Cth−/−) pregnant mice display any features of preeclampsia. Cth−/− females developed normally but showed mild hypertension (~10 mmHg systolic blood pressure elevation) in late pregnancy and mild proteinuria throughout development/pregnancy. Cth−/− dams had normal numbers of pups and exhibited normal maternal behavior except slightly lower breastfeeding activity. However, half of them could not raise their pups owing to defective lactation; they could produce/store the first milk in their mammary glands but not often provide milk to their pups after the first ejection. The serum oxytocin levels and oxytocin receptor expression in the mammary glands were comparable between wild-type and Cth−/− dams, but the contraction responses of mammary gland myoepithelial cells to oxytocin were significantly lower in Cth−/− dams. The contraction responses to oxytocin were lower in uteruses isolated from Cth−/− mice. Our results suggest that elevated homocysteine or other unknown factors in preeclampsia-like Cth−/− dams interfere with oxytocin that regulates milk ejection reflex.Elevated plasma homocysteine levels are considered as a risk factor for cardiovascular diseases as well as preeclampsia—a pregnancy disorder characterized by hypertension and proteinuria. We previously generated mice lacking cystathionine γ-lyase (Cth) as cystathioninuria models and found them to be with cystathioninemia/homocysteinemia. We investigated whether Cth-deficient ( Elevated plasma levels of homocysteine, a sulfur-containing amino acid intermediate in methionine metabolism, are widely known as an independent risk factor for cardiovascular diseases (CVDs) such as myocardial infarction, stroke, and venous thromboembolism ,3,4,5,6.Cbs−/− survivors  were found to be infertile (our unpublished observation), pregnancy complications could not be investigated. In our previous study, we generated mice lacking cystathionine γ-lyase (Cth), which is another essential transsulfuration enzyme downstream of Cbs, as a model for cystathioninuria (MIM 219500)  219500) . Our hom 219500) N-acetylcysteine administration [2S) levels and increased the blood pressure/irregular branching of the fetal vasculature of the placenta, both of which were reversed by the application of GYY4137, a slow-releasing H2S donor [Cth−/− dams. In this study, we report preeclampsia-like hypertension/proteinuria during late pregnancy as well as partial lactation (milk ejection) failure due to impaired oxytocin responses in Cth−/− mice. Our results shed light on the novel, clinically relevant roles of Cth and homocysteine in the systemic circulation.Previous studies have also shown that the Cth expression was upregulated in the livers of lactating mouse  or rat  dams, anstration . Moreovestration  while Ct2S donor . TherefoCth+/− and homozygous Cth−/− female mice grew in a similar manner as wild-type (WT: Cth+/+) females during juvenile development, gestation, and lactation periods  than those of the respective WT females (left). Systolic blood pressures during lactation periods and diastolic blood pressures in all periods  were indistinguishable between the Cth genotypes . Urinary protein levels  were significantly higher (p < 0.05) in virgin Cth−/− mice than those in the respective WT mice, which lasted to G15.5 , the females C left. Sto G15.5 A. Urinar+/− mice A. Serum Cth+/− pups born to Cth−/− dams  were significantly higher (p < 0.01) than that in Cth+/− pups born to WT dams   in nearly half of the litters of Cth−/− dams while not all the Cth+/− littermates in any of the litters of WT dams died (data not shown). In the remaining half of the litters born to Cth−/− dams, all the Cth+/− pups survived to adulthood even though they showed slightly retarded growth in early development  in − males) A. Comparf hunger B. Althoual hours F. In conCth−/− dams was equivalent to that of the mammary glands in WT dams . Histological analyses localized Cth expression in the myoepithelial cells of WT mouse mammary glands, but this finding was absent or unclear in those of Cth−/− mouse mammary glands , its levels were indistinguishable between WT and Cth−/− dams , WT uteruses markedly responded to oxytocin (0.1 unit/mL) with increased frequency (×4.50) and higher peak values  (Cth−/− uteruses were much milder (×1.95) in contraction frequency (Cth−/− mice show homocysteinemia [Cth−/− uterus. Although the application of high concentrations of methionine (2 mM) failed to affect both the contraction frequency and magnitude, homocystine (200 µM) showed a trend toward lower contraction magnitudes in both spontaneous and oxytocin-induced conditions  without altering the contraction frequency (Cth was expressed in the uterus A and oxyificant) A–C. In crequency A–C. As Cteinemia , we nextrequency A–C.Cth−/− female mice, their responses to vasopressin, another peptide hormone that resembles oxytocin (only two amino acid substitutions in a nine amino acid peptide) and that acts on the Oxtr-ortholog receptor, vasopressin receptor 2 (V2r) [Cth−/− female mice .If the systemic oxytocin signaling is impaired in  2 (V2r) , could a 2 (V2r) ; howeverCth−/− dams. To evaluate another oxytocin-mediated signaling in the CNS [Cth−/− male mice. When a male mouse is exposed to an “unfamiliar” female in his home cage, he spends much of his time in the anogenital inspection of the novel individual during the brief social encounter. Upon the repetitive exposures to the “familiar” female, the time spent for investigation usually declines with a full recovery following the introduction of a new female [Cth−/− male mice displayed declines in the investigation time upon the repetitive exposures to the “familiar” females , and the recoveries of the time upon new females in a similar fashion  that can be regulated by oxytocin within the central nervous system (CNS) as well as substantially impaired peripheral oxytocin-mediated responses, milk ejection and uterine contraction, in  the CNS , social w female ,37,38. WCth−/− virgin adult female mice exhibited serum amino acid profiles that were different from those of the respective WT mice as we previously reported in 2-week-old female/male-mixed pups  Cth−/− mice display (hyper)homocysteinemia/cystathioninemia [Cth−/− mice require dietary cyst(e)ine as an essential amino acid (unlike WT mice) [Cth−/− mice show reduced endogenous H2S levels [Cth−/− mice display increased vulnerability to paraquat [Cth−/− mice appeared normal under normal condition and were fertile [Cth is a transsulfuration enzyme essential for homocysteine clearance, cysteine biosynthesis, endogenous production of the gasotransmitter Honinemia ; (2) CthWT mice) ; (3) CthS levels ,40,41, aparaquat , dietaryparaquat , acetamiparaquat ,44, cardparaquat , unilateparaquat ,46, and paraquat . However fertile . Patient fertile . TherefoCth−/− dams showed hypertension during pregnancy and mild proteinuria  during development/pregnancy  and in the initial nursing  [Cth−/− and heterozygous Cbs+/−  dams, as previously reported in Cbs+/− dams by Sonne et al. [Cth−/− dams  . We did e et al. . In addi−/− dams  that couResponses or behaviors displayed by the female that specifically support the development and growth of her pups constitute a set of responses termed as maternal behavior. These include (1) parturitional responses such as neonate stimulation, amniotic fluid consumption, and placentophagia; (2) offspring-directed responses such as retrieval, licking/grooming, nursing/crouching (warming), and nest building; and (3) offspring-related responses, including maternal aggression, increased food consumption, and reduced anxiety to enhance exploratory activities . Among tq-coupled Oxtr and thereby activates phospholipase C and intracellular calcium signaling, which triggers various downstream signaling pathways. Previous studies using Oxt−/− and mice lacking Oxtr (Oxtr−/−) revealed that both Oxt−/− and Oxtr−/− females have normal fertility, pregnancy, and parturition, but they were unable to eject milk; therefore, all pups born to these mice die within 24 h of birth due to the deficiency of milk [Cth−/− dams could survive  reduced uterine contraction magnitudes  although both levels were much higher than those in their respective WT mice  [Cth−/− dams influenced on (high) tHcy in their whey  oxytocin signaling less active in ctively) . This coeir whey  but not eir whey , and mayIn conclusion, this study revealed preeclampsia-like features and partial lactation failure in cystathioninuria model mice. Cth-deficient cystathioninuria patients are currently buried because they are considered free of apparent clinical manifestations and, unlike Cbs-deficient homocysteinemia patients, they are not detected in the current newborn screening that detects hypermethioninemia . As lactCth+/− mice were generated and backcrossed for 10 generations to C57BL/6J inbred strain  [Cth+/− males and females were bred to obtain Cth−/− mice. Mice were housed in an air-conditioned room , kept in a 12-h dark/light cycle, and allowed free access to a CE-2 standard dry rodent diet (CLEA Japan) and water. Before the surgeries, mice were anaesthetized with isoflurane. All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals, 8th Edition published by the US National Research Council and were approved by the Animal Care Committees of Showa Pharmaceutical University ., Japan) . The N10Blood pressure was measured using a BP-98A tail-cuff manometer  ,28. BrieBlood and urine were collected from isoflurane-anaesthetized mice through cardiac and urinary bladder punctures, respectively. Serum (or urinary) levels of total protein, creatinine, urea nitrogen, AST, and ALT were measured using commercial colorimetric assay kits from Fujifilm-Wako . For the blood oxytocin measurements, blood was collected from the retro-orbital plexus. Serum was prepared and its protein components were denatured by 0.05% trifluoroacetic acid and removed by the MonoSpin C18 column  with acetonitrile elution. After acetonitrile evaporation, oxytocin contents were measured using the Oxytocin ELISA Kit  according to the manufacturer’s instruction.For examining the nesting activity score, postpartum mice were monitored using an infrared camera, and pictures were taken every 10 min before the birth and until 6 h after birth, or during 6–12 h after birth. When the dam crouched over more than half of her pups in one place, the dam was judged to have