However, DCD hearts undergo in vivo ischemia with a variable “ischemic period.” In addition, the DCD hearts are exposed to an intense catecholamine surge that is not seen with ex vivo perfused hearts. Thus, the severity of mitochondrial damage in in vivo ischemia hearts could differ from the ex vivo ischemia hearts even following the same period of ischemia. The aim of our current study is to identify the mitochondrial dysfunction in DCD hearts and propose strategies to protect mitochondria. Adult Sprague Dawley rat hearts underwent in vivo or ex vivo ischemia for 25 min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from hearts following ischemia. We found that both ex vivo and in vivo ischemia led to decreased oxidative phosphorylation in SSM and IFM compared to time control or DBD hearts. The proportion of damage to SSM and IFM, including proton leak through the inner membrane, was higher with ex vivo ischemia compare to in vivo ischemia. Time control hearts showed a decrease in SSM and IFM function compared to DBD hearts. The calcium retention capacity (CRC) was also decreased in SSM and IFM with ex vivo and in vivo ischemia, indicating that ischemic damage to mitochondria sensitizes mitochondrial permeability transition pores (MPTP). Our study found differential mitochondrial damage between the in vivo ischemia and the ex vivo ischemia setup. Therefore, consideration should be given to the mode of ischemia while evaluating and testing myocardial protective interventions targeting mitochondria to reduce IR injury in hearts.Heart transplantation is the ultimate treatment option for patients with advanced heart failure. Since hearts from donation after brain death (DBD) donors are limited, donation after circulatory death (DCD) donor hearts could be another source for heart transplantation. DCD process involves ischemia-reperfusion (IR) injury. Mitochondrial dysfunction contributes to IR and is well established in the  Heart transplantation is the ultimate treatment option for patients with advanced heart failure; however, this option is limited due to the available donor hearts, which primarily come from donation after brain death (DBD) donors . Donatioc and apoptosis-inducing factor (AIF), are released into cytosol leading to apoptosis in caspase-dependent and caspase-independent manner  ischemia model  or ex vivo (buffer perfused) ischemia models. Knowledge gained from such a study will lay the foundation for future interventions aimed at protecting DCD hearts and preserving their function.There are two populations of cardiac mitochondria: subsarcolemmal mitochondria (SSM) located underneath the plasma membrane and interfibrillar mitochondria (IFM) that exist between the myofibrils . In the l models . An IFM Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health   . The stuIn vivo global ischemia model adult male Sprague Dawley rats  were randomly assigned to a DBD (n = 6) or an in vivo ischemia group (n = 5). The rats were placed on a heating pad to maintain the body temperature of 37°C, anesthetized using sodium pentobarbital (100 mg/kg) administered intraperitoneally, followed by endotracheal intubation with 14-gauge angio-cath for respiratory support. Heart activity was monitored by continuous EKG recording. Heparin (300 U) was administered intraperitoneally and allowed to circulate for 1 min, followed by intramuscular administration of a paralytic agent  and allowed to circulate for 5 min. Rats in the DBD group underwent a thoracotomy, and hearts were procured while beating. Hearts were then processed for mitochondrial isolation, as described previously  administered intraperitoneally. Hearts were then isolated under general anesthesia and mounted on the Langendorff setup , oxygenated with 95% O2 – 5% CO2 to maintain a pH of 7.4. Rat hearts were assigned to time control (n = 11) or ex vivo ischemia (n = 9) groups. In the ex vivo ischemia group, hearts were perfused for 15 min equilibration with K-H buffer followed by 25 min of global ischemia at 37°C. In the time control group, hearts were only buffer perfused for 40 min without ischemia (ff setup . Hearts in vivo ischemia hearts once procured fresh were placed into cold (4°C) buffer A . Mitochondria were isolated using our previously published protocol  for 10 min at 4°C. The homogenate was centrifuged at 500 × g for 10 min, and the supernatant was further centrifuged at 3,000 × g for 10 min to sediment IFM. The SSM and IFM were washed twice and then suspended in 100 mM KCl, 50 mM MOPS, and 0.5 mM EGTA. Mitochondrial protein content was measured by the Lowry method , succinate (20 mM) plus 7.5 μM rotenone (complex II substrate), and N,N,N′,N′ tetramethyl p-phenylenediamine, 1 mM (TPMD) ascorbate  + 7.5 μM rotenone were used  at 30°C, as previously described . Mitochoere used .c at 550 nm on the addition of 40 μM reduced decylubiquinone and 1 mM cytochrome c to 3 μg/mL mitochondrial protein with or without 100 μM antimycin A. Complex III activity was expressed as antimycin A sensitive rate (complex III activity without antimycin A subtracted complex III activity with antimycin A). Citrate synthase (CS) activity was quantified in cholate-solubilized mitochondria by measuring the rate of 5,5-dithiobis(nitrobenzoic acid)-reactive reduced coenzyme A  at 37°C as previously described (Frozen and thawed SSM/IFM were solubilized in 5% cholate (pH 7.2), then diluted with KME  buffer to 0.1% protein concentration. Hewlett-Packard model 8453 spectrophotometer  was used to measure respiratory enzyme activities. Complex II activity was measured as the rate of oxidation of dichlorophenol-indophenol (DCPIP) at 600 nm in the presence of mitochondria (2 μg/mL), 1 mM oxidized decylubiquinone, and 1 M succinate. Complex II activity was expressed as the thenoyltrifluoroacetone (TTFA) sensitive rate (complex II activity without TTFA subtracted complex II activity with TTFA). Complex III activity was measured as the initial rate of the reduction of cytochrome escribed .Mitochondrial cytochrome contents  were determined using our published method . SSM/IFM2PO4, 5 mM succinate in 20 mM Tris/HCl, pH 7.4. MPTP was induced by sequential pulses of calcium (5 nmol/min). Succinate was used as a substrate in that the CRC is much higher in mitochondria oxidizing a complex II substrate compared to a complex I substrate  was used to assess calcium-induced MPTP opening in isolated mitochondria using LS-5 fluorimeter  . Mitochoubstrate . Extra-m2O2 production in freshly isolated SSM/IFM was determined using the oxidation of the fluorogenic indicator amplex red in the presence of horseradish peroxidase. The concentrations of horseradish peroxidase and amplex red in the incubation were 0.1 U/mL and 50 μM, respectively. Fluorescence was recorded in a fluorimeter  with 530 nm excitation and 590 nm emission wavelengths. Standard curves obtained by adding known amounts of H2O2 to assay medium in the presence of the reactants (amplex red and horseradish peroxidase) were linear up to 2 μM. In a typical experiment, SSM/IFM were incubated at 0.1 mg of protein/mL at 30°C. H2O2 production was initiated in mitochondria using glutamate (20 mM) or succinate (20 mM) + rotenone (7.5 μM) as complex I and complex II substrates, respectively  based on parametric one-way analysis of variance (ANOVA)  for SSM with succinate as substrate for state 3 respiration, 2 mM ADP-stimulated respiration and ADP/O ratio; SSM TMPD-ascorbate oxidation, and IFM respiratory control ratio (RCR) with glutamate as substrate failed the test of normality and were compared using the Kruskal–Wallis one-way ANOVA followed by Dunn’s test for multiple groups. Data in in vivo ischemia in t-test (two-tails) after data passed tests of normality. Comparison of SSM cytochrome c content between DBD and in vivo ischemia groups in in vivo ischemia group: one determination of SSM state 4 respiration with glutamate as substrate, aa3 content, Continuous variables were expressed as a mean ± standard error of the mean, or a median with interquartile range (IQR) if appropriate. Data were tested for normality and equal variance before performing statistical comparisons between four groups  . Alternaustments . Specifiin vivo ischemia, ex vivo ischemia, and time control groups compared to the DBD hearts. In particular, the protein yield in the ex vivo ischemia group was significantly less compared to the in vivo ischemia group. Compared to DBD hearts, the rate of state 3 respiration (ADP-stimulated) in SSM oxidizing complex I substrate (glutamate) was significantly decreased by 33 and 44% for in vivo and ex vivo ischemia groups, respectively. Compared to the DBD group, state 4 respiration (ADP-limited) in SSM was not significantly different in in vivo ischemia group. Interestingly, time control perfusion alone led to a significant increase in state 4 respiration in SSM compared to DBD hearts. Ex vivo ischemia significantly increased state 4 respiration in SSM compared to in vivo ischemia group. RCR was calculated as a ratio of state 3/state 4 respiration. Compared to DBD SSM, all other groups had significantly reduced RCR  was equally decreased in in vivo ischemia and ex vivo ischemia groups compared to DBD or time control groups. ADP-stimulated OXPHOS was also decreased in in vivo ischemia SSM oxidizing complex II substrate (succinate) in a manner similar to what is observed with complex I substrate  was used. DNP transfers proton across the inner membrane collapsing the inner membrane potential and essentially bypassing complex V  stimulated respiration was also decreased in IFM with ex vivo ischemia compared to in vivo ischemia for the oxidation of complex I substrate but not with complex II substrate.There was no significant difference in the protein yield from IFM of D hearts . In partia group . CompareD hearts . Ex vivotrol IFM . In IFM ischemia . The stain vivo ischemia IFM oxidizing complex I  and IFM (35% less) following in vivo ischemia compared to DBD control hearts. The in vivo ischemia did not alter the contents of aa3 (an index of cytochrome oxidase protein content). b and c1 (components of complex III) in SSM compared to DBD hearts. There were no differences in the contents of cytochrome b and c1 in IFM between DBD and in vivo ischemia hearts. The in vivo ischemia led to decreased aa3 content in IFM compared to DBD hearts.Mitochondrial cytochrome in vivo ischemia hearts was decreased compared to DBD hearts, indicating an increased susceptibility to MPTP opening in in vivo ischemia heart mitochondria  vs SSM-in vivo ischemia (50.7 ± 1.9), p = NS; IFM-DBD (56.6 ± 2.9) vs IFM-in vivo ischemia (51.2 ± 2.0), p = NS].The amount of Hin vivo ischemia model based on current clinical DCD practice and the time-tested ex vivo ischemia model where most of the published data on ischemia and reperfusion emanated from. The main findings of our study are, (a) both ex vivo and in vivo ischemia damages OXPHOS function of SSM and IFM, (b) the proportion of damage to SSM and IFM including proton leak through the inner membrane are significantly higher with ex vivo ischemia, (c) this excess damage to mitochondrial ETC from ex vivo ischemia was not apparent when DNP was used to exclude the complex V from ETC, (d) time control hearts showed a decrease in SSM function compared to DBD hearts driven by greater state 4 respiration; and lastly, (e) the CRC was decreased in SSM and IFM with ex vivo and in vivo ischemia.Heart transplantation is limited by the available donor hearts, and DCD hearts have the potential to expand the donor pool. There are several steps involved in clinical DCD heart transplantation to ensure a successful outcome. Our study is focused primarily on identifying and quantifying the damage to mitochondria in the DCD set-up. It is essential to develop a reliable and reproducible animal model to study the effects of warm ischemia in a DCD heart to propose potential interventions to mitigate the ill effects of IR injury, which are inherent to the DCD process. Our study quantified the differences that exist between the in vivo ischemia model, we carefully examined the correlation between the in vivo ischemia times with derangements in mitochondria (OXPHOS) and heart function quantified through direct measurement of left ventricle developed pressure (LVDP). With a shorter duration of ischemia, the myocardium suffered less damage. With increasing duration of in vivo ischemia hearts sustained greater damage, in these hearts, when the initial LVDP was <50% compared to control hearts, they did not show any signs of functional recovery upon reanimation over 60 min. However, when the residual heart function was >50% of