Cancer-related fatigue (CRF) is a major symptom experienced by lung cancer patients receiving chemotherapy and radiation therapy. Since CRF has a multidimensional influence on cancer patients, they may experience physical weakening, a decline in cognitive function, and depression from emotional consequences. Kyung-Ok-Ko is used for improving fatigue or weak physical constitution. It is known to be effective in immune activation, reducing fatigue, and enhancing cognitive function. Although Kyung-Ok-Ko is clinically used for the treatment of CRF, its efficacy and safety against CRF in lung cancer patients are yet to be studied. Therefore, we aimed to investigate the efficacy and safety of Kyung-Ok-Ko.This is a randomized, placebo-controlled, patients-assessor blind, parallel-group, single-center clinical trial. Lung cancer patients with CRF, after termination of chemo or radiation therapies, are randomized in a 1:1 ratio to receive either Kyung-Ok-Ko or placebo for 6 weeks. The primary outcome is Brief Fatigue Inventory (BFI). The secondary outcomes include Visual Analog Fatigue Scale (VAFS), Functional Assessment of Cancer Therapy (FACIT) Fatigue scale, Hospital Anxiety Depression Scale (HADS), Montreal Cognitive Assessment Korean version (MoCA-K), and Korean pattern identification questionnaire. Adverse events are evaluated by Common Terminology Criteria for Adverse Events (CTCAE). All outcomes and adverse events are assessed at the baseline, mid-treatment, post-treatment, and at 1-month follow-up.This study investigates whether Kyung-Ok-Ko can alleviate CRF in lung cancer patients. The results of this study will provide clinical evidence for the application of Kyung-Ok-Ko in the treatment of CRF in lung cancer patients.http://cris.nih.go.kr; registration number: KCT000666).Korean Clinical Trial Registry (Trial status: Currently, participant recruitment is ongoing. Lung cancer treatments involve multimodality approaches, including surgery, radiotherapy, and cytotoxic chemotherapy, or molecularly targeted agents. During or post-treatment, a number of cluster symptoms, such as anxiety, fatigue, and dyspnea, appear. About 60% to 96% of patients who receive anticancer treatments complain of fatigue.Lung cancer is the leading cause of deaths due to cancer in both men and women worldwide. Since the influence of CRF on cancer patients is multidimensional, they may experience physical weakening, a decline in cognitive function due to deteriorating mental strength, and depression from emotional consequences.Cancer-related fatigue (CRF) has been referred to by a panel of the National Comprehensive Cancer Network (NCCN) as a distressing, persistent, subjective sense of physical, emotional, and/or cognitive tiredness or exhaustion related to cancer or cancer treatment that is not proportional to recent activity and interferes with usual functioning. It decreases the patient's quality of life, resulting in reduced compliance with chemotherapy, and ultimately has a negative effect on the survival of the patient; it is an index for the decreasing survival rate of cancer patients.,9 An 8-year long cohort study, comprising 2405 lung cancer patients, was conducted and among the symptoms of fatigue, coughing, and dyspnea that the patients frequently complained of, only fatigue was identified as a significant predictor of the poor 4-year and 5-year survival rate. In addition, according to a recently reported study, not only improving the quality of life but also self-evaluation of the symptoms affecting the quality of life alone was shown to increase the survival rate in patients receiving cancer treatment.CRF is experienced more by lung cancer patients compared to other types of cancer, and many patients were reported to experience CRF with disabilities. Although CRF is induced by various factors, many studies have reported inflammation as the common factor of the mechanism.The major reasons for CRF are the side effects of cancer treatment. Although the exact mechanism is not known, all treatments, including chemo and radiation therapy, molecular targeted agents, and surgical treatment can induce CRF.Since CRF is the main reason for the interruption in treatment or exacerbation of the disease, much effort has been focused on the management of CRF. However, it remains underdiagnosed and undertreated, without any standard therapy until now.vitro and clinical studies that Kyung-Ok-Ko activates the immune system, reduces fatigue,,14 and enhances the cognitive function. Anti-inflammatory effects have also been reported, as a supporting mechanism.–18Kyung-Ok-Ko is a prescription, which is described in Donguibogam, a traditional Korean medicine book, and consists of Ginseng Radix, Poria Sclerotium, Rehmanniae Radix Crudus, and honey. It has been widely used for improving fatigue or weak condition. It has been developed as a traditional Korean medicine based on national standards approved by the MFDS (Ministry of Food and Drug Safety). It is reported via in Although Kyung-Ok-Ko is used for alleviating fatigue, research on CRF in lung cancer patients is limited. Therefore, we aimed to identify the efficacy and safety of Kyung-Ok-Ko on lung cancer patients experiencing CRF.22.1This is a randomized, placebo-controlled, patients-assessor blind, parallel-group and single-center clinical trial. This study was designed to evaluate the efficacy and safety of Kyung-Ok-Ko in lung cancer patients who have received cancer treatment and are experiencing CRF. The trial will be conducted for a total of 10 weeks, as patients who experienced CRF 2 weeks after completing cancer treatment will take the clinical trial drug for 6 weeks, followed by a 4-week observation period. A flow diagram of the study is presented in Figure 2.2Subjects are randomized in a 1:1 ratio to the experimental group (Kyung-Ok-Ko group) and control group (placebo group). For randomization, a statistician independent of this clinical trial will use SAS ver.9.1.3  for Microsoft Windows to randomly assign numbers to create a randomization table. The experimental group or placebo group assignments according to the table of random numbers and random numbers generated using SAS are kept by a third party until termination or completion of the trial, and investigators do not require them. The study drug and placebo are assigned and managed by a code and a patients-assessor blind method, where the investigators and subjects cannot distinguish study drug and placebo, was followed. For this purpose, the placebo was manufactured similar to the study drug in terms of appearance, flavor, and scent. The manufacturing company conducted code labeling assigned for the study drug and placebo, and a third party matches the random number and code. When registering each study subject, the third party provided the management number of the study drug or placebo that has been matched with the random number to the investigator via telephone, text message, or mobile communication application. Clinical trial drug and placebo were kept in the pharmacy of Kyung Hee University Korean Medicine Hospital Clinical Trial Center (K-CTC) and was delivered to the subjects by a pharmacist who was kept blind. If the blinding should be broken prior to completion of the clinical trial due to serious adverse events (SAEs) during the clinical trial period, the reasons for breaking blindness will be reviewed and approved before breaking the blindness by the principal investigator.2.32.3.1(1)Men and women aged above 19 years.(2)Lung cancer patients who received cancer treatment (chemo and radiation therapy).(3)Patients who have completed cancer treatment >2 weeks ago and do not have any further cancer treatment plans.(4)Patients experiencing fatigue, with brief fatigue inventory (BFI) ≥4.(5)Patients with Eastern Cooperative Oncology Group (ECOG) scale ≤2.(6)Patients who voluntarily decided to participate in this clinical trial and signed the consent form.2.3.2(1)Patients suspected of relapse or progression according to chest CT or chest X-ray.(2)Patients with hemoglobin level <9 g/dL, or platelets <50 000/mL, or absolute neutrophil count <1000/mL.(3)Patients with uncontrolled thyroid disorder .(4)Patients with moderate liver and kidney impairment (increase in aspartate transaminase (AST), alanine transaminase (ALT) by >3 times, or increase in creatinine by >2 times).(5)Patients who do not expect >6 months of life expectancy after cancer diagnosis.(6)Patients diagnosed and treated for severe dementia , severe depression .(7)Patients who are deemed difficult for clinical trial performance.(8)Early-stage lung cancer patients who are treated with surgery alone and late-stage lung cancer patients who are not receiving cancer treatment.(9)Patients who received oriental medicine or pharmacotherapy  for the purpose of improving CRF in the last 4 weeks.(10)Patients who are pregnant or breastfeeding.(11)Patients who did not agree to contraception during the clinical trial period.(12)Patients who participated in a clinical trial for the same disease, in the last 3 months.(13)Patients who are not eligible to participate in this trial at the discretion of the investigator.2.3.3(1)Subjects who received treatment that could affect the outcome without the investigator's instructions during the study period.(2)Subjects who do not follow clinical study protocol or have compliance <80%.(3)Subjects who cannot continue clinical trial due to adverse events (AEs) or if serious AEs occurred.(4)Subjects who withdraw consent.(5)Subjects who use methylphenidate, modafinil, bupropion, and oriental medicines during clinical trial, which can influence fatigue improvement.