect to COVID-19 variables, due to significant under-reporting; yet our analysis is defensible unless there were systematic errors in measurement of variables. However, given all these limitations, we make our best effort to bring the several pieces of data together to provide an exploratory analysis of COVID-19 vaccination in India.Our study attempted to explain the variation in the observed COVID-19 vaccination rates across districts in India during the first few months of the COVID-19 vaccination drive. With vaccination decisions being a complex interplay between the demand and supply of a vaccine, it is difficult to identify the causal factors that might influence observed vaccination rates, as the observed vaccination outcome could indicate lack of supply, demand or both. Therefore, our exploratory analysis provides suggestive evidence surrounding observed vaccination rates, based on which we suggest possible reasons that might have led to the observed empirical relationships.We used district-level data from the first few months of the COVID-19 vaccination drive in India, which we combined with several other administrative data to create a unique data set that facilitated heterogeneity analysis across different vaccination phases and districts. We found evidence of past reported infection rates positively correlated with first doses COVID-19 vaccination. Past cumulative COVID-19 deaths as a proportion of district population was associated with lower COVID-19 vaccination, but the percentage of past cumulative reported infections was positively correlated with COVID-19 vaccination. A higher population burden per health centre was likely to indicate a lower COVID-19 vaccination rate. Districts with larger rural populations had lower vaccination rates, whereas the association between COVID-19 vaccination and literacy rate was positive. Higher percentages of fully immunized children were positively associated with COVID-19 vaccination, whereas vaccination rates were lower in districts with higher percentages of wasted children. COVID-19 vaccination was lower among pregnant and lactating mothers. Higher COVID-19 vaccination rates among populations with higher blood pressure and hypertension (some of the comorbidities of COVID-19 infection) was observed.Our research makes an important contribution to the area of vaccine research in the context of LMICs by using data from India, which implemented the largest COVID-19 vaccination drive in the world. We uncovered several associations between vaccination rates and health and demographic variables that provide insights regarding COVID-19 vaccination and vaccination in general that can be used to design future research and investigations surrounding vaccination in LMICs.


To enhance the properties of polyamide 12 (PA12/Nylon 12) manufactured by the selective laser sintering (SLS) process, micron-sized glass beads are used as a filler, and the resulting composite is known as glass bead-filled PA12 (PA 3200 GF). Despite PA 3200 GF basically being a tribological-grade powder, very little has been reported on the tribological properties of laser-sintered objects based on this powder. As the properties of SLS objects are orientation-dependent, this study is devoted to investigating the friction and wear characteristics of the PA 3200 GF composite sliding against the steel disc in the dry-sliding mode. The test specimens were aligned in the SLS build chamber along five different orientations . Additionally, the interface temperature and the friction-induced noise were measured. The pin-shaped specimens were examined using a pin-on-disc tribo-tester for 45 min to investigate the steady-state tribological characteristics of the composite material. The results revealed that the orientation of build layers relative to the sliding plane was a ruling parameter that determined the dominant wear pattern and the wear rate. Accordingly, where build layers were parallel or inclined to the sliding plane, abrasive wear predominated, and wear rate became 48% higher than that of specimens with perpendicular build layers, for which adhesive wear predominated. Interestingly, a noticeable synchronous variation of adhesion and friction-induced noise was observed. Taken together, the results from this study can efficiently serve the goals of fabricating SLS-functional parts with customized tribological properties. The selective laser sintering (SLS) process is an additive manufacturing (AM) technique which allows the fabrication of polymer-based complex 3D objects. SLS is a layer-by-layer manufacturing technique in which the 3D CAD file of the object is sliced into discrete successive layers of predetermined thickness. During fabrication, the polymer powder is heated to a temperature close to its glass transition temperature (in case of amorphous polymers) or close to the melting temperature , and a fresh layer of powder is spread over the build area. Then, the laser beam scans a selected area corresponding to the