Thymomas consist of neoplastic thymic cells intermixed with variable numbers of non-neoplastic lymphocytes. Metastatic thymomas are typically managed with non-curative chemotherapy to control tumor-related symptoms; no prolonged survival is expected. Metabolic-based approaches, such as fasting and ketogenic diets, target cancer cell metabolism by creating an increased reliance on ketones while decreasing glucose, glutamine, and growth factor availability, theoretically depriving cancer cells of their metabolic fuels while creating an unfavorable environment for cancer growth, which may be beneficial in metastatic thymoma. We report the case of a 37-year-old woman with myasthenia gravis, diagnosed with an inoperable type AB, stage IVA thymoma, who pursued a metabolic intervention consisting of periodic fasting , combined with a modified ketogenic diet on feeding days, for 2 years. Fasting-related adverse effects included cold intolerance, fatigue, and generalized muscle aches, all of which resolved during the second year. She experienced two myasthenia relapses, each associated with profoundly reduced oral intake, marked weight loss, and tumor regression-the first relapse was followed by a 32% decrease in tumor volume over 4 months, the second relapse by a dramatic 96% decrease in tumor volume over 4 months. The second relapse also required prednisone to control the myasthenia symptoms. We hypothesize that 2 years of fasting and ketogenic diet therapy metabolically weakened the neoplastic thymic cell component of the thymoma, “setting the stage” for immune activation and extreme energy restriction to destroy the majority of cancer cells during both relapses, while prednisone-induced apoptosis eradicated the remaining lymphocytic component of the thymoma during the second relapse. This case is unique in that a metabolic-based fasting and ketogenic diet intervention was used as the primary management strategy for a metastatic cancer in the absence of surgery, chemotherapy, or radiotherapy, culminating in a near-complete regression. Nearly 3 years after being diagnosed with inoperable metastatic cancer, our patient shows no signs of disease and leads a full and active life. Thymomas, the most common tumor of the anterior mediastinum, are composed of neoplastic thymic epithelial cells intermixed with variable numbers of non-neoplastic lymphocytes , 2. ThymCancer is generally regarded as a primarily genetic disorder, yet it may also be perceived as a primarily metabolic disorder, with most of the genetic abnormalities arising as secondary phenomena . Cancer Metabolic interventions, such as fasting and ketogenic diets, target cancer cell metabolism and may be effective alongside medical therapies in treating advanced cancers , 13. FasWe report the case of a 37-year-old, 37 weeks pregnant marketing consultant who presented with 2 months of eyelid weakness worsening on activity as well as 1 month of pleuritic chest pain and dyspnea. She was diagnosed with myasthenia gravis at age 26, during which time a coexisting mediastinal mass was laparoscopically resected and diagnosed as a World Health Organization (WHO) type B2 invasive thymoma. Although she had been intermittently managed with pyridostigmine, prednisone, and intravenous immunoglobulin (IVIg) in the interim, her only current medication was azathioprine 50 mg orally daily. She weighed 53 kg. A neurological examination revealed subtle bilateral asymmetric ptosis and mild eyelid fatiguability. Examination findings regarding upper and lower limb power, reflexes, plantar responses, and sensation were normal.3); several masses invaded the left pleura, and there was a small left pleural effusion. Our patient was treated with IVIg  to ensure the uncomplicated delivery of a healthy baby girl 2 weeks later. She underwent a left pleural percutaneous needle biopsy the day after delivery and was diagnosed with a WHO type AB , Masaoka stage IVA  thymoma [A computed tomography (CT) scan of the neck and chest revealed several large soft tissue masses in the left lung, the largest being 10 × 5 × 14 cm  combined with a modified ketogenic diet  on feeding days . She mon3, representing a 32% decrease in diagnosis volume), moderate bilateral pleural effusions, and a small pericardial effusion. Given that the myasthenia symptoms persisted combined with the possibility that the IVIg may have contributed to the partial regression, the azathioprine was stopped and the IVIg frequency increased to 3-weekly.The first 3 months of the combined metabolic intervention was uneventful, but during months 4–7, our patient experienced reduced appetite and oral intake in the setting of several weeks of diarrhea (up to 10 motions per day), resulting in 6-kg weight loss; the diarrhea was extensively investigated and thought to be a thymoma-associated autoimmune enteropathy . CoincidDuring months 9–12, our patient's diarrhea ceased, she regained her weight, and the myasthenia symptoms resolved. However, the 12-month CT demonstrated that the thymoma had regrown to its original diagnosis volume. Given her ongoing myasthenia control and lack of IVIg inhibitory effect on the growth of the tumor, the IVIg frequency was decreased to 6-weekly.3, representing a 13% increase in diagnosis volume), and all effusions had disappeared.During months 13–24, our patient remained largely free of diarrhea and maintained her diagnosis body weight, and the myasthenia symptoms remained controlled. Her IVIg frequency was decreased to 8-weekly and then 12-weekly. By the time of the 25-month CT, the thymoma showed minimal growth  were measured at 4.98 ± 0.55 and 3.50 ± 1.27 mmol/L, respectively. During each 7-day fasting period, she lost an average of 2.9 ± 0.72 kg of body weight, with mean blood glucose and BHB levels during the fasting periods measured at 3.92 ± 0.73 and 6.31 ± 1.55 mmol/L, respectively. There were several fasting-related adverse effects including cold intolerance, fatigue, and generalized muscle aches, all of which peaked during the first 3 days of each fasting period and progressively resolved over the first year such that they no longer occurred by the second year. No adverse effects occurred in relation to the ketogenic diet.3, representing a 96% decrease in diagnosis volume).During months 25–29, our patient experienced several weeks of drastically reduced oral intake and diarrhea, resulting in 15-kg weight loss. She also developed a second, more severe myasthenia relapse resulting in bilateral ptosis, dysarthria, four-limb weakness with fatiguability, and respiratory failure requiring 2 weeks of intubation and mechanical ventilation in the intensive care unit. She was treated with plasma exchange followed by 3- to 4-weekly IVIg and 10 days of octreotide 1.5 mg subcutaneously daily (followed by a single dose of long-acting octreotide 20 mg intramuscularly 1 month later). Despite the previous adverse effects experienced by our patient in relation to prednisone, we decided to commence prednisone 40 mg orally daily. Her myasthenia symptoms resolved, and the 29-month CT revealed a near-complete regression of the thymoma , whereas during the first and second relapses, she lost 6 kg (11% of body weight), and 15 kg (28% of body weight), respectively. In both cases, such a degree of weight loss would have created drastic alterations in ketone, glucose, glutamine, and growth factor levels, fostering a hostile physiological environment for metabolically inflexible cancer cells. Thus, it is possible that an extreme metabolic-induced regression occurred in our patient.Third, one or more of the medications used to treat our patient's myasthenia symptoms may have contributed to the regressions. The possibility that IVIg contributed to the first regression was considered and partially formed the rationale for increasing the IVIg frequency from 4- to 3-weekly in week 9. Despite this adjustment, the thymoma regrew to its original size by week 12, which suggests that IVIg did not contribute to either regression. In the second and more pronounced regression, octreotide and prednisone were additionally used to placate the myasthenia gravis and autoimmune enteropathy. Octreotide inhibits somatostatin receptors, dampening angiogenesis and growth factor availability, which can produce objective response rates of 10–37% in thymic tumors , 33. HowTaken together, it is likely that several factors culminated in the near-complete regression of our patient's metastatic thymoma. It is difficult to ignore 2 years of periodic fasting combined with a ketogenic diet, which would have immersed the tumor in ketones while depriving it of glucose, glutamine, and growth-promoting factors. It is equally difficult to ignore the two myasthenia relapses in which immune activation and extreme energy restriction may have contributed to both regressions; in the case of the second relapse, it is possible that prednisone-induced apoptosis of the lymphocytic component of the tumor contributed to the dramatic second regression. Overall, we hypothesize that 2 years of a combined fasting and ketogenic diet intervention metabolically weakened the neoplastic thymic cell component of the thymoma, “setting the stage” for immune activation and extreme energy restriction to destroy the majority of cancer cells during the relapses, with prednisone-induced apoptosis destroying most of the lymphocytic component of the thymoma during the second relapse, culminating in the virtual eradication of the tumor.Given that this study involved one patient, its major limitation is obvious, and it is difficult to draw definitive conclusions. Additional potential limitations include concerns that periodic fasting and ketogenic diets may produce unwanted weight loss and other adverse effects in patients with metastatic cancer. It is therefore important to note that our patient did not experience weight loss after 2 years undergoing both metabolic interventions. Furthermore, although she experienced several fasting-related adverse effects, these were all transient and improved as she adapted to each successive fasting period.In conclusion, this case is unique in that a metabolic-based fasting and ketogenic diet intervention was used as the primary management strategy for a metastatic cancer in the absence of surgery, chemotherapy, or radiotherapy, culminating in the near-complete regression of an inoperable metastatic thymoma, with our patient experiencing only transient, fasting-related side effects. Nearly 3 years after being diagnosed with inoperable metastatic cancer, our patient shows no signs of disease and leads a full and active life. Although we cannot be certain of the mechanism underlying this remarkable outcome, the most plausible explanation is that 2 years of fasting and ketogenic diet therapy metabolically weakened the thymoma, setting the stage for a combined immunity-induced, metabolic-induced, and prednisone-induced near-complete regression. Despite our uncertainty, the extraordinary outcome in our patient highlights the importance of exploring metabolic-based therapies in advanced cancer cases, in the hope that more options may be offered to patients in the years to come.Written informed consent was obtained from the patient in this case report for the publication of any potentially identifiable images or data included in this article.MP: conception, design, interpretation, and write-up of final article. DM: diet implementation and advice, and proof-reading of final article. SS: histology analysis and advice, and proof-reading of final article. BM: imaging analysis and advice, and proof-reading of final article.DM was employed by the company Healthy Kitchen Christchurch Ltd. The remaining 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.