a nest. When the dam had a nest in 0–9, 10–18, 19–27, and 28–36 shots among a total of 36 shots, the nesting activity scores were evaluated as 0, 1, 2, and 3, respectively. For the retrieving activity, the latency to retrieve 1–4 pups after 5 pups were placed in the diagonal position of a rectangle cage (225 mm × 338 mm × 140 mm high) was examined. When more than half of the pups headed to their dam under her arms or belly in 0–9, 10–18, 19–27, and 28–36 shots among the total 36 shots, the breastfeeding activity scores were evaluated as 0, 1, 2, and 3, respectively. Namely, the breastfeeding activity score becomes higher when the dam has likely more chance to breastfeed their pups. The actual confirmation of breastfeeding was not executed not to stimulate lactating dams.Mammary glands were isolated from lactating dams after isoflurane anesthetization. For whole-mount Carmine alum staining , isolateFor Cth immunostaining, after the deparaffinization with xylene/ethanol, the samples (on the slide glasses) were immersed in Immunosaver  at 98 °C for 45 min for antigen activation and blocked with 10% normal donkey serum, 1% casein, and 0.1% Tween 20 in phosphate buffered saline (PBS). The samples were incubated with anti-CTH rabbit polyclonal antibody ,59 (1:10w/v) Triton X-100, 0.1% (w/v) phenylmethylsulfonyl fluoride, and Complete EDTA-free Protease Inhibitor Cocktail ) using a MicroSmash-100R (MS-100R) homogenizing system  and zirconia beads . Homogenates were centrifuged at 10,000× g for 10 min at 4 °C, and the supernatants were recentrifuged in the same way. After heating  with the sample buffer, samples (20 µg/lane) were resolved on 10% pre-cast PAGE gels, and then transferred to the Immobilon PVDF membrane (Millipore) and subjected to western blotting. Cth was detected with anti-CTH mouse monoclonal antibody ; 1:5000 in Can Get Signal immunostain Solution 1 (Toyobo)) and mouse IgGκ BP-HRP -Tween 20). Oxr was detected with anti-Oxr rabbit monoclonal antibody ; 1:5000 dilution in Can Get Signal immunostain Solution 1) and anti-rabbit IgG, HRP-linked whole Ab donkey antibody . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected with anti-GAPDH rabbit monoclonal antibody ; 1:10,000 in TBS-Tween 20) as a loading control. Chemiluminescence detection was performed using Chemi-Lumi One Ultra  and the ATTO WSE-6100 LuminoGraph I imager .Each tissue aliquot (~100 mg) was homogenized in a lysis buffer (50 mM Tris-HCl (pH7.4), 150 mM NaCl, 1 mM EDTA, 1% ) at 37 °C. After 5 min of holding, methionine , homocystine , or vehicle (AEF) was applied to the chamber. After 30 min of incubation, oxytocin  was applied. After 5 min of recoding the contraction frequency, the chamber was washed once with AEF and filled with AEF containing 25 mM KCl to obtain the maximal contraction. The contraction (/relaxation) frequency per min and average peak heights (magnitudes) relative to those of 25 mM KCl were recorded.Adult female mice (8–10-week-old) were subcutaneously injected with 10 µg of 17β-estradiol (in 100 µL of 50% DMSO/50% PBS) on their backs and, after 2 days, uteruses on both sides were dissected out from anaesthetized mice. The uterus (~1.5 cm long) was mounted between two steel hooks in an isolated tissue chamber of the conventional Magnus apparatus containing artificial extracellular fluids  was placed in the home cage of each male mice (n = 5 each) for a 1-min confrontation. Then the stimulus female was removed to an individual cage for 10 min. We repeated this sequence on the same male and female for four trials with 10-min intervals. In the 5th dishabituation trial, we introduced a new unfamiliar female. Behavior was video-recorded and the duration (s) spent for anogenital inspection was counted during each 1-min confrontation.Social memory/recognition test was performed as described previously . Both 8–g for 10 min at 4 °C to remove the fat contents, and then at 150,000× g for 10 min at 4 °C to collect the whey samples. The serum and whey samples were analyzed for their amino acid concentrations using amino acid/thiol-derivatization reagents as previously described in detail [Lactating dams (whose pups were removed before 2 h) were subcutaneously injected with oxytocin (4 units) and breast milk was collected by nipple massaging. The breast milk was centrifuged at 2300× n detail .n: sample numbers). Statistical comparison was performed using the two-tailed unpaired Student’s t-test, Mann–Whitney U test (only in p values less than 0.05 denoted a significant difference.Data were expressed as mean ± SD ( only in E,G, or K


We investigate the effectiveness of network attack strategies when the attacker has only imperfect information about the network. While most existing network attack strategies assume complete knowledge about the network, in reality it is difficult to obtain the complete structure of a large-scale complex network. This paper considers two scenarios in which the available network information is imperfect. In one scenario, the network contains link errors  due to measurement errors, and in the other scenario the target network is so large that only part of the network structure is available from network sampling. Through extensive simulations, we show that particularly in a network with highly skewed degree distribution, network attack strategies are robust against link errors. Even if the network contains 30% false links and missing links, the strategies are just as effective as when the complete network is available. We also show that the attack strategies are far less effective when the network is obtained from random sampling, whereas the detrimental effects of network sampling on network attack strategies are small when using biased sampling strategies such as breadth-first search, depth-first search, and sample edge counts. Moreover, the effectiveness of network attack strategies is examined in the context of network immunization, and the implications of the results are discussed. The network attack problem has received much attention, and several network attack strategies have been proposed –4. The nWhile most existing network attack strategies assume complete knowledge about the network , 2, 4, iMelchionna et al.  performeWhile the effects of link errors on network attack strategies have been studied, the effects of network sampling, which is a typical cause of errors introduced in the networks, on network attack strategies have not been studied before. The networks of interest nowadays are huge, and network sampling is unavoidable to analyze such large-scale networks –19. HoweIn this paper, we aim to reveal the effectiveness of network attack strategies when the attacker has only imperfect information about the network, extending the work by Melchionna et al. . We consThe main contributions and findings in this paper can be summarized as follows.We evaluate the effectiveness of CI, a state-of-the-art network attack strategy, under imperfect information. Our results show that CI is effective when the complete knowledge on the network is available whereas CI is sensitive against network uncertainty. When the level of network uncertainty is large, the effectiveness of CI is comparable with that of the conventional Degree strategy.We investigate how the network sampling affects the effectiveness of network attack strategies. We show that the attack strategies are far less effective when the network is obtained from random sampling, whereas the detrimental effects of network sampling on network attack strategies are small when using biased sampling strategies such as breadth-first search, depth-first search, and sample edge counts.We evaluate the effectiveness of network attack strategies in the context of network immunization , 24. OurG =  when a proportion p of all nodes was removed based on the attack strategy. For this study, we assumed that perfect information about network G was unavailable and the attackers only know network G′ = , a network with unknown errors. We used the attack strategies to determine which nodes of network G′ to attack. We then investigated the connectivity of the true network G with the same nodes removed.We investigated the connectivity of the undirected network G. One is a network that expresses connections between autonomous systems (AS) , and the other is a network that expresses connections between peers, namely a peer-to-peer (P2P) network [http://snap.stanford.edu/data/p2p-Gnutella04.html). We also used two types of synthetic network for network G. For generating synthetic networks, we used the Barabási–Albert (BA) model [m = 2 for generating networks with similar average degree with the AS network. For the GRG model, the radius for connecting nodes was 0.015 to generate connected networks while making the average degree be not far from other networks. BA graphs and GRG graphs were generated with R igraph package (https://igraph.org/). The characteristics of each network are shown in We used two types of real network for network  network , 26 (avaA) model  and the G′ from network G: a network with link errors and a sampled network. We created the network with link errors using the same procedure as in Melchionna et al. [E|δ (0 ≤ δ ≤ 1) randomly selected links from network G and added |E|α (0 ≤ α ≤ 1) links to randomly selected node pairs within the network to create incomplete network G′. Here, α and δ are the parameters that controlled the magnitude of the incompleteness. To create the sampled network, we followed the procedure outlined by Tsugawa and Kimura [ps of nodes from network G to create incomplete network G′. Sampling node v enabled us to obtain the associated links. In incomplete network G′ = , V′ is the set of sampled nodes, and E′ is the set of links between the set of nodes in V′.We created two incomplete networks a et al. . Specifid Kimura . Specifid Kimura , and  a strategy based on degree centrality (Degree) [G and then evaluated its connectivity by using the size of its GC as the evaluation index.Using incomplete network (Degree) , (ii) a (Degree) , and (ii(Degree) . We remoG′ to determine which nodes to attack. We immunized the nodes to be attacked in network G and simulated the virus diffusion based on the susceptible–infected–removed (SIR) model [t = 0, we randomly selected one non-immunized node in network G and changed its state to infected. The remainder of the non-immunized nodes are susceptible (but not infected). At any given time t, infected nodes v cause adjacent susceptible nodes u to become infected at infection rate β. In addition, infected nodes transition to the removed state at recovery rate γ. To serve as an index to quantify the extent of virus spread, we determined the number of non-infected nodes (susceptible and immunized nodes) at the time when the aforementioned stochastic processes converge and node states stop changing. The stochastic processes were realized using the random number generator in the Python NumPy module (https://www.numpy.org/).Furthermore, in this study we simulated the spread of viruses to evaluate the effectiveness of attack strategies when applied to the immunization problem. We used incomplete network R) model . The SIRG′ for each parameter used to determine the magnitude of incompleteness. For each incomplete network G′, we determined the nodes to attack, and calculated the size of the GC, and the average of the GCs were obtained. In addition, we conducted 1,000 independent virus spread simulation runs for each incomplete network G′ to determine the average number of non-infected nodes. We used l = 1 and l = 2 as parameters for the CI attack strategy. In addition, we used an infection rate of β = 0.1 and a recovery rate of γ = 0.01 in the virus spread simulations.For the ensuing experiments, we created 100 incomplete networks p of nodes removed from the AS, P2P, BA, and GRG networks and the proportion of the largest connected components when the nodes are removed. These figures compare different magnitudes of incompleteness.First, we investigated how link errors affected the effectiveness of each attack strategy. Figs In the AS network, the magnitude of incompleteness has hardly any effect on the size of the largest connected components under each of the attack strategies . In contl = 2) is more effective than other strategies when the complete network is available. By contrast, when the magnitude of incompleteness is large, the attack strategies deliver similar effects. Even in the GRG network, the difference between CI and other strategies is small. These results suggest that CI, which is a state-of-the-art attack strategy, experiences a decline in effectiveness similar to that with other strategies when the magnitude of incompleteness is large.Next, we compared the attack strategies under the same magnitudes of incompleteness Figs , 8 and 9G and node ranking obtained from incomplete network G′. To investigate how the node rankings based on the attack strategies are changed by link errors, we investigated the Spearman’s rank correlation between the node ranking obtained from the ground-truth network Next, we investigated how node sampling affects the effectiveness of the attack strategies. Figs Our results show that random sampling has an extremely strong effect on the Degree strategy in each network. However, the effects of other sampling methods are much smaller than those of random sampling. Next, we compared different attack strategies under the same sampling methods and sample sizes Figs  and 18. G and node ranking obtained from sampled network G′ to investigate how the node rankings based on the attack strategies are changed by node sampling. For calculating the rank correlation, we considered that non-sampled nodes have the lowest rank. We again investigated the Spearman’s rank correlation between the node ranking obtained from the ground-truth network Finally, we investigated the effectiveness of the attack strategies when applied to the network immunization problem. The size of the GC in the experiments thus far expresses the extent of the damage from virus spread in the worst case. For this section, we conducted virus spread simulations based on the SIR model with immunized nodes, and we evaluated the average extent of the damage from virus spread. Figs l = 2) in the GRG network, we confirmed that the effects of link errors are not so large.These results show that in most networks, the effectiveness of each attack strategy at controlling virus spread is essentially the same, for networks with link errors as well as complete networks. Except for the results of CI Click here for additional data file.S2 Fig(EPS)Click here for additional data file.S3 Fig(EPS)Click here for additional data file.S4 Fig(EPS)Click here for additional data file.S5 Fig(EPS)Click here for additional data file.S6 Fig(EPS)Click here for additional data file.S7 Fig(EPS)Click here for additional data file.S8 Fig(EPS)Click here for additional data file.S9 Fig(EPS)Click here for additional data file.S10 Fig(EPS)Click here for additional data file.S11 Fig(EPS)Click here for additional data file.S12 Fig(EPS)Click here for additional data file.


The fabricated passive probe is tested under a 3-coil inductive link to evaluate power transfer efficiency (PTE) and power delivered to a load (PDL) for feasibility assessment. The minimum PTE/PDL at 137 MHz were 0.76%/240 μW and 0.6%/191 μW in the air and lamb head medium, respectively, with coil separation of 2.8 cm and 9 kΩ receiver (Rx) loading. Six hermetically sealed probes went through wireless hermeticity testing, using a 2-coil inductive link under accelerated lifetime testing condition of 85 °C, 1 atm, and 100%RH. The mean-time-to-failure (MTTF) of the probes at 37 °C is extrapolated to be 28.7 years, which is over their lifetime.A new class of wireless neural interfaces is under development in the form of tens to hundreds of mm-sized untethered implants, distributed across the target brain region(s). Unlike traditional interfaces that are tethered to a centralized control unit and suffer from micromotions that may damage the surrounding neural tissue, the new free-floating wireless implantable neural recording (FF-WINeR) probes will be stand-alone, directly communicating with an external interrogator. Towards development of the FF-WINeR, in this paper we describe the micromachining, microassembly, and hermetic packaging of 1-mm As neural interfaces strive to more effectively interact with the brain, neural recording and modulation of the future will need the ability to simultaneously interface with multiple sites distributed across large areas of the brain ,2. TheseA major barrier on the path to clinical viability of the intracortical BMIs is their failure in neural signal acquisition over extended periods in the order of the patients’ lifetime . MultiplTo replace the existing centralized, and thus large and anchored, neural interfaces, a new class of stand-alone BMIs are under development based on the idea that sufficiently small untethered implants with smooth surface and rounded corners, which are free-floating with the brain, can reduce these effects, improve biocompatibility, and extend the electrodes effective lifetime ,22. TensL1), the resonator coil underneath dura (L3), and the receiver coil around the ASIC (L4) [L4, will be used as an antenna to transmit raw neural data across a short distance to a headstage on top of the head. The headstage relays the neural data to a common server for post-processing through a microcontroller unit (MCU) with built-in Bluetooth Low Energy (BLE). The battery-powered headstage also includes an efficient power amplifier (PA) that supplies power to tens to hundreds of FF-WINeRs, distributed over wide cortical areas of interest, through the 3-coil inductive link.SIC (L4) . The samThe fabrication and assembly of the FF-WINeR probe are key aspects of the design that must be addressed at the early stages of development because they directly affect the design decisions made for the other components, such as area and power available for the active circuitry. Exploring the assembly and packaging of the mm-sized passive floating probe, which are similar to the final probes in every aspect, except for the ASIC, is necessary to discover the recording failure mechanisms due to hermetic sealing, manual handling, surgical procedure, or the adverse effect of foreign body reaction and scaring in the brain tissue. Even though there have been many studies concerning brain tissue response to insertion and presence of implantable neural interfacing devices, these were primarily constrained to traditional tethered electrodes ,25,26,27In this paper, we focus on microfabrication, assembly, wireless powering, and wireless hermeticity testing of the mm-sized FF-WINeR probes. Hermetically sealed passive probes can be utilized to explore their longevity and robustness in-vitro for future experiments toward the actual FF-WINeR system implementation and in-vivo testing. 2 IMDs via 2-coil inductive link with 1 cm transmitter-to-receiver (Tx-Rx) coil separation is in the order of 1% [Q) resonator , which offers considerably wider coverage and higher PTE [A comparative study shows that inductive power transfer has the highest power density among several power sources for mm-sized implantable medical devices (IMDs) . To enerer of 1% ,32. The gher PTE .The minimum required power delivered to a load (PDL), based on power consumption of a recent state-of-art neural recording ASIC is ~80 μW per channel: 2.6 μW for analog front-end (AFE) , 2.4 μW Vs and Rs represent the PA and its output resistance, respectively. The PA drives the Tx coil (L1) that is coupled to the resonator coil (L3) with coupling coefficient k13, which is in turn encompassing and coupled to one or more Rx coils (L4) with coupling coefficient k34. R1, R3, and R4 represent parasitic resistances of L1, L3, and L4, respectively, and RL represents the load resistance. Using tuning capacitors, C1, C3, and C4, all coils are tuned at ω0, the frequency of the sinusoidal carrier signal that is generated by VS at the PA output.L1 to the resonator coil, L3, can be determined as [Q1 = ω0L1/R1, Q3 = ω0L3/R3, and Q4L = Q4QL/(Q4 + QL), in which Q4 = ω0L4/R4 and QL = RL/ω0L4. Q1,Q3, and Q4 are the quality factors of the coils and QL is the load quality factor. PTE between L3 and the Rx coil(s), L4, can be approximated as [The PTE from mined as , (1)η13=total = η13 × η34, L3-L4 link should be optimized first, followed by L1-L3 link. More specifically, we optimize Q4L with pre-determined geometry of L4, which is defined by the size of ASIC in this application. k34 is highly dependent on the geometries of L3 and L4, and less so on their number of turns since both their mutual- and self-inductances of L3 and L4 increase with their