(6)Subjects who are determined by the investigator to be ineligible for participation in the clinical trial.2.42.4.1The experimental group is administered Kyung-Ok-Ko, which is a dark-brown paste preparation (23.5 g per package), twice a day for a total of 6 weeks. Kyung-Ok-Ko is manufactured by Kwang Dong Pharmaceutical Co., Ltd , which is approved by the MFDS in accordance with the Korean Good Clinical Practice (GCP) regulations. The 23.5 g Kyung-Ok-Ko is composed of 7.98 g of Rehmanniae Radix Crudus, 2.48 g of Poria Sclerotium, 1.24 g of Ginseng Radix, and 8.3 g of honey and other excipients. The subjects receive a 14-day package per visit.2.4.2The placebo group is administered placebo Kyung-Ok-Ko (23.5 g per package) twice a day for a total of 6 weeks. The placebo is manufactured by Kwang Dong Pharmaceutical Co., Ltd, similar to the physical traits of the clinical trial drug. The placebo that is very similar in size, flavor, scent, and color without the key ingredients is used. The placebo is composed of glucose, sucrose, lactose, corn starch, D-sorbitol, xanthan gum, and caramel color. With a 2-week follow-up, the subjects received a 14-day package per visit.2.4.3The clinical trial drug is kept in airtight containers at room temperature (21–30°C). The clinical trial drug is administered by the pharmacist in accordance with the investigator's prescription, and the pharmacist accurately records the clinical trial drug provided to the study subjects and management status. Since this is a double-blind clinical trial, study subjects are only identifiable through their identification number, and information about the prescribed and collected clinical trial drug should be appropriately recorded. Upon termination or completion of the clinical trial, the pharmacist must return the unused clinical trial drug to the sponsor, retain the proof of return, and the sponsor shall dispose of the returned clinical trial drug.2.52.5.12.5.1.1 It is composed of 9 questions, with 3 questions for the degree of CRF and 6 questions for the effects of CRF. Each question is scored from 0 to 10 points, where a higher score indicates severe symptoms; 1 to 3 point is mild, 4 to 6 point is moderate, and 7 to 10 point is severe. Measurements are taken at the baseline, 2 weeks, 4 weeks, 6 weeks, and 10 weeks after treatment  to 10 (very tired) and classified as: Grade 1 or mild , Grade 2 or moderate , and Grade 3 or severe  using National Cancer Institute Common Toxicity Criteria version 4.03. Measurements are taken at the baseline, 2 weeks, 4 weeks, 6 weeks, and 10 weeks after treatment.2.5.2.2 The 13 questions of this measurement tool are based on the cancer patient's feelings and experience with fatigue, eating and sleeping with fatigue, activity status, and fatigue experienced in the past week. It is scored from 0 points for ‘Strongly disagree’ to 4 points for ‘Strongly agree’, where a higher score indicates a high degree of fatigue. Measurements are taken at the baseline, 2 weeks, 4 weeks, 6 weeks, and 10 weeks after treatment.The FACIT Fatigue scale is composed of 13 questions, and the cancer patient is required to give a score to the statement that best represents his or her weekly experience.2.5.2.3 It is composed of 2 subcategories, anxiety and depression, with a total of 14 questions, consisting of 7 questions for anxiety and 7 questions for depression. Each question is based on a 3-point scale, with a total of 21 points possible for each subcategory of anxiety and depression. A higher score indicates a high degree of anxiety and depression, where 0 to 7 points indicate no anxiety and depression, 8 to 10 points suspect anxiety and depression, and >11 points indicate anxiety and depression. Moreover, the total scores refer to a combination of anxiety and depression scores and classified as mild, ranging from 8 to 10, moderate from 11 to 14, and severe from 15 to 21 points. Measurements are taken at the baseline, 2 weeks, 4 weeks, 6 weeks, and 10 weeks after treatment.The HADS is an efficient screening tool generally used to assess the anxiety and depression of hospitalized cancer patients.2.5.2.4 It consists of 30 points in total, consisting of short-term memory test (5 points), clock drawing for space-time function (3 points) and 3-dimensional block drawing tests (1 point), trail-making test B for performance (1 point), language fluency test (1 point) and 2-item verbal abstraction task (2 points), concentration maintenance test to test concentration (1 point), subtraction in order (3 points), digit span forward and backward (2 points), animal names (3 points) and following speaking complex phrases (2 points), and disorientation for time and place (6 points). If the total score is <22, mild cognitive impairment can be suspected. Moreover, mild cognitive impairment score ranges from 18 to 26 points, moderate from 10 to 17 points, and severe if <10 points. Measurements are taken at the baseline, 2 weeks, 4 weeks, 6 weeks, and 10 weeks after treatment.The Korean version of Montreal Cognitive Assessment is a tool developed to examine mild cognitive impairment.2.5.2.5 It was developed as a tool to assess chronic fatigue, and studies on its validity have been conducted. Measurements are taken at the baseline, 2 weeks, 4 weeks, 6 weeks, and 10 weeks after treatment.Qi Blood Yin Yang Deficiency Questionnaire is based on a 4-point Likert scale, composed of 15 questions.2.5.2.6–30 In this regard, we aimed to examine the inflammatory markers as one of the objective indicators of the efficacy of Kyung-Ok-Ko in improving fatigue. Inflammatory markers included C-reactive protein, erythrocyte sedimentation rate, and cytokines, such as interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, IL-1ra, IL-8, IL-10, and interferon (IFN)-γ. Measurements are taken at the baseline and 6 weeks after treatment.Many researchers have reported changes in the immune and inflammatory responses as a mechanism of CRF and inflammation-related cytokine studies, in relation to CRF, have been conducted.2.6AEs refer to undesirable, unintended signs, symptoms, or diseases that occur during clinical trials and do not necessarily have causal relationships with treatments used in the trial. The presence of AEs should be checked at every visit. The severity of AEs will be evaluated by Common Terminology Criteria for Adverse Events (CTCAE). An SAE refers to one of the following AEs occurring in a clinical study participant during a clinical trial:(1)Death or danger to life;(2)Hospitalization or extension of hospital stay due to the adverse event;(3)Permanent or significant failure or degradation of function;(4)Development of fetal malformations or abnormalities;(5)Other medically important situations.In the case of SAEs, the investigators should report to the sponsor very quickly . This would ensure patient safety in the clinical trials and meet the reporting requirements of the Korean MFDS. In addition to reporting AEs to the sponsor, the investigator also reports to the Institutional Review Board (IRB). To assess the safety, laboratory tests determined the AST, ALT, alkaline phosphatase, total bilirubin, gamma glutamyl transferase, blood urea nitrogen, and creatinine. Measurements are taken at the baseline, 2 weeks, 4 weeks, 6 weeks, and 10 weeks after treatment.2.7 the mean and standard deviation of BFI were 3.4 ± 1.6 and 5.2 ± 1.9 for the experimental group and placebo group, respectively. Using a 0.05 significance level, 90% power, and allowing for a 10% dropout rate, a sample size of 50 participants was estimated, 25 in each group.The sample size was estimated following a similar clinical research study because the exact same study design has not been employed before. The primary endpoint is the BFI, and the sample size is calculated based on the effect size of the primary endpoint. According to the previous literature,2.8Among lung cancer patients receiving cancer treatment, patients who experienced CRF are recruited from the division of pulmonology and radiation oncology of the Kyung Hee University Medical Center. This clinical trial is to be conducted at K-CTC. The purpose of this study is explained to the subjects, and screening is conducted among those who signed the informed consent form.2.9Data collection is conducted based on the approved protocol. The collected data is recorded in the case report. The quality of the study will be managed by regular monitoring. An independent monitoring supervisor would be assigned to contact and visit the researchers regularly, and thus, supervise the trial process. All investigators will be trained and will comply with GCP regulations. All documents related to the clinical trial will be archived by the principal investigator and the manager. Data will be stored in a separate place with security. The participants’ information will be maintained in storage for a period of 3 years after study completion. In case of a change in the protocol, it will be immediately reported to the IRB, and all investigators will be informed2.10This trial has been authorized by the IRB of the Kyung Hee University Korean Medicine Hospital (KOMCIRB 2018-05-017-001). The protocol complies with both the Declaration of Helsinki and GCP Guidelines. Signed informed consent forms will be obtained from all eligible participants before enrollment.This trial is registered with the Korean Clinical Trial Registry (KCT0003115).2.11Statistical significance is based on the significance level 0.05 with a power of 0.8.ITT: Includes all subjects who were administered with the study drug or placebo at least once, and performed and returned the evaluation sheet for BFI.PP: Includes all subjects who were administered with >80% of the assigned study drug or placebo, and completed the evaluation sheet for at least 34 days  of BFI.For all analyses, ITT analysis is the major analysis, and PP analysis is simultaneously performed to present the results together.U test is performed. Categorical variables are presented as frequency and ratio, and Chi-square test or Fisher's exact test  is performed.Continuous variables are presented as mean and standard deviation, and independent t test or Mann–Whitney t test is performed if normality test is satisfied, and Mann–Whitney U test is performed if the normality test is not satisfied. If a significant difference between groups is found for the baseline values, ANCOVA is performed. The missing value will be replaced by the last measured value of each subject, according to the Last Observation Carried Forward (LOCF) method.Primary efficacy evaluation is the comparison of the mean of BFI score assessed at week 6 (42 days after administration) between the groups. The mean difference between the baseline and week 6 of VAFS, FACIT-F, MoCA-K, HADS and inflammation markers are to be compared. Independent 3–18 A study had demonstrated its effect in lung cancer, and the antifatigue effect has also been studied.,13 However, a study to investigate the efficacy and safety of Kyung-Ok-Ko in CRF, which is not easily alleviated, and thus, affects the quality of life and prognosis of cancer patients, is yet to be conducted.This study is the first research protocol to clarify the efficacy and safety of Kyung-Ok-Ko in CRF in lung cancer patients. Kyung-Ok-Ko is widely used in East-Asian countries, especially Korea, for recovery from fatigue and the reinvigoration of physical strength. In addition, Kyung-Ok-Ko is, clinically, the most frequently used drug to improve CRF. Kyung-ok-ko has been proven to have anti-inflammatory effects in a variety of illnesses. Observational research has found that fatigue is induced by the presence of inflammatory markers, and a pilot study found that fatigue was improved by blocking TNF-α in cancer patients who had experienced fatigue. Based on the previous studies, which have shown the anti-inflammatory effects of Kyung-Ok-Ko, this