Irritable bowel syndrome (IBS) is a common functional bowel disorder. The global incidence of IBS is as high as 9% to 23%, accounting for about 50% of outpatients in gastroenterology, and the new case detection rate is 0.2% every year. IBS has become a global gastrointestinal functional disease. Although IBS is not a life-threatening disease, it seriously affects the quality of life of patients, causing huge economic and mental burden to individuals, society and families. Lipi Guben decoction (LPGBD) is an important auxiliary treatment for IBS, but lack of robust Evidence-based medicine evidence proving its efficacy. Therefore, we designed a randomized controlled trial to evaluate the efficacy and safety of LPGBD in the treatment of IBS.In this randomized controlled trial, a total of 100 eligible patients will be allocated to the blank control group or LPGBD group in a ratio of 1:1. The treatment period was 12 weeks. The primary outcome measure will be the total clinical effective rate. The Secondary outcomes will include IBS clinical symptom scores, IBS-Severity Scoring System, IBS-Quality of life, Hamilton Rating Scale for Anxiety, Hamilton Rating Scale for Depression, and Bristol Stool Form Scale. The safety outcome will include Echocardiogram, blood examination , urine routine test and stool routine test. The evaluation indicators and all safety results will be performed at baseline, week 4, week 8 and week 12.This study will be helpful to evaluate the efficacy and safety of LPGBD in the treatment of IBS.LPGBD may improve the clinical efficacy of patients with IBS, which has important value in practical applicationChictr20000039617, registration time: November 3, 2020 IBS has become a growing global public health issue. Although IBS is not a life-threatening disease, it seriously affects the quality of life of patients, and creates a huge economic and mental burden for affected individuals, society and families. IBS have been ranked as a common cause of illness-related absenteeism, second only to the common cold. According to the study, the direct and indirect costs of each IBS patient in the United States reach US $4527 per year.Irritable bowel syndrome (IBS) is a common intermittent or durative gastrointestinal functional disease characterized by abdominal pain, abdominal distension with defecation habit change, abnormal stool characteristics without organic causes.–6 Clinical studies have found that diarrhoea-predominant IBS (IBS-D) is the most common type.–8 In modern medicine, drugs that regulates intestinal movement, reduces visceral hypersensitivity, improve central emotion, and regulate intestinal flora are commonly used to relieve IBS-D symptoms.–12 Alosetron, a selective 5-hydroxytryptamine (5-HT) receptor blocker, was once considered to be the most effective drug for IBS-D, which was approved by Food and Drug Administration (FDA). However, due to its serious side effects, it was limited to female IBS patients with severe symptoms. Although the newly developed new drugs for IBS, such as zeamak and Trevor, have certain curative effect, their clinical application is limited due to their high side effects and high price. At present, modern medicine lacks specific and effective curative drugs for the treatment of IBS. The efficacy and mechanism of traditional Chinese medicine in the treatment of IBS have attracted more and more attention.–15The symptoms of IBS are complex and diverse, and the condition is complicated with an unknown etiology and pathogenesis. It is generally believed that the occurrence of IBS is the result of a combination of multiple factors, including mental and psychological disorders, visceral hypersensitivity, gastrointestinal motility disorder and intestinal infection.,17 Integrated TCM and Western medicine therapy for IBS patients could reduce symptoms, anxiety, depression, and improve quality of life.–23 However, due to the small sample size and unscientific research methods, the conclusions are limited. Therefore, we conducted a randomized controlled trial (RCT) to evaluate the efficacy of safety of Lipi Guben decoction (LPGBD) in the treatment of IBS.Integrated traditional Chinese medicine (TCM) and Western medicine therapy has been the most distinctive methods in the treatment of IBS in China.22.1This is a RCT. Participants will be recruited from the outpatient clinics of the First Affiliated Hospital of Guizhou University of TCM. The Figure http://www.chictr.org.cn/edit.aspx?pid=63618&htm=4. And the registration number is ChiCTR2000039617.This protocol has been registered in the Chinese Clinical Trial Registry on November 3,2020, 2.2Inclusion criteria:(1)Patients meet the the Rome III diagnostic criteria for IBS-D;(2)Those who agree to participate in this study and are willing to sign informed consent.(3)The age ranged from 18 to 65 years old.Exclusion criteria:(1)Under 18 years old or over 65 years old;(2)A woman who is pregnant or is preparing for pregnancy, and lactating.(3)Intestinal organic diseases, such as inflammatory bowel disease, intestinal tuberculosis, colon polyps;(4)Patients with severe cardiovascular and cerebrovascular diseases, liver, kidney, hematopoietic system diseases and tumors;(5)There were records of digestive tract operation;(6)Patients with severe primary diseases and mental disorders;(7)Patients with allergic history or drug and food allergy during the test period;(8)Those who do not sign informed consent(9)Those who do not meet the inclusion criteria, fail to use drugs according to the regulations, cannot judge the curative effect or incomplete data, which affects the evaluation of efficacy or safety.(10)The researchers consider it inappropriate to conduct this clinical trial.2.3 we hypothesized that the total clinical effective rate of the blank control group and LPGBD group at 12 weeks will be 73% and 93%, respectively. Set a=0.25 (2-sided) and b = 0.10. We use PASS 15 software  to calculate the size of the blank control group and LPGBD group N1 = N2 = 41. Assuming that 20% of patients may be lost during follow-up, a total of 100 patients will be recruited.The sample size was calculated based on the total clinical effective rate at 12 weeks. According to the previous literature,2.4Patients who met the inclusion criteria will be randomly assigned to blank control group and LPGBD group in the ratio of 1:1. Measures to Reduce the Risk of Bias Concealed randomization prevents selection bias. The random allocation sequence listing was generated by a statistician who will not participate in the clinical study using SAS v9.4 .5-10 minutes before treatment, the doctor will inform the patient about the randomization procedure, telephone number and name; then, the doctor will be informed of the random number of patients and the treatment group. The randomization information will be concealed until the end of the trial.2.5Due to the nature of TCM, doctors and patients will know the grouping information. However, principal investigator, statisticians, and outcome evaluators will be blinded to group allocation.2.6Five principal investigator with TCM background will be responsible for data collection. The supervisors and analysts who do not know the regulatory data will monitor the entire experiment. The principal investigator, outcome evaluators, and statisticians will be strictly trained before trial.2.7Patients will be recruited in the gastroenterology outpatient clinics of the First Affiliated Hospital of Guizhou University of TCM. The poster will briefly describe the trial and provide treatment details, as well as contact information. If a potential participant is interested in taking part, they can directly contact the study team. We will provide potential participants with detailed information about benefits and possible risks of the trial. If they decide to participate, they will be asked to sign written informed consent. If a participant withdraws from the trial, the reason for the withdrawal will be recorded. In