number of turns [L1-L3 and L3-L4, represented by d13 and d34, respectively, are the other key parameters affecting PTE and PDL, which nominal values  are selected based on the human head and brain anatomy, as shown in To maximize ηrms. Under these constraints, the paramters in the Rx coil that can be optimized are the coil pitch, wire diameter, and number of turns. With the choice of an insulated bonding-wire , wire diameter is fixed at 25 μm. The coil pitch is somewhat difficult to control when the coil is wound using a manual a wire-bonding machine. Finally, the optimal number of turns is chosen to maximize Q4L using the High Frequency Structural Simulator .The size of Rx coil is often limited by constraints of the application ,36. For L4 is shown in Q4L vs. number of turns of L4 at 135 MHz, which varies from 11.5 to 17.2, indicating that the optimal number of turns is 7.The HFSS simulation model for 34. Here we have considered the outer resonator diamer, Do3 = 3.6 cm, to provide enough room for about 100 FF-WINeRs inside. The outer diameter of the Tx coil, Do1, is derived considering the distance between the Tx and resonator coils, d13 = 2.8 cm [There is a trade-off between the number of FF-WINeRs that fit inside each resonator, set by the diameter of the resonator and η= 2.8 cm , (3)Do1=L1 and L3 (d13), and L4 horizontal misalignment from the center of L3 (x34), as shown in The 3-coil inductive link is implemented based on the above design considerations to explore the achievable PTE and PDL versus the coil distance between L1 to L4 in the presence of L3 on the same plane with L4 using a ZVB4 vector network analyzer . The twisted red-white feeding line to L4 in L4, and can diminish the measurement accuracy. Therefore, we have adopted a calibration method, known as de-embedding, which is described in [x34 and d13 in the air is depicted in x34 = 0 mm, the min and max S21 are −24.45 and −21.2 dB when d13 = 42 mm and 28 mm, respectively. At d13 = 28 mm, the nominal Tx-resonator coils’ distance that maximizes S21, the min and max of S21 are −21.2 dB and −15.1 dB when x34 = 0 mm and 12 mm, respectively. Similarly, the minimum S21 is measured −22.26 dB in a lamb head medium, as shown in x34 = 0 mm and d13 = 28 mm. Based on the measured S21 and S11, the PTE can be derived as [Z0 is the characteristic impedance, 50 Ω. The achieved minimum PTE (with no rotation) in the air and lamb head medium using Equation (4) are 0.76% and 0.6%, respectively. Since the source power of the vector network analyzer is fixed at 15 dBm, the achieved minimum PDL in the air and neural tissue medium can be calculated as 240 and 191 μW, respectively. This level of PDL is sufficient to operate the state-of-art neural recording ASICs with small number of channels [The S-parameters are measured from ribed in . The de-rived as , (4)PTE=channels .L4 to be perfectly aligned with L3. This is expected to reduce the achievable minimum PTE and PDL of the 3-coil inductive link due to angular misalignment between L3 and L4. We measured the PTE and calculated PDL of the proposed link while rotating L4 from 0° to 90° in the lamb head tissue medium. The measurement was repeated 6 times for better accuracy because of difficulty in setting the angular rotation of L4 in the lamb head. L4 angular misalignment at x34 = 0 mm and d13 = 28 mm. With 15° L4 angular misalignment, PTE and PDL decease to 0.54% and 171 μW, respectively, with ~10% measurement error. They drop further down to 0.2% and 60 μW, respectively, with 45° misalignment. Therefore, for state-of-art operating power of 80 μW in [The brain surface is curved and filled with gyri and sulci, which make it unlikely for the small mm-sized 80 μW in ,33, the L4 is only 191 µW, its temperature elevation is negligible. On the other hand, circuit simulation using the fabricated coil parameters in L3 under abovementioned operating conditions. Considering the model and measurements presented in [2 surface area, is will result in ~1.5 °C. However, the surface area of L3 is 15 times larger than the implant presented in [For chronic implantation, the thermal elevation in the brain tissue surrounding the proposed 3-coil inductive link should be considered, even though a detailed modeling and measurement of thermal effects are out of the scope of this manuscript. Since PDL to ented in , if thisented in , and it Fabrication of the FF-WINeR probes requires a novel process flow due to the extremely small size of the device. The process includes micromachining, microassembly, and hermetic packaging. Fabricated probes should undergo a hermeticity testing to estimate their lifespan under harsh operating conditions inside the body.2 dice on a blank Si wafer, based on previously developed processes described in [For passive Si die fabrication, ultraviolet (UV) photolithography is used to define an array of 1 mmribed in ,41,42,43The procedure to microassemble the FF-WINeR probes is illustrated in L4 is one of the key steps in passive probe fabrication. One way is to utilize a fully-automated wire-bonder which bonding tip is programmed to move along a pre-defined trajectory around the core [C4, is a tuning capacitor to resonate with L4 at the operating frequency and the other is the load capacitor, CL, low-pass filtering the output of a voltage doubler for AC-to-DC conversion.Implementing the mm-sized the core . For proTo hermetically seal the FF-WINeR against the harsh environment in the body and maintain biocompatibility, a 5-μm thick parylene-C coating is applied on the assembled passive probe by vapor deposition . Even though parylene-C is biocompatible, a layer of Sylgard 184 PDMS coating  is added over parylene-C to extend the probe life-span and create a smooth and soft surface. The uncured PDMS mixture has many trapped air bubbles, which should be removed in a dessicator connected to a vacuum pump until the air bubbles are removed. The passive probe is then dipped in bubble-free PDMS, and hung upside down from a hook with the electrode tip inserted in a piece of styrofoam for at least 24 h to cure at the room temperature. Since biological fluids contain NaCl, KCl, phosphates, carbonates, enzymes, and other proteins, they create a harsh environment for the probe, while the surface of the probe can create a harmful environment for the surrounding cells . In suchiL, attached to a network analyzer with the device-under-test (DUT), which is the FF-WINeR Rx L4C4 tank coupled by the coupling coefficient, k14, in this case, placed in proximity. The basic principle is measuring the changes in reflected impedance of L4 and its tuning capacitance, C4, plus its parasitic capacitance, ΔC4p DUT due to water ingress in the package. iR and R4 are the parasitic resistance of iL and L4, respectively. The DUT reflected impedance onto the interrogator coil side is depicted in iL and L4. Moisture in the package can either nullify C4 by shorting the SMD capacitor or increase C4p by increasing dielectric constant. The relative dielectric of water is 80.1, 29.9 times larger than that of PDMS, which is 2.68 [L4 since the permeability of water is very close that of vacuum. The electrical characteristics of iL and L4 in the current FF-WINeR prototype are summarized in Our hermeticity testing method was inspired by the wireless capacitive sensing presented in . Figure  is 2.68 . Water lL1, the frequency at which it peaks, fZpeak can be estimated by solving Equation (6) for ω [i and ω4 are the resonant frequencies of iCiL and L4C4, respectively. Variations in fZ11peak, based on solving Equation (6) in Mathematica , are plotted in C4p, shifting fZ11peak by less than in a few 100 kHz, which is very small compared to the original fZ11peak, 384 MHz. On the other hand, the short circuited C4 can cause considerable frequency shift from 384 MHz down to 379.8 MHz. To estimate C4p when C4 is shorted, the FF-WINeR coil impedance, Z44, was measured when it was soaked in water. C4p can be estimated ~1 pF when L4 = 92 nH.If we calculate Z11, the input impedance seen through 6) for ω , (6)1− in years and describes as [hour is the failure rate per hour that can be defined as, F and T are the number of failures and test hours per device. The MTTF in this case is estimated to be 28.7 years. The small amount of water penetration into the hermetic package may be considered as a hermetic sealing failure. It should be noted that since the passive FF-WINeR has two layers of polymer coating, PDMS over parylene, water ingress into the PDMS layer does not necessarily lead to device failure despite causing a small change in the fZ11peak. The 5 μm parylene-C coating layer is considerably more resistant to water than the PDMS coating, and maintains functionality of the active circuit even though the L4C4 resonance frequency may shift as a result. This shift can be remedied, however, using the auto resonance tuning (ART) method, described in [In a widely used model for accelerated lifetime testing, the acceleration factor (AF) is defined based on the Arrhenius equation as , (7)AF=MTTFribed in .We have presented the structure, microfabrication, and assembly steps of a mm-sized implant toward a distributed free-floating wireless implantable neural recording (FF-WINeR) system, which can be used for safer and less invasive neural interfacing with potentially less damage to the neural tissue. The silicon chip that houses the ASIC is micromachined after standard CMOS processing to also acts as a substrate and provide mechanical support for the microwire electrodes and the bonding-wire coil, which is wound around the ASIC to offer higher Q compared to on-chip coils. Wireless power transmission to the FF-WINeR is designed based on 3-coil inductive link, using a high-Q planar implantable resonator in the same plane as the FF-WINeR probes and encompassing them. Preliminary experiments show that the required PTE and PDL for the desired functions in the ASIC are achievable with Tx coil separation of 2.8 cm. A simple method is developed for wireless hermeticity testing of the dual polymer coated FF-WINeR probes based on accelerated lifetime testing technology. We are now developing the ultra-low power and area efficient active circuitry for this probe and further improving the hermeticity testing methodology to achieve more accurate estimates of the FF-WINeR probe lifetime. Future steps also include in-vivo experiments on rat animal model.