control hearts, there was a trend toward slow but steady improvement in LVDP over 60 min of reanimation  ex vivo ischemia  process but are not seen with the ex vivo ischemia model. While the ex vivo model allows for an abrupt cessation of oxygen delivery in an isolated heart via non-blood based perfusate, the in vivo ischemia process is relatively gradual and occurs in the setting of residual blood. There are distinct hormonal and hemodynamic changes that occur with the brain and circulatory death process  along with a drop in arterial PO2 from 107 to 23 mmHg. It is clear from this seminal study that in less than 2 min from the withdrawal of ventilation, effective tissue hypoxia sets in. By magnetic resonance imaging, the right ventricle size increased by 18% and left ventricle size decreased by 12% from baseline values. Since mitochondria are responsive to mechanosensitive stimuli  but the supply of small amounts of oxygen may also have promoted the production of ROS . These findings were also corroborated in the porcine DCD model, where only three out of sixteen hearts developed ventricular tachycardia or ventricular fibrillation and the rest maintained an organized ventricular activity through the 20 min of observation. This is an intriguing finding as pig hearts are very prone to arrhythmias with both hypoxia and acidosis  using H+/Na+ exchanger inhibitors  cychibitors , and (d)hibitors ,an inhibhibitors . The effin vivo ischemia and ex vivo ischemia model. Reperfusion injury further augments the ischemic injury to mitochondria, which was not studied here. Contact with blood during in vivo ischemia process may have afforded protective mechanisms to mitochondria that are not accounted for in ex vivo ischemia model. Isolated mitochondria are commonly used to assess mitochondrial function in the hearts following ischemia-reperfusion. Since mechanical forces are required to permeabilize the cell membrane to release mitochondria, some physical damage to mitochondrial cannot be avoided. Ischemia may increase cell and mitochondrial fragility and potential damage. The decreased protein yield in mitochondria from ischemic hearts suggests the potential loss of mitochondria during the isolation process. Had those mitochondria remained in the analyzed samples, the differences between the study and control samples would have been more pronounced. Measurement of respiratory enzyme activities from whole heart homogenate may be an alternative approach to address this question in the future. The use of different substrates is an important tool to localize defects at the ETC. Glutamate, succinate, and TMPD-ascorbate are used in this study as complex I, II, and IV substrates, respectively. Duroquinol (DHQ) is a complex III substrate used in our previous study (in vivo ischemia group of rats; however, studies from previous DCD animal models confirmed the onset of tissue ischemia in less than 2 min from the withdrawal of ventilation. Lastly, ethical aspects of organ transplantation from DCD donors differ from that of the DBD donors and are not within the scope of this basic science study. An interested reader may find discussions on this subject in relevant publications (A small sample size may not have the power to uncover differences that may further exist between the us study . Using Dications .ex vivo ischemia compared to in vivo ischemia. In particular, the oxidative phosphorylation function of SSM are severely impaired with ex vivo ischemia compared to in vivo ischemia. CRC is equally decreased with both forms of ischemia, even without reperfusion. Possible reasons for the relatively less damage to mitochondria in the setting of in vivo ischemia could be due to gradual onset of ischemia, exposure to low concentrations of oxygen during ischemia, myocytes remaining in contact with blood, and a complex interplay between metabolic acidosis, myocyte stretch, and excess catecholamine levels.Injury to mitochondria is greater with Dr. Toldo is PI of investigator-initiated studies from Olatec Therapeutics LLC and Kiniksa Pharmaceuticals.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by the Hunter Holmes McGuire Veterans Administration Hospital, Virginia Commonwealth University, and Institutional Animal Care and Use Committee.MQ, OA, ST, EL, and QC participated in the research design. OA, QC, and RC conducted the experiments. MQ, OA, EL, QC, LK, and RC performed the data analysis. MQ, EL, OA, LK, and QC wrote and contributed to the writing of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The behavioural impacts of prenatal exposure to ethanol include a lower IQ, learning problems, anxiety and conduct disorders. Several components of the neurochemical network could contribute to the long-lasting effects of ethanol embryonic exposure. Adenosine is an important neuromodulator, that has been indicated to be affected by acute and chronic exposure to ethanol. Here, embryos of zebrafish exposed to 1% ethanol during the developmental stages of gastrula/segmentation or pharyngula exhibited anxiolytic effect, increased aggressiveness, and decreased social interaction. The exposure during pharyngula stage was able to affect all behavioural parameters analysed at 3 months-post fertilization (mpf), while the treatment during gastrula stage affected the anxiety and social interaction parameters. The aggressiveness was the only behavioural effect of early ethanol exposure that lasted to 12 mpf. The use of a specific inhibitor of adenosine production, the inhibitor of ecto-5′-nucleotidase (AMPCP/150 mg/kg), and the specific inhibitor of adenosine degradation, the inhibitor of adenosine deaminase, EHNA (100 mg/kg) did not affect the effects over anxiety. However, AMPCP at 3 mpf, but not EHNA, reversed aggressive parameters. AMPCP also recovered the social interaction parameter at 3 mpf in animals treated in both stages, while EHNA recovered this parameter just in those animals treated with ethanol during the gastrula stage. These results suggest that long-lasting behavioural effects of ethanol can be modulated by intervention on ecto-5′-nucleotidase and adenosine deaminase activities. The term alcohol-related neurodevelopmental disorder (ARND) describes a particular pattern of disordered behaviour and impaired cognitive development in children and young people who did not develop physical and morphological features related to alcohol exposure2. The behavioural impacts of prenatal exposure to ethanol include a lower IQ, learning problems, anxiety and conduct disorders4. Several animal models, especially rodent-based models and more recently zebrafish models, were developed to contribute to a trustworthy picture of the neurobiology of the behaviour sequelae of ARND7. The cellular targets underlining the long-lasting cognitive and behavioural outcomes of ethanol exposure include components of serotoninergic, GABAergic, glutamatergic and dopaminergic systems. Data from human and animal studies indicate that ethanol exposure during early brain development is able to block NMDA receptors, to exacerbate GABAA receptor action, to decrease 5-HT-binding and to decrease dopamine levels11.Worldwide, the percent of pregnant women who consume ethanol via alcoholic beverages is estimated to be around 10%13. Adenosine is able to affect the synaptic activity by pre-, post- and non-synaptic actions, especially through control of neurotransmitter release, receptor-receptor interaction and post-synaptic impact on membrane depolarization15. The ability of adenosine to modulate the major neurotransmitter systems related to common cognitive and behavioural consequences of early ethanol exposure brings the attention to the possible involvement of this nucleoside on these long-term effects. The activation of adenosine receptors and, therefore, the adenosine neuromodulation, are a reflection of the control of the extracellular levels of this nucleoside, which are a result of several enzymatic activities, such as ecto-5′nucleotidase and ecto-adenosine deaminase, and of equilibrative and concentrative transport mechanisms that control the availability of adenosine for the P1 receptors and, consequently, the neuromodulation of major neurotransmitter systems16. The ecto-adenosine deaminase (E.C. 3.5.4.4) catalyzes the deamination of adenosine producing inosine and NH317. The ecto-5′-nucleotidase (E.C. 3.1.3.5) catalyzes the dephosphorilation of AMP producing adenosine and inorganic phosphate18. In fact, the purinergic system is also a direct target of ethanol exposure24. Both enzymes are affected by acute or chronic exposure to ethanol in mature CNS23.Besides the role of adenosine in energy metabolism, epigenetic mechanisms and the genetic transmission of information, this nucleoside exerts an important neuromodulatory effectDanio rerio) model27. The embryonic zebrafish has some advantages as a model of early ethanol exposure, since zebrafish shows easy manipulation and clear differentiation of the developmental stages through a transparent egg28. Zebrafish exposed to alcohol concentrations ranging between 0.25 and 1.00% shows no apparent morphological changes29. The use of doses above 1% may cause significant decrease in survival and morphological deformities such as pericardial edema, axial malformations, yolk sac edema, cyclopia30 and micropthalmia32. In that sense, in order to give base to studies of subtle effects of ethanol that could contribute to behavioural effects, the use of doses bellow 1% ethanol is suitable. Zebrafish neuroanatomy has many similarities to that of mammals, especially in areas such as the spinal cord and hindbrain, in which zebrafish and mammals share major neurotransmitter and neuromodulator pathways, including the expression and functionality of major enzymes and receptors of purinergic system35. An increasing number of studies have demonstrated that ethanol exposure affects a variety of zebrafish behaviours, such as anxiety36, aggressiveness37, memory38 and social interaction39, some of which are long-lasting39. Therefore, because adenosine is a potent neuromodulator and ethanol is able to affect several neurotransmitters modulated by adenosine and the purinergic system itself, the objective of this research was to investigate and understand the possible effects of interventions on enzymes involved in adenosine production and degradation over the long-lasting behavioural effects caused by ethanol exposure in different stages of early development.Behaviour impairments caused by ethanol have been widely studied over the years in the zebrafish  = 3.75; P = 0.028]   = 3.81; P = 0.028]  or survival rate (data not shown) outcomes when evaluated at 3 or 12 mpf. Locomotor parameters (distance travelled and mean velocity) had few effects of the factors evaluated  = 23.48; P < 0.0001]. Ethanol treatment in both developmental stages produced an anxiolytic effect, since the 3 mpf animals increased their time in the upper zone by 79% in the gastrula/segmentation group and 92% in the pharyngula group, compared to water/saline group  = 0.5721; P = 0.57] , a measure to assess aggressiveness, at 3 mpf animals   = 7.597; P < 0.0001]. At 3 mpf, the time spent in the zone 1 was significantly increased in animals treated with 1% ethanol during pharyngula stage when compared to control/saline animals (P = 0.048) and animals treated with 1% ethanol during gastrula/segmentation stage (P = 0.002) , but not by EHNA  = 10.31; P = 0.0001]. The time spent in the zone 1 was significantly increased in 12 mpf animals treated with 1% ethanol during pharyngula stage when compared to control/saline animals (P = 0.014) and animals treated with 1% ethanol during gastrula/segmentation stage (P = 0.015)  Fig.  indicateethanol treatment , purinergic intervention , as well as, the interaction between these factors  in animals at 3 mpf . Ethanol given to animals at pharyngula stage decreased the time spent on the stimulus side of the aquarium by 29% when the animals were at 3 mpf in comparison to control/saline group (P < 0.0001). These alterations were recovered by AMPCP and EHNA in those animals treated with 1% ethanol at the gastrula/segmentation period, while only AMPCP recovered this parameter in those animals treated with 1% ethanol during the pharyngula stage  = 4; P = 0.03], while multiple comparison of means did not indicate differences between control/saline group and ethanol-treated groups  had significant effects from factors 42. Those long-term effects of ethanol related to aggression and social interaction were recovered by adenosine metabolism manipulation.Here, we identified that ethanol exposure in the gastrula/segmentation and in