order to improve the compliance of patients, we will strengthen doctor-patient communication and timely health education for patients.2.8Potential adverse events associated TCM include rash, skin allergy and liver and kidney function damage. Clinicians record the occurrence of any adverse events and the intervention will be stopped immediately. The independent safety supervision committee composed of 3 experts from different fields in the First Affiliated Hospital of Guizhou University of TCM has the right to terminate the study.33.1Subjects in the blank control group will receive conventional treatment: Pinaverium Bromide Tablets (50 mg), orally 3 times daily for 12weeks; Bifidobacterium triple viable capsule (420 mg), orally twice times daily for 12weeks.3.2Subjects in the LPGBD group will be given LPGBD, based on the conventional treatment in the blank control group. The herbal formula of LPGBD comprises the following TCM: fried Atractylodes macrocephala 15 g, Poria cocos 10 g, tangerine peel 6G, Rhizoma Pinelliae 10 g, Shenqu 10 g, malt 15 g, roasted licorice 6G, Astragalus 20 g, Paeonia Alba 10 g. The medicinal materials were decocted twice at 100 °C under a normal pressure, each for 40 minutes. Each decoction (150 ml) should be taken twice a day.3.3The duration of treatment was 12 weeks.3.4During the trial, Participants are prohibited from taking any other TCM, western medicine or therapeutic measures . If a concomitant intervention is used, the name, dosage, and frequency of the accompanying interventions should be recorded in detail.44.1Disease outcomes include cure, significant effect, effective, and ineffective. The main outcome will be the total clinical effective rate of the 2 groups after treatment (week 12), that is, that is, the sum of cure rate, apparent efficiency and effective. Outcome indicators will be collected at the 12th week.4.2(1)IBS clinical symptom scores: abdominal pain, diarrhea, abdominal distension will be scored as follows: 0, none;2, light;4, moderate; 6, heavy. IBS clinical symptom scores will be measured on week 0, 4,8, 12.(2)IBS-Quality of life (IBS-QOL): IBS-QOL consists of 8 domains: dysphoria, interference with activity, body image, health worry, food avoidance, social reaction, sexual function and impact on relations. The IBS-QOL score ranged from 0 to 100 points and a higher score indicated a better QOL. IBS-QOL will be measured on week 0, 4,8, 12.(3)IBS-Severity Scoring System (IBS-SSS): The IBS-SSS score ranged from 0 to 500 points and a higher score indicated a worse SSS. The IBS-SSS are scored as follows: mild, ≤175 points; moderate severity, >175 points and ≤300 points; severe, >300 points. IBS-SSS will be measured on week 0, 4,8, 12.(4)Hamilton Rating Scale for Anxiety (HAMA): HAMA are scored as follows: normal, <7 points; suspected anxiety, 7 to 14 points; definite anxiety, 14 to 21 points; obvious anxiety, 21 to 29 points; serious anxiety, ≥29 points. HAMA will be measured on week 0, 4,8, 12.(5)Hamilton Depression Scale: HAMA are scored as follows: normal, <8 points; suspected depression, 8-20 points; definite depression, 20 to 35 points; severe depression, >35 points. Hamilton Rating Scale for Depression will be measured on week 0, 4,8, 12.(6)Bristol Stool Form Scale: Bristol Stool Form Scale will be measured on week 0, 4,8, 12.4.3Safety results will include echocardiogram, blood tests , urine routine tests and stool routine tests. All outcomes be measured on week 0, 4,8, 12. If an adverse event occurs, the investigator will record the details  in the form of a case record form (CRF). Serious adverse reactions will be reported to the ethics committee of the First Affiliated Hospital of Guizhou University of TCM, and relevant rescue procedures will be started immediately.4.4Test data need to be recorded in paper-based and electronic CRF. An independent data-monitoring committee reviewed the data every 3 months. During the trial, only data managers and principal investigators will have access to the electronic CRF. At the end of the trial, the database will be locked by the data management team, after which the principal investigators cannot modify the data. Paper and electronic documents will be kept for at least 5 years. If readers and reviewers have any questions, they can contact the appropriate author to obtain the original content data.5This study has been approved by the ethics committee of the First Affiliated Hospital of Guizhou University of TCM  and registered in the China clinical trial registration center on November 3, 2020. The trial strictly followed the Helsinki Declaration (2000 EDITION). Only patients with informed consent can participate in the trial.6The results of the study will be communicated to healthcare professionals, participants and the public through peer-reviewed publications, scientific conferences. Moreover, the data of this study can be obtained from corresponding authors according to reasonable requirements.7All personal information of the participants will be kept strictly confidential and the anonymized individual patient data will be shared on request.8In case of direct injury related to this study, corresponding medical and nursing care can be obtained. Emergency contact number will be Provided for each subject so that they can contact the researcher whenever they have any questions or questions.9P values less than .05 will be considered to be statistically significant.The clinical results will be analyzed by SPSS 22.0 statistical software . In this study, the intention-to-treat analysis and per-protocol analysis will be conducted. Primary outcomes will be based on the per-protocol analysis, with the intention-to-treat analysis analysis conducted to assess robustness of the results. The measurement data will be expressed as mean ± standard deviation, and the counting data will be expressed as percentage, will be tested by a t test and chi-square test respectively. All statistical tests will be 2-sided and 10The main clinical manifestations of IBS-D are abdominal pain and pathogenic factors, which seriously reduce the quality of life of patients and affect their work. The main therapeutic target of IBS-D is to reduce abdominal pain and diarrhea, improve gastrointestinal function and the quality of life of patients. There is no good treatment for IBS-D in modern medicine. Therefore, alternative therapy has been welcomed and focused on. TCM has been used in the treatment of IBS-D for hundreds of years a certain effect. However, in terms of evidence-based medicine, the quality of these studies was moderate. Therefore, in order to evaluate the efficacy and safety of LPGBD on IBS-D, we designed a RCT for 12 weeks to evaluate the efficacy and safety of LPGBD in the treatment of IBS-D.RCT is the gold standard to evaluate the efficacy and safety of drugs. Of course, this study also has some shortcomings, mainly reflected in the following 2 aspects:(1)Due to the long observation period of this study, it can be concluded that the shedding rate is high, which will affect the results of the study. Therefore, we should maintain a good relationship between doctors and patients in the research process, and the scale involved in this study is a self-evaluation scale, which can be conducted subjectively by patients, so as to better improve the quality of research data;(2)The patients in this study are limited to Chinese patients, so the results of this study have some limitations and may not be extended to other regions.11Protocol version number V1.0, September 10, 2020. The trial is currently in the recruitment phase of participants. Recruitment begins on November 10, 2020 and is expected to end on December 31, 2022.We would like to thank all the patients who will participate in the trial and the staff for their support.Conceptualization: Hongmei Yang, Xin Liu, Wei Peng, Rong Chen.Investigation: Xin Liu.Supervision: Yang Chen.Writing – original draft: Hongmei Yang.Writing – review & editing: Hongmei Yang, Yang Chen.