Treatment of Class II malocclusion accompanied with a skeletal discrepancy is challenging. The approach of correction depends on several factors such as the status and pattern of growth, severity of the malocclusion, and patient cooperation. This case report describes a successful management of a 12-year-old young adolescent boy that was presented with a Class II division 1 malocclusion with an underlying skeletal discrepancy in horizontal and vertical dimensions. Growth modification was achieved by means of bite opening and unlocking the mandible together with Class II elastics and mechanics. Treatment was highly effective and efficient by achieving all treatment goals within a period of 18 months. Class II dental malocclusion considered as the second most prevalent malocclusion after Class I, with a prevalence range of 13%-24% . As descTreatment approach and modality for Class II division 1 with an underlying skeletal problem depend on several factors that should be considered by the clinicians such as chronological age, growth potential, skeletal maturity, severity of the condition, and patient motivation and cooperation , 11. TheSeveral studies showed that over the long-term treatment results achieved with Class II elastics are similar or comparable to those achieved with functional appliances, given that treatment started at an appropriate timing –17. BothIn the current case report, we discuss the treatment of a Class II division 1 malocclusion accompanied with a severe Class II skeletal discrepancy and hypodivergent growth pattern in a growing adolescent.12 years and 8 months young boy referred to the orthodontic specialty clinic with a chief complaint of “protruded front teeth”. Medical and dental history indicated no significant findings. Extraoral clinical examination revealed a symmetrical euryprosopic face type, convex soft tissue profile, retruded mandible, deep mentolabial sulcus, decreased lower facial third, and on smiling lower lip was trapped by the upper central incisors .st and 2nd molars. Mild anterior crowding with rotated canines . Patient had poor oral hygiene, plaque accumulation, and mild gingival inflammation. All permanent teeth are erupted while the 3rd molars are still developing  impinging overbite, deep curve of Spee (COS) of 3 mm, and unilateral posterior cross-bite on the upper left 1Cephalometric analysis revealed a severe skeletal Class II relationship (ANB = 7.8°) with horizontal growth pattern tendency , and anterior facial height is markedly reduced. Cervical vertebral maturation (CVM) indicates cervical stage 1 (CS1). Lower incisors are proclined (L1 − MP = 99.3°), and both lips are anterior to the E-line by 1 mm .The following were the list of treatment objectives: (1) improve the sagittal and vertical skeletal relationships; (2) enhance and guide mandibular growth; (3) resolve increased overjet; (4) establish Class I canine and molar relationship; (5) resolve the impinging overbite, deep COS, and traumatic occlusion; (6) resolve the left posterior cross bite; (7) resolve crowding and rotations; and (8) improve facial profile and smile esthetics.Given the patient's age and problem list, a nonsurgical and nonextraction treatment plan based on growth modification to enhance mandibular growth was developed. The primary plan presented to the patient and his family was to start with growth modification using a removable Class II functional appliance or cervical pull headgear with anterior bite plane followed by leveling and aligning with a fixed edgewise appliance. The option was not considered by the family due to increased cost from the use of additional device. In addition, the treatment duration could be prolonged especially if it was divided into two phases. Therefore, an alternative plan addressing those concerns was presented which involved bonding a fixed edgewise appliance, anterior bite turbo, and simultaneous growth modification using intermaxillary elastics taking advantage of the patient's early adolescence and prepubertal stage of development.The patient demonstrated a poor oral hygiene and few carious lesions, so before starting orthodontic treatment, oral hygiene instructions were given, and patient was referred for periodontal and restorative clearance.″ × 0.028″ MBT was bonded in both arches. Leveling and aligning lasted for 6 months, which were achieved by the following NiTi wire sequence  then followed by stainless steel (SS) working wires . SS arch wire expansion and criss-cross elastic from palatal of upper left first molar to buccal of lower left molar were used to correct the unilateral posterior cross bite on left molars.Once cleared, a full-fixed straight wire appliance of slot size 0.022In addition, to address the vertical and sagittal skeletal problems, anterior bite turbos palatal to upper central incisors were cemented ; gradualAfter 10 months in treatment, the antro-posterior canines and molars relationships were corrected. Final detailing and finishing included arch wire adjustments and power chain use for some remaining spaces closure. The fixed appliance was debonded after 18 months of active treatment. Clear vacuum retainers were delivered, and after 1 month, they were replaced by Hawley retainers with anterior bite plate on the upper appliance.The overall outcome of treatment was successful, and the patient's chief complaint was addressed. Treatment objectives were achieved in an efficient treatment duration (18 months and 14 visits). The skeletal and dental relationships were significantly improved. Facial profile showed great improvement as facial proportions were restored, mandible was advanced, convexity was reduced, and restored normal mentolabial sulcus. Moreover, smile esthetics were improved, and lip position was corrected with no entrapment . Intraory-axis 64.7°) to (y-axis 68.1°). Maxillary incisors were retroclined, and the deep bite was corrected by relative intrusion of a combination of molar extrusion and mandibular incisors intrusion and proclination. In addition, mandibular molars moved mesially while maxillary molars slightly distalized  to posttreatment , facial convexity decreased from 12.9° to 6.9°, mandibular plane angle increased from  to , total face height (Na-Gn) increased from 105.2 mm to 116.9 mm, lower face height (ANS-Gn) increased from 59.7 mm to 65.5 mm, and the hypodivergent growth pattern improved from ( Figures .At 1-year follow-up, the occlusion was stable with no significant relapse, and the patient demonstrated poor oral hygiene with mild gingival inflammation . LateralThis case report showed a successful management of a 12-year-old adolescent boy presented with a Class II division 1 malocclusion accompanied with a severe skeletal Class II discrepancy. Several key factors contributed to the success of this treatment mainly including early intervention at prepubertal stage, pattern of skeletal growth, patient's cooperation, and mechanics applied.The time of intervention in this case was of great impact in correcting the skeletal discrepancy. The treatment started when the patient was in the prepubertal stage of growth. The patient was at early stage of CVM CS1, , secondaIn the current case, upon opening the patient's bite and releasing the locked mandible from the impinging overbite, a great improvement in sagittal skeletal relation was achieved. Angle indicated that in Class II malocclusion, the lower dentition is locked in distal occlusion, and thereby, the mandible will be locked as well . SeveralIt was well noticed that most of the sagittal discrepancy was improved after removing the restriction from the impinging deep bite and unlocking the mandible, while the use of Class II intermaxillary elastics was for a short period still effective to add to the final sagittal correction. Class II elastics carried out further guidance and enhancement of mandibular growth toward normal skeletal relation, added considerable restriction to the growth of the maxilla, and improved the vertical skeletal relationship . DespiteThe vertical skeletal relationship improved significantly from the correction of the deep bite by use of reverse COS wires, Class II elastics, and bite turbos. Those mechanics helped to extrude the molars, intrude and procline the incisors, and facilitate further mandibular growth. As mentioned earlier, with Class II elastics, there is a clockwise rotation of the occlusal plane and mandible with a final outcome of increase in lower anterior face height , 20, 22.The skeletal growth pattern of the patient was a favorable adjunct in the management of his case and helped in accommodating the current treatment plan. The vertically hypodivergent mandibular plane angle, the forward rotation of the mandible, and the underdeveloped anterior face height facilitated in toleration of the applied extrusive mechanics and the resulted backward rotation of the mandible. On the opposite, cases of vertical growth pattern with high mandibular plane angle are challenging and difficult to treat, and applying such extrusive mechanics should be avoided as they would deteriorate the final results and will not work in favor of the patients while more control of the vertical dimension should be considered .Among the challenges that were faced in this case was to maximize the orthopedic changes using Class II elastics while minimizing elastics possible side effects. Proclination of lower incisors was significantly increased  from ClaClass II elastics considered a viable orthodontic option for correcting Class II skeletal discrepancy in growing patients, given the careful case selection based on timing of growth spurt, pattern of skeletal development, severity of the malocclusion, and patient's cooperation and motivation.Moreover, orthodontists should consider the benefits and potential side effects of Class II elastics use, plan that in the treatment, and be able to handle and direct them to the patient's favor once executed in treatment.


Penile Mondor’s disease is a rare condition characterized by thrombophlebitis of superficial penile dorsal veins. We report a case of a 26-year-old male who developed a lump on the dorsal surface of the penis following intensive masturbation after prolonged sexual abstinence. Physical examination revealed a firm, cord-like swelling with beaded texture with no localized regional lymphadenopathy. The patient was successfully managed with non-steroidal inflammatory drugs and was symptom-free on a follow-up. Mondor’s disease (MD) is a self-limiting and benign condition, recognized clinically by the presence of palpable cord-like induration on the body surface. The first case was reported in 1939 as a result of generalized thoracoabdominal phlebitis and as an isolated superficial penile veins thrombosis known as Penile Mondor’s disease (PMD) in 1958 . Other sA 26-year-old male with no significant past medical history and sexual abstinence of more than six months presented in the outpatient department with a cord-like swelling on the dorsal surface of the body of the penis for one day. He first noticed a small lump on the dorsal surface at the distal shaft that suddenly progressed to swelling, extending distally and wrapping like a cord around the shaft of the penis just proximal to glans. The patient had mild pain at rest and moderate discomfort on erection. He reported masturbating three times a day in the last seven days. He denied any recent systemic symptoms such as fever, urinary frequency/urgency, dysuria, hematuria, urethral discharge, local trauma, or any history of current or past sexually transmitted diseases. Physical examination revealed a firm cord-like swelling with beaded texture on the dorsum of the distal penis, extending and wrapping around the shaft just proximal to glans Figure . No locaMD is a rare clinical entity with a reported incidence of 1.39%. It is described as sclerosis of superficial thrombophlebitis of the veins of the anterior thoracoabdominal wall. PMD or Mondor’s cord has been historically reported as affecting the superficial veins on the dorsal surface of the penis. Table PMD is usually a clinical diagnosis. History and physical examination are reliable diagnostic tools that can reveal a palpable, firm cord-like lesion on the dorsal aspect of the penis. Doppler ultrasound can be utilized as next for differential diagnosis or if the physical examination is unclear. We used clinical judgment to diagnose our patient. The presence of thrombosed segment and uncompressible vein with no signal flow on ultrasound plus high resistance, low-velocity blood flow in cavernous arteries on Doppler are supporting features for disease and helps to differentiate it from sclerosing lymphangitis . MRI useThe condition is usually self-limiting resolving within four to six weeks. Treatment is generally supportive and limited to the use of mostly non-steroidal inflammatory agents orally or anti-coagulants in topical forms, with a focus on relief of pain and inflammation -9. OtherPMD is a rare and underreported condition that manifests as cord-like swelling on the dorsal surface of the penis. Attention should be given to secondary underlying causes, such as thrombophilia, malignancy, or sexually transmitted diseases. Ultrasonography with Doppler is the imaging of choice for diagnosis, and invasive or expensive investigations should be avoided. Management should be focused on reassurance about the condition and pain alleviation. Surgery should be reserved for only refractory cases.