geometry of the sliced CAD model. The supplied laser energy causes the powder particles to fuse together, forming the first layer of the object. After that, the build platform is lowered by a layer thickness, and a new, fresh powder layer is spread over the build area. The process is repeated until the whole object is fabricated as consolidated successive layers . FinallySeveral thermoplastic powders are now available for SLS; however, polyamide 12 (PA12) is the most commonly used powder for SLS, due to its favorable sintering properties, which include good powder flowability, low melt viscosity, and a wide range between the melting and crystallization temperatures . ConsequDespite PA 3200 GF being mainly a tribological-grade powder, most research on glass-bead-filled PA12 focus on evaluating the mechanical properties of laser-sintered objects ,12,13,14The glass bead-filled PA12 test specimens, in the form of pins with a 4 mm diameter and a 25 mm length, were fabricated by the SLS technique in an EOS P380 system using EOS-PA 3200 GF tribological-grade powder. Fabrication parameters are given in In this work, the tribological properties of PA 3200 GF pin-shaped specimens were examined on a TM 260 pin-on-disc wear test apparatus see . The pinThe frictional heating temperature of disc surface close to the contact zone was measured using an infrared laser sensor (KEYENCE-FT-H30), along with a digital amplifier unit FT-50AW. The infrared laser sensor detected the emitted frictional heat from a circular area with a 6 mm diameter around the sliding track after about 40 ms of disengagement.As polymer friction results in the formation of a polymer transfer film on the countersurface, which in turn affects the performance of tribo-contacts, several stainless-steel discs of 50 mm diameter were manufactured with almost similar surface roughness, so a fresh disc was used with every test specimen. The surface roughness of all discs was measured using SURFTEST SJ-210, and the measured (Ra) roughness value normal to the sliding track (r = 18–22 mm) was around 0.35 ± 0.03 microns see . In the The friction-induced noise was measured using a digital sound level meter (GM1357), with 30–130 dB measurement range and an accuracy of ±1.5 dB. Measurements were carried out in a semi-anechoic chamber constructed as a wooden box lined with a high-density and echo-friendly polyurethane foam. The acoustic foam was capable of damping out low-to-mid noise frequencies inside the room. The recorded background noise, before running the experiments, was 35–37 dB. During the experiments, several measures were taken to exclude noise from external sources.The pin-on-disc wear test apparatus was equipped with a double-bending beam force sensor (ME-KD60). The output signal of the KD60 load cell was fed into the NI-9237 module through NI 9949 . The module accessed the data at a rate of 50 kS/s and was connected to the NI-compactDAQ controller. A moving average function with Labview was used to smooth out the signal and record the friction force value at 500 ms intervals. On the other hand, an Arduino Uno microcontroller board was used to convert the 4–20 mA output of the infrared laser temperature sensor into 0–5 V signal, and a real-time averaging function was used to smooth out the sensor signal and record the temperature at 500 ms time intervals. Similarly, the DC output signal of the digital sound level meter of 10 mV/dB was handled with an Arduino Uno board, and the friction-induced noise level was recorded after smoothing at time intervals of 500 ms.For tribo-surface characterization, the worn surfaces of PA 3200 GF test specimens were coated with a 100 Å thin film of gold in a JFC-1100E ion sputtering device and then scanned with a JSM-5400LV scanning electron microscope (SEM).Throughout the present experiments, the pin-shaped PA 3200 GF specimens were loaded against the rotating steel disc by means of a loading lever in such a way that the normal load on the pin was 50 N, and the disc was rotated at 120 rpm. To experimentally simulate more realistic operating conditions, the experiments were run for a duration of 45 min, which was sufficient enough to attain steady-state tribological properties of the composite material. As the diameter of the test specimen was 4 mm, the average contact pressure (P) was about 4 MPa. Indeed, the real contact pressure was much higher than the average value, since the real contact area was much smaller than the apparent area of contact; the real contact area was 10–30% of the apparent contact area . AdditioThe effects of build orientation on the properties of fabricated objects were mainly attributed to two main factors: the delay time between scanning vectors (hatching pattern) and the layer orientations relative to the loading direction. Indeed, the wear depth in the current experiment was less than the layer thickness (150 μm), and hence, the effect of the former factor on the tribological properties of test specimens became