the pharyngula developmental stages promotes long-term behavioural effects, such as increased anxiety, increased aggressiveness and decreased social interaction. These results resemble those seem in rodent studies and reinforce the zebrafish as an excellent animal model to evaluate long-term effects of early exposure to ethanol43. While the methods of exposure to ethanol are a sensitive variable in the literature, these data suggest that none of the long-term behavioural outcomes were affected by locomotor or morphological impairment. Our results showed that, at least in behaviour alterations, the pharyngula stage (24–48 hpf) seems to be the period more affected by ethanol exposure. At the pharyngula stage, the heartbeat, vascularization and circulation in the yolk occur and some studies performed with rodents and zebrafish have demonstrated that ethanol can affect vasculogenesis and angiogenesis development, which can affect neuronal activity, a prerequisite to growth and behaviour development, which could contribute to the results seem here46.Our results demonstrated that the exposure to 1% ethanol during the gastrula/segmentation or pharyngula stages did not affect morphology and had minor effect on locomotor activity of animals, in agreement with the literature39. The exposure to moderate levels of ethanol appears to be more correlated to negative consequences in the social domain including decreased investigation and interaction, increased aggressive behaviour and altered responses to social stimuli47. In zebrafish, this behaviour could be related to the decrease in cortisol levels, since the hypothalamic–pituitary–interrenal axis is fundamental to stress and anxiety responses and involves the cortisol hormone36. While the involvement of the hypothalamic–pituitary–interrenal was not evaluated in this work, some studies showed that zebrafish exposed to low doses of ethanol during development had reduced the cortisol levels when evaluated at 6 months of age48.Our results reinforce the long-term effect of ethanol on anxiety parameters. Ethanol exposure in the gastrula/segmentation and pharyngula stages caused an increase in the time spent in the upper zone of the tank at 3 mpf, indicating an increase of exploratory behaviour, which did not persist until 12 mpf. Recently, a similar study showed that 2-h-long exposure of zebrafish embryos to 1% ethanol at 24 hpf was also able to promote anxiogenic-like behaviour in adult zebrafish36. To differentiate these behaviours, in addition to the mirror test, we observed increasing of aggressive behaviour by observation of biting, sprinting, attack behaviour and changes in colour pattern. Here, the social interaction was decreased, while aggressiveness was increased, at the young adult phase of animals exposed to ethanol during early phases of development. While the social interaction reduction did not continue until 12 mpf, the aggressive behaviour did persist until 12 mpf in the animals exposed to ethanol during pharyngula stage. The dose and time of exposures appears to be a determinant point to the long-term effects of ethanol exposure, since absent to mild and severe aggressive behaviour have been suggested in the literature47. Although in our results the social interaction reduction was not persistent until 12 mpf, studies have previously demonstrated that zebrafish embryos exposed to 1% ethanol for 2 h at 24 hpf had impaired social interaction responses at 24-month-old fish, not due to altered motor function or visual perception, but likely instead due to a central nervous system alteration affecting social behaviour itself43.The zebrafish is a social species, but the preference for being close to a conspecific can also be a result of aggressive behaviour, both of which may be influenced by ethanol51. As all the major neurotransmitter systems are modulated by adenosine16, and adenosine appears as a target of ethanol20, some of the long-term effects of ethanol could also be related to disruption of adenosine modulation.The literature suggests that behavioural abnormalities resulting from similar embryonic ethanol exposure may be primarily due to altered dopaminergic and serotoninergic mechanisms, while amino acid neurotransmitters, such as glutamate and GABA, seem also be somewhat related to long-term neurochemical effects of ethanol1 receptors displayed increased aggressiveness52. Also, inhibition of nucleoside transporters antagonizes aggressive behaviour in mice, implying that endogenous adenosine seems to play a role in influencing aggressive behaviour53. In this way, the reduction of aggressiveness in animals treated with ethanol by AMPCP could be a reflex of the reduction of adenosine modulatory effects. The reduction on social interaction by ethanol treatment regardless developmental stage, seems to be also affected by intervention in adenosine metabolism, since the inhibition by AMPCP reverted this effect in animals treated in both developmental stages with ethanol, and by EHNA in the animals treated with ethanol at gastrula/segmentation stage.Studies suggest that the adenosinergic system can be involved in aggressive behaviour. Behavioural assessment of mice lacking adenosine A54.From all behavioural parameters evaluated, only the increase of aggressiveness was persistent to 12 mpf. Differences observed between juvenile adults and aged adults are hard to explain. However, the natural modifications of neurotransmission during lifetime could be enough to the rescue of cognitive impairment. In fact, situations when adenosine level increases, such as neonatal hypoxia, have been related to better cognition in aged rats50. The other possibility is that the adenosine modulation could be one of the primary targets of ethanol, with long-lasting effects. In fact, previous works from our group41, using the same methodologic approach, demonstrated that ecto-5′-nucleotidase activity is increased in a persistent way when zebrafish is exposed to ethanol during gastrula/segmentation stage. The increasing ecto-5′-nucleotidase activity could elevate adenosine production, while we did not prove that41. So, in this sense, the inhibition of ecto-5′-nucleotidase by AMPCP, could reduce a persistent increased ecto-5′-nucleotidase, or the basal ecto-5′-nucleotidase, both contributing to alter the adenosine inhibitory modulation over major neurotransmitters related to social behaviour domain, such as dopamine and serotonin50. The participation of ADA is still unclear. The effects seem here could be a complex response to the systemically injections of EHNA over basal ADA activity, especially because ADA displays several isoforms with important intracellular effects55. Although, in vitro and ex vivo studies indicated that intra and extracellular adenosine deaminase are targets of ethanol56, a recent study did not register persistence to adulthood of impacts of early exposure to ethanol over adenosine deaminase activity and expression from zebrafish41.Our results showed that inhibition of ecto-5′-nucleotidase and adenosine deaminase were able to recover social interaction and inhibition of ecto-5′-nucleotidase recovered aggressive behaviours, both affected by early ethanol exposure. One possibility to explain these results is that the inhibition of these enzymes, especially ecto-5′nucleotidase, promotes an adjustment of adenosine modulation over several major neurotransmitters, since some of them were already identified as ethanol targetsIn conclusion, the long-term effects over behaviour from early ethanol exposure can be reversed by inhibition of ecto-5′-nucleotidase and, lesser, by inhibition of adenosine deaminase in adult zebrafish. The reflex of these results over adenosine modulation should be expanded by further studies conducted to identifying the contribution of nucleoside transporters, adenosine receptors functionality and impact over major neurotransmitters modulated by adenosine in regard to these long-lasting consequences of ethanol.D. rerio) (Tübingen background) from the colony held at our laboratories  were used for obtaining fertilized eggs following the protocols of our laboratory as already published41. Briefly, animals were housed in a re-circulating system equipped with mechanical and biological filtration at a temperature of 28 °C and pH of 7.4. After mating, eggs were collected and kept in water for maintenance  on a 14:10 h light/dark cycle. After the beginning of the swimming phase, larval zebrafish were transferred to aquariums and kept until 3 or 12 months of age at proper fish densities . Animals were fed three times a day with commercial flakes  and supplemented with live brine shrimp.Adult zebrafish  Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) and were approved by the Ethics Committee for the animal use (CEUA) from the Pontifical Catholic University of Rio Grande do Sul (15/00468—CEUA PUCRS).41. The 1% ethanol solution (171 mM) could be considered high in comparison to human levels, which often reach 66 mM57. However, some investigators identified that the egg chorion acts as an important barrier, since the ethanol concentration inside the egg (6–48 hpf) is 2.7–6.2-fold lower than ethanol media levels59. The animals were exposed to ethanol in groups of 50 embryos, kept in Petri dishes. After the gastrula/segmentation or pharyngula stages, animals were maintained in drug-free water until they reach 3 or 12 mpf, two ages when the animals are mature and present preserved locomotor activity59.Embryos of zebrafish were exposed to 1% (v/v) ethanol (diluted in the maintenance water) in two distinct developmental stages: gastrula/segmentation [5–24 hpf (hours post-fertilization)] or pharyngula (24–48 hpf)60. Prior to the injection, the adult animals were anesthetized in tricaine solution 61. Behavioural analysis was performed 30 min after drug exposure. Control animals received saline.To investigate the effect of adenosine metabolism over behavioural impacts of early ethanol exposure, we used the specific inhibitors of adenosine deaminase, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride), which could avoid adenosine degradation, and AMPCP  an inhibitor of 5′-nucleotidase, which could inhibit adenosine formation by AMP catabolism. The pharmacological treatments (AMPCP at 150 mg/kg or EHNA at 100 mg/kg) of adults were performed by intraperitoneal (ip) injection with the use of a Hamilton microliter syringe in a volume of 10 μL per animal (20 mL/kg), following literature using zebrafish60 . After 30 s of adaptation, the zebrafish activity was recorded for 5 min and analysed using the video tracking system Ethovision XT8 . The locomotor parameters analysed were distance travelled (m) and mean speed . Exploratory behaviour, an indicator of anxiety, was assessed as the time spent in the top section of tank and was virtually divided into two horizontal lines (bottom and upper zones)62 . Additionally, observations of aggressive behaviour, like biting, sprinting and changes in colour pattern, were also made. Usually, attack behaviour is a characteristic short bout of fast swimming directed towards the opponent and is sometimes accompanied by opening the mouth and biting62. Randomized and independent groups of 10 animals were used for aggressiveness test.At 3 or 12 mpf, aggressive behaviour was analysed in control and ethanol-treated animal 30 min after injection of AMPCP, EHNA or saline (n = 10). Individually, the fish were placed in a single tank  with a mirror (22 cm) positioned in the back of the tank forming a 22.5° angle36 . Three tanks  were placed side by side: the far-left tank was left empty, the one in the middle held the test animals and the far right held 15 stimuli conspecific fishversus ethanol on gastrula stage versus ethanol on pharyngula stage = factor ethanol treatment) and the purinergic drug exposure (no drug versus AMPCP versus EHNA = factor purinergic intervention) at adult life. The effect of water versus ethanol treatment at 12 mpf animals for time on upper zone (anxiety measure) and time in the stimulus side  was analysed by One-Way ANOVA, since the AMPCP and EHNA were not tested for this analysis at this age. The multiple analyses of the means were analysed by Tukey’s multiple comparisons test in behaviour and locomotors parameters when appropriated. The significance levels were attributed at P < 0.05. Data were expressed as mean ± standard deviation (SD).The normality of data was checked using the D’Agostino & Pearson Omnibus normality test. Two-Way Analysis of Variance (ANOVA) was used to compare groups considering as factors the ethanol exposure (no ethanol Supplementary Figure.Supplementary Table.