Aenigmetopia Malloch is described for the first time, along with the first instar larvae of three other Australian species representing the genera Amobia Robineau-Desvoidy and Protomiltogramma Townsend. Larval morphology was analysed using a combination of light microscopy, confocal laser scanning microscopy and scanning electron microscopy. The following morphological structures are documented: pseudocephalon, antennal complex, maxillary palpus, facial mask, modifications of thoracic and abdominal segments, anal region, spiracular field, posterior spiracles and details of the cephaloskeleton. Substantial morphological differences are observed between the three genera, most notably in the labrum and mouthhooks of the cephaloskeleton, sensory organs of the pseudocephalon, spinulation, sculpture of the integument and form of the spiracular field. The first instar larval morphology of Aenigmetopia amissa Johnston, Wallman, Szpila & Pape corroborates the close phylogenetic affinity of Aenigmetopia Malloch with Metopia Meigen, inferred from recent molecular analysis. The larval morphology of Amobia auriceps (Baranov), Protomiltogramma cincta Townsend and Protomiltogramma plebeia Malloch is mostly congruent with the morphology of Palaearctic representatives of both genera.The first instar larva of a species of the Australian endemic genus  During the past decade much information about the larvae of this subfamily has been accumulated e.g.4, resulting in the availability of morphological data for almost all Palaearctic genera. In contrast to this, the preimaginal instars of most non-Palaearctic taxa remain unknown, and the Australasian region stands out in that morphological information on the immature stages of its miltogrammine fauna is completely lacking.The first larval instar of Miltogramminae show diverse and peculiar morphologies, which most likely reflect adaptations to locating suitable food sources by digging through sand, often combined with different types of kleptoparasitism6. Most of the known species occur in dry habitats, particularly the deserts and semideserts of Asia and Africa7. Recent targeted taxonomic research has revealed a remarkable miltogrammine diversity in Australia (currently 28 named species), with the most speciose genera being Protomiltogramma Townsend (12 spp.) and Aenigmetopia Malloch (5 spp.), while the genera Amobia Robineau-Desvoidy (3 spp.), Macronychia Rondani (1 sp.), Metopia Meigen (2 spp.), Miltogramma Meigen (4 spp.) and Senotainia Macquart (1 sp.) are more modestly represented12. The Australian fauna constitutes only a small part of the global miltogrammine diversity6, with a noteworthy high proportion for Protomiltogramma , modest for Amobia (21.4%), and very low for the other genera: Macronychia (3.8%), Metopia (4.9%), Miltogramma (3.4%) and Senotainia (1.5%)14,. However, endemism is high (89.3%) as only three species, one in each of Amobia, Metopia and Senotainia, are found outside Australia.Subfamily Miltogramminae is a relatively diverse group with more than 600 described speciesAenigmetopia amissa Johnston, Wallman, Szpila & Pape, Protomiltogramma cincta Townsend, Protomiltogramma plebeia Malloch; and (2) Amobia auriceps (Baranov), widely distributed in the Oriental and Australasian-Oceanian regions. The newly discovered larval morphology of Australian species is compared with existing data across the global fauna of Miltogramminae. We also discuss conflicts and congruence between larval morphology and both former and current phylogenetic hypotheses presented for the genera in question. Special focus is on the systematic position of Aenigmetopia, the only endemic genus of Australian miltogrammines.The present study fills an important gap in the morphospace of a large taxon of Diptera by providing the first morphological documentation of the preimaginal stages of four Australian miltogrammine species: (1) endemic Ae. amissa, Am. auriceps, P. cincta and P. plebeia follows the general pattern for the Calyptratae, with the body being divided into a bilobed pseudocephalon (pc) equipped with antennal and maxillary sensory organs, three thoracic segments (t1–t3), seven abdominal segments (a1–a7), and an anal division (ad) carrying the posterior spiracles (ps)  Fig. A,B,E,F.Aenigmetopia amissa Johnston, Wallman, Szpila & Pape, 2020.Figs. A,C, 6A,BMaterial examined: 17 larvae from a single female: /Australia, NT, 585/Litchfield NP/Lost City/-13.21652 130.73544/10 Nov 2017/leg. FG-NT Expedition/.Pseudocephalon. Antennal complex (an) large, antennal dome (and) oval but with slightly conical tip, antennal basal ring (abr) high  elongated, slender anterior part of labrum shorter than broader basal part, anterior part of labrum curved downward and with pointed tip  with from 3–4  to 11–12  rows of spines, spines arranged separately from each other, mono- or rarely bi-cuspid  on a1–a7 with from 1–2 to 8–9 rows of spines, all bands complete  incomplete, without spines on the middorsal surface .Figs. B,D, 6C,DMaterial examined: 18 larvae from a single female: /Australia, NT/Litchfield NP/Lost City/-13.190533, 130.774565/10 Nov 2017/leg. FG-NT Expedition/.Pseudocephalon. Antennal complex (an) large, antennal dome (and) oval but with rounded tip, antennal basal ring (abr) high  long, curved downward, gradually tapering to pointed anterior tip  large, antennal dome (and) oval, with rounded tip, antennal basal ring entirely reduced  elongated, slender anterior part of labrum shorter than broader basal part, anterior part of labrum curved downward and with pointed tip  to 8–11  rows of spines  on a1–a7 with from 1–2 to 8–9 rows of spines, all bands complete  incomplete, without spines laterally on the lateral surfaces  very large, antennal dome (and) oval, with slightly conical tip, antennal basal ring entirely reduced  elongated, slender anterior part of labrum shorter than broader basal part, anterior part of labrum curved downward and with pointed tip  to 6–11  rows of spines  on a1–a7 with from 1–2 to 8–9 rows of spines, all bands complete  incomplete, without spines laterally 12. An interesting similarity is observed between Ae. amissa and Senotainia conica 2, which share the following character states: (1) antennal complex with high basal ring, (2) club-like first sensillum basiconicum situated close to the central cluster of sensilla, (3) ventral organ sensilla placed on a small fleshy lobe, (4) oral ridges present, (5) mouthhook with serrated tip, and (6) cuticular sculpture in the form of regular ridges. However, most of these character states cannot be considered to provide a strong phylogenetic signal as they are also present in taxa situated in different clades of the miltogrammine phylogenetic tree, and they are symplesiomorphic at this level12. Additionally, the shape of the oral ridges and the general morphology of the anal division of Ae. amissa and S. conica are different, but the autapomorphic condition in Ae. amissa makes phylogenetic deductions premature. Searching for larval synapomorphies between Aenigmetopia and Metopia is made complicated by the diverse larval morphology of Metopia16. Despite this, Ae. amissa and the five species of Metopia for which larvae have been described to date , M. campestris , M. grandii Venturi, M. italiana Pape) share a uniquely shaped apical part of the mouthhook, which is serrated with multiple teeth orientated perpendicular to the main axis of the mouthhook16. Additionally, the mouthhook in Ae. amissa has teeth with rounded tips, which is a character state expressed in all species of Metopia except M. campestris. Overall, the morphological similarities between Ae. amissa and Metopia spp. support a monophylum consisting of Aenigmetopia and Metopia as suggested by Johnston et al.12. Unfortunately, the first instar larva of M. nudibasis, the sister species to Aenigmetopia in the phylogenetic tree provided by Johnston et al.12, remains unknown. Therefore, the possible paraphyly of Metopia cannot be tested by larval morphology, and formal synonymization of Aenigmetopia under Metopia is considered premature and should await further analyses based on additional taxa and more data.While the morphology of first instars of Am. auriceps is almost identical to those of the Palaearctic species in most characters2, including: (1) long and slender first sensillum basiconicum situated close to the central cluster of sensilla, (2) ventral organ sensilla placed on a large fleshy lobe, (3) oral ridges short, (4) labrum long, curved downward, gradually tapering to pointed anterior tip, (5) broad basal part of mouthhook, (6) cuticular sculpture on abdominal segments in the form of warts, (7) abdominal segments ventrally with locomotory protuberances/prolegs, (8) abdominal segments dorsally with transverse row of large triangular cones, (9) peristigmatic tufts small, and (10) spiracular field without encircling spines. The first instar of Am. auriceps differs from the majority of Palaearctic Amobia in two key characters, which henceforth should be considered to be intragenerically variable: (1) the antennal basal ring of Am. auriceps more pronounced than the low and flat structure described in the larvae of Palaearctic species; and (2) the entire lateral surface of thoracic segments in Am. auriceps covered by cuticular warts  basal ring in antennal complex absent, (2) first sensillum basiconicum shifted to the inner periphery of maxillary palpus, and (3) posterior surface of anal division between spiracular field and anal complex with circular depression. This set of features is not observed in larvae of other miltogrammines, and Eremasiomyia, Protomiltogramma and Pterella probably form a monophylum. The morphology of the first instar larvae of Australian Protomiltogramma is almost identical to that of the Palaearctic species P. fasciata (Meigen) in all aspects, such as the form and distribution of the sensory organs on the pseudocephalon, the cephaloskeleton, the cuticular sculpture and the shape of the anal division. The presence of a large circular depression on the pseudocephalon and on the anal division represent two intriguing character states of Protomiltogramma larvae. Szpila2 did not mention these depressions in his description of the larva of P. fasciata, but they are plainly visible in his Fig. 56, and they have also been observed in larval material of Protomiltogramma from the United Arab Emirates . These depressions have been described only in