inferior. Furthermore, current SLS systems evolved to the degree that the optimum hatching pattern was set automatically to maximize object density and reduce the porosity of fabricated objects. On the other hand, by excluding the effect of the hatching pattern, the layer orientations relative to the loading direction became the ruling factor that affected the tribological properties of PA 3200 GF-fabricated objects. Therefore, the discussions in the following sections will be focused on the effects of layer orientations relative to the sliding plane on the tribological properties of PA 3200 GF composites.The dry friction at the polymer/metal interface was attributed mainly to two processes: (1) polymer surface deformation as a result of the ploughing and/or cutting action of hard surface asperities into the polymer surface and (2) the shearing of adhesive junctions formed at regions of real contact between the polymer and the hard surface . In factThe COF of PA 3200 GF specimens sliding against steel was obtained by dividing the measured friction force by the applied normal load. The evolution of the COF with times for specimens with different build orientations is shown in 3 vs. 1.02 g/cm3), the glass beads tended to precipitate in the layer during the laser-sintering process, motivated by the low melt viscosity of PA12.  had a higher density compared to PA12  . At highThe measured friction-induced noise levels of specimens with different build orientations are shown in It was estimated that most of the work performed against friction is converted into heat energy at the sliding interface . AdditioThe results clearly showed that the interface temperature gradually increased for all specimens. However, it is noteworthy that the measured temperature was affected by the reflectivity of the disc surface in the vicinity of the contact zone, which, in turn, was affected by the thickness and homogeneity of the transfer film. Additionally, as the pin slid over the disc, the real contact area increased, making room for a more adhesive junction to be formed and sheared away, which resulted in a gradual increase in the interface temperature. Therefore, both the change in disc surface reflectivity and the increase in real contact area contributed to the shown increase in the measured interface temperature.sK) in (mm3/N.m) was calculated as [3), P is the applied load (N), and L is the sliding distance (m). The wear of specimens’ tribo-surfaces occurred as a result of the abovementioned modes of friction. The observed modes of wear in the dry sliding of PA 3200 GF against the steel disc were abrasive wear, adhesive wear, and surface fatigue wear. The specific wear rate Ks=ΔThe average values of the tribological parameters of PA 3200 GF sliding against the steel disc for a duration of 40 min (after 5 min of running-in stage) are shown in Characterization of the tribo-surface is an extremely important tool for controlling the friction and the wear behavior of polymer composites. Therefore, the sliding surfaces of PA 3200 GF specimens built along the five different orientations were investigated using a scanning electron microscope (SEM), and the results are shown in In addition, Similarly, the SEM of the YZ-oriented tribo-surface, shown in In contrast to the Z-oriented tribo-surface, the SEM of the XY-oriented sliding surface see  showed cThe selective laser sintering (SLS) technique has evolved to the point that it is being used extensively in the manufacturing of functional parts and with moderate production capacity. However, the properties of laser-sintered parts are orientation-dependent. Therefore, the present study aimed to investigate the orientation-dependent tribological properties of glass bead-filled PA12 (PA 3200 GF) composite to facilitate the SLS fabrication of parts with customized tribological characteristics. The friction and wear properties of pin-shaped specimens with five different build orientations were examined on the pin-on-disc tribo-tester. Additionally, interface temperate and friction-induced noises were measured. Experiments were carried out under a normal load of 50 N, a sliding speed of 0.25 m/s, and for 45 min. The results suggest that the layers’ orientation relative to the sliding plane was a ruling parameter that determined the dominant wear pattern. Accordingly, where the build layers were parallel or inclined to the sliding plane, abrasive wear predominated and wear rate increased. By contrast, adhesive wear dominated when the build layers were perpendicular to the sliding plane, and wear rate decreased. In addition, the results elucidated a noticeable synchronous variation pattern of adhesion and friction-induced noise. Moreover, the SEM of the PA 3200 GF sliding surface suggests that where adhesion occurred and polymers transferred to the countersurface, transfer film could not adhere efficiently to the steel disc, and eventually, back-transfer occurred; the process continued, thereby causing an increase in the wear rate.