Today, we face difficulty in generating new hypotheses and understanding oral lichen planus due to the large amount of biomedical information available. In this research, we have used an integrated bioinformatics approach assimilating information from data mining, gene ontologies, protein–protein interaction and network analysis to predict candidate genes related to oral lichen planus. A detailed pathway analysis led us to propose two promising therapeutic targets: the stromal cell derived factor 1 (CXCL12) and the C-X-C type 4 chemokine receptor (CXCR4). We further validated our predictions and found that CXCR4 was upregulated in all oral lichen planus tissue samples. Our bioinformatics data cumulatively support the pathological role of chemokines and chemokine receptors in oral lichen planus. From a clinical perspective, we suggest a drug (plerixafor) and two therapeutic targets for future research. Histopathological features of OLP include hyperkeratosis, acanthosis, degeneration by liquefaction of the basal keratinocyte layer, and prominent lymphocyte infiltration at the connective tissue–epithelium interface2. OLP is not a homogeneous clinical entity, but a complex disease. A recent review proposes a new classification of this disease, taking into consideration a set of lesions with similar characteristics, including oral lichenoid contact lesions, oral lichenoid drug reactions, and lichen planus pemphigoides, among others3.The clinical findings vary from white reticular plaques to erythema, erosions, ulceration, and hyperkeratotic plaques in the oral mucosa4. Although reticular lesions are often asymptomatic, burning or pain often accompanies erosive/ulcerative manifestations of OLP. The severity of pain may vary from mild to severe and may even interfere with patients’ speech, feeding, and swallowing4. According to the WHO, this disease has a potential for cancerization5. This potential is presented in about 1% of patients6. Despite the efforts in biomedical research, the etiology of OLP is unknown, and its molecular basis is still unclear. This may explain why the treatment is palliative. For these reasons, exploration of the mechanisms that govern the disease’s pathophysiological process is needed.Oral lichen planus (OLP) is a chronic immunologically mediated disease7. Today, we face difficulty in generating new hypotheses and understanding OLP due to the large amount of biomedical information available. Encapsulating that information in an understandable way is one of the current challenges of network-based approaches to the study of human diseases8. The heterogeneity of pathologies, the difficulty of finding a gene or protein in a certain localization, and the cost involved in experimental studies have led to the development of several in silico approaches for the identification of genes and proteins associated with a disease9. These bioinformatic approaches address complexity by simplifying systems, summarizing them as interaction maps or interactomes, and containing components (nodes) and lines (interactions) between them7. To explore biological systems, analyses involve data mining10, protein–protein interactions11, gene ontology (GO)12, gene regulatory networks, and pathways for gene identification candidates13. Several publications have reported on the successful use of such techniques to prioritize disease-related genes and propose therapeutic targets16. However, these technologies have not been applied to the study of OLP.A disease is rarely the result of an abnormality in a single gene, but it reflects disturbances in the complex intracellular and intercellular network that links tissue and organ systemsIn order to better understand the molecular basis of OLP and propose new therapeutic targets, we use data mining to obtain a large number of protein coding genes associated with this disease. Then, we describe the biological attributes of proteins and prioritize a small group of them using protein–protein interaction networks. With the most relevant proteins, we defined the OLP interactome, which allowed us to propose a new treatment for the disease and two therapeutic targets. Finally, we check our computational predictions by immunohistochemistry.17. The Ethics Committee of UTALCA (protocol #2017-02-CR) approved this research (10.5281/zenodo.3576206).Figure 18. This tool analyzes relationships between genes and biomedical topics in all abstracts available on MEDLINE/PubMed. The topic of interest was defined by the search : “lichen planus, oral” [MeSH] and “biomarkers” [MeSH] and (risk ratio [Title/Abstract] or relative risk [Title/Abstract] or odds ratio [Title/Abstract] or risk [Title/Abstract]) and (“humans” [MeSH Terms]). With these criteria, 23 abstracts were identified as a training set. In order to find only significant coding genes, we established as a cut-off p-value of <0.01 for abstracts and a false positive discovery rate (FDR) of <0.01 for genes. We use Fisher’s exact test to define the relationship between genes and search topics.To identify genes encoding proteins related to OLP from a large volume of information, we use the Génie web toolhttp://www.pantherdb.org)19 version 13.1 to determine classifications based on biological processes, cellular components, molecular functions, and metabolic pathways. We select the coding genes from the most overrepresented classifications.The gene list was subjected to a gene ontology analysis using the PANTHER representation test (20 and the STITCH web tool (http://stitch.embl.de/)21. CHAT identifies the genes of greatest relevance within a list. For this, it builds a molecular network composed of (i) the list of genes (each gene is a node), (ii) the numerical values for each gene , and (iii) a database with the genes that will establish interactions. The smallest p-values represent the centers of greatest activity within the biological network (hubs). We assign a numerical value of +3 (context attribute) to all coding genes included in the overrepresented PANTHER categories, and as a database for the interactions, we select IntAct22. To establish whether the resulting hubs corresponded to a biologically connected network, we used STITCH. These base reports known interactions as well as establishing predictions of interactions between proteins and chemicals. In STITCH, we choose to establish interactions with the maximum confidence level (0.9) using all available resources. We call the resulting network the “OLP interactome.”To build the OLP interactome, we use the Contextual Hub Analysis (CHAT) application of the Cytoscape programn  =  10), head and neck cancers (n = 4), pemphigoid (n = 2), pemphigus (n = 2) and OLP lesions (n  =  12) in paraffin-embedded tissues stored in the UTALCA Biobank/Oral Pathology Laboratory . The information of patients is provided in Supplementary Table 2. Immunostaining of 3 μm histological sections was performed using EnVision FLEX target retrieval solution  according to our previously published protocols24. The primary antibody used was a CXCR4 antibody , used overnight at 4 °C. Two pathologists blinded to the clinical data provided a consensus opinion of staining patterns. We use oral cancer cases as positive controls. Also, evidence that CXCR4 antibody represent a specific staining can be consulted in our previous studies24.We retrospectively collected focal fibrous hyperplasias n  =  0, head aWith Génie, we obtained 872 statistically significant protein-coding genes extracted from 1,075,776 articles  that interact with more “contextual” nodes . CHAT calls these nodes “contextual hubs.” These hubs have topological and functional relevance, since their elimination causes great damage in a network, so they are the best representatives of a biological systemTo confirm our bioinformatic predictions, we used immunohistochemistry. We detected higher expression of CXCR4 in OLPs than in other lesions , CCL19, CCL20, CCL21, CXCR1, CXCR2, CXCR3, CXCR4, CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, IL-8, MTHFR, MTR, MTRR, and MTHFD1. The main cluster includes chemokines and chemokine receptors participating in dendritic cell chemotaxis. In addition, these proinflammatory molecules are produced by cells primarily to recruit leukocytes at sites of infection or injuryThe switch that starts the mechanisms of oral lichen planus is still unknown. Revealing the molecules that constitute the major centers of activity or disturbance points in the OLP network can provide a chance to find new therapies. Surprisingly, using STITCH, we predict that plerixafor has two therapeutic targets, CXCR4 and CXCL12.42. This axis modulates effects on cells in an autoimmunity context, which may be important for the development or severity of various diseases, including psoriasis, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, idiopathic inflammatory bowel diseases, and type 1 diabetes43. Recent experimental evidence shows that the CXCL12-CXCR4 axis participates in inappropriate retention of activated innate inflammatory cells at inflammatory sites. This is highly relevant for chronic diseases, such as chronic obstructive pulmonary disease and asthma44. The broad participation of the CXCL12-CXCR4 axis in several diseases justifies the recognition of antagonistic drugs.The CXCL12-CXCR4 axis regulates leukocyte chemotaxis in inflammatory conditions and autoimmune diseases. It has significantly been studied in numerous cancers and autoimmune diseases45. Plerixafor has proven to be useful for inhibiting the CXCL12-CXCR4axis in patients with leukemia46. Although there is no evidence of the usefulness of this drug in OLP, a biological basis supports this therapeutic use.Plerixafor, also known as AMD3100, was originally developed as a drug against human immunodeficiency virus and then characterized as a CXCR4 antagonist47. In addition, our immunohistochemistry corroborated the predictions. We tested the expression of CXCR4 for two reasons: first, because our research group has previous studies with this protein24 and second, the immunohistochemistry method is poorly quantitative and can suffer from low sensitivity for detection of secreted proteins 48.Previous evidence shows that the microdissected oral epithelium of OLP patients presents an increase in gene expression of 258% for CXCL12 and 629% for CXCR450. Since dendritic cells are highly activated in OLP, this may be an important event for disease development. Dendritic cells are the most potent antigen presenting cells for lymphocytes52. These cells present the antigen to memory T cells, the predominant phenotype in OLP53. It is known that CXCR4 is highly-expressed in resting T cells, including naïve and memory T cells, and is downregulated during T cell activation54. We believe that the presence of high amounts of CXCR4 may represent populations of dendritic cells, memory T cells, or a combination of both.We observed that CXCR4 is highly expressed in the connective tissue of OLP patients. It is known that CXCR4 and CXCL12 are expressed in dendritic cells (Langerhans cells)1. Therefore, it is logical to think that an intervention that interrupts the triggering of lymphocytic infiltrate could ensure epithelial integrity. Although topical steroids are considered the first-line treatment for symptomatic oral lichen planus, there is no evidence to support the effectiveness of these drugs56, which we believe is an invitation to explore new options.An initial event in the disease mechanism may involves the expression or presentation of the keratinocyte antigen (still unknown) to trigger the immune responseThe initial search for our design does not distinguish between the clinical variants of OLP; however, our analyses are inspired by the erosive/ulcerative forms that are accompanied by painful symptomatology. Our results are limited to the performance of our applications in silico, and new studies are needed to provide experimental data for our analysis. In the future, we should test whether the high expression of CXCR4 can be verified using other techniques, such as PCR experiments (after tissue microdissection) or salivary ELISA.In this investigation, bioinformatics data cumulatively support the pathological role of chemokines and chemokine receptors in OLP. From a clinical perspective, we suggest a drug and two therapeutic targets for future research. Additionally, we demonstrate that it is possible to comprehensively analyze a large volume of biomedical information in order to better understand the molecular complexity of the OLP, even confirming a bioinformatics prediction using immunohistochemistry. We believe in the need for future studies to verify and monitor – experimentally and clinically – the role of the proteins listed in this research to move towards more precise therapies.Supplementary Table 1.upplementary Dataset 1.upplementary Dataset 2.upplementary Dataset 3.Supplementary Figure S1.