research was designed to investigate the effect of Kyung-Ok-Ko in the regulation of inflammatory cytokines to reveal the treatment mechanisms of CRF.Most of the major causes of CRF are side effects of cancer therapy, and it is commonly found that the fatigue mechanism is associated with inflammation. It has been reported that inflammation occurs because the cytokines affecting the brain induce many neurological, physiological, and behavioral changes, causing fatigue.This study has the following strengths. First, we unified only lung cancer to examine the efficacy and safety of Kyung-Ok-Ko in fatigue. Second, the study could contribute to the identification of a mechanism in association with inflammation, which is considered as a fatigue mechanism, by observing cytokine level before and after Kyung-Ok-Ko prescription. Third, we developed a dark-brown placebo paste preparation. To achieve this, the researchers had several meetings and tested various prototypes to develop the placebo. Although it was difficult to match the unique fragrance and viscosity of the traditional Korean medicine, we finally made a similar placebo, which made it possible to “blind” the patients and the researcher. Fourth, the study protocol was established closely following the Guidelines for GCP and the Consolidated Standards of Reporting Trials (CONSORT).In conclusion, the study is aimed to examine the efficacy and safety of Kyung-Ok-Ko in treating CRF in lung cancer patients and establish high-quality evidence for clinicians and patients who are willing to use Kyung-Ok-Ko for CRF.Conceptualization: BJLData curation: MKInvestigation: SHLMethodology: MK, SHLProject administration: MK, SHLSupervision: BJLWriting – original draft: KIKWriting – review & editing: KIK, BJL


The lateral septal nucleus (LS) plays a critical role in emotionality, social behavior and feeding processes, through neural connections with the hippocampus and hypothalamus. We investigated the neural circuits of LS by using herpes simplex virus 1 strain H129 (H129) and pseudorabies virus stain Bartha (PRV). Virus H129 indicates that LS directly projects to some cerebral nuclei , part of the hypothalamus , thalamus  and the pontine central gray. Then the LS has secondary projections to the CA3 and CA1 field of the hippocampal formation, lateral and medial preoptic area, and the mammillary body. PRV tracing shows that LS are mainly receiving primary inputs from the amygdala, hippocampus, hypothalamic, thalamus, midbrain and hindbrain, and secondary inputs from dorsal and central linear nucleus raphe, the lateral part of the superior central nucleus raphe, the ventral anterior-lateral complex, the intermediodorsal nucleus, the central medial nucleus, the rhomboid nucleus, and the submedial nucleus of the thalamus. The neural circuit data revealed here could help to understand and further research on the function of LS. Summary: We identified the sequence of projections from the lateral septal nucleus by virus tracing and expanded the data on neural circuits, which could help to understand brain function. The lateral septal nucleus (LS) is the rostrodorsal part of the septal region, dorsolateral adjacent to the medial septal complex. Usually, the LS is cytoarchitectonically sectioned into dorsal, intermediate and ventral divisions . AccordiExtensive studies have been focused on the functions of LS, such as its critical role in emotionality, social behavior, and feeding processes. The neurons connections in the CA2-LS-ventromedial hypothalamic area can control aggressive behavior . The neuEven though conventional neuroanatomical tracing techniques show the connections of LS with other brain areas , they arNeurotropic viruses have been developed as effective trans-neuronal tracers and to analyze central nervous system networks involving chains of two or more functionally connected neurons. The pseudorabies virus (PRV) strain Bartha is an excellent retrograde trans-neuronal tracer, and the herpes simplex virus 1 strain H129 (H129) is an anterograde tracer . These vThis study’s purpose was to determine the cascade of projection from the LS. Our results show that a low dose of virus H129 enables the tracing of explicit second-order projection sites in the medial septal nucleus, hypothalamus and hippocampus, and PRV enables the tracing of specific second-order projection sites in the thalamus and raphe nuclei.9 pfu/ml) and PRV-EGFP virus (2×1010 pfu/ml) are shown in Figs S1 and S2, respectively. Red fluorescence of cholera toxin subunit B (conjugated Alexa Fluor™ 594) was used in the cocktail to help identify the injection sites. We excluded the unsatisfactory samples, and the injection sites of the remaining animals were highly circumscribed within LSr, specifically around the medial and/or dorsolateral zone of LSr (LSr.m and LSr.dl) as inferred from the subdivision of LSr in rat  A, the veIn the amygdala and adjacent areas , the projection appeared in the lateral amygdala nucleus, basolateral amygdala nucleus, central amygdala nucleus and substantia innominate at 40 h after inoculation E. In theAt 40 h after inoculation in the thalamus, the labeled neurons could be found in the polymodal association cortex related area, including the paraventricular nucleus, the parataenial nucleus, the nucleus of reuniens, the anterior group of the dorsal thalamus A and medIn the hippocampal area, no trace of infection could be found at 40 h after inoculation. At 56 h after injection, the field of CA3 received input in the pyramidal layer D. MoreovThe middle of pontine central gray C,D, whicAt 40 h after inoculation, the distribution of PRV could be found in similar areas infected with H129 virus, with the field of CA2 and the entorhinal area in hippocampal formation, and the parvicellular reticular nucleus labeled by PRV only B.Fig. S2). At 40 h after virus injection, the trace of infection was found at the lateral septal complex  and medial septal complex , nucleus accumbens and bed nuclei of the stria terminalis ; the dorsal part of taenia tecta , substantia innominata  and magnocellular nucleus .The site of PRV injection was also identified within the LSr.m and LSr.dl , periventricular zone , periventricular region  and adjacent areas such as lateral preoptic area, were infected by PRV. In the hypothalamic medial zone and adjacent area, the medial preoptic nucleus  and anterior and ventromedial hypothalamic nucleus (VMH)  had labeled neurons. Nearby, scattered labeled neurons were distributed in the amygdala .In the rostral part of the hypothalamic area at 40 h after virus injection , disperse labeled neurons were identified. In addition, labeled neurons were found in the stria medullaris and stria terminalis . At 56 h after inoculation, labeled neurons in the ventral anterior-lateral complex, the mediodorsal nucleus, central medial nucleus, rhomboid nucleus and submedial nucleus of the thalamus could be found .At 40 h after virus injection in the thalamus areas, such as the paraventricular nucleus, the nucleus of reuniens, and medial habenula  and the amygdala   also have labeled neurons at 40 h after virus injection.The parts of hippocampus, such as field CA1, CA2, CA3, the dentate gyrus, the ventral part of the subiculum, the lateral part of the entorhinal area .In the midbrain, the motor related part, including the periaqueductal gray, the intermediate gray layer of the motor-related superior colliculus, the midbrain reticular nucleus and the retrorubral area of midbrain reticular nucleus have scattered labelled neurons and a weak signal at 40 h after virus injection , also have labeled neurons.In the hindbrain, the motor-related area, including the tegmental reticular nucleus and pontine central gray, in addition to the behavioral-state related pons area, including the pontine reticular nucleus, the medial part of the superior central nucleus raphe, locus ceruleus and nucleus incertus , and in the pons, the lateral part of the superior central nucleus raphe, received input at 56 h after virus injection .Also, the dorsal nucleus raphe and central linear nucleus raphe in the midbrain  some cerebral nuclei, including the nucleus accumbens, bed nuclei of the stria terminalis and the amygdala; (2) part of the hypothalamus, such as the median preoptic nucleus area, the paraventricular hypothalamic nucleus, the lateral hypothalamic area and the dorsomedial nucleus of the hypothalamus. (3) Part of the thalamus, containing the paraventricular nucleus, the parataenial nucleus, the nucleus of reuniens, the medial habenula, and midline nuclei; (4) the pontine central gray. Then secondarily projects to the LSv, the medial septal nucleus, the field CA3 and the field CA1 of the hippocampal formation, lateral and medial preoptic area, and the mammillary body.PRV tracing shows LS receive many more direct projections from the above area. Then secondary projection from several raphe , the lateral part of superior central nucleus raphe in the pons, and part of the thalamus .Our results suggest that the secondary projections cross through medial brain fiber tracts (such as the fimbria) to reach the field CA3 and CA1, and traveling along the supramammillary decussation to the mammillary body, might come from the subsequent projection of subnucleus in the LS . The forAs the dorsal subnucleus of the lateral septal nucleus sent the largest number efferent fibers to the medial septal nucleus than other subnuclei , that maRetrograde tracing results were more complicated at the nucleus accumbens, some nuclei in the amygdala and hypothalamus may provide mainly secondary projection to the thalamus and raphe nuclei, those projections may be involved in emotion, cognitive and sensorimotor processes  and 40 nl 0.05% recombinant cholera toxin subunit B conjugated Alexa Fluor™ 594  or cocktail containing PRV-CMV-EGFP (2×1010 pfu/ml) and 40 nl 0.05% cholera toxin subunit B was injected into the medial zone on rostral lateral septal nucleus with coordinate AP/DV/ML: 0.6/0.3/3.3 mm using a pulled glass pipette on a speed of 30 nl/min . After the injection, the pipette was left in place for 15 min and then was drawn out gently. Four mice at 40 h and four mice at 56 h were euthanized. All operations with the virus were conducted in a Level-2 Biosafety laboratory.The herpes simplex virus 1 strain H129 (H01001 HSV-EGFP) and pseudorabies virus strain Bartha (P01001 PRV-CMV-EGFP) were supplied by BrainVTA Co. Ltd . 100 nl of cocktail containing 60 nl HSV-EGFP  and adjusted by NIS-Elements. The locations of the labeled neurons and outlines of the brain nuclei were manually defined according to the Allen Mouse Brain Atlas , and the