the genera Protomiltogramma , Pterella Robineau-Desvoidy  and Phrosinella Robineau-Desvoidy 17. They are of a very similar morphology and therefore unlikely to be artefacts. However, the function of these circular depressions remains unknown and needs further study using other approaches, e.g. anatomical methods.The first instar larva of species representing the genera 12 and the global Miltogramminae18. This alignment between larval morphology and molecular phylogeny in the Australian Miltogramminae provides evidence for both the stability of these traits in the Sarcophagidae and their potential value for the inference of evolutionary relationships in this family.The present study fills an important gap in our knowledge both of the preimaginal instars of miltogrammine flies and the morphological diversity of endemic Australian taxa. Congruence was established between larval morphology and the current phylogenetic hypotheses presented for the Australian MiltogramminaeProtomiltogramma) or preserved in 95% ethanol . Females were identified using the keys of Malloch19, Johnston8 and Johnston et al.11 in combination with comparative studies of voucher specimens from the Australian National Insect Collection, Canberra, Australia (ANIC); the Australian Museum, Sydney, Australia; the Department of Ecology and Biogeography, Nicolaus Copernicus University, Toruń, Poland (DEB); and the Natural History Museum of Denmark, University of Copenhagen, Denmark.Adult female miltogrammine flies were collected from various locations in the Northern Territory, Australia  to avoid deformation and stored in 70% ethanol. Preserved larvae were slide-mounted in Hoyer’s medium for light microscopy. Preparation for scanning electron microscopy (SEM) involved dehydration through 80, 90 and 99.5% ethanol and critical-point drying in COAe. amissa and Am. auriceps were similar in size and examined under a 63× objective lens with a numerical aperture (NA) of 1.4, and the first instar of P. cincta was examined under 40× with a NA of 1.3. Effective 3D visualization requires collecting more data than necessary to obtain acceptable 2D images20, and differences in the thickness of the individual larvae required optimization of the amount of data collected from each frame. Individual z-step size settings were defined for each sample manually. The larger size in the z-plane imposed the collection of fluorescence signals from different focal planes, which allowed for full visualization of the examined structures. After the acquisition of the preliminary image series, the 3D model was built in the LAS X 3D Viewer program. When the received model was of low quality acquisition of image series was repeated with manually increased number of z-steps. The respective number of z-frames was individually adjusted for each of the three specimens; for Ae. amissa there were 123 z-frames, for Am. auriceps 146 z-frames and for P. cincta 224 z-frames.Material intended for visualization with confocal laser scanning microscopy (CLSM) was freshly prepared from whole available larvae. Specimens were cleared initially by immersion in 10% potassium hydroxide for over 48 h, until tissue digestion was satisfactorily obtained. The material was then dehydrated in 99.5% EtOH for 20 min and the specimens were embedded in Euparal on a microscope slide and covered with a coverslip. Due to the small size of the examined material, flat microscope slides were used to avoid damaging the structures of interest. Prepared slides were scanned using a Leica TCS SP8 Confocal Laser Scanning Microscope. In order to obtain optimal fluorescence, only the excitation wavelengths of 561 nm and 633 nm were used. A strong fluorescence signal was also obtained with an excitation wavelength of 488 nm, but this was omitted as it simultaneously revealed some contamination and undigested tissue, which significantly disturbed the resulting image. First instars of P. cincta and P. plebeia, all larvae, and associated microscope slides have been deposited in DEB. Females of Ae. amissa and Am. auriceps have been deposited in ANIC. Larval terminology follows Courtney et al.21, with a few modifications proposed by Szpila and Pape22 and Szpila2. Nomenclature follows Pape6 and Johnston et al.11, with the generic names of Aenigmetopia and Amobia abbreviated as Am. and Ae., respectively, to avoid confusion. Label data of specimens are given verbatim using a slash (/) for the start and end of a line, and a double slash (//) for the end of a label and the beginning of the next (from top to bottom on the same pin) (Fig. The females of


Background—This study aimed to explore whether living alone or with a spouse only affects the short-term prognosis of acute ischemic stroke patients. Methods—We conducted a retrospective cohort study of patients with a diagnosis of acute ischemic stroke from April 2014 to February 2019 in Japan. The primary outcome was defined as worsening by at least one grade on the modified Rankin Scale (mRS). The secondary outcome was set as the degree of worsening on the mRS. The outcomes were compared between three groups of patients: (1) those living alone (ALONE), (2) those living with their spouse only (SPOUSE), and (3) OTHERs. Results—In total, 365 patients were included in this study: 111 (30%) ALONE, 133 (36%) SPOUSE, and 121 (33%) OTHERs. Cardiogenic embolisms were observed more frequently in ALONE than in OTHERs. The primary outcome occurred in 88 (79.3%) patients in ALONE and in 96 (72.2%) patients in SPOUSE, both of which were higher than the 72 (59.5%) in OTHERs. After adjusting with 19 variables, the risk of worsening was higher in ALONE (odds ratio (OR): 2.90, 95% confidence interval (CI): 1.50–5.58) and SPOUSE  compared with OTHERs. Conclusions—In patients with acute ischemic stroke, not only living alone but also living with a spouse only may be associated with a worse short-term prognosis, independent of other cardiovascular risks. Low socioeconomic status is considered to increase stroke mortality, as well as its incidence . Recent The data that support the findings of this study are available from the corresponding author on reasonable request. This retrospective cohort study was based on data obtained from the medical records of inpatients treated at St. Luke’s International Hospital in central Tokyo. Patients who presented by ambulance and were admitted to the hospital with a suspected acute ischemic stroke between 1 April 2014 and 28 February 2019 were included. All patients with clinical symptoms were diagnosed as having had an ischemic stroke by a neurologist or neurosurgeon, and confirmation was made based on findings from magnetic resonance imaging with fluid-attenuated inversion recovery and diffusion-weighted imaging. Among these patients, cases with other diseases, namely, a transient ischemic attack, epilepsy, and peripheral vertigo were excluded. The requirement for informed consent was waived because the data were anonymized and retrospective. All data were subject to strict privacy policies and all patients or their family members were given the chance to opt out. This study was approved by the Institutional Review Board at St. Luke’s International Hospital.Cohabiting family members, including a spouse (wife/husband) or partner, daughter/son (including daughter/son-in-law), and another family member(s)  who were living with the patients were prospectively and routinely interviewed at admission. Patients who had been living in a nursing home were counted as living alone, unless they were living with a family member at the nursing home. In terms of mutually exclusive factors, we classified patients into the following three groups: (1) patients living alone (ALONE); (2) patients living with their spouse only (SPOUSE); (3) patients living with at least one other family member, e.g., spouse and child (OTHERs).The covariates originally selected were age, sex, date of admission, date of discharge, discharge condition , current smoking status, comorbid conditions , drug usage , mRS score before onset as judged by a family member(s) and at discharge as judged by a physical therapist or doctor, National Institute of Health Stroke Scale (NIHSS) score at admission, cardiogenic embolism, wake-up stroke, treatment  and execution of mechanical thrombectomy), time from onset to the ambulance call, time from ambulance call to arriving at the hospital, and laboratory data at admission , hemoglobin (Hb), C-reactive protein (CRP), total protein (TP), albumin (Alb), blood urea nitrogen (BUN), and creatinine (Cr)).The primary outcome was set as a worsening by at least one grade on the mRS at discharge compared with before the stroke onset. The mRS measures functional independence on a seven-grade scale, from 0 (no symptoms) to 5  and 6 (dead) . Patientp-values < 0.05 were considered statistically significant. All analyses were conducted using STATA 14.0 software .A logistic regression model and an ordered logistic regression model were applied to compute the odds ratios (ORs) and 95% confidence intervals (95% CIs) for the primary binary outcome and secondary ordered outcomes, respectively, with or without an adjustment using the following 19 variables: (1–4) age quartiles; (5) sex; (6) current smoking status; comorbidity of (7) hypertension, 8) dyslipidemia, (9) diabetes, and (10) atrial fibrillation; (11) usage of anticoagulants; (12) antiplatelets; (13) NIHSS at admission; (14) cardiogenic embolism; (15) wake-up stroke; (16) administration of tPA; (17) execution of mechanical thrombectomy; (18) time from onset to the ambulance call; (19) time from the ambulance call to arriving at the hospital. Age and creatinine and albumin levels were not normally distributed; therefore, the Kruskal–Wallis test was applied. Sex, hypertension, diabetes, atrial fibrillation, and current smoking were binary data; therefore, the chi-squared test was applied. When the 95% CI did not include 1, the OR or risk ratio (RR) was considered statistically significant. Time and laboratory data were compared between the three groups using the Kruskal–Wallis test and a test for trends [ dyslipidFrom April 2014 to February 2019, 394 patients were presented by ambulance and were hospitalized because of initial suspicion of an ischemic stroke. However, 29 patients were excluded because their diagnosis turned out to be a different disease: 19 cases of transient ischemic attack, six cases of epilepsy, and four cases of