BackgroundVitamin B12 deficiency is linked to impaired cognition and memory along with a sensation of tingling and numbness, an outcome of poor myelination. Elevated methylmalonic acid and serum homocysteine levels are markers of Vitamin B12 deficiency. Elevated homocysteine levels are also often associated with Alzheimer’s disease and stroke. We conducted this study to determine the effect of vitamin B12 replacement therapy on vitamin B12-deficient patients with noted cognitive impairment.MethodsWe conducted a cross-sectional, multicenter study of patients with minimal cognitive impairment (MCI) to assess for Vitamin B12 and homocysteine levels. All patients found to be deficient in vitamin B12 underwent replacement therapy and were assessed again after three months via the Mini-Mental State Examination (MMSE) and a review of symptoms.ResultsA total of 202 patients were included in the study. Of those, 171 (84%) patients reported marked symptomatic improvement after vitamin B12 replacement while MMSE scores improved in 158 (78%) patients. Of the remaining 44 patients who reported no symptomatic improvement, MMSE scores improved in 26 while 18 patients showed no MMSE score improvements.ConclusionsVitamin B12 deficiency is linked to cognition, and replacement therapy may be an option to improve patient cognition outcomes. Further studies are needed to confirm and refine the observed associations over a larger scale and to determine whether these findings will translate to a reduction in cognitive decline. Vitamin deficiencies have long been known to cause various effects on human health. Vitamin B12 deficiency has been correlated to various neurologic problems; however, research has suggested that abnormally low levels of vitamin B12 can be the cause of significant cognitive dysfunction . Low vitIn general, vitamin B12 levels decline with age, and therefore, the prevalence of vitamin B12 deficiency increases in the elderly population . As noteDietary deficiency of vitamin B12 plays a major role in the causation of a state of clinical deficiency. In the developing world, poor socioeconomic status plays a major role in inadequate levels of nutrition. In young adults, a major cause of vitamin deficiency is the inadequate consumption of animal-source food. In older patients, malabsorption may be the main cause of the deficient state . A studyHowever, in developed countries, the causes of vitamin deficiency vary beyond consumption. A French study showed that among 172 patients with vitamin B12 deficiency, only 2% accounted for an inadequate dietary intake; therefore, the deficiency arises from other factors such as malabsorption, postgastrectomy state, bacterial overgrowth, and long-term use of medications for some comorbidities  .In Pakistan, no such study had been conducted on the causes and effects of vitamin B12 deficiency in general, especially through the lens of mental health. We conducted this study in a low socioeconomic area of Karachi in patients attending a neurology outpatient department with a variety of neurologic manifestations.According to Lavretsky, minimal cognitive impairment (MCI) is defined as a cognitive decline that is greater than expected for an individual’s age and educational level but that does not interfere with the activities of daily living . It can This study was a cross-sectional, multicenter project conducted at Jinnah Medical College Hospital in Korangi and Medicare Cardiac and General Hospital in Karachi. Patients at these two centers who presented in the neurology outpatient department from January 2017 to December 2017 and provided written informed consent were selected for inclusion. Although vitamin B12-deficient patients have a wide variety of vague neurologic manifestations, our present study was specifically focused on cognitive impairment. All patients above the age of 18 years who presented with concerns of forgetfulness, poor focus and concentration, memory decline, generalized tiredness, lethargy, paresthesias, and tingling sensation, and poor balance were included in this study. As our main focus in this study was on MCI, we assessed patients for clinical signs of MCI and used the Mini-Mental State Examination (MMSE), Backward Digit Span test , and the East Boston Memory Test (EBMT).All patients who fulfilled the clinical criteria of MCI were further assessed with a detailed neurologic examination. Patients with any focal neurologic deficit were sent for a preliminary brain imaging study . Finally, only those patients were selected for this study who had MCI but for whom no focal cause was found.All selected patients were further assessed for any metabolic cause of memory impairment by blood sampling. Patients with no other major metabolic derangement except for having low vitamin B12 levels or high serum homocysteine levels were considered for inclusion.Biochemical samplingBlood samples from the selected patients were taken and analyzed for complete blood picture, urea, creatinine, serum electrolytes, blood glucose levels, liver function tests, thyroid hormone levels, and serum vitamin B12 and homocysteine levels.Patients with MCI and low serum levels of vitamin B12 without any evidence of focal brain lesion or any major metabolic derangement and who had gradual development of cognitive impairment without any premorbid history of similar complaints were ultimately selected for inclusion into the study.This population of patients was offered vitamin B12 replacement therapy and was kept on a three-month follow-up. The mode of administration was planned according to the deficiency state . These patients were reassessed clinically and by biochemical analysis of vitamin B12 and homocysteine levels after six months.Almost 8000 patients visited the neurology outpatient department over 12 months, among whom 1470 (18.3%) patients presented with the relevant concerns of poor focus, gradual memory decline or recent forgetfulness, lethargy without weight loss, and sensory paresthesias. Selected patients were older than 18 years, and most patients were older than the age of 50 years. These patients were further assessed with a detailed history, clinical examination, MMSE, EBMT, and backward counting tests. Only 281 (19%) of 1470 patients were selected to be included in this study, as these patients fulfilled the clinical criteria of MCI and had an MMSE score below 24. The lowest MMSE score in this population was 12, which was considered in the dementia category.Among these 281 patients, 19 had a focal neurologic deficit. After neuroimaging , 14 patients were found to have a stroke, while five had a space-occupying lesion. Among these five patients, three had a brain tuberculoma and two were finally diagnosed to have brain tumors. These 19 patients were not included in this study. Thirteen more patients were excluded from the study group because of being diagnosed as metabolic derangements (chronic kidney disease in five and chronic liver disease in eight cases).Finally, 249 (16.9%) of 1470 patients were selected to participate in this study, as these patients had MCI, but no discernable focal or metabolic cause for their condition. Among this patient population, 202 (13.7%) of 1470 patients had a low or lower normal range of vitamin B12 levels with elevated homocysteine levels.Of these 202 patients, 126 were women and 76 were men. Considering the age range, 137 of 202 were older than age 55 years, 47 patients were aged between 30 and 55 years, and 18 patients were aged between 18 and 30 years.Among these patients, 58 (28.7%) of 202 had vitamin B12 levels between 200 and 350 pg/mL and were considered to be mildly deficient, while 113 (56%) of 202 patients had a level between 100 and 200 pg/mL (moderate deficiency); 31 (15.3%) of 202 had a vitamin B12 level between 50 and 100 (severe deficiency). The minimum vitamin B12 level was 51 pg/mL.Forty-three patients had a serum homocysteine level above 20 µmol/L, 101 patients had levels between 15 and 20 µmol/L, and 58 patients had levels between 8 and 15 µmol/L.All the vitamin B12-deficient patients were offered vitamin B12 replacement therapy by parenteral route initially to make sure that there was no issue with absorption, followed by oral replacement therapy for three months.These patients were reassessed after three months of therapy by detailed history and the same cognitive assessments, as well as by a repeat vitamin B12 level assessment. All the patients had vitamin B12 levels between 400 and 800 pg/mL. A total of 171 (84%) of 202 patients reported marked symptomatic improvement, while MMSE results were improved in 158 (78%) of 202 patients. Of the remaining 31 patients who reported no symptomatic improvement, MMSE outcomes were improved in 13, while 18 patients did not show any improvement in MMSE.These 18 patients  were further assessed for any other cause of symptoms  and were continued on a low-dose oral supplement of vitamin B12. All results are shown in Table  The precise definition of MCI has long been controversial, but a diagnosis of MCI follows similar methods that are used to diagnose clinical dementia and Alzheimer’s disease. An algorithm assists clinicians in identifying the subjects and classifying them into various subtypes of MCI . A key sThe long-running lack of a gold standard value denoting vitamin B12 deficiency presented a challenge to investigators, but a cut-off of 203 pg/mL was finally set, and neurologic manifestations become apparent between 298 pg/ml and 350pg/mL , 19. CliIn our studied population, 202 (13%) patients were found to be in a vitamin B12-deficient state; 31 (15%) of them had an absolute deficiency state with serum B12 levels between 50 pg/mL and 100 pg/mL. This population presented with significant symptoms of memory worsening, poor focus and concentration, and lethargy affecting their activities of daily living. Most of these patients had a persistent need for a caregiver to maintain their balance and carry out their routines. When replacement therapy was given in these patients, almost all of them showed a correction of serum biomarkers, but only 13 patients showed improvement of MMSE outcomes, and 18 showed no improvement in their signs and symptoms. Among the patients with serum vitamin B12 levels above 100 pg/mL, 171 (84%) had significant symptomatic improvement and 158 patients showed improvement in their MMSE score. This finding can be the basis of a hypothesis that chronic, very low levels of vitamin B12 could be the cause of permanent, or at least refractory, changes in cognition and memory, which