The hippocampus plays a crucial role in memory and cognition. Because of the associated toxicity from whole brain radiotherapy, more advanced treatment planning techniques prioritize hippocampal avoidance, which depends on an accurate segmentation of the small and complexly shaped hippocampus. Purpose: To achieve accurate segmentation of the anterior and posterior regions of the hippocampus from T1 weighted (T1w) MRI images, we developed a novel model, Hippo-Net, which uses a mutually enhanced strategy.The proposed model consists of two major parts: 1) a localization model is used to detect the volume-of-interest (VOI) of hippocampus. 2) An end-to-end morphological vision transformer network is used to perform substructures segmentation within the hippocampus VOI. The substructures include the anterior and posterior regions of the hippocampus, which are defined as the hippocampus proper and parts of the subiculum. The vision transformer incorporates the dominant features extracted from MRI images, which are further improved by learning-based morphological operators. The integration of these morphological operators into the vision transformer increases the accuracy and ability to separate hippocampus structure into its two distinct substructures.A total of 260 T1w MRI datasets from Medical Segmentation Decathlon dataset were used in this study. We conducted a five-fold cross-validation on the first 200 T1w MR images and then performed a hold-out test on the remaining 60 T1w MR images with the model trained on the first 200 images. The segmentations were evaluated with two indicators, 1) multiple metrics including the Dice similarity coefficient (DSC), 95th percentile Hausdorff distance (HD95), mean surface distance (MSD), volume difference (VD) and center-of-mass distance (COMD); 2) Volumetric Pearson correlation analysis.In five-fold cross-validation, the DSCs were 0.900±0.029 and 0.886±0.031 for the hippocampus proper and parts of the subiculum, respectively. The MSD were 0.426±0.115mm and 0.401±0.100 mm for the hippocampus proper and parts of the subiculum, respectively. Conclusions: The proposed method showed great promise in automatically delineating hippocampus substructures on T1w MRI images. It may facilitate the current clinical workflow and reduce the physicians’ effort. In radiaTo determine whether the hippocampus is atrophic, clinicians often need to segment the bilateral hippocampus on MRI scans and analyze their shape and volume., 27 ThisThe existing automatic hippocampal segmentation methods can be categorized into two main types: atlas-based methods and machine learning-based methods. Atlas-based methods can be further divided based on the number of atlases used in the segmentation process into single-atlas-based, average-shape atlas-based, and multi-atlas-based approaches. For instance, Haller et al. first proposed to use the single-atlas-based approach for hippocampal segmentation., 10 HoweMachine learning-based methods can be further classified into traditional machine learning-based approaches and deep learning-based approaches. Traditional machine learning-based approaches mainly include support vector machine (SVM), Markov random field (MRF), principal component analysis (PCA), et al., 17 For Because convolutional neural network (CNN) models can automatically extract the pixel feature information from images, they have been widely used in multiple medical image analysis tasks. For examHowever, most existing deep learning-based methods ignore the spatial information of the hippocampus relative to the entirety of the human brain. As a result, they cannot effectively fuse the shape features and the semantic features, which leads to lower segmentation accuracy. Hippocampal tracing began from anterior where the head is visible as an enclosed gray matter structure inferior to the amygdala, and continued posteriorly using surrounding white matter or CSF as boundaries. Subiculum (posterior parts of hippocampus) was included in the hippocampus. Delineation stopped when the wall of the ventricle was visibly contiguous with the fimbria. The subiculum occupies a portion of the para-hippocampal gyrus in the mesial temporal lobe and is a component of the medial temporal memory system. Therefore, in this work, we aim to develop a novel deep network framework to segment the hippocampus by introducing a spatial attention mechanism to capture the spatial location information of the hippocampus relative to the brain. We also designed a cross-layer dual encoding shared decoding network to extract the semantic characteristics of the hippocampus. By combining the spatial location information and semantic characteristics of the hippocampus, we enhanced the segmentation accuracy of the hippocampus. In this study, we trained a novel morphological visual transformer learning-based hippocampus substructure segmentation for accurate segmentation of the anterior and posterior regions of the hippocampus from T1 weighted (T1w) MR images.22.1The proposed model consists of two deep learning-based subnetworks, i.e., a localization model and a segmentation model. The localization model is a hippocampus) detection network that is used to detect the volume-of-interest (VOI) for both the hippocampus proper and parts of the subiculum from theDuring inference, the trained localization model takes a hippocampus T1w MR image as input and detects the VOI of hippocampus