A piezoelectric nanogenerator (PNG) that is based on c-axis GaN nanowires is fabricated on flexible substrate. In this regard, c-axis GaN nanowires were grown on GaN substrate using the vapor-liquid-solid (VLS) technique by metal organic chemical vapor deposition. Further, Polydimethylsiloxane (PDMS) was coated on nanowire-arrays then PDMS matrix embedded with GaN nanowire-arrays was transferred on Si-rubber substrate. The piezoelectric performance of nanowire-based flexible PNG was measured, while the device was actuated using a cyclic stretching-releasing agitation mechanism that was driven by a linear motor. The piezoelectric output was measured as a function of actuation frequency ranging from 1 Hz to 10 Hz and a linear tendency was observed for piezoelectric output current, while the output voltages remained constant. A maximum of piezoelectric open circuit voltages and short circuit current were measured 15.4 V and 85.6 nA, respectively. In order to evaluate the feasibility of our flexible PNG for real application, a long term stability test was performed for 20,000 cycles and the device performance was degraded by less than 18%. The underlying reason for the high piezoelectric output was attributed to the reduced free carriers inside nanowires due to surface Fermi-level pinning and insulating metal-dielectric-semiconductor interface, respectively; the former reduced the free carrier screening radially while latter reduced longitudinally. The flexibility and the high aspect ratio of GaN nanowire were the responsible factors for higher stability. Such higher piezoelectric output and the novel design make our device more promising for the diverse range of real applications. The emergence of portable/wearable electronic gadgets that may accompany human body day-in and day-out has attracted worldwide attention of researchers due to their potential for a variety of applications. Among these, a variety of potential energy sources, mechanical sources, such as vibrations ,2, movem31, e33, and e15 while the latter has only one independent piezoelectric coefficient e14. The strongest piezoelectric semiconductor materials are from wurtzite family of crystal structure such as GaN, ZnO, InN, and AlN. The piezoelectric effect of semiconductors was studied in early 1960s to develop acoustic electronics in Bell Telephone laboratories [Traditionally, ceramic materials were used to fabricate PGs, but their applications remain limited due to very low output power density, such as quartz for watches and electric cigarette lighters ,11,12. Tratories . A boom ratories . The aliThe semiconductor-based PGs can be fabricated using thin film or quasi one dimensional (1D) nanostructure; both the structures carry their pros and cons simultaneously. The fabrication of semiconductor thin films is easy, but the piezoelectric output is relatively low along with low compliance values. On the other hand, the fabrication of quasi 1D nanostructure is difficult, but it is accompanied with several advantages. Semiconductor NWs are the best candidate to fabricate compact and efficient PGs , becauseAmong wurtzite semiconductor materials, ZnO is the most researched material for piezoelectric applications. GaN has a slightly lower piezoelectric coefficient, but it is considered to be a superior material due to its biocompatibility, mechanical robustness. In order to harvest high piezoelectric output from a semiconductor PG, suppression of internal screening by rectifying contact is an essential requirement. Forming a p-n homojunction and heterojunction is an efficient route to harvest high piezoelectric output by suppressing the free carrier screening and junction screening ,22,23,24In this study, we have fabricated the GaN NW-based PNG using Ni as metal contact. The effect of MDS interface was studied to enhance piezoelectric output using Polydimethylsiloxane (PDMS) as dielectric medium between Ni electrode and GaN NWs. In order to use PNGs for real applications, it is necessary to fabricate them on flexible substrate ,33,34,353 were 300 µmol/min and 450 mmol/min, respectively, which were the respective precursors for gallium and nitrogen; moreover the V/III ratio was kept constant at 1500. The growth was carried out at 1175 °C at the reactor pressure of 200 torr for 5500 s. In order to reduce the stress in GaN thin film due to lattice mismatch of substrate and the grown thin film of GaN, a low temperature buffer layer of GaN, was also grown at 600 °C. In order to demonstrate piezoelectricity from GaN NWs, only c-axis GaN NWs are feasible due to non-centrosymmetric behavior of crystal structure upon straining. In order to eliminate the stresses inside NWs due to lattice mismatch of NWs and substrate, 3 µm thick c-axis GaN thin film was grown on c-plane sapphire substrate. The flow rates of trimethylgallium (TMGa) and NH−6 torr on GaN thin film. Then, the samples were loaded inside the reactor of MOCVD. Then, the samples were annealed at 880 °C for 500 s in H2 environment, so that the agglomeration can take place. After the agglomeration of the catalyst, the precursors of gallium and nitrogen were introduced. TMGa and NH3 were used as precursors of gallium and nitrogen, respectively. The flow rate of TMGa and NH3 was 39.5 µmol/min and 6.7 mmol/min, respectively, with V/III ratio of 170. The growth of GaN NWs under VLS mode was carried out for 5000 s at 850 °C at the reactor pressure of 60 torr.In order to grow c-axis GaN NWs, vapor-liquid-solid (VLS) technique was adopted. VLS technique requires a metal catalyst. Thus, 0.8 nm thick Au thin film was deposited by e-beam evaporation at vacuum of 5 × 10After the growth of high aspect ratio c-axis GaN NWs, PDMS was deposited by spin coating on GaN NWs and NWs embedded in PDMS is called matrix of NW-arrays. The spin coating was performed at 7000 rpm for 300 s. To harden PDMS, the samples were baked at 150 °C for 600 s. In order to complete the fabrication of PNG, ITO was deposited on Si-rubber substrate using sputtering. Then, the PDMS matrix embedded with GaN NW was cut using doctor blade, flipped and transferred on ITO coated Si-rubber substrate. Subsequently, PDMS was deposited to sandwich the matrix between PDMS and ITO-coated Si rubber. Then, Ni metal was deposited, followed by ITO deposition by sputtering to fabricate the top electrode of PNG.In order to evaluate the morphology of GaN NWs, field emission scanning electron microscope  was used. After the successful fabrication of PNGs, the piezoelectric performance of PNGs was measured by a high speed current-voltage measurement unit , while PNGs were actuated by a periodic stretching-releasing agitation driven by a linear motor.In the case of semiconductors, the piezoelectric effect can be observed only from the crystal structures, which exhibit non-centrosymmetry upon straining. The wurtzite crystal structure is non-centrosymmetric along c-axis only. In this regard, c-axis GaN NWs were grown by the VLS mode using Au as catalyst. Due to the c-axis orientation of GaN thin film, GaN NWs were grown well aligned as depicted from the schematic illustration in The piezoelectric performance of the device was evaluated after connecting the GaN NW-based PNG to potentiostat, while the device was actuated by a cyclic stretching-releasing agitation mechanism that was driven by a linear motor. At actuation frequency of 8 Hz, the open circuit output voltages and short circuit current were found 15.4 V and 64 nA, respectively, as shown in The work function of Ni metal is 5.22 eV, while the electron affinity of GaN is 4.53 eV. After the fabrication of the device, the Fermi level of GaN and metal are aligned, as a result, the Fermi level of all regions, including PDMS, aligned at the same level. The top surface of c-axis GaN NWs is the Ga-polar plane, thus under compressive stress, negative piezoelectric charges appear at the top of NWs towards c-axis direction, while positive charges appear at the bottom that is the—c-axis direction. The top and bottom ITO electrodes are not in direct contact, thus a capacitive coupling is achieved between ITO/Ni metal contact and GaN NWs through PDMS dielectric layer on the top side of PNG, further, the capacitive coupling is also formed between the bottom side ITO contact and GaN NWs. Moreover, NWs matrix was flipped and was then transferred, thus, the negative charges appear on top ITO contact of PNG and negative on bottom contact upon compressing.The c-axis GaN NWs were intrinsically n-type, thus, due to the upward bending of valance band and the conduction band near surface of NWs, electrons are spatially separated from holes due to surface band bending ,37. ThusThe piezoelectric performance of this GaN NW array based PNG is compared with NW array based PNG that is composed of other material systems. The piezoelectric output of ZnO NW arrays was reported to be 20 V and 58 V in the literature ,39, and In order to evaluate the compatibility of our device in a real application, a long-term stability test was performed for 20,000 cycles under an actuation frequency of 8 Hz, as shown in Well aligned c-axis GaN NWs were grown for the fabrication of flexible PNG. The transfer of the PDMS matrix embedded with c-axis GaN NWs on Si-rubber substrate was carried out using doctor’s blading, followed by the completion of device fabrication. The piezoelectric performance was measured, while the flexible PNG was actuated by a cyclic stretching-releasing agitation mechanism driven by a linear motor. A maximum open circuit output voltages and short circuit current of 15.4 V and 85 nA were measured, respectively. Moreover, the piezoelectric output of flexible PNG was evaluated as a function of actuation frequency. The actuation frequency was varied up to 10 Hz; piezoelectric output voltages remained constant throughout the range of actuation frequency, while a significant increase of piezoelectric current was observed by increasing the actuation frequency. The constant output voltages of 13.6 V were measured for all of the actuation range while the output current was found 34 nA, 64 nA, and 85 nA at actuation frequencies of 4.5 Hz, 8.0 Hz, and 10 Hz, respectively. Such high piezoelectric output was attributed to the surface Fermi-level pinning of GaN NWs, which reduced the free carriers inside GaN NWs, ultimately reducing free carriers screening. Moreover, the suppression of free carrier screening was enhanced due to the MDS interface, which inverts the surface of GaN NWs accordingly. At the end, the feasibility of our device for a real application was also checked by performing the long term stability test for 20,000 cycles, and the device performance was degraded less than 18% of its initial output. The design and the piezoelectric output make our device intriguing for a variety of applications in the field of sensors and ambient energy harvesting applications.