Streptomyces sp. strain BR123, isolated from rhizospheric soil that exhibited promising antimicrobial properties, was sequenced and assembled. Here, we report an 8,157,040-bp genome sequence with a G+C content of 72.63%. This genome sequence enlightens the genes responsible for the production of secondary metabolites and antimicrobial compounds by this strain.The genome of  Streptomyces sp. strain BR123, isolated from rhizospheric soil that exhibited promising antimicrobial properties, was sequenced and assembled. Here, we report an 8,157,040-bp genome sequence with a G+C content of 72.63%. This genome sequence enlightens the genes responsible for the production of secondary metabolites and antimicrobial compounds by this strain.The genome of  Streptomyces members are aerobic, Gram-positive filamentous bacteria belonging to the order Actinomycetales of Actinobacteria. Bacteria in this genus are well reported for their production of bioactive secondary metabolites that have great biofunctional diversity and different applications, such as antibacterial, antifungal, antiviral, anticancer, immunosuppressant, insecticidal, and herbicidal, which make them suitable for use as pharmaceuticals and in agricultural industries  medium (https://sourceforge.net/projects/samtools/files/samtools/1.9/), BEDtools 2.28 (https://bedtools.readthedocs.io/en/latest/), and BWA-MEM 7.12 (de novo assembly was performed (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/) was used to assemble metrics.Paired-end reads yielding 30× coverage were checked for quality control, adapted reads were trimmed by using Trimmomatic 0.30 with a sliding window quality cutoff of Q15 , and quaMEM 7.12 . By the erformed , and annN50 value of 22,797 bp, and the largest contig has 109,551 bp. The genome of Streptomyces sp. strain BR123 contains 8,157,040 bp with a G+C content of 72.63%, a value highly similar to those reported in Streptomyces spp. (The genome assembly yielded 723 contigs with an ces spp. , 14. TheJACBGN000000000, BioProject number PRJNA643667, SRA number SRR12527047, and BioSample accession number SAMN15423153. The 16S rRNA gene sequence has been deposited at DDBJ/ENA/GenBank under the accession number MT799988.This whole-genome shotgun project has been deposited at GenBank under whole-genome sequencing project number


There is now good evidence that small group teaching provides a fruitful academic environment, which optimises learning, particularly in the healthcare setting, and especially when compared to lectures. An individual student’s understanding of knowledge is increased when they are able to actively compare and build on their own understanding in conjunction with their peers. Small group teaching provides opportunities for learners to work collaboratively, and promotes team-building skills – skills that are essential to work within healthcare settings. The aim of this paper is to provide health professional students and early career health professionals involved in peer and near peer teaching, with an overview of approaches and tips to improve learner engagement when facilitating small groups. Health professional education occurs in a variety of contexts, including those within university, hospital, community-based and clinical settings. Curricula activities at the university target development of students’ knowledge of the basic sciences of healthcare , which are then integrated into the clinical setting, thus contextualising this knowledge. The clinical setting also plays a crucial role in developing students’ clinical skills, communication skills, and professionalism. The clinical application of the basic sciences also occurs in case scenario based small group teaching methods, such as problem based learning (PBL), Team-based learning (TBL), Case based learning (CBL), in the university setting –7; and cAn individual student’s understanding of knowledge is increased when they are able to actively compare and build on their own understanding in conjunction with their peers –16. Smalactive participation, ‘face-to-face’ contact between participants, and purposeful activities. When implemented with all three elements in play, the small-group context offers many benefits, and enhances students’ learning experiences in many ways. For example, small group learning has the potential to: . Af. Af31]. What went well during the lesson?What can be improved?Did the lesson cover the learning objectives I set at the start of the lesson?Was my questioning technique effective?How well did I engage learners?Where can I improve next time?Critical reflection is considered an essential step to effective education in healthcare, and should be practiced by facilitators . This in◦ Be open to the feedback being given as it is intended to be helpful◦ Avoid instantly dismissing feedback that does not match self-reflection◦ Avoid becoming defensive - instead engage in constructive discussion◦ Ask for specific examples to explain the feedback being given.Gaining feedback from learners can also assist in self-reflective practices . This caStudents being reluctant to engage in discussion with each otherStudents are not prepared for small group activitiesIndividual ‘free riders’ failing to contribute (may be shy or disinterested)Individual students dominating discussion or being disruptiveAttention being directed towards the facilitator, who is expected to provide answersFacilitator’s questions don’t go beyond the level of recallFacilitator’s lack of attempt to get students to answer their own questionsFacilitators providing insufficient/poor feedbackFacilitators talking too much, lecturing rather than facilitating.Although small group teaching offers many advantages, it may pose some difficulties and limitations for the facilitator and students. Group problems commonly stem from , 26, 32:The facilitator should reflect on why the problem is occurring, what can be done differently to help overcome the problem, and how accountability for success can be shared with the student group . DependiIndividual dominant students: summarise points and divert the discussion to others; indicate time pressure; give the group specific tasks.Quiet students: give them time to respond; divide the group into pairs for a task; positively reinforce any contribution.Attention being directed towards the facilitator: build on students’ responses to a limited extent by sharing clinical experiences or provide a clinical context where appropriate to heighten the relevance of the topic.Students receiving insufficient feedback: schedule a time for student feedback throughout the session, or afterward.Students attending unprepared for small group activities: ensure student accountability to their team members by including short tests at the beginning of class, which may also help to prevent late arrivals [arrivals .Small group teaching can be very rewarding, for both the learners and facilitators. However, successful small group facilitation requires appropriate facilitation methods to encourage active and purposeful participation, and enhance student learning. The facilitator’s role is crucial in encouraging the learners to interact with the content, and with their peers. Self-reflective practices and the use of feedback provide a valuable means to improve small group facilitation skills.


We discuss the impact of a Covid-19–like shock on a simple model economy, described by the previously developed Mark-0 Agent-Based Model. We consider a mixed supply and demand shock, and show that depending on the shock parameters (amplitude and duration), our model economy can display V-shaped, U-shaped or W-shaped recoveries, and even an L-shaped output curve with permanent output loss. This is due to the economy getting trapped in a self-sustained “bad” state. We then discuss two policies that attempt to moderate the impact of the shock: giving easy credit to firms, and the so-called helicopter money, i.e. injecting new money into the households savings. We find that both policies are effective if strong enough. We highlight the potential danger of terminating these policies too early, although inflation is substantially increased by lax access to credit. Finally, we consider the impact of a second lockdown. While we only discuss a limited number of scenarios, our model is flexible and versatile enough to accommodate a wide variety of situations, thus serving as a useful exploratory tool for a qualitative, scenario-based understanding of post-Covid recovery. The corresponding code is available on-line. The Covid-19 pandemic has buffeted the world economy and induced one of the most abrupt drops in output ever recorded. What comes next? Will the economy recover quickly as lockdown measures are lifted, or will the damage inflicted by the massive waves of layoffs be more permanent? In pictorial terms, will the economic crisis be V-shaped (quick recovery), as commentators were initially hoping for, or U-shaped (prolonged drop followed by a quick recovery), or perhaps W-shaped, with a relapse due either to further lockdowns, or to premature lifting of the economic support to households and firms? The possibility of an L-shaped crisis, with a permanent loss of output, is also considered. Or else, maybe, a “swoosh”, with a rapid drop followed by an excruciatingly slow recovery?  express the sensitivity of the firm’s target production to excess demand/supply. We posit that the coefficients i  are given by:x〛 = 1 when x ≥ 1 and 〚x〛 = 0 when x ≤ 0. The factor Γ > 0 measures how the financial fragility of firms influences their hiring/firing policy, since a larger value of Φi then leads to a faster downward adjustment of the workforce when the firm is over-producing, and a slower (more cautious) upward adjustment when the firm is under-producing. Γ itself depends on the inflation-adjusted interest rate and takes the following form:αΓ is a free parameter, similar to αc that captures the influence of the real interest rate.f thumb” , 18. In Price update. Following the initial specification of the Mark series of models  random variables and γ is a parameter setting the relative magnitude of the price adjustment. The factor f models , prices Wage update. The wage update rule follows the choices made for price and production. Similarly to workforce adjustments, we posit that at each time step firm i updates the wage paid to its employees as:t, U random variable and g modulates how wages are indexed to the firms’ inflation expectations. If i at time t with this amount of wages would have been negative, Wi(t + 1) is chosen to be exactly at the equilibrium point where i, is an increasing function of Wi. Hence, firms that want to produce more (hence hire more) do so by increasing Wi, as to attract more applicants.Note that within the current model the productivity of workers is not related to their wages. The only channel through which wages impact production is that the quantity The above rules are meant to capture the fact that deeply indebted firms seek to reduce wages more aggressively, whereas flourishing firms tend to increase wages more rapidly:If a firm makes a profit and it has a large demand for its good, it will increase the pay of its workers. The pay rise is expected to be large if the firm is financially healthy and/or