can lead to dementia.Research conducted in 2016 revealed that metabolic vitamin B12 deficiency was present in 10% to 40% of the population and is frequently missed, this could be easily treated if recognized early, and it may be an important opportunity to prevent dementia and stroke . ElevateGiven our study’s cross-sectional design, a direct association between low vitamin B12 level and cognitive impairment reflects the adverse effect of this common nutritional deficiency on mental health in all age groups. Although vitamin B12 deficiency is prevalent in elderly populations, a significant number of patients in our study were of a comparatively younger age group. Possible causes of such a common deficiency state could be gastritis-associated poor absorption, excessive use of other drugs , pernicious anemia, or nutritional deficiency due to poor socioeconomic status. Signs and symptoms of this deficiency are usually vague and are easily missed. The effects of treatment have shown that early identification and replacement therapy causes a significant reversal of symptoms, which is an important possible step toward a healthy mental state. As Wong reported, classical treatment for vitamin B12 deficiency is parenteral administration, usually an intramuscular injection to rebuild the tissue stores . It is bThe strength of our study was clear control over key confounders, exclusion of other possible causes of worsening cognition to the best of our ability, and results that supported our idea of improved cognition with replacement therapy. While some data were self-reported , these were not proven according to our selected scales. Our main challenges were the lack of a gold standard indicator of low vitamin B12 level causing cognitive decline and any gold standard test of MCI; however, the strong association found between low MMSE scores and low vitamin B12 status attests to its ability to capture the cognitive impairment due to this cause. This study was not carried out on a large scale; however, considering that a significant percentage of the population suffers from such a nutritional deficiency that can affect the quality of life but can be easily corrected, there is a need for a similar study to be conducted in a larger population.We found a clear association between low vitamin B12 levels and progressive cognitive impairment; therefore, further studies are needed to confirm and refine the observed associations over a larger scale and to determine whether this change will translate to a reduction in cognitive decline. Moreover, we are in favor of screening for vitamin B12 deficiency, at least in the elderly population, to find and prevent the possible causes of this deficiency state, as this can be an easily preventable cause of impending dementia before the cognitive decline becomes irreversible.


Polydora websteri Hartman have resulted in the collapse of oyster aquaculture industries in Australia, New Zealand, and Hawaii. These worms burrow into bivalve shells, creating unsightly mud blisters that are unappealing to consumers and, when nicked during shucking, release mud and detritus that can foul oyster meats. Recent findings of mud blisters on the shells of Pacific oysters (Crassostrea gigas Thunberg) in Washington State suggest a new spionid polychaete outbreak. To determine the identity of the polychaete causing these blisters, we obtained Pacific oysters from two locations in Puget Sound and examined them for blisters and burrows caused by polychaete worms. Specimens were also obtained from eastern oysters (Crassostrea virginica Gmelin) collected in New York for morphological and molecular comparison. We compared polychaete morphology to original descriptions, extracted DNA and sequenced mitochondrial (cytochrome c oxidase I [mtCOI]) and nuclear  genes to determine a species-level molecular identification for these worms. Our data show that Polydora websteri are present in the mud blisters from oysters grown in Puget Sound, constituting the first confirmed record of this species in Washington State. The presence of this notorious invader could threaten the sustainability of oyster aquaculture in Washington, which currently produces more farmed bivalves than any other US state.Invasions by shell-boring polychaetes such as  Polydora websteri Hartman, commonly known as a “mud worm” or “mud blister worm”1, which bores into the shells of molluscs2. By creating unsightly blisters on the shells of their commercially important hosts, these pests have led to significant economic losses for shellfish aquaculture3. Polydora websteri can infest a variety of mollusc hosts (see reviews6), including oysters10, mussels13, scallops16, and abalone17.The most notorious invasive species simultaneously compromise the function of ecosystems and jeopardize the human societies that depend upon those ecosystems. Among these invaders is the spionid polychaete Polydora websteri and related polydorins  have compromised and collapsed oyster aquaculture industries around the world. In the late 1800s, the introduction of Polydora websteri with translocated oysters caused subtidal oyster beds in New South Wales, Australia to disappear22. When oyster transplants from Kaneohe Bay brought Polydora websteri to Kakuku, Hawaii, the introduction caused extensive damage to shellfish production24. Oyster farms on the east coast of the United States have been plagued with Polydora websteri infestations since the 1940s, resulting in substantial oyster farm losses27. In addition, high mortalities of the Japanese scallop Mizuhopecten yessoensis (Jay 1857) in British Columbia, Canada were attributed to Polydora websteri28. These examples attest to the ability of Polydora websteri to successfully invade new locations and, once established, to significantly impact aquaculture production.Polydora websteri infestations are detrimental to oyster aquaculture because the worms result in unsightly blisters on oyster valves, decreasing market value. The mud worm typically has a pelagic larval stage, after which the larvae settle onto the external side of a calcareous shell30. The worm then forms a U-shaped burrow with two exterior openings31. As they grow, burrows breach the inner surface of the valve, causing the host to produce a brittle layer of nacre that walls off the burrow34. The worm continues to expand this burrow beneath the thin, calcareous layer produced by its host; as this space fills with detritus, mud, and worm feces, a “mud blister” is formed35. Blisters can be irregular in shape and darkly colored, compromising the presentation of oysters served on the half-shell 36. Moreover, if a blister is nicked during oyster shucking, the mud and feces will foul the oyster meat, rendering it inedible3. This is particularly problematic for oyster-growing areas where a large proportion of production goes to the half-shell market.37. When infested with Polydora ciliata Johnston, the gastropod Littorina littorea (Linnaeus) has significantly reduced shell strength relative to uninfested individuals, making the infested gastropods more vulnerable to predation38. Pacific oysters (Crassostrea gigas) infested by the polydorids Polydora hoplura Claparède, Polydora cornuta Bosc, and Boccardia semibranchiata Radashevsky grow more slowly and have poorer body condition than do uninfested oysters39. Glycogen, protein, and lipid content relative to the shell cavity volume are lower in infested compared to uninfested Crassostrea sp oysters40. Additionally, polydorins have been shown to increase mortality rates in Pacific oysters that are heavily infested42. These negative effects on growth and survivorship may be caused by the energetic demands of worm-induced nacre production46; that is, infested hosts may need to invest energy into isolating their tissue from the worm by building multiple costly shell layers instead of investing that energy into their own growth and reproduction47. Given these impacts on the growth and reproduction of the host, Polydora websteri outbreaks may affect more than just the bottom line of the shellfish industry; they may also compromise the important ecosystem services provided by filter-feeding shellfish species48.In addition to their detrimental impact on aquaculture production, heavy mud worm infestations can also impact shell integrity, growth, and survivorship of mollusc hostsPolydora websteri has been reported from locations all over the world (see reviews52), but due to its complex taxonomic history (see55), many records remain to be confirmed. Some historical reports of Polydora ciliata (a non-boring species) have been re-identified as other, shell-boring polydorin species, including Polydora websteri9, and additional erroneous historical reports might exist. Polydora websteri is believed to be of Asian origin, and genetic homogeneity among North American, Hawaiian, and Asian specimens suggests that human-mediated transport produces high levels of connectivity among populations10. Although Polydora websteri has been predicted to be present in Washington, USA56 based on records of its presence to the north in British Columbia57, and to the south in Oregon and California (e.g.60), it has never before been described from Washington. Its potential absence is a fortunate circumstance; as the United States’ leading producer of bivalve shellfish, Washington State’s bivalve aquaculture brings in over $92 million dollars in revenue annually61. Of Washington State’s cultured shellfish production, Pacific oysters (Crassostrea gigas) contribute 38% by weight and 38% by value61. Pacific oysters are also culturally important to local communities, Native American tribes, family-owned farms, and recreational farmers and collectors62. As the industry has evolved in recent years, producers have shifted to the lucrative half-shell market, where the shell is presented to the consumer61. Utilizing the half-shell market, Washington’s oyster industry is structured in such a way that a Polydora websteri outbreak could cause extensive damage if infested oysters result in lost value.personal communication). However, in recent years, one of us (TK) began noticing mud blisters on the valves of Pacific oysters (Crassostrea gigas) grown in Puget Sound  from Long Island, New York , were collected for morphological and molecular comparisons. Worms were recovered from shell blisters and burrows, and identified to species using morphological traits, as well as mitochondrial cytochrome c oxidase subunit I [mtCOI] and nuclear small subunit 18S rRNA [18S rRNA] gene sequences. Our results constitute the first formal report of a shell-boring polychaete from Puget Sound, and the first report of the notorious pest Polydora websteri in Washington State.Washington State oysters have long been prized for the consistent color of their inner valves, in contrast to the mud-blister-blemished valves of oysters grown in other parts of North America , redescription54, and more recent reports63. Although the caruncle was described as extending to end of chaetiger 2 in the lectotypes of Polydora websteri54, others have found it reaching mid-chaetiger 39, end of chaetiger 363 or to chaetiger 410. In the present specimens the caruncle extended to mid-chaetiger 2 in some and through end of chaetiger 3 in others. As noted by54, in lateral view the caruncle can appear to extend further posteriorly because the middorsal boundaries between chaetigers are displaced backward in comparison to lateral boundaries . After fixation in formalin and preservation in ethanol, the differences in palp pigmentation patterns are retained . The methyl green staining pattern is similar to that observed by Read9, although he noted granular staining in anterior branchiae. Major