There is only limited information on mortality over extended periods in hospitalized patients with pneumococcal community-acquired pneumonia (CAP). We aimed to evaluate the 30-day mortality and whether is changed over a 20-year period among immunocompetent adults hospitalized with pneumococcal CAP.We conducted a retrospective observational study of data that were prospectively collected at the Hospital Clinic of Barcelona of all adult patients hospitalized with diagnosis of pneumococcal CAP over a 20-year period. To aid analysis, results were divided into four periods of 5 years each . The primary outcome was 30-day mortality, but secondary outcomes included intensive care unit (ICU) admission, lengths of hospital and ICU-stays, ICU-mortality, and need of mechanical ventilation.From a cohort of 6,403 patients with CAP, we analyzed the data for 1,120 (17%) adults with a diagnosis of pneumococcal CAP. Over time, we observed decreases in the rates of alcohol consumption, smoking, influenza vaccination, and older patients (age ≥65 years), but increases in admissions to ICU and the need for non-invasive mechanical ventilation. The overall 30-day mortality rate was 8%  and did not change significantly between periods (p = 0.33). Although, we observed a decrease in ICU-mortality comparing the first period (26%) to the second one (10%), statistical differences disappeared with adjustment (p0.38).Over time, 30-day mortality of hospitalized pneumococcal CAP did not change significantly. Nor did it change in the propensity-adjusted multivariable analysis. Since mortality in pneumococcal pneumonia has remained unaltered for many years despite the availability of antimicrobial agents with proven in vitro activity, other non-antibiotic strategies should be investigated. Community-acquired pneumonia (CAP) continues to have high global morbidity and mortality rates, and is associated with considerable health costs,2. Some Streptococcus pneumoniae (pneumococcus) remains the most common pathogen in CAP[en in CAP,17, beinen in CAP. In Cataen in CAP. The 7-ven in CAP. In Cataen in CAP,22. Hoween in CAP.In a large homogeneous cohort of critically ill patients with pneumococcal CAP, Mongardon et al. observedWe hypothesized that mortality among patients hospitalized with pneumococcal CAP had decreased over time because of improvements in early detection, antibiotic therapy, clinical management, support bundles and improvement of health care systems. Thus, we aimed to analyze 30-day mortality rate and the trend in mortality for pneumococcal CAP in a large hospitalized adult population over a 20-year period. We also analyzed the main factors related to overall mortality from pneumococcal CAP.S. pneumoniae pneumonia between January 1996 and December 2016 were included. To aid analysis, we divided the study population into four cohorts by time period: 1997–2001, 2002–2006, 2007–2011, and 2012–2016. For the purpose of publication the Ethics Committee of the Hospital Clinic of Barcelona approved the study (Register: 2009/5451), and the need for written informed consent was waived because of the non-interventional design.This was a retrospective observational study of data that were prospectively collected over 20 years in the Hospital Clinic, Barcelona. All adults with a clinical diagnosis of We included all adults with a clinical diagnosis of pneumococcal pneumonia, including those coming from nursing homes. We excluded patients with active tuberculosis or immunosuppression, including those taking >20 mg/day of prednisone  for at least 2 weeks, those receiving cytotoxic therapy, those known to have human immunodeficiency virus infection.Demographic variables, comorbidities, and physiological parameters were collected in the emergency department within 24 hours of admission. We also calculated the pneumonia severity index (PSI), the CURB-65 , and the sequential organ failure assessment (SOFA) scores at admission–27. DuriS. pneumoniae; bacterial growth exceeding 103, 104, or 105 colony forming units (CFU)/mL in protective brush sample, bronchoalveolar lavage cultures or tracheobronchial aspirate, respectively. Definition and additional details are shown in Supplementary Material.The etiology of pneumococcal pneumonia was determined by positive results in any of the following: valid sputum culture; valid blood culture; pleural fluid and transthoracic needle aspiration cultures; urinary antigen for The primary outcome was 30-day mortality, and the secondary outcomes were the lengths of hospital and ICU stays, admission to ICU, mortality in ICU, and need for mechanical ventilation. Early mortality was defined as the mortality in the first 72 hours of admission and late mortality was defined as the mortality after 72 hours to 30 days of admission.in < 0.05, pout > 0.10). We calculated the hazard ratios (HRs) and their 95% CIs. We calculated the area under the receiver operating characteristic curve (AUC) for the multivariable model to predict 30-day mortality. Internal validity of the prediction model was assessed by ordinary nonparametric bootstrapping with 1000 bootstrap samples and bias-corrected, accelerated 95% CIs[We report the number and percentage of patients for categorical variables, the mean and standard deviation (SD) for normally distributed data, and the median and interquartile range (IQR) for non-normally distributed data. To reduce the variability and random noise in year-by-year data we divided the study into four time periods of five years each, defining 1997–2001 as the reference period. We estimated the overall 30-day mortality and the mortality in each of the four periods, reporting them with 95% confidence intervals (CIs). Trends d 95% CIs. Also, tWe used multiple imputation method for missing data for handling missing data in multivariable analyses. The levOf the 6,403 patients with CAP admitted during the study period, 1,120 (18%) were diagnosed with pneumococcal pneumonia and included in the study “The main clinical characteristics of the study population are summarized by study period in “Regarding severity, there was a progressive increase in presentations with ARDS  and patients with septic shock .The prevalence of pneumococcal CAP did not change significantly over time, only varying between 39% and 42% (p = 0.98) “The most frequent causes of death were acute respiratory failure secondary to pneumonia , multiorgan failure associated with septic shock , and acute cardiac events related to pneumonia .The overall 30-day mortality rate among all 1,120 patients was 8% . By subgroup, the 30-day mortality rates were as follows. In the 701 patients aged ≥65 years, it was 10%  compared with 4%  in the 419 patients aged <65 years (p < 0.001). In the 406 cases with invasive pneumococcal CAP it was 10%  compared with 6%  in the 579 patients with non-invasive pneumococcal CAP (p = 0.035). Finally, the 30-day mortality among 815 patients hospitalized to conventional wards was 4% , but this increased to 18%  among the 302 patients admitted to ICU (p < 0.001).The 30-day mortality rate also changed with the number of comorbidities. In 779 patients with at least one comorbidity, the rate was 9%  compared with 4%  among 338 healthy patients (p = 0.002). As expected, compared with the 30-day mortality of 7%  among the 1,080 patients receiving adequate antibiotic therapy, mortality increased to 25%  among the 32 patients receiving inadequate antibiotic therapy (p < 0.001).The frequency of mortality in the first 72 hours of admission  and the frequency of mortality after 72 hours to 30 days  did not change over time. Outcomes by study period are shown in “The overall 30-day mortality rate did not change significantly between periods (p = 0.33), ranging from 8% in the first period to 10% in the last period “In the subgroup of patients receiving NIV, ICU mortality changed non-significantly from 0% in the first period to 9% in the last period (p = 0.23). By Kaplan–Meier analysis, time to death in the ICU and time to death of patients receiving NIV in the ICU did not differ significantly between periods  .The univariable Cox regression analysis revealed several variables to be significantly associated with 30-day mortality “The AUC was 0.70  for the model predictive of 30-day mortality . The senThe univariable Cox regression analysis revealed several variables significantly associated with ICU mortality among the subset of patients admitted to the ICU “There are three main findings to this study. First, it was striking that the 30-day mortality of patients hospitalized for pneumococcal CAP did not change significantly over time . Second, although we observed a progressive increase in the number of patients admitted to ICU and receiving NIV between periods, the mortality of patients receiving NIV in ICU did not change over that time. Third, mortality in ICU fell from 26% in the first study period to 10% in the second period, and remained stable thereafter; however, when we adjusted by propensity-adjusted multivariable analysis, study period was not associated with higher mortality.Recently, there have been few studies about the changes in CAP-related mortality over time,6,37, wiS. pneumoniae, which they attributed to vaccination programs. However, they relied entirely on retrospective data extracted from an administrative database, which would be subject to potential biases, especially in the case of S. pneumoniae diagnosis. By contrast, our data acquisition was prospective and relied on a confirmed microbiological diagnosis of S. pneumoniae. In addition, our 30-day mortality was 3%–4% lower than that reported by the Canadian group.This study is the first, to our knowledge, to describe trends in 30-day mortality over a 20 year period in patients hospitalized with confirmed pneumococcal CAP. It was remarkable that we found no evidence of 30-day mortality for pneumococcal CAP decreasing in hospitalized adult patients over that period. Nevertheless, our results are consistent with those of epidemiological research in Canada (excluding Quebec) over a 6 year period. In thatS. pneumoniae but does seem related to the introduction of virulent clones expressing non-vaccine serotypes, especially serotype 1[2, NO), and dry and cold air increased the incidence of pneumococcal infections[Although we did not observe a fall in 30-day mortality over time, an interesting finding was the decrease in the presence of comorbidities, and also the decrease in the proportion of current smokers over time. One possible explanation is the change of pneumococcal serotypes over the course of the study period. Since the beginning of 2000, serotype evolution in pneumococcus has been in constant progress, influenced by the introduction of pneumococcal conjugate vaccines (PCVs),38. Desperotype 1. On the erotype 1–43 as reWe observed an increase in patients admitted to ICU, as well increases in the proportions of patient with ARDS, with septic shock, and requiring NIV. These data are consistent with results of a UK study in which it was reported that a growing number of CAP cases have required ICU admission in the last 20 years, regardless of age groups. The reaICU mortality was 26% in the first period, dropped to 10% in the second period, and then remained stable. However, we could not confirm this downward trend and subsequent stabilization when adjusting the results by propensity-adjusted multivariable analysis. We think that the reduction in ICU mortality between the first and the second periods of our study was related to the 2001 and 2007 CAP guidelines, recommeIt is noteworthy that our data are in partial disagreement with the results of Gattarello et al., who conThe major strengths of our study are the large cohort, long study period, consecutive enrollment, and inclusion of only cases with microbiologically confirmed pneumococcal CAP. However, a potential limitation of the study is that data were collected from a single academic teaching hospital in Spain, which has extensive experience in the management of CAP; consequently, it may not be possible to extrapolate our results to patients admitted to other types of hospitals in other countries. Other potential limitations are the changes in the diagnosis, treatment, and prevention of CAP during the 20 years of study for which we did not account. However, our antibiotic treatment protocol has not changed substantially for CAP during these years, and we have followed the 1993, 1998, 2001, and 2007 guidelines for managing CAP.In conclusion, we observed increases in the rates of patients admitted to ICU and in the rates of ARDS, septic shock, and requirement for NIV. We did not find a decrease in mortality from pneumococcal CAP. Given that mortality from pneumococcal pneumonia has not changed for many years despite treatment with appropriate antimicrobial therapy, there is a growing need for non-antibiotic strategies to be investigated.