as the first step. Then, the cropped image within the VOI is sent to the segmentation model, i.e., morphological visual transformer, to segment the substructures. Finally, based on the detected coordinates derived by the localization model, the segmented contour is converted back to its original coordinates to obtain the final segmentation.2.2The aim of the localization model is to crop the image to a VOI that only covers the hippocampus to ease computational task of substructure segmentation. In order to preserve the spatial information of substructure, the coordinate the detected VOI is recorded during testing. Thus, the localization of ground truth hippocampus is used to supervise the localization model. To derive it, the manual contour is needed. For a set of MR images The localization model design is inspired by a recently developed focal modulation network, which is used in object detection. The locaGiven input MRI function. After seThen, by using a neural network layer, we aim to derive the estimation of C, labeled as 2.3For the next step, the MRI  is used. AfterwarAfter the MVT step, consolidation can be used to transform the segmentation back to the original coordinate system To supervise the MVT, a combination of two loss functions is used, which are generalized cross entropy loss The 2.4In total, 260 T1w MR images from Medical Segmentation Decathlon were used in this study.[We conducted a five-fold cross-validation study on the first 200 T1w MR images. Then, a hold-out test was performed on the remaining 60 images using a model trained on the first 200 images. The segmentation was evaluated with multiple quantitative metrics including the Dice similarity coefficient (DSC), 95th percentile Hausdorff distance (HD95), mean surface distance (MSD), volume difference (VD) and center-of-mass distance (COMD). A Bland-Altman analysis and volumetric Pearson correlation analysis were also performed.2.5The investigated deep learning networks were designed using Python 3.6 and TensorFlow and implemented on a GeForce RTX 2080 GPU that had 12GB of memory. Optimization was performed using the Adam gradient optimizer. The learning rate was 2×10–4. With the batch size setting of 20 during training, the percentage of utility of GPU memory is 96%. Once the network was trained, it only takes 1.5 mins for hippocampus segmentation. To demonstrate the utility of morphological operator, an ablation study was conducted. Namely, we tested the performance of the proposed method of with and without using morphological operator. To further demonstrate the significance of the proposed work, we compared the proposed method with another popular segmentation models, cascaded U-Net (CasU) and visu33.1The visual comparison between the proposed method and comparing methods are shown in The linear correlation coefficient calculated as target volume of ground truth and segmentation, is shown in The quantitative metrics of the proposed method and the alternate methods from the 200 cases’ cross-validation and 60 cases’ hold-out test are listed in In the hold-out test using external datasetthe proposed model is significantly superior to the alternate approaches, as shown in 4A novel hippocampus segmentation method  is proposed by introducing a localization mechanism to aid segmentation and designing the morphological visual transformer network for substructures segmentation. The localization model is used to detect the VOI of hippocampus. The end-to-end morphological vision transformer network is used to perform substructures segmentation within the hippocampus VOI. The substructures include the anterior and posterior regions of the hippocampus, which are defined as the hippocampus proper and parts of the subiculum. The vision transformer incorporates the dominant features extracted from MRI images and is improved by learning-based morphological operators. The morphological operators integrated into the vision transformer enhance the ability to separate the hippocampus structure into two substructures.Due to limited computational resources, our method focused on domain incremental learning with a cropped region for analysis. We plan to test the performance of our method in a class incremental setup. As the visual transformer contains several orders of magnitude larger number of parameters due to the self-adapting process as compared to the traditional CNNs, it is essential to investigate an effective optimization method to reduce the amount of GPU memory allocation as well as simplify the overall ViT U-Net architecture.Our MVT is a supervised method, which means it still requires accurate manual contours as training labels. Currently, there are semi-supervised learning methods that can learn features from unlabeled data. We will extend the proposed method with the ensemble approach to improve its generalization performance by integrating the supervision learning and semi-supervised learning methods from the limited labeled data and large-scale unlabeled data of MRIs in a future study.The auto-segmentation of substructures of hippocampus has significant clinical relevance. For example, in hippocampal sparing whole brain radiation therapy (HA-WBRT), current 5We have developed a novel deep learning-based method to accurately segment the anterior and posterior of hippocampus. Our results showed good performance in terms of DSC and VD between the segmentation result and the ground truth.