spines are falcate, with a shallow lateral flange  were present on chaetigers 1 and 2, but presence/absence on posterior chaetigers should be confirmed based on specimens fixed in glutaraldehyde. One of the specimens from WA  were infested with at least one blister or burrow. Among oysters from Oakland Bay, in South Puget Sound Fig. , 53% werPolydora websteri by morphological analysis were confirmed as belonging to that species by molecular analyses. Of the 13 specimens collected from Oakland Bay sequenced at 18S rRNA, 12 were identified as Polydora websteri  and valuable ecosystem services to Washington State61.Our findings constitute the first report of es Table  and 6. BBay Fig.  matched Bay Fig. ; the samPolydora websteri has never before been documented in Washington State oysters suggests a recent introduction, but it is also possible that the species has been present in the region for some time and has undergone a recent increase in prevalence perhaps associated to the aquaculture industry or environmental changes. Extensive exchange of shell and live oysters among regions in Washington continues to the present day, and to such an extent that Polydora websteri populations are genetically homogenous across broad swathes of their contemporary range10. Washington State has a long history of exchange with other oyster-growing regions65 and polydorin pelagic larvae may also have been introduced through ballast water67. Although it is likely that Polydora websteri is native to Asia and exotic to North America10, we suggest that Polydora websteri be considered cryptogenic in Washington State68 until further research can resolve its origins. Considering the species is distributed north and south of Washington (e.g.60), it is likely that the species has been present in this region but has never before been reported because it occurred only at low prevalence until recently. The prevalence of Polydora websteri is sensitive to environmental change. For example, increasing siltation can increase the susceptibility of Crassostrea virginica to Polydora websteri69. In contrast, reducing pH actually decreases susceptibility to infestation70. Because Polydora websteri can recruit to both live and dead oyster shells30, the expansion of the oyster aquaculture industry, oyster restoration, and increased density of oysters in beds across the state might have promoted an increase in transmission and prevalence if the polychaete was already present. Whatever their origin or how affected they are by changing conditions, the blister-forming polychaetes we document here are a new challenge for Washington State oyster growers and the government agencies charged with management of shellfish stocks.The fact that Polydora websteri is a generalist pest33, it may impact other shellfish species of ecological, economic, and cultural importance to Washington State. An important example is the Olympia oyster (Ostrea lurida), an overexploited native species that is the focus of intensive restoration efforts71. Mussels13, scallops16, and abalone (17see review in4) are also at risk. Given the important ecosystem services provided by filter-feeding shellfish species48, a polydorin outbreak could affect more than just the bottom line of the shellfish industry; ecosystem functioning is also at risk.Because Polydora websteri, in commercially farmed Pacific oysters, providing the first formal documentation of this globally distributed pest in Washington State. The pathology caused by shell-boring mud worms results in unsightly blisters that reduce the market value of infested oysters, especially those served on the half-shell. Washington’s Pacific oyster industry is dominated by the half-shell market61, and given the high prevalence of infestation found in this study, these pests have the potential to threaten the valuable Pacific oyster aquaculture operations in Washington State.We were not able to definitively identify the majority of worms collected from Totten Inlet Fig.  using ouCrassostrea gigas) and to confirm the species identity of these worms, we purchased 183 commercially grown oysters from retail shellfish farms in Washington State, USA. Of these, 69 individuals came from Oakland Bay  from North Sea Harbor, Long Island, New York, USA .To assess whether shell-boring polychaetes were present in Washington Pacific oysters  were photographed and measured (height and length of the shell) using a digital caliper  for storage. For examination with a scanning electron microscope (SEM), the specimens were dehydrated in an ascending ethanol series through 100% EtOH. Drying was accomplished with a Samdri 795 Critical Point Dryer. Once dried, the specimens were mounted on aluminum stubs, coated with gold using an EMS-550 Sputter coater, and viewed with a FEI Quanta 250 SEM. Voucher specimens , and 0.5 µl MgSO4 in a 10-µl reaction. 18S rRNA was PCR-amplified with an initial activation step of three minutes at 98 °C, followed by 35 cycles of denaturation (30 seconds at 98 °C), annealing (30 seconds at 54 °C), and extension (30 seconds at 72 °C) with a final extension step (10 minutes at 72 °C). Only the first of the three regions for 18S rRNA (18S-1F1/18S-1R632) was used for analysis because the other two did not amplify consistently. mtCOI was PCR-amplified with an initial activation step of 98 °C, followed by 30 cycles of: denaturation (30 seconds at 98 °C), annealing (30 seconds at 45 °C), and extension (60 seconds at 72 °C) with a final step of five minutes at 72 °C. The size of the PCR amplicons was checked in a 1.5% agarose gel. PCR products were sequenced in both directions using the amplification primers at Molecular Cloning Laboratories .For a subset (n = 27) of the total number of worms vouchered (n = 107) and for four additional worms collected from Long Island, New York, we extracted DNA using DNeasy 96 Blood & Tissue Kit  following the manufacturers’ instructions. We used two genes for molecular identification: the nuclear 18S rRNA [18S rRNA] and the mitochondrial cytochrome c oxidase I [mtCOI]. For the 18S rRNA gene, three regions were amplified: 18S-1F1/18S-1R632, 18S-2F576/18S-2R1209, and 18S-3F1129/18S-R1172Polydora websteri and other species in the genus Polydora, obtained from GenBank  to create consensus sequences. The consensus sequences were submitted to NCBI and registered in GenBank (accession nos. in Table nk Table . For thies Table  and nucles Table , were alt Tables  and 4.TaSupplementary Dataset 1.Supplementary Table 1.


The accuracy and precision of the predictions were improved compared with the Chinese healthy volunteers and Caucasian renal impairment population models. The PBPK population models were preliminarily developed and the first‐step validation results of four antibacterial drugs following intravenous administration showed acceptable accuracy and precision. The population models still need more systematic validation by using more drugs and scenarios in future studies to support their applications on dosage recommendation for Chinese renal impairment patients.To preliminarily develop physiologically based population models for Chinese renal impairment patients and to evaluate the prediction performance of new population models by renally cleared antibacterial drugs. First, demographic data and physiological parameters of Chinese renal impairment patients were collected, and then the coefficients of the relative demographic and physiological equation were recalibrated to construct the new population models. Second, drug‐independent parameters of ceftazidime, cefodizime, vancomycin, and cefuroxime were collected and verified by Chinese healthy volunteers, Caucasian healthy volunteers, and Caucasian renal impairment population models built in Simcyp. Finally, the newly developed population models were applied to predict the plasma concentration of four antibacterial drugs in Chinese renal impairment patients. The new physiologically based pharmacokinetic (PBPK) population models can predict the main pharmacokinetic parameters, including area under the plasma concentration–time curve extrapolated to infinity (AUC The PBPK population models of Chinese renal impaired patients were preliminarily developed by employing demographic and physiological parameters of Chinese patients and results of first steps towards verification by four antibacterial drugs following IV administration showed acceptable accuracy and precision in Chinese patients with mild, moderate, and severe renal impairment. According to a national report, up to 834,600 discharged patients multiplied by an average number of hospitalization on average admitted to 166 hospitals with 36.9% average utilization rate of antibacterial drugs, indicating that antibacterial drugs are widely used in clinical practice in China with high frequency  model combines the complex interplay of physiological parameters  with drug‐related properties  and thus can serve as a mechanistic approach to quantitatively predict the drug PK in different populations , peak concentration of drug in plasma (Cmax), and renal clearance (CLr), were compared with the observed data to assess the predictive performance of PBPK population models.Figure 2.12), moderate (GFR 30–59 ml/min/1.73 m2), and severe (GFR 15‐29 ml/min/1.73 m2) renal impairment were first developed based on the default Chinese healthy volunteers' model in Simcyp by recalibrating age distribution, height, weight parameters, and mean serum creatinine concentration values. The demographic information and serum creatinine in Chinese patients with various renal impairment were collected from the China Kidney Disease Network (CK‐NET) 2015 annual data report and published data , incorporating serum creatinine concentration along with demographic data, is the most commonly used index of overall kidney function and used to define the various levels of renal impairment in this study  of these variables from simulated 4000 subjects  were compared to observed data.2.2Vss) of ceftazidime, cefodizime, cefuroxime, and vancomycin was initially predicted  were selected. The literature retrieval strategy was shown in Figure 2). The other demographic and physiological parameters were kept the same as the Caucasian healthy volunteers' model  model built in Simcyp as a template and increasing concentration of serum creatinine by 1.5‐fold considering that the GFR lower bound cutoff value of mild renal impairment patients is about 1.5‐fold lower than healthy subjects  was additionally applied to examine and compare the precision and bias of simulations in PBPK models:Trial designs in Simcyp Simulator were set to match the observed clinical studies. The demographic information and study designs for all trials were summarized in Table 2.3In the combination of the verified drug models, the newly developed Chinese renal impairment population models were then applied to predict plasma concentrations of ceftazidime, cefodizime, vancomycin, and cefuroxime, respectively. The methods of simulation setting and assessment were the same as that of drug parameter verification in the above text.inf versus observed one and MAPE values, were compared.To further confirm the necessity of the newly developed population models, the Chinese healthy volunteers and Caucasian mild, moderate, and severe renal impairment population models were applied to simulate the same clinical designs of model drugs in Chinese renal impairment patients. Predictive performance among the three models, which was reflected by the ratio of predicted AUCBesides, the drug ceftazidime was chosen to simulate plasma concentration profiles over time in renal impairment with different levels . Three administration regimens  based on recommended dosage were designed and simulated to demonstrate the application of Chinese renal impaired population models.33.1Chinese renal impairment population models were first developed in Simcyp, for which age, body weight, height, and serum creatinine in Chinese patients with mild, moderate, and severe renal impairment were collected. The age limitation and sex ratio were represented by observational data. The summarized data and equation of system‐dependent parameters of the population models were shown in Tables −1 .The 2015 annual data report of CK‐NET included 887,816 hospitalized patients with CKD in 2015, which provided sufficient data for us to determine the sex ratio of Chinese mild and moderate renal impairment patients  were within 0.8‐1.25‐fold of the observed value; however, none of the predicted CLr was within 0.8–1.25‐fols of the observed values. As for subjects with moderate renal impairment, 67% of predicted AUCinf, 33% of predicted Cmax, and 50% of predicted CLr were within 0.8–1.25‐fold of the observed values. For patients with severe renal impairment, 25% of predicted AUCinf. 