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The acute respiratory distress syndrome (ARDS) is a critical illness with high mortality and a worse prognosis. Mechanical ventilation (MV) is currently considered to be one of the most effective methods of treating ARDS. In this meta-analysis, we discussed the efficacy of airway pressure release ventilation (APRV) in treating ARDS.Following the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA), Ovid Medline, Embase, and PubMed were systematically searched with the keywords of “ARDS” and “APRV”. The studies containing the treatment of APRV in ARDS were included. According to the MV protocol used in the studies, the comparison was undertaken between the APRV group vs low tidal volume (LTV) group and synchronized intermittent mandatory ventilation (SIMV) group. The relative risk (RR) and the standard mean difference with 95% confidence intervals (CI) were used for the comparison between groups.2/FiO2 was 75.4% in the APRV group vs 44.1% in the non-APRV group. No significant differences were found in mortality and duration of ICU stay between APRV vs LTV (P = .073 and P = .404) and APRV vs SIMV (P = .370 and P = .894).Fourteen studies with 2096 patients were included in the meta-analysis. The average increasing rate of PaO2/FiO2 ratio, which was a safe protocol with a compatible effect comparing to LTV and SIMV.The APRV protocol would have a higher increase in the PaO The clinicopathological aspects, which includes severe inflammatory injury to the alveolar-capillary barrier, surfactant depletion, and loss of aeratable lung tissue, immediately leads to profound hypoxemia, decreased lung compliance, and increased intrapulmonary shunt and dead space. Currently, it is suggested that ARDS is characterized by acute diffuse pulmonary inflammation and increased alveolar permeability during trauma, stress, and injury. However, the pathogenesis of ARDS is not fully understood, resulting in relatively limited treatment, which ultimately leads to a high mortality rate as high as 40% to 50%.–7Acute respiratory distress syndrome (ARDS) is a common critical illness that might lead to the multiple organ dysfunction syndrome (MODS) and even death. In the early stage of development of this technique, the standard MV mode was adopted, which utilize a normal tidal volume and conventional positive end-expiratory pressure (PEEP). However, for patients with ARDS, it was found that the suboptimal PEEP levels in MV can cause ventilator-induced lung injury, which is associated with higher mortality, extended ICU stay and high cost.,10 Thereafter, the low tidal volume ventilation is widely used and considered as the current standard MV strategy.In the clinical development of ARDS disease, refractory hypoxemia is the primary physiological feature. Therefore, providing active and adequate oxygen therapy has a positive effect on the prognosis of patients with ARDS. Invasive mechanical ventilation (MV) is currently considered to be one of the most effective methods of ARDS treatment.,11 It has a purported advantage than conventional MV in alveolar recruitment, preservation of spontaneous breathing, improving oxygenation and hemodynamics, and potential lung-protection. Although it has been developed for almost 20 years, APRV is still not used as a routine for patients in clinical practice in several countries. Up to now, APRV is mainly used as rescue therapy for the patients with ARDS who cannot be properly oxygenated by MV. More recently, several studies demonstrated that the early application of APRV in ARDS patients could reduce the duration of ventilation and intensive care unit (ICU) stay with a improving oxygenation outcome.,12 However, the efficacy of APRV in the patients diagnosed with ARDS is still controversial. Thus, we designed this systematical review and meta-analysis.Airway pressure release ventilation (APRV) is a pressure controlled, intermittent mandatory ventilation mode with a short intermittent release phase, allowing the release of partial lung volume and spontaneous breathing throughout the high level of pressure.2 and this study was in accordance with the ethical guidelines of the Declaration of Helsinki.The PRISMA guidelines were used for designing this study,2.1The Ovid Medline, Ovid Embase, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and PubMed were systematically searched. The Google Scholar and related websites were also searched for grey literatures. The searching keywords were “airway pressure release ventilation”, “Bi-Vent”, “APRV” and “respiratory distress syndrome”. All the database was searched from its inception to 23rd January 2019. All the studies containing abstracts and titles were imported into Endnote  for duplicate papers exclusion and literature screening.2.2All the studies mentioned the use of APRV in ARDS in the database we searched were involved in our study. The inclusion criteria were:(1)the study mentioned the approach of APRV;(2)the dataset of patients containing the disease of APRV ;(3)the data of intensive care unit ventilation data or outcome could be extracted in the study;(4)study design was limited in observational study, case-control study, cohort studies, and randomized control trials.The meta-analysis, review, and comments were reading for further inclusion of studies. Only articles written in English were involved in this systematic review.The exclusion criteria included(1)animal experiment;(2)the studies not mentioning ARDS or not mentioning the percentage of ARDS patients;(3)case reports, conference abstract, and non-English studies;(4)no available data of outcome or respiratory assessment;(5)containing pediatric ARDS patients.2.3Two investigators (XS and LY) independently screened the titles and abstracts according to the inclusion and exclusion criteria. If the inclusion and exclusion of the literature could be determined based on the criteria, the full text was further evaluated. The third reviewer (LN) was adapted for discussion if there were any disagreement existed between the former two investigators.O2/FIO2, and etc.) and outcome assessment  were extracted and collected.The following information was collected based on original studies. The study characteristics , the ventilation mode (APRV and following mentioned mode), the characteristics of patients in different ventilation group , the ventilator setting and respiratory measurement in baseline and Day 3  was used for assessing the quality.Two investigators (YD and ZY) independently assess the quality of the included papers. For those randomized controlled trials (RCT), the Cochrane Collaboration tool for assessing the risk of bias in RCTs was used to evaluate the quality of the literature.2.42/FiO2 ratio ≤ 200 mmHg without the evidence of arterial hypertension or cardiogenic edema. Currently, the Berlin definition was accepted globally in 2013, which defined the ARDS as an acute diffuse, inflammatory lung injury, with PaO2/FiO2 ratio ≤300 mmHg while ratio >200 mmHg was categories as mild ARDS. Thus, in our study, studies with acute lung injury (ALI) patients were included.Until recently, the most common definition for ARDS was proposed by American-European Consensus Conference (AECC) in 1994, in which ARDS was defined as the acute onset of respiratory failure, bilateral infiltrates on chest radiograph, hypoxemia with the PaO,18 The primary modes, such as volume-control ventilation (VCV), pressure-control ventilation (PCV), and pressure support ventilation, were used to compare the efficacy with APRV. Moreover, the combination ventilation mode including assist-control ventilation (A/C), synchronized intermittent mandatory ventilation (SIMV), continuous positive airway pressure, and low tidal volume (LTV) ventilation were also compared. Generally speaking, for both LTV ventilation and APRV, the mechanical ventilation goals were to achieve adequate oxygenation with required PaO2 and can be weaned to less than 55 mmHg as tolerated, and arterial pH > 7.25.The different ventilation modes and their definitions were summarized in Table 2.5 The heterogeneity was evaluated using the I2 statistic and χ2 test was used for statistical heterogeneity (I2 ≥ 50% indicating the presence of heterogeneity). When the heterogeneity existed, the random-effects model was used, while on the contrary, the fix-effect model was used. Finally, the forest plots were drawn, and the funnel plots were used for evaluating the publication bias.The relevant outcomes of this meta-analysis were performed using Stata . The relative risk (RR) was used for statistical analysis for categorical variables, while for continuous variables, the standard mean difference (SMD) was used. Both were reported with 95% confidence intervals (CI) and the P value was set as 0.05. If there were data provided as medians and range (or interquartile range), we would convert the data into means and standard deviation (SD) using the formula provided by Hozo et al.33.1,12,20–28A total of 2135 studies were found by search strategy. The flowchart was shown in Figure 3.2,9,12 Four studies compared APRV vs SIMV protocol.,21,25,26 The rest 3 studies compared ARPV vs A/C, PSV, and PCV, respectively.,22,27 The median age of APRV group patients were 51.5 years old (range 40–70 years old), while non-APRV group patients were 52 years old (range 42–63 years old). The median acute physiology and chronic health evaluation II (APACH II) score were 21 vs 20 in APRV and non-APRV group. The baseline median PaO2/FiO2 was 113 (range 78–215) in APRV group vs 131 (range 96 to 220) in the non-APRV group. And on Day 3, the median PaO2/FiO2 was 190 (range 150–280) in APRV group vs 180 (range 161–212) in the non-APRV group. The average increasing rate of PaO2/FiO2 was 75.4% in APRV group vs 44.1% in the non-APRV group.The characteristics of the included studies were summarized in Table 3.32/FiO2 would increase significantly after 24 hours which could improve oxygenation and reduce the duration of ventilation and ICU stay. The forest plot comparing the mortality was shown in Figure P = .073, Fig. P = .370, Fig. The outcomes and findings in included studies were summarized in Table P = .404, Fig. P = .849, Fig. The forest plot comparing the duration of ICU stay were plotted in Figure 42/FiO2 ratio significantly in ARDS patients. However, no evidence was demonstrated that APRV could decrease the mortality and duration of ICU comparing to SIMV and LTV.This is the first systematic review and meta-analysis focusing on comparison the impact of APRV on patients with ARDS. Our study demonstrated that the APRV could increase the PaO Intra-pulmonary factors mainly include pneumonia and aspiration in patients; extra-pulmonary factors are severe infections caused by trauma or surgery, severe multiple injuries, and shock. Due to the acute onset of ARDS, patients usually have shortness of breath, accompanied by chest tightness, cough, and other symptoms. The patients with advanced disease may have altered consciousness and even a outcome of death. An increase in the permeability of the membrane was found with the recruitment of neutrophils and other inflammation factors, which resulted in the pulmonary edema. Some studies suggested that the pathogenesis of ARDS may also be associated with systemic inflammatory response syndrome (SIRS). When the patients suffer from infection, trauma, and stress, inflammatory factors, such as interleukin and tumor necrosis factor, are released, forming a “waterfall effect”, causing uncontrollable symptoms of inflammation in patients, and eventually leading to the occurrence of ARDS. Besides, the excessive activation of complement leads to an increase in alveolar vascular permeability and aggravation of exudation. Furthermore, ventilation/perfusion (V/Q) dysregulation is also an essential feature of ARDS. Because ARDS lesions tend to be heterogeneous, when the ventilator is used, alveolar ventilation is insufficient due to fibrosis. V/Q is down regulated which causing a physiological