if unemployment is low because pressure on salaries is high.Conversely, if the firm makes a loss and has a low demand for its good, it will attempt to reduce the wages. This reduction is more drastic if the company is close to bankruptcy, and/or if unemployment is high, because pressure on salaries is then low.In all other cases, wages are not updated.Profits and dividends. Finally, the profits of the firm δ of their cash balance θ is the Heaviside step-function. These dividends are then reduced from the firms’ cash balance and added to the households savings in ρ0 via a Taylor-like rule:ϕπ modulates the intensity of the central bank policy, ρ* is the baseline interest rate and π* is the inflation target for the central bank. The banking sector then sets interest rates for deposits ρd (for households) and loans ρl (for borrowing by firms). Defining f then determines how the impact of these defaults fall upon lenders and depositors—f interpolates between these costs being borne completely by the borrowers (f = 1) or fully by the depositors (f = 0). The total amount of  money M in circulation is kept constant and the balance sheet of the banking sector reads:The banking sector in Mark-0 consists of one “representative bank” and a central bank which sets baseline interest rates. The central bank also has an inflation targeting mandate. The central bank sets the base interest rate R (hiring/firing ratio) and Θ (bankruptcy threshold for firms) are important Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: PartlyReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: N/AReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: NoReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The Results section should be presented at the end, after describing the model and all approximations. In this section, the authors describe the parameters of the model before describing the model itself.99-106 and table 1 shows the empirical parameters of the model. However, their significance is not substantiated.The Politics section offers different scenarios for overcoming the crisis, but without prior formalization of the mathematical model, it is impossible to understand these scenarios.ABM models - depend on random values. Therefore, the simulation results may be unstable. It is not clear from Figures 4 and 5 this is the result of the work of an ensemble of models or a single simulation.The authors do not provide an analysis of the sensitivity and stability of the model from changes in various factors.The Model section should be moved to the beginning of the article.In this section it is expedient to formalize the model in the form of the scheme of interaction of various components-blocks of systemIt would be expedient to justify the choice of values of empirical parameters of the model to the characteristics of a particular country.It would be interesting to compare the effectiveness of the strategies of different countries in comparison with the results of the modelReviewer #2: The short title is better than long title that creates confusion.The study is interesting with the aim to suggest guidelines for economic recovery after COVID-19 pandemic crisis and shock of lockdowns.Introduction has to provide more theoretical background about COVID-19 and risk factors current and future in society to know the problem for health and economies (see suggested readings that have to be read and used in the text).The study is based on simulations and without empirical evidence. Of course, the recovery depends on countries. I suggest to clarify better research setting because countries are different …a recovery in Italy is different from a recovery in Sweden because of different structural indicators…. for instance, Italy has a high public debt and a similar shock generates different effect than other countries. Moreover, also the lockdown generates different effects according to the duration…longer lockdown generates a higher deterioration of economic systems than short one (see new literature).How these aspects are considered in the model?The contraction of some economies is higher than others.In short, there are manifold variables to control and I suggest whenever possible to discriminate the design of the model for countries that have some similarities, such as Spain and Italy, Germany and France, Scandinavian countries etc. to be more consistent with the reality and reduce the heterogeenity between countries and provide more reliable policy implications.As you know the applications of policies generates different effects according to the countries and the structure of their economies… I would like to know how it is possible a match between these different shapes L, W, etc. of recovery and different countries, and policy implications…. I know it is necessary a time consuming revision, but authors can provide more indications to provide a main contribution for political economy of growth post COVID-19.Conclusion has to present better manifold limitations of this study for the complexity of factors and heterogeneity of countries and in particular for the methods of inquiry based on simulations done in a computer lab.Suggested readings of relevant papers that have to be read and all inserted in the text and references to improve the study.Leach, M., MacGregor, H., Scoones, I., Wilkinson, A. 2021Post-pandemic transformations: How and why COVID-19 requires us to rethink development, World Development 138,105233Coccia M., 2020. National lockdown to cope with COVID-19 pandemic: effects (contradictory) on public health and (negative) on economic system, Working Paper CocciaLab n. 56D/2020, CNR -- National Research Council of Italy. Available at Research Square, DOI is: 10.21203/rs.3.rs-115665/v1Abuselidze, G., Slobodianyk, A. 2021 Pandeconomic crisis and its impact on small open economies: A case study of COVID-19 Advances in Intelligent Systems and Computing, 1258 AISC, pp. 718-728Coccia M. 2020. An index to quantify environmental risk of exposure to future epidemics of the COVID-19 and similar viral agents: Theory and Practice. Environmental Research, Article number 110155, DOI: 10.1016/j.envres.2020.110155Yoshino, N., Hendriyetty, N. 2020 The COVID-19 Crisis: Policy Recommendations for Japan. Economists' Voice , 17(1),20200017Iuga, I.C., Mihalciuc, A. 2020Major crises of the XXIst century and impact on economic growth. Sustainability (Switzerland), 12(22),9373, pp. 1-20Reviewer #3: The article is well written and provides interesting insights about the response of the economy to the pandemic due to COVID-19. In general, the authors did a good job explaining the purpose of the study, and described properly the instruments they used to develop the study. I have some minor comments for the authors to be addressed before the study can be accepted for publication.1. I suggest including some literature in the introduction (Lines 40-44) about using ABM to understand the impacts of COVID. For example, please review the following article.https://doi.org/10.1016/j.ssci.2020.1050222. Line 204The authors could explain better why they selected 2 policies. Are there different policies that simulate the same effects from the policies that the authors used in the study in the literature? If so, explain why other policies are not selected.3. I suggest to the authors to include in the study some discussion about how the results were validated and verified. If not, please also state that clearly.4. I would also recommend to the authors to include the word economy in the title.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  4 Feb 2021We thank the reviewers for their comments which have helped to improve the manuscript. All points raised by the reviewers have been addressed in the Rebuttal letter attached with the resubmission.Attachmentrebuttal_letter.pdfSubmitted filename: Click here for additional data file. 15 Feb 2021V -, U -, L - or W-shaped economic recovery after COVID-19: Insights from an Agent Based ModelPONE-D-20-37904R1Dear Dr. Sharma,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. In this vein, there are required a couple of revisions regarding references.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Stefan Cristian Gherghina, PhD. Habil.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressedReviewer #3: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The author took into account the comments of the reviewer. Therefore, I believe that the article can be published in a journalReviewer #2: V -, U -, L - or W-shaped economic recovery after COVID-19: Insights from an Agent Based ModelI have read thoroughly the revised version of paper. The authors have done considerable additional work, and addressed all concerns and criticisms in the revised manuscript, which I believe has improved substantially in the theoretical framework, study design and discussion of results.Finally, I suggest to revise some citations of papers to update them and correctly cite some articles in references, such as:https://ssrn.com/abstract=3560337--Fornaro, Luca and Wolf, Martin, 2020. Covid-19 Coronavirus and Macroeconomic Policy (March 2020). CEPR Discussion Paper No. DP14529, Available at SSRN: https://doi.org/10.1016/j.scitotenv.2021.145801--Coccia M. 2021. The relation between length of lockdown, numbers of infected people and deaths of Covid-19, and economic growth of countries: Lessons learned to cope with future pandemics similar to Covid-19. Science of The Total Environment, Available online 12 February 2021, 145801. --Eichenbaum M. S., Rebelo S., Trabandt M., 2020. The Macroeconomics of Epidemics, NBER Working Papers 26882, National Bureau of Economic Research, Inc. doi = 10.3386/w26882After that, the paper is OK.Reviewer #3: Thank you for preparing a response to my comments. Since this is a very current and interesting topic, I recommended the article for publication.**********what does this mean?). If published, this will include your full peer review and any attached files.7. 