33% of predicted CLr, and none of Cmax were within 0.8–1.25‐fold of the observed values. Overall, these results suggested that drug‐dependent parameters were adequately inputted in our drug models.The drug models for ceftazidime, cefodizime, vancomycin, and cefuroxime were developed to predict plasma drug concentration following IV administration. The models were then verified with PK data from Caucasian healthy subjects, Chinese healthy subjects, and Caucasian renal impairment subjects. As shown in Figure 3.3inf for model drugs following single IV administration in Chinese renal impairment patients were within twofold of the observed values. Specifically, 50%, 100%, and 40% of prediction on AUCinf were within 0.8–1.25‐fold of the observed values in mild, moderate, and severe renal impairment patients, respectively. Only the observed CLr of cefodizime and observed Cmax of cefuroxime were available in Chinese severe renal impairment patients, which were in the range oftwofold and 0.8–1.25‐fold of the predicted values, respectively. Also, all predicted plasma concentration profiles were in good agreement with observed data that were available in the public domain and illustrated in Figure As was presented in Table inf simulated by Chinese renal impairment population models were much closer to the observed data, indicating improved precision and accuracy by our new population models.As shown in Table Figure 4Antibacterial drugs are frequently used in China. Although many of them are predominantly eliminated via renal and may have altered drug exposure in Chinese renal impairment patients, dose adjustment is usually not conducted in these patients due to a lack of corresponding PK data Xu & Wu, . To fill2; every 10 ml/min/1.73 m2 decline in GFR was associated with a mean 2.3 kg  weight decline  is below 60 ml/min in men and 40 ml/min in women   were within twofold of the observed data. Besides, the MAPE was used to evaluate the predictive ability and systematic prediction biases. The Chinese healthy volunteers model generally underestimated the exposure of model drugs in patients with various renal impairments. Particularly, the predicted values of the model in patients with moderate and severe renal impairment were lower than 50% of observed data, indicating that renal function loss exerts great effect on drug clearance. Although the predicted AUCinf values in Caucasian renal impairment population models were within twofold of the observed value as that in the Chinese renal impairment population models, the Chinese renal impairment population models exhibited higher accuracy and precision. Among these four drugs, ceftazidime and cefodizime, which were primarily excreted by glomerular filtration, showed better predictive performances. For vancomycin, it was reported that 25% of the drugs had (Table In this study, the antibacterial drugs ceftazidime, cefodizime, vancomycin, and cefuroxime, which are predominantly cleared via renal elimination, were used as model drugs to explore the predictive performance of developed population models. Compared with the observed data of four model drugs in Chinese patients with renal impairment, all predicted PK parameters (i.e., AUCad Table  renal tuad Table , whereasad Table  with 28%ad Table  of drugsThough the predicted PK profiles and parameters for these different drugs were generally in agreement with the observed data, our study had some limitations. First, due to the lack of hematocrit, serum albumin, and kidney volume data in the Chinese population, these data were assumed to be the same as Caucasian renal impairment patients. This assumption requires further verification and confirmation even though our model presented reasonable prediction on the PK of all four drugs in Chinese patients with mild, moderate, and severe renal impairment. Second, for drugs with high degree of active transport, the current model is insufficient to accurately predict their PK profile and parameters, and further development of mechanistic kidney models based on Chinese data is needed (Scotcher, Jones, Posada, Galetin, & Rostami‐Hodjegan, 5The PBPK population models by employing demographic and physiological parameters of Chinese patients with renal impairment were preliminarily developed and the results of first step toward verification by four antibacterial drugs following IV administration showed acceptable accuracy and precision in Chinese patients with mild, moderate, and severe renal impairment. In future studies, the model should be systematically validated using more drugs with various elimination mechanisms and diverse administrations to support its application in dosage recommendation in Chinese renal impairment patients even in the absence of a dedicated PK study.The authors have no conflict of interest to disclose.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


Homo naledi recovered from the 2013–2014 excavation season. From this assemblage, we attribute 16 postcranial elements and a partial mandible with some dentition to a single juvenile Homo naledi individual. The find includes postcranial elements never before discovered as immature elements in the sub-equatorial early hominin fossil record, and contributes new data to the field of hominin ontogeny.Immature remains are critical for understanding maturational processes in hominin species as well as for interpreting changes in ontogenetic development in hominin evolution. The study of these subjects is hindered by the fact that associated juvenile remains are extremely rare in the hominin fossil record. Here we describe an assemblage of immature remains of  Australopithecus afarensis [Australopithecus sediba [Homo erectus [Our knowledge of maturational processes and life history stages in human evolution is hindered by how extremely rare immature remains are in the hominin fossil record. Further complicating our understanding is that hominin fossil remains rarely combine teeth, cranial and associated postcranial bones, and only a handful of such partial skeletons represent immature individuals. Yet such remains provide important evidence about the maturation of extinct hominins. Apart from recent modern humans and Neanderthals , only tfarensis , 3, Austs sediba ,5 and Ho erectus –9. AugmeHomo naledi represent individuals of many juvenile and adult life stages, presenting a unique opportunity to identify and document in particular a range of immature individuals of an extinct species, and contribute data to the study of comparative hominin ontogeny [The immature individuals from the Dinaledi and Lesedi assemblages of ontogeny –13. The ontogeny . The Dinontogeny ,11,13,14The geology and taphonomy of the Dinaledi Chamber material has been described previously by Dirks et al. . To sum 2, excavated in two sessions in 2013 and 2014, to a depth of c20 cm below the surface. This unit lies midway inside the chamber and was targeted for excavation after fossil material was observed eroding through the surface. In total more than 1800 fossil specimens have been recovered from the Dinaledi Chamber.When the Dinaledi Chamber was first assessed, approximately 300 fossil fragments were found scattered across the surface of the chamber floor . The majen bloc in 2014 (U.W. 101–1477) appears to contain multiple fragments of an infant cranium. However, analysis of this thin and fragile material has only recently begun and identifications and descriptions are still underway.Within the primary excavation unit, material is commingled. Detailed analyses of the taphonomy and distribution of the hominin bone accumulations suggested at least some of the bodies were deposited while still fleshed ,17,18,19While the comingled nature of the assemblage makes it challenging to associate skeletal parts to single individuals, several instances of postcranial elements in direct articulation have been noted ,17,18. Tin situ articulated elements excavated from the southwest portion of the excavation unit, approximately 41–100 mm below surface (depths 1 and 2). The articulated elements include an almost complete left tibia (U.W. 101–1070), recovered with unfused epiphyses in place  was previously assessed from the 2013–2104 season based on dental remains: nine immature individuals, and six adult individuals ,13,20 F. The parThe DH7 associated lower limb represents a ‘Late Juvenile’ in skeletal maturation stage, with unfused but well-developed epiphyses of the hip, knee and ankle, a maturational skeletal pattern that is consistent across apes and hominins ,25–29.H. naledi anatomy [Criteria for including or excluding elements from association with DH7 include: duplication of element; developmental stage; size difference; spatial proximity (depth and span); refitting of specimens into a single element; and articulation/anatomical position of elements . Skeleta anatomy ,34,35,36 anatomy . If dept anatomy ,19, deptin situ articulation of the left lower limb elements provides certain evidence that these elements represent a single individual, deposited with soft tissue still intact. The U.W. 101–996 tibia meets this standard on the evidence of its mirror-image anatomy to U.W. 101–1070 [In this commingled sample we set a very high evidentiary standard to accept the association of different elements. Anatomical 101–1070 , its ideH. neanderthalensis) recovered mostly from a 1 m2 unit, together with multiple elements from at least one younger and one older immature individual. The consistent developmental stage and some direct articulations support the interpretation that the J1 material represents a single individual [2, along with developmental consistency and lack of element duplication [The evidence for skeletal association here meets the standards linking immature partial skeletons at other hominin sites with commingled individuals ,4,8,9. E2 unit, in addition to a small amount of material collected from the chamber surface. While the surface area of the chamber consists of approximately 20 m2, the depth of the sediments within is currently unknown. Consequently, we cannot predict what other remains of this individual may yet be recovered. We further caution that many elements in the collection are too fragmentary to accurately assess developmental stage.The 2013–2014 Dinaledi excavations involved a single 0.8 mWe identified additional skeletal specimens that are developmentally consistent in maturity indicators with the remains of DH7, recovered within 2–20 cm of the articulated lower limb Figs ; Table 4in situ ankle, at depth 2  was recovered from depth 1 . The unfh 2 Figs  and 12. h 2 Figs .en bloc at depth 2, and processed together in the lab . H. nal