shunt. At the same time, due to severe hypoxemia, stasis of the microcirculation in the lungs, microthrombus formation, the hemodynamic changes leading to a relative upregulation of V/Q, which resulting in the increase of “dead space”.The pathophysiology of ARDS results from acute inflammation affecting the alveolar-capillary membrane, which is related to intra-pulmonary and extra-pulmonary factors. Since firstly proposed in 1987, the APRV experienced a flourishing development with pros and cons. APRV produces exhalation by releasing pressure from high pressure (Phigh) to low pressure (Plow), with a higher baseline pressure favors oxygenation, and intermittent pressure release promotes CO2 excretion. By setting Phigh, APRV does not lead to an excessive alveolar pressure, but maintains the alveolar complex tension for a few seconds, avoiding excessive inspiratory lung volume resulting in excessive traction and physical damage to the alveoli; setting Plow in a short release phase, with a longer time to remain constant alveoli open, avoiding repeated opening and closing of the damaged alveoli. Thigh is the time to use the CPAP to recapture the collapsed alveoli, while preventing lung damage caused by small airway periodic respiration to achieve optimal oxygenation and improve lung compliance. Usually, the Thigh is set to 80% to 95% of the entire respiratory cycle, that is, the lowest value is 4.0 seconds. Tlow allows both adequate ventilation and complete exhalation of constant alveolar, which results in PEEP facilitating alveolar recruitment. Tlow is determined by the time constant of the expiratory flow rate rather than artificially set, which is more consistent with lung physiology and respiratory mechanics according to the expiratory flow rate and peak flow rate. The static pressure-volume curve can reflect the elastic characteristics of the lung. The low inflection point is considered to be the beginning of the collapse of alveolar in patients with ARDS. The high inflection point is the beginning of the collapse of alveolar recap, and the beginning of excessive alveolar expansion. Thereafter, APRV is also considered to be a “protective lung ventilation” strategy for preventing ventilator-induced lung injury (VILI). APRV is based on the “pulmonary open strategy”, which progressively retracts the collapsed alveoli by a higher airway pressure while preserving spontaneous breathing.In the early stage of MV application, several studies realized that the conventional MV could lead to the excessive expansion of the alveolar ventilation area, while the collapse area cannot be expanded as the ideal state. Moreover, the junction area is in an alternating state of expansion and collapse. As a result, conventional MV may not improve the patient's oxygenation, and sometimes even promote the progress of the disease. Afterward, the low tidal volume with high PEEP ventilation mode was introduced for ARDS.,39,40 Roy et al designed a rat model of trauma and found that the group using APRV had a significant decrease of PaO2/FiO2 in histopathology comparing to conventional MV group. Moreover, the bronchoalveolar lavage fluid total protein was decreased with a substantial increase of surfactant protein B concentration and epithelial cadherin tissue expression. In terms of VILI, Emr et al demonstrated that the APRV could prevent the VILI and ARDS comparing to the conventional MV and PEEP in healthy rats. Besides, APRV has little effect on the cardiovascular system, which could improve heart function to some extent, restore or approaching normal V/Q ratio, increase systemic blood flow, and improve the perfusion of the whole body and organ.A large number of animal experiments and clinical studies have shown that APRV can use a slightly higher airway pressure, a smaller peak inspiratory pressure, lower minute ventilation and fewer sedatives, to reduce dead space ventilation, increase alveolar ventilation, and improve oxygenation.2/FiO2 was higher in APRV group which means after 3 days of APRV application, patients would get more oxygenation. Because of the limited number of clinical data, some results cannot be shown in the forest plot. However, in the findings of different studies, sedative agents are also decreasing with a reduction in peak airway pressure, and thereafter, it potentially reduces the time of ventilation and the duration of ICU stay. But unfortunately, no evidence could be demonstrated to support that APRV could decrease the mortality and the duration of ICU stay. However, APRV is still regarded as a relatively safe ventilation mode for patients with ARDS, and the application of appropriate parameters at specific disease stages can help improve outcomes. Even though there were several prospective clinical trials undergoing, the number of cases reported in the relevant research is currently small, which may be related to the lack of clear definition criteria.,43 Therefore, it is urgent to establish a unified APRV parameter setting standard in the clinical settings, which is to maximize lung recruitment, improve oxygenation, and avoid VILI.In our study, the average increasing rate of PaOThere were still some limitations in our study. First, the whole sample of the ARDS patients using APRV was still scarce, and the meta-regression, meta-network cannot be undertaken in terms of lack of comparable studies. A large sample, multi-center, prospective randomized clinical trials are still needed. Secondly, due to the different definition of ARDS and ALI, and a different set of ARDS mode, some bias and heterogeneity cannot be avoided. The standardized setting mode of ARPV needs to be recommended. Thirdly, despite the data we included covering several countries, the studies we included were limited in English, and more European, African, and Asian studies should be searched in the local database.52/FiO2 ratio, which may improve the oxygenation and thereafter potentially improve the symptoms of ARDS patients. APRV was a safe MV protocol with a compatible effect comparing to LTV and SIMV. Further investigation should be undertaken to investigate the standardized APRV setting and detect the ventilation mechanism in ARDS using the standardized definition.In conclusion, the APRV protocol would have a higher increase in PaOConceptualization: Xuri Sun.Data curation: Xuri Sun, Yuqi Liu, Neng Li.Formal analysis: Neng Li.Methodology: Neng Li, Deyuan You, Yanping Zhao.Software: Yanping Zhao.Supervision: Xuri Sun.Writing – original draft: Xuri Sun.Writing – review & editing: Yuqi Liu, Neng Li, Deyuan You, Yanping Zhao.


To investigate the effects of childbirth age on maternal and infant outcomes in pregnant women.The clinical data of 4552 singleton parturient women and their newborns treated in the Second People’s Hospital of Liaocheng, China from June 2015 to June 2017 were retrospectively analyzed. They were divided into group A (<20 yr old), group B , group C (30–<35 yr old), group D (35–<40 yr old), group E (≥40 yr old) according to the age of the parturient women. The incidence rates of pregnancy complications and adverse pregnancy outcomes of the pregnant and parturient women and their newborns in each group were compared.P=0.028, 0.038, 0.042, 0.025, 0.012). The incidence rates of adverse pregnancy outcomes were increased gradually with the increase of childbirth age . After correction of factors including pre-pregnancy body mass index (BMI), parity, gravidity and educational level, the incidence rate of cesarean section was reduced and the incidence rate of premature birth was increased in group A compared with those in group B. The incidence rates of cesarean section, premature birth, postpartum hemorrhage of pregnant women and the transference of newborns into NICU in group C, D and E were higher than those in group B .With the increase of childbirth age, the incidence rates of pregnancy complications in pregnant women were increased gradually (Both low and high age pregnancy can increase the incidence rate of adverse pregnancy outcomes. Elder pregnant and parturient women refer to the parturient women aged 35 yr or above at the expected date of childbirth. The childbirth age has now become a global problem, the fertility rate of Chinese pregnant and parturient women aged between 25–29 yr old has reduced to about 96%, while the fertility rate has increased to about 17% in women at the age of 35–39 and increased to about 5.74% in women at the age of 40–44  yr old. The number of pregnancy was 1–6 times with an average of (1.86 + 0.79) times. The number of birth was 0–4 times with an average of (0.51 + 0.44) times.This study was approved by the Ethics Committee of the Second People’s Hospital of Liaocheng. Signed written informed consents were obtained from the patients.Inclusion criteria: 1) Singleton pregnancy and parturient women who had a normal delivery in our hospital, 2) pregnant and parturient women aged 18 yr old or above, and 3) pregnant and parturient women with complete clinical data and follow-up data.Exclusion criteria: 1) Pregnant and parturient women complicated with a variety of acute or chronic diseases, 2) pregnant and parturient women complicated with a serious immune system disease, or 3) pregnant and parturient women complicated with a serious mental sickness.All pregnant and parturient women were followed up from the first antenatal examination in our hospital, and the incidence rates of pregnancy complications and adverse pregnancy outcomes of pregnant women and their newborns in each group were recorded and compared. 1) Pregnancy complications include intrahepatic cholestasis of pregnancy, pre-eclampsia, hypertensive disorder complicating pregnancy, adherent placenta, premature rupture of fetal membranes, postpartum hemorrhage, placenta praevia, abnormal amniotic fluid, gestational diabetes, placental abruption and anaemia, 2) adverse pregnancy outcomes include asphyxia neonatorum, low birth weight infants (LBWI), the transference of newborns into neonatal intensive care unit (NICU), neonatal birth defects, stillbirth, giant baby, postpartum hemorrhage, premature birth and cesarean section, 3) the clinical data of the pregnant and parturient women and their newborns were retrospectively analyzed and Logistic multifactor regression analysis was performed to analyze the relationships between the childbirth age and the occurrence risk of adverse pregnancy outcomes.x2 test was used for the enumeration data. After the correction of confounding factors, statistically significant adverse pregnancy outcomes were subjected to Logistic multifactor regression analysis, and P<0.05 suggested that the difference was statistically significant.Statistical analysis was conducted by using Statistical Product and Service Solutions (SPSS) 20.0 , and P<0.05)  .P =0.006, 0.026, 0.010, 0.028). There was no statistically significant difference in the incidence rate of premature birth of newborns in group A compared with that in group E   .P =0.002, 0.019, 0.043, 0.015)  .With the rapid development of China’s economy, most childbearing-age women choose to postpone their childbirth age. Besides, with the continuous application of assisted reproductive technology, the pregnancy rate of some primary or secondary infertile patients has increased, which also increases the average maternal childbirth age. The elderly women are characterized by ovarian dysfunction and diminished fertility, and some of them are combined with chronic diseases such as hypertension and diabetes, which lead to a higher incidence rate of adverse pregnancy outcomes ,5. At thP <0.05). The incidence rates of the transference of newborns into NICU, premature birth, postpartum hemorrhage and cesarean section were increased gradually with the increase of childbirth age, and the differences were statistically significant (P<0.05). There was no statistically significant difference in the incidence rate of premature birth of newborns in group A compared with that in group E (P>0.05). For young pregnant and parturient women, their own growth and development have not been completed, and the fertility is relatively low. The fetus needs a lot of nutrient supplies during the pregnancy, so the young pregnant women are difficult to meet the needs of their own and their fetuses during the pregnancy. Therefore, the fetal growth and development are limited and even the incidence rate of premature birth is relatively high  have been completely observed by the authors.