peripheral vertigo. Finally, a total of 365 patients (238 (65%) male, median age: 74 years, interquartile range (IQR): 63–82 years) were enrolled in this study . A compaWe hypothesized that cohabiting family members would notice signs of a stroke and call an ambulance, and thus, that patients living alone may be associated with a longer time from onset to call. However, no differences were found between the three groups in the time from the onset to the ambulance call or in the time from the ambulance call to arriving at the hospital . Overallp < 0.001) and CRP (p = 0.01), but not with any other factors. On the other hand, the TP, Alb, BUN, and Cr values were not significantly different between the three groups based on the Kruskal–Wallis test for trends.The WBC, CRP, and Hb values measured at admission were the highest in the ALONE group, followed by the SPOUSE and OTHERs groups . The NIHIn total, 17 (4.7%) patients died after admission. The risk of death was higher in the ALONE group (9 (8.1%)) than in the OTHERs group (2 (1.7%); RR: 0.20, 95% CI: 0.05–0.92), but not significantly different compared with the SPOUSE group (6 (4.5%)). In total, 109 (29.9%) patients were discharged from the hospital without a reduction in the mRS score; in other words, the mRS scores were worse by at least one grade at discharge in the other 256 (70.1%) patients. The primary outcome, i.e., worsening by at least one grade on the mRS, was seen in 88 (79.3%) patients in the ALONE group and 96 (72.2%) in the SPOUSE group, both of which were significantly higher compared with the 72 (59.5%) in the OTHERs group a. RegardEven after adjusting with 19 variables, the risk of worsening by at least one grade on the mRS was significantly higher in the ALONE  and SPOUSE groups  than in the OTHERs group . In a muTo our knowledge, this is the first study in the world that was designed to examine the influence of cohabitation status, i.e., to conduct a comparison between patients living alone, with a spouse only, or with others, in terms of the short-term prognosis of an acute ischemic stroke. The primary outcome, i.e., worsening by at least one grade on the mRS at discharge compared with before the stroke onset, was observed in approximately 80% of patients in the ALONE group and 70% of those in the SPOUSE group, both of which were higher than that in the OTHERs group (60%). The secondary outcome, i.e., degree of worsening on the mRS, was also higher in the ALONE and SPOUSE groups than in the OTHERs group. Even after multivariate analyses, being in the ALONE or SPOUSE groups remained risk factors for both outcomes. Previous studies have reported that living alone is associated with increased long-term mortality after an ischemic stroke ,10. AnotIn this study, the time from the stroke onset to the ambulance call did not differ between the three groups. Patients living alone were found to be less likely to arrive at the hospital within 2.5 h, to receive thrombolysis, or to be discharged home , but we The WBC, CRP, and Hb values were the highest in the ALONE group, followed by the SPOUSE and OTHERs groups. A review of several previous studies indicated that high CRP values resulted in unfavorable long-term functional outcomes . Indeed,This study had several limitations. First, this was a retrospective cohort study. Although almost no data were missing, variables associated with cardiovascular disease, such as body mass index, were limited. Moreover, patients were not always transported from home; they were sometimes transported from their workplace, although we were able to collect data on cohabitation status. Second, only the prognosis during admission was determined in this study; additional studies involving long-term follow-up are therefore warranted. Third, socioeconomic statuses, including education, occupation, and income, were not measured. Fourth, confounders such as excessive alcohol intake, smoking, obesity, and a sedentary lifestyle were not measured. Finally, due to the uniqueness of the Japanese healthcare system and the central Tokyo area, caution should be taken when generalizing the results of this study to other countries or even other areas of Japan.The results of the present study suggest that not only living alone but also living with a spouse only, may be associated with a worse short-term prognosis, independent of other cardiovascular risks, in Japanese patients after acute ischemic stroke. However, to improve the generalizability of these findings, a multicenter, prospective, cohort study is needed.


Papillary adenoma of the lung is a rather rare tumor. We will present a case of papillary adenoma in the lung with malignant transformation in a 65-year-old male patient. A high dense soft tissue mass was detected in the lateral segment of the right middle lobe by CT examination. Cytologically, the tumor contained the benign cells similar to normal alveolar epithelium and the malignant cells which were significantly enlarged and irregular, crowded, or overlapping. Immunohistochemical staining showed that the epithelial cells were diffusely positive for TTF-1, napsin-A, and CK7, but were negative for p63, p40, CK5/6, CgA, Syn, CD56, and TG. The Ki67 index was about 5%. All of these evidences indicated that it was a case of papillary adenoma with malignant transformation. Thus, it should be noted that more active treatment measures should be taken to treat pulmonary papillary adenoma. Papillary adenoma of the lung is a very rare tumor. It was first reported by Spencer et al. in 1980 –5. At prA 65-year-old male patient came to our hospital for “dizziness for 1 week, chest CT showing right lung neoplasm, and head CT showing brain metastasis.” CT imaging of the chest showed a 42 × 34mm, in size, high dense soft tissue mass in the lateral segment of the right middle lobe . Bronchoμm thick. HE staining and immunohistochemical staining were performed. The envision two-step method was used for immunohistochemistry. Thyriod transcription factor 1 (TTF-1), napsin-A, cytokeratin 7 (CK7), P63, P40, P53, CK5/6, Ki67, chromogranin A (CgA), synaptophysin (Syn), CD56, thyroglobulin (TG), and secondary antibody were purchased from the Roche company.The Thinprep Cytologic Test (TCT) and pap staining were used for cytology. Histological specimens were fixed with 4% neutral formaldehyde, embedded in paraffin, and sectioned 4 Cytological observations under microscope showed that the cells were arranged in flat sheets/nests, papillary architecture, acini formation, and three-dimensional cluster. Most of the cells comprised of cohesive sheets of uniform cells reminiscent of type II pneumocytes or Clara cells, which were columnar or cuboidal cells with moderate amounts of cytoplasm and evenly distributed chromatin. The cells had slightly larger nuclei than those of the surrounding alveolar cells, an equal amount of eosinophilic cytoplasm, and smooth or slightly irregular nuclear membrane. Single to multiple small nucleoli were seen, and no mitotic figures or necrosis was found. The cells were streaming arrangement, similar to metaplasia or reparative cells. Some cells had more irregular nuclear membrane and crowded or overlapping nuclei with moderate cytologic atypia, which were 1.5-3 times the size of the contrastive cells with the same shape. The nucleoli were about 2-3 times the size of the contrastive cells. Some obvious eosinophilic deviated nucleoli were discovered. A small number of cells formed a three-dimensional spherical structure, similar to adenocarcinoma cells. No cilia and goblet cells were found Figures –7.Histologically, most of the tumor cells showed papillary fronds with fibrovascular cores. A large number of neutrophils, lymphocytes, and plasma cells infiltrated in the stroma of the fibrovascular cores. The epithelial cells were columnar or cubic and arranged in single layer. Cilia, goblet cells, and basal cells were not found. Most of the epithelial cells had minimal cytological atypia, and no mitotic figures were identified. A few cells had obvious and eosinophilic nucleoli. The cell adhesion was poor, and the cell polarity disappeared. A small number of atypical cells were arranged in a glandular pattern, and a single heterotypic cell could be seen infiltrating in the fibrotic stroma Figures –10. Immu11Papillary adenoma of the lung is a rare tumor that mainly occurs in the peripheral part of the lung. At present, there are more than 30 cases reported in the literature, with the age spectrum ranging from 2 months to 78 years old and an average age of about 40 years. Men are slightly more common than women. The maximum diameter of the tumor is 0.4-6 cm . The patMalignant transformation is the process by which cells acquire the properties of cancer. This may occur as a primary process in the normal tissue, or secondarily as malignant degeneration of a previously existing benign tumor. In our case, the tumor demonstrated a wide spectrum of cells across both benign and malignant components, and head CT showed brain metastasis. These findings indicated the process of malignant transformation.At present, it is believed that papillary adenoma originates from primitive multipotential respiratory epithelium, which shows bidirectional differentiation into type II alveolar epithelium and Clara cells , 10–12. In this case, we observe the cytological features of the tumor. One part is the benign components close to the normal alveolar epithelium. The benign cells are typically mild and uniform with fine chromatin, arranged in a flat sheet pattern. The other part is the malignant components. The malignant cells are significantly enlarged and irregular, crowded, or overlapping. The nucleoli are eosinophilic. Some cells formed a three-dimensional spherical structure. Histologically, the benign components present a simple papillary structure, without branching papillae or budding. The malignant components show glandular arrangement or infiltrative growth of tumor cells in the fibrous stroma. All of these evidences indicate that this is a case of papillary adenoma with malignant transformation. From this case, we not only have a profound understanding of the cytological characteristics of pulmonary papillary adenoma with malignant transformation but also obtain important guiding information for clinical practice. As to pulmonary papillary adenoma, we should take more active treatment measures, and clinical follow-up observation should be performed.