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Thermally treated watermelon juice (TW) presents a strong unpleasant smell, resulting in poor consumer acceptance. It is necessary to identify the key off-flavor compounds in TW. Solid-phase microextraction (SPME) and solvent-assisted flavor evaporation (SAFE) coupled with gas chromatography–olfactometry–mass spectrometry (GC–O–MS) were applied to the extraction and analysis of the volatile compounds in TW. Five aroma-active compounds and seven off-flavor compounds were quantitatively analyzed by the standard curve method. Based on the flavor dilution factor (FD), odor attribute, odor activity value (OAV) of volatile compounds, and partial least-squares regression (PLSR) analysis, seven key off-flavor compounds were preliminarily identified as follows: (E)-2-heptenal, decanal, octanol, diisopropyl disulfide, hexanol, (E)-2-decenal, and (E)-2-octenol. Aroma recombination proved that these off-flavor compounds above had a negative impact on the overall flavor in TW. Omission experiments were taken to confirm them further. Finally, octanol, diisopropyl disulfide, and (E)-2-decenal were identified as the most potent off-flavor compounds in TW. In China, the annual planting area and yield are approximately 1.85 million hmHeating watermelon juice considerably affects its quality owing to its thermo-sensitive nature . HoweverTo identify the off-flavor compounds in thermally treated watermelon juice (TW), it is necessary to understand the mechanism of flavor change. Current studies have focused on the aroma of fresh watermelon (FW) or its juice. The flavor compounds in watermelon juice are mainly C6 and C9 aldehydes, ketones, and alcohols, such as nonanal, (E)-6-nonenol, -2,6-nonadienal, (Z)-3-nonenal, -2,6-nonadienal, and -3,6-nonadienal ,8. DevelIn the present study, the aroma profiles of FW and TW were compared to identify the off-flavor compounds. The key off-flavor compounds in TW were determined using gas chromatography–olfactometry–mass spectrometry (GC–O–MS), odor attributes, and partial least-squares regression (PLSR) analysis. Furthermore, aroma recombination and omission experiments were performed to verify the key off-flavor compounds.2SO4 used for extraction and separation of flavor substances were all analytical reagent and provided by Banxia Scientific Instruments Co. Ltd. .n-Alkanes (C7-C30) for the retention index (RI) calculation, 2-methyl-3-heptanone , -2,6-nonadienal (95%), (E)-2-heptenal (97%), decanal (≥98%), octanol (≥99%), -2,6-nonadienol (≥95%), (E)-2-octenal (≥95%), diisopropyl disulfide (≥96%), hexanol (≥99%), (E)-2-nonenal (97%), (E)-2-decenal (≥95%), nonanol (98%), and (E)-2-octenol (97%) were purchased from Sigma-Aldrich . Hexane, diethyl ether, n-pentane, and anhydrous NaQilin is the main cultivar with a large yield in China. Twenty watermelons  were one-time selected and purchased from Beijing Yonghui Supermarket randomly on June 10th, 2018. The pulp was blended , and quickly filtered through a nylon mesh (200 mesh). FW was analyzed immediately. Batches of 100 mL juice were vacuum-packed with an odorless vacuum packaging bag with aluminum foil immediately and frozen using liquid nitrogen. All the packed juices (100 mL for each bag) were stored at −80 °C for further analysis. TW was processed using a water bath at 70 °C for 20 min according to the pasteurization method (the juice flavor changed greatly under this condition through the pre-experiments. A thermometer was inserted into the juice through the bag and timing began when the center temperature of juice reached 70 °C) .Volatile organic compounds of a 10 mL sample of watermelon juice with 1 μL internal standard  added in a 40 mL headspace vial  were extracted manually with SPME using a 2 cm, 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane fiber . Samples were allowed to equilibrate for 20 min at 40 °C before collection for 40 min with continuous stirring at 100 rpm  .v/v) and stirred for 8 h. Fifty microliters of 2-methyl-3-heptanone (0.816 μg/μL) were added as the internal standard. Then, volatiles were extracted from the solvent extracts by distillation for 2 h at 10−4 torr. The solvent layer was concentrated to 2 mL with a Vigreux column  after being dried through an anhydrous Na2SO4 column. The volume further reduced to 0.2 mL under a flow of nitrogen [One hundred milliliters of watermelon juice was mixed with 150 mL diethyl ether-pentane mixture  with an olfactometer  was used to analyze the volatile compounds. DB-WAX and DB-5 chromatographic columns  were employed to separate these compounds .−1 scan rate. Compounds were identified according to NIST 14.0  mass spectra libraries.Mass spectra in electron ionization mode were recorded at 70 eV and a mass/charge range of 50–350 amu at a 2.0 scan sGC–O analysis was carried out on polar and non-polar columns by three well-trained panelists. Before analysis, the panelists were trained by smelling the odors of the model solutions of reference compounds at different concentrations. The aroma descriptor, intensity value, and retention time were recorded by the panelists during analysis . If two v/v). The process was ceased when aromas could not be smelled. FD factor could be expressed as the ratio of the initial and final concentration of the flavor compound of juice. The compounds with FD higher than 1 were identified as key flavor compounds.Two types of dilution analysis were used to identify key flavor compounds, including headspace dilution analysis (HDA) for SPME and aroma extraction dilution analysis (AEDA) for SAFE, as described by Zhang et al. . For SPMThe compound identification was carried out by NIST 14.0 mass spectrum database, the retention index (RI), and odor properties. The key flavor compounds were confirmed further compared with the standard compounds. RI was calculated using Equation (1) and compared with the references.and Cn+1 .Y-axis represented the peak area ratio of analyte to the internal standard, and the X-axis represented the concentration of reference standards of the analyte [C0 is the concentration of the compound before reference standard being added, C1 is the detected concentration after reference standard being added, and C2 is the reference standard concentration being added [GC conditions were the same as mentioned above, and a selected ion monitor (SIM) was selected as the mass acquisition mode. Both extract methods were used to quantify different ions. Reference standards with a series of concentrations were prepared. The mixed reference standards (1 μL) were added to the sample gathered with internal standard 1 μL, 0.816 μg/μL). The extraction procedures were the same as the methods of SPME and SAFE as described above. Standard curves were established based on the peak area and concentration of each compound. The  analyte . In orde μg/μL. TiC is a compound concentration and iOT is the odor threshold of this compound. Compounds with OAV ≥1 were considered to contribute to the juice flavor [The equation to calculate OAV was as below: e flavor .Twelve panelists  were recruited from the Molecule Sensory Laboratory of Beijing Technology and Business University. Members of the sensory panel were trained for 2 months to familiarize the watermelon aroma characteristics. Sensory evaluation was strictly in accordance with To verify the obtained result of off-flavor compounds, the aroma recombination system was prepared and compared with the actual watermelon juice flavor . A modelMixture models were produced by omitting one kind of key off-flavor compounds from the aroma recombination system. The same sensory panels evaluated the flavor similarity between the omission and recombination models in a triangle test. Every sensory evaluation was conducted in triplicate .Analysis of variance was carried out to determine the significance at a 95% confidence interval using SAS 9.3 software . Partial least-squares regression (PLSR) was implemented using SIMCA-P 11.5 software . All experiments were performed in triplicate.In order to extract and analyze the flavor compounds of watermelon juice comprehensively, the extract methods of SPME (non-solvent extract) and SAFE (solvent extract), the chromatographic columns of DB-Wax (strong polar) and DB-5 (weak polar)  were applied in this study. Thus, the aroma-active compounds and key off-flavor compounds could be identified accurately and thoroughly. Fifty-seven compounds in FW and TW were extracted and identified . Five grAmong the 17 compounds identified in FW and TW, aldehydes were the most prevalent and potent aroma compounds in watermelon or its juice . Of the Eleven alcohols, six of which  were identified with FD >1 in both FW and TW. Hexanol, octanol, (E)-2-octenol, nonanol, -3,6-nonadienol, and -2,6-nonadienol could be formed due to reduction of the corresponding aldehydes. All identified compounds were previously reported for watermelon aroma, and presented fatty, fruity, floral, green, or cucumber-like smell ,23. AmonAmong the seven identified ketones, geranyl acetone was found with FD >1 in both FW and TW. They were reported in the melon fruits with different rootstocks, seedless watermelon, and watermelon juice treated by high-intensity pulsed electric fields ,14,23. GFive sulfides, diisopropyl disulfide, and dipropyl trisulfide were identified with FD >1 both in FW and TW. Few reports have described sulfides in watermelon or its juice, although these compounds exist in Jiashi muskmelon . These tAs shown in Thermal treatment promoted the formation and release of flavor compounds with higher FDs in TW. After thermal treatment, watermelon juice produced a strong unpleasant flavor. As shown in As shown in As shown in p < 0.05). In summary, these seven volatile compounds were preliminarily identified as the key off-flavor compounds in TW.The odor attributes of “fatty” and “cooking flavor” concentrated on the left side of the loading plot, which presented significant positive correlations with compounds: (E)-2-heptenal, diisopropyl disulfide, (E)-2-decenal, decanal, (E)-2-octenol, octanol, and hexanol  was calculated to guarantee the accuracy of the quantitative results . The staOAV was another index that contributed aroma compounds to the overall flavor . As showp < 0.05) being observed. This indicated that the identification and quantitation experiments were accurate, and that the aroma-active and key off-flavor compounds were precisely identified [According to the quantitative results of TW, the aroma recombination was carried out to verify the contribution of the seven key off-flavor compounds to the overall flavor of TW. As shown in entified ,43. Therp ≤ 0.001). This result revealed that octanol played a very important role in the overall flavor in TW. Among them, the absence of diisopropyl disulfide and (E)-2-decenal showed significant differences (p ≤ 0.05 and p ≤ 0.01), which agreed with the higher FDs and OAVs, respectively. Therefore, these two compounds also had a significant influence on the overall flavor of TW. The omission experiments: hexanol and (E)-2-octenol (OAV ≤1 but higher FDs) also showed significance differences (p ≤ 0.01 and p ≤ 0.05). However, no significant differences were observed when (E)-2-heptenal and decanal were omitted from the recombination in spite of their higher FDs and OAVs. Comprehensive consideration of significance difference of omission experiment, FD, and OAV, octanol , diisopropyl disulfide , and (E)-2-decenal  were identified as the most potent off-flavor compounds in TW.In order to further verify and rank the contribution level among the seven key off-flavor compounds, omission experiments were divided into seven groups . When ocThe above three compounds were previously reported as off-flavor compounds. Octanol contributes to the off-flavor of whey protein concentrate during storage of 45 °C for 15 weeks, and it changes the organoleptic properties of packaged food ,45. In bIn conclusion, seven key off-flavor compounds in TW were preliminarily identified by concentration variation, odor attributes, and PLSR analysis. Five aroma-active compounds and seven key off-flavor compounds were quantified by the standard curve method. They were further confirmed by both OAV and FD. The aroma recombination was employed to verify the contribution of the seven key off-flavor compounds to the overall aroma profile. In addition, the omission experiment from the recombination system was carried out to confirm the results. Octanol, diisopropyl disulfide, and (E)-2-decenal were identified as the most potent off-flavor compounds in TW.