ALDH18A1 gene resulting in the deficiency of the encoded enzyme  have been pathogenetically linked to HSP. P5CS is a critical enzyme in the conversion of glutamate to pyrroline-5-carboxylate, an intermediate that enters in the proline biosynthesis and that is connected with the urea cycle. Interestingly, two urea cycle disorders, Argininemia and Hyperornithinemia-Hyperammonemia-Homocitrullinuria syndrome, are clinically characterized by highly penetrant spastic paraplegia. These three diseases represent a peculiar group of HSPs caused by Inborn Errors of Metabolism. Here we comment on these forms, on the common features among them and on the hypotheses for possible shared pathogenetic mechanisms causing the HSP phenotype.Hereditary Spastic Paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by a progressive rigidity and weakness of the lower limbs, caused by pyramidal tract lesions. As of today, 80 different forms of HSP have been mapped, 64 genes have been cloned, and new forms are constantly being described. HSPs represent an intensively studied field, and the functional understanding of the biochemical and molecular pathogenetic pathways are starting to be elucidated. Recently, dominant and recessive mutations in the  Hereditary Spastic Paraplegias (HSPs) represent a heterogeneous group of neurodegenerative conditions characterized by a progressive inability to walk due to length-dependent axonal degeneration of the pyramidal tract . A simplDespite the fact that more than 80 forms have been mapped, many patients remain without a genetic diagnosis, suggesting that more genes or undefined causes are involved with HSP .Among the Inborn Errors of Metabolism (IEM), spasticity represents a common finding in many forms with pyramidal tract involvement . Indeed,ALDH18A1 as new HSP-disease gene (SPG9) (SLC25A15 in HHH syndrome and ARG1 in Argininemia) cause syndromes where spastic paraplegia is present and highly penetrant. This metabolic pathway involves the metabolism of glutamate connected to the urea cycle, thus identifying a subgroup of HSP caused by IEM affecting a common metabolic pathway (The identification of e (SPG9) , 6 point pathway .From a clinical point of view, it is important to promptly identify these conditions since for some IEM therapeutic options are available. Furthermore, from a scientific perspective, studying this group of diseases may give a deeper understanding of possible converging mechanisms resulting in the spastic paraplegia phenotype, allowing to design a tailored therapy.ALDH18A1 gene maps on chromosome 10 and it encodes for delta-1-pyrroline-5-carboxylate synthetase (P5CS), an enzyme that localizes in the mitochondria inner membrane.The Mutations in this gene cause P5CS deficiency, a condition first described in 1998 and molecularly characterized in 2000 –9.P5CS is a bifunctional ATP and NADPH dependent enzyme, converting glutamate into L-glutamate-5-semi-aldehyde (GSA) in two steps, catalyzed sequentially by the L-glutamate 5-kinase domain (G5K) (EC2.7.2.11) and by the L-glutamyl-5-phosphate reductase domain (G5PR) (EC1.2.1.41). GSA is in tautomeric equilibrium with delta-1-pyrroline-5-carboxylate (P5C) and is then converted into proline by pyrroline-5-carboxylate reductase or it is directed toward the urea cycle where it is involved in the biosynthesis of ornithine, arginine and citrulline .Two transcriptional variants of this gene have been described, differing only by two amino acids on protein level. The long form is expressed in several tissues, while the short form is highly expressed in the intestine and it is inhibited by ornithine .ALDH18A1 mutations (“ALDH18A1-Related Disease”), at least two distinct conditions exist.Despite the variable clinical presentations among patients with ALDH18A1 can cause dominant  and recessive  forms of HSP. These forms are mainly characterized by spasticity of the lower limbs, and the clinical picture can be complicated by low plasma levels of proline, arginine, citrulline, and ornithine associated with hyperammonemia, developmental delay, persistent vomiting, hypotonia, early cataracts and connective tissues abnormalities  or autosomal recessive  disease. Clinical features may include early cataracts, connective tissues abnormalities, progeroid features, vessels tortuosity, and neuropathy. So far, only two residues have been found mutated in the dominant form . This gene maps on 13q14.11, and it encodes for the mitochondrial ornithine/citrulline antiporter ORC1. Mutations in this gene result in a defect of ornithine transport through the mitochondrial membrane , an autosomal recessive disease caused by biallelic mutations in membrane , causingHHH can occur at any age –17. The Most patients develop neurological dysfunction mainly characterized by pyramidal tract signs with spastic gait, associated with cerebellar symptoms .ARG1, a gene mapping on chromosome 6q23.2. This gene encodes for arginase, the last enzyme of the urea cycle, which is necessary for the transformation of arginine into urea and ornithine (EC3.5.3.1) (Arginase deficiency (MIM#207800) is a recessive condition caused by mutations in 3.5.3.1) . Arginas3.5.3.1) .Usually urea cycle disorders present symptoms at birth, but in case of arginase deficiency, first symptoms are often noted between 2 and 4 years of age and consist of a variable association of progressive spastic paraplegia, intellectual disability and seizures , 21. ShoEngineered organisms are indispensable tools to model genetic conditions in order to dissect their pathological mechanism and to test innovative therapies at the entire organism level .As for HHH syndrome, the disease does not seem to be only an exclusive prerogative of the human species, since a spontaneous animal model exists. A report describes two consanguineous weanling foals, presenting a subacute encephalopathy in the early post-weaning period . The cliALDH18A1-related disease have not yet been described. The research in the field would greatly benefit from the generation and characterization of such models.To this day, engineered animal models for HHH syndrome have not been reported, and models for ARG1 is the cytoplasmic form, mainly expressed in the liver. ARG2 is expressed in the mitochondria and it is expressed mainly in non-hepatic tissues.Two isoforms of arginase exist: Arg1 and Arg2 are the mouse orthologs of the human ARG1 and ARG2 genes.Arg1-KnockOut (KO), and double Arg1 and Arg2-KO mice have been described  . The endSimilarly to other urea cycle defects , treatmePharmacological and dietary treatments are the standard clinical approach for these disease and reduce the risk of metabolic decompensation. The progression of spastic paraplegia, however, is unaffected , 21.The pathogenesis of neurological manifestations in patients with P5CS deficiency, Arginase deficiency and HHH syndrome, is not completely understood and may be related to different mechanisms.Arginase deficiency is characterized by very high plasma levels of arginine and decreased ornithine recycling. Both these biochemical features can be responsible for the phenotype. It has been suggested that increased levels of arginine can be responsible for spasticity and other severe cerebral and motor neurological signs. Arginine and its metabolites, including guanidino compounds, are reported to act as neurotoxins . GuanidiIn HHH syndrome, hyperammonemia occurs due to the inability to import ornithine from the cytosol into the mitochondria resulting in a functional impairment of the urea cycle at the level of ornithine transcarbamoylase. In the absence of intramitochondrial ornithine, accumulating carbamyl phosphate either condenses with lysine to form homocitrulline, leading to homocitrullinuria, or is shunted through the cytosolic pyrimidine biosynthetic pathway leading to increased excretion of orotic acid and uracil in the urine .In HHH syndrome, abnormal mitochondria are often seen, suggesting a role of a functional defect at mitochondrial level.per-se is solely responsible for the pathophysiology of this disorder, since also affected individuals who are diagnosed early and maintain good metabolic control and normal plasma ammonia levels develop progressive neurological dysfunction years after the initial diagnosis  presents some similarities with P5CSD . PYCR1  is the biggest organelle in cells and formation and maintenance of ER morphology are regulated by a series of proteins controlling membrane fusion and curvature. Some of these regulators have been demonstrated to be involved in HSPs, in particular Reticulons (RTNs) family. RTNs are a group of membrane associated proteins involved in shaping the tubular endoplasmic reticulum network, membrane trafficking, inhibition of axonal growth, and apoptosis. , 48. ConThe development of a neurological phenotype in HHH syndrome, ARG1 and P5CS deficiency, can be induced by the formation of toxic compounds, resulting from the accumulation of substrates, or alteration in mitochondria, where ornithine is low or absent in these conditions. These observations point toward an impairment of the ornithine/arginine metabolism as a common mechanism for the development of the neurodegenerative phenotype observed in all three metabolic HSPs.Moreover, a link between autophagy and HSP has been demonstrated . Strong in vivo metabolite concentrations in the central nervous system.To test this hypothesis it would be ideal to perform a standardized analysis of these patients including an accurate clinical evaluation , plasma aminoacid profile analysis in fasting and non-fasting conditions, and H-RMS to check for alterations in In order to dissect the pathogenetic mechanism of these conditions, it will also be essential to generate organisms and cell models.In particular, testing and comparing the metabolomics profile in normal and stress conditions of either patient's primary cell lines or engineered cell lines obtained taking advantage of genome editing technologies will be relevant to understand deregulated pathways. Through a metabolomic approach, it will be possible to demonstrate common abnormalities, pinpointing to a possible therapeutic target.in vivo.Finally, the generation of model organisms will be essential in order to test and evaluate the efficacy of the therapeutic approach EP, DM, CD, and MS conceived the study. EP, PM, and DM collected and interpreted the literature data. EP, DM, CD, and MS wrote the manuscript.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.