Bioluminescence reactions are widely applied in optical in vivo imaging in the life science and medical fields. Such reactions produce light upon the oxidation of a luciferin (substrate) catalyzed by a luciferase (enzyme), and this bioluminescence enables the quantification of tumor cells and gene expression in animal models. Many researchers have developed single-color or multicolor bioluminescence systems based on artificial luciferin analogues and/or luciferase mutants, for application in vivo bioluminescence imaging (BLI). In the current review, we focus on the characteristics of firefly BLI technology and discuss the development of luciferin analogues for high-resolution in vivo BLI. In addition, we discuss the novel luciferin analogues TokeOni and seMpai, which show potential as high-sensitivity in vivo BLI reagents. This reaction proceeds by two-steps: DLH2 is first adenylated with ATP, and then it is oxidized by O2, to generate excited oxyluciferin, which produces yellow-green light (λmax = 560 nm) [oplophorus luciferase (Oluc) in the presence of O2 to generate coelenteramide, which produces blue light (λmax = 454 nm) [There are many bioluminescent organisms ,2,3, suc 560 nm) ,5,6,7. I 454 nm) ,9,10. Recently, Oba et al. published a report on the evolutionary history of firefly bioluminescence . They reluc+, luc2). The luc2 gene, which is developed by adjustment of codon and reducing of the transcription factor binding site from wild-type luciferase sequence, enables the high expression of luciferase in mammalian cells [Firefly luciferin- and luciferase-based bioluminescence imaging (BLI) is widely utilized in the life science and medical science fields ,14, for an cells .® [® reaction is independent of the concentration of ATP [On the other hand, many researchers have synthesized CTZ analogues for application to optical imaging ,46,47,48® ,50,51. Sn of ATP , it is an of ATP . This isn of ATP , as it in of ATP . In contn of ATP .14C labeled DLH2 revealed that, after 15 min of intraperitoneal administration, DLH2 is distributed mainly at the skin and does not reach the brain or lungs [Firefly BLI methods are based on the detection of optical signals and are, thus, more suitable for imaging shallow tissues, such as subcutaneous tissue, than for imaging deeper tissues, such as those in the brain or lungs ,58. Thisor lungs .Accordingly, numerous luciferin analogues ,64 and mSince different luciferin analogues and luciferase mutants have their own strengths and weaknesses, it is important to choose the right combination for a particular in vitro and in vivo experiment. Most previous research has used single-color in vivo BLI. However, multicolor bioluminescence reactions have been developed for application to in vivo imaging.In this review, we present a brief introduction to the characteristics of firefly BLI technology and discuss advances in the development of luciferin analogues and luciferase mutants for high-resolution in vivo BLI.Luciferin analogues have been synthesized as a means to overcome the two weaknesses of DLH2 mentioned above, i.e., low light permeability and biodistribution, thus realizing higher sensitivity and utility for in vivo imaging . A viablKm = 0.10 and 1.06 µM in N-hetero ring [Miller et al. developed CycLuc1, in which a pyrrolidine moiety is fused to the benzothiazole moiety of DLH2. The wavelength of CycLuc1 is 599 nm . NIRLuc2 exhibits wavelength at 683 nm , in which the π-conjugation is extended with an olefin moiety . The wavositions . NH2-NpL2-NpLH2, or TokeOni) and luciferase  [2-NpLH2/CBR2 at 20 min, and TokeOni/AkaLuc at 10 or 15 min. Additionally, they measured the bioluminescence wavelength of each combination in vivo, using bandpass filters. Interestingly, the wavelength was very different for each combination. That for DLH2/Luc2 was 610 nm, DLH2/CBG99 was 540 nm, NH2-NpLH2/CBR2 was 730 nm, and NH2-NpLH2/CBG99 was 620 nm. Each combination for DLH2 and NH2-NpLH2 shifts by approximately 100 nm, depending on the luciferase. Conversely, the wavelength for CycLuc1/AkaLuc was 600 nm, and that for TokeOni/AkaLuc was 660 nm. Although the wavelengths for these two substrates change with different luciferases, the degree of the shift is approximately 20–40 nm. Thus, the combination of luciferin and luciferase is very important, and if the right combination is not used, it will be difficult to detect the observed tissues with high sensitivity.Zambito et al. investigated the bioluminescence characteristics of each possible combination of luciferin  . The bioThese results demonstrate that bioluminescence intensity and wavelength can be changed significantly by developing luciferase mutants specialized for luciferin analogues. Furthermore, orthogonal combinations with characteristic properties can be expanded the variation of applications. For instance, Prescher et al. developed orthogonal combinations of luciferin analogues and luciferase mutants ,95,96. TMaki et al. reported a series of luciferin analogues B that reCaenorhabditis elegans, using a dual-luciferase system based on Eluc  and SLR (stable luciferase red) [Indeed, dual-color BLI technology has already been explored in some animal studies. For instance, Aswendt et al. performed dual-color in vivo BLI of mouse brain tissues, allowing them to monitor both stem cell survival and differentiation in one imaging session simultaneously . Furtherase red) . Dual/muThe solubilities of AkaLumine in PBS and ultra-pure water are very poor, and thus usability was limited for in vivo imaging ,83. TherAccordingly, Iwano et al. developed a mutant luciferase named AkaLuc, which was tailored for TokeOni, and termed their new TokeOni/AkaLuc combination method as AkaBLI . The intKm = 6.2 and 44.9 µM, respectively in Two characteristics of TokeOni were improved by seMpai; since the pH value of TokeOni is acidic , acidosiRecent studies have provided important information for the selection of in vivo BLI systems. For instance, Fukuchi et al. performed a comparative analysis of DLH2, TokeOni and seMpai by monitoring the expression of brain-derived neurotrophic factor (BDNF) in Bdnf-luc transgenic mice. They reported the interesting finding that TokeOni BLI shows a different luminescence than those for DLH2 and seMpai, which show the same pattern in brain BLI . FurtherThe fact that bioluminescence patterns change depending on the tissue permeability of the luciferin analogues used is a serious problem because it means that the chemical and physical properties of luciferin analogues have significant effects on biological discovery. Nevertheless, since seMpai BLI reduces hepatic background signals, it is very useful for the study of systemic mouse models, such as those used to study metastasis  Figure . Thus, iIn this review, we have summarized the development and application of luciferin analogues for in vivo BLI and NIR imaging technology. We suggest that the development of luciferin analogues should not just focus on bioluminescence activity, but also consider improved pharmacokinetics for animal safety. The informed combination of luciferin analogue and mutant luciferase can be used to tailor luminescence wavelength and intensity, as well as dynamics in vivo. Thus, researchers using such techniques for life science and medical research must be aware of the influence of BLI system selection.


A 44-year-old male without any significant past medical history presented to the emergency department (ED) with the chief complaint of severe constant epigastric pain for three hours. On physical examination, the abdomen was distended and tender, particularly in the epigastric region. The lab work showed an elevation of the lipase  and triglycerides . An abdominal CT scan with contrast was ordered, which revealed non-necrotic pancreatitis. In addition, the liver ultrasound showed no evidence of gallstones. Subsequently, fluid infusion, meropenem, pain medication, and an insulin drip were started, and the patient was transferred to the intensive care unit (ICU). After six hours in the ICU, he complained of abdominal pain despite taking a high hydromorphone dose.On further physical examination, the abdomen was tender and distended but without rebound tenderness. The gastric distention on kidneys, ureter, and bladder (KUB) and a bladder pressure of 34 mmHg raised the suspicion for intra-abdominal hypertension (IAH), which led us to place a nasogastric tube (NGT) and consult the surgical team. The patient's symptoms and bladder pressure were closely followed and showed significant improvement. On day seven in the ICU, the patient responded well to medications; feeding through the Dobhoff tube was started, and his triglycerides decreased to approximately 1,000 mg/dL. Despite his general improvement and meropenem regimen, the patient spiked a fever of 38.5 °C. Due to the possibility of pancreatitis complications, a CT abdomen with contrast was ordered, which showed partial portal vein thrombosis (PVT). Subsequently, enoxaparin was started, and the patient was closely observed for gastrointestinal bleeding. Eventually, after 17 days in the ICU, the patient was transferred to the floor and then discharged from the hospital with normal lab tests and without evidence of portal thrombosis on abdominal CT.In this report, we illustrate and discuss a case of hypertriglyceridemia (HTG)-induced pancreatitis (HTGP), which progressed to PVT and IAH. Physicians should be aware that patients with HTG are inclined to have severe pancreatitis. In addition, the degree of triglyceride elevation is correlated with the severity of acute pancreatitis. Acute pancreatitis is diagnosed when two of the following three criteria are met in a patient: acute severe onset epigastric pain usually radiating to the back, serum lipase or amylase level elevation to three times or more than the upper limit of normal, and characteristic signs of pancreatitis on radiography  . HypertrThe risk of developing HTG-induced pancreatitis (HTGP) increases with higher triglyceride levels. The risk of HTGP with serum triglycerides of >1,000 mg/dL (11.3 mmol/L) is approximately 5% in comparison to 10-20% with triglyceride levels of >2,000 mg/dL 22.6 mmol/L) . In addi mmol/L .In this report, we present a case of HTG-induced severe acute pancreatitis (SAP) that progressed to portal vein thrombosis (PVT) and intra-abdominal hypertension (IAH) during the course of admission.The patient was a 44-year-old male without any significant past medical history who presented to the emergency department (ED) with complaints of severe 10/10 epigastric pain for approximately four hours. The patient described the pain as sharp, stabbing, epigastric abdominal pain that radiated to the back. The examination was notable for generalized abdominal tenderness mostly on the epigastric region without any masses or noted rebound. The patient did not have a history of smoking or alcohol consumption and took no home medications. Initial laboratory workup was significant for white blood cell (WBC) count of 19.2 K/uL , hematocrit of 50% , potassium of 5.4 mmol/L , glucose of 269 mg/dL , hemoglobin A1c of 8.3% , lipase of 12,405 U/L , C-reactive protein (CRP) of 18.7 mg/dL , cholesterol of 452 mg/dL , triglyceride of 5,837 mg/dL , lactate dehydrogenase (LDH) of 412 U/L , calcium of 5.9 mg/dL , total bilirubin of 6.9 mg/dL , and a calculated Ranson’s score of 8, indicating severe pancreatitis. An abdominal CT scan with contrast revealed non-necrotic pancreatitis with an association of duodenitis  and was started on 250 mL/h of lactated Ringer’s solution. The pain was controlled with intravenous hydromorphone, and the patient was transferred to the ICU for close monitoring. After six hours, the abdominal pain worsened, and the patient developed abdominal distension and increased bladder pressure (34 mmHg). A kidney, ureter, and bladder (KUB) X-ray revealed significant gastric distention Figure . Given tAfter five days, feeding through a Dobhoff tube was initiated. Despite the improvements in abdominal tenderness and triglyceride levels, one episode of fever 38.5 °C) was observed. A repeat CT scan of the abdomen with contrast revealed partial PVT , comprehensive metabolic panel (CMP), and triglyceride levels, were within normal limits, and abdominal CT with contrast showed complete resolution of PVT. The plan after discharge was to repeat abdominal CT in six weeks and continue apixaban for six months.PVT and abdominal hypertension were two complications of HTG-induced acute pancreatitis that were seen in our patient. Pancreatitis can cause a range of venous and arterial vascular complications. Major vascular complications of pancreatitis occur with a frequency of 1.2-14%, with a greater incidence seen in chronic pancreatitis 7-10%) than in acute pancreatitis (1-6%). Of these complications, venous thromboses are more commonly reported in the splenic vein and less frequently in the portal vein or superior mesenteric vein . Accordi% than inIn a retrospective study by Mortele et al. on 100 patients with acute pancreatitis, the presence of splenic and superior mesenteric vein thrombosis was significantly correlated with the severity of pancreatitis. However, thrombosis of the portal vein was not correlated with pancreatitis severity. This finding is important since it indicates that clinicians and radiologists should be aware of this complication even in patients with “moderate” pancreatitis . Acute PThe incidence of IAH in patients with SAP is approximately 60-80%. It is usually an early phenomenon and partly related to the effects of the inflammatory process, which can lead to the development of retroperitoneal edema, fluid collections, ascites, and ileus. This can also be partly iatrogenic, resulting from aggressive fluid resuscitation . IAH hasAs ileus and gastroparesis are often present in acute pancreatitis, reducing the intraluminal volume of the gastrointestinal tract is a logical first step. In the case of gastric dilatation, nasogastric decompression can easily be done and may have a significant impact on IAP . Our casPancreatitis in the setting of severe HTG is a unique and often underdiagnosed disease with significant morbidity and mortality rates. When suspected, prompt diagnosis and treatment should be initiated. Supportive therapy should be initiated to treat acute pancreatitis, glucose levels need to be controlled, and subsequently, triglyceride levels need to be reduced with the aid of lifestyle modifications and pharmaceutical agents. Additionally, physicians should be aware of the potential complications of pancreatitis such as venous thrombosis and IAH, even in the setting of non-necrotic pancreatitis, as was presented in our case.


COVID-19 has speedily immersed the globe with 72+ million cases and 1.64 million deaths, in a span of around one year, disturbing and deteriorating almost every sphere of life. This study investigates how students in Pakistan have coped with the COVID-19. Zung’s self-rating anxiety scale (SAS) was used for measuring anxiety and the coping strategies were measured on four strategies i.e., seeking social support, humanitarian, acceptance, and mental disengagement. Among 494 respondents, 61% were females and 77.3% of the students were in the age group of 19–25 years. The study findings indicate that approximately 41 percent of students are experiencing some level of anxiety, including 16% with severe to extreme levels. Seeking social support seemed to be the least preferred coping strategy and that female students seek social support, humanitarian, and acceptance coping strategies more than males. Students used both emotion-based and problem-based coping strategies. The variables of gender, age, ethnicity, level and type of study, and living arrangement of the students were associated with usage of coping strategies. Findings showing that students do not prefer to seek social support. The study outcomes will provide basic data for university policies in Pakistan and the other countries with same cultural contexts to design and place better mental health provisions for students. The diseases transmitted to humans through animals are known as zoonotic diseases. Jones  suggestsMak et al.  mention p < 0.01).” This study was done in Karachi, the first city in Pakistan to report the COVID-19 cases.To contain the COVID-19 epidemic, the government of Pakistan had announced country-wide closure of all schools, colleges, and universities on 13th March 2020 . The eduCarver  defines Coping strategies are defined as “as efforts to regulate emotions, behaviors, cognitions, psychophysiology, and environmental aspects in response to the stress of everyday events” . It is aIndividuals engage in coping strategies to manage the stress, specifically when confronting a significant life change or traumatic event. Brooks  reportedA self-administered validated questionnaire was used that was initially pilot tested. Subsequently, the final version of the questionnaire was sent via WhatsApp and email to university students, using Google Forms. Respondents from 50 universities and colleges participated in the study from all the four provinces/states of Pakistan. The major institutions were Bahria University, Baqai Medical University, Bolan University of Medical and Health Sciences, Dow University of Health Sciences, National University of Computer and Emerging Sciences, Federal Medical and Dental College, IBA, International Islamic University, Iqra University, Jinnah Medical & Dental College, Jinnah Sindh Medical University, Lahore Garrison University, Liaquat University of Medical & Health Sciences, Mehran University of Engineering & Technology, Peoples Medical University, Peoples Medical University of Health Sciences for Women, Quaid-i-Azam University, SMBB Medical University, University of Sindh, University of Karachi SZABIST, and Ziauddin University. Data were collected during the lockdown period from 1 June to 15 June 2020. The questionnaire was unnamed, and informed consent was sought. Ethical approval was obtained from the institutional review boards (IRB) of the partaking universities.The research instrument included basic personal and demographic data, and Zung’s self-rating anxiety scale (SAS) . The quet-test, analysis of variance (ANOVA) and multivariate analysis of variance (MANOVA) procedures were used. For ANOVA and MANOVA, the p-values were adjusted using Tukey’s multiple comparisons procedure. The level of significance was 0.05 for all tests.The data were analyzed using SPSS 22.0 software. Quantitative variables were summarized as means and standard deviations, while qualitative variables were described as frequencies and percentages. For the data analysis, The ethical approval was obtained Prince Sultan University Saudi Arabia , and Khairpur Medical College, Pakistan .A total of 510 responses were received. After data cleaning, 494 responses were found to be usable. From the 494 respondents, 60.9% were females and 39.1% were males. A percentage of 77.3% of the students were between 19–25 years, 13.5% were above 26 years, and the remaining 9.1% were below 18 years old. Most of the respondents were studying health sciences (39.1%) followed by social sciences (28.5%), sciences (19.8%), management (6.5%), and engineering (6.1%). Almost three-quarters of the respondents were pursuing a professional program, whereas 20% were at the undergraduate level and 5% were at the postgraduate level. Notably, 37.9% of the respondents were enrolled in their first year, 19.6% in their second year, and 9.5% were in their third year. The remaining 33% were enrolled in their fourth year and beyond. Majority of the students (69%) responded that they had transitioned towards virtual learning during the pandemic. In terms of accommodation, 91% of the students were staying at their family homes, 6.6% were staying outside campus, and only 2.4% were living in college residencies.In the 20-item Zung’s SAS, each item is scored on a scale of 1 to 4. The summary scores for the 20 items are shown in Among the respondents in the sample, 125 (25.3%), 45 (9.1%), and 34 (6.9%) experienced mild to moderate, marked to severe, and most extreme levels of anxiety, respectively. For further analysis, cases with marked to severe anxiety and most extreme anxiety were grouped together and named as “Severe to Extreme” level of anxiety; This study aimed to know what strategies the students used in coping with anxiety during the COVID-19 pandemic. Four coping strategies, “Seek social support,” “Acceptance,” “Mental disengagement,” and “Humanitarian” were tested. The items were measured on a scale of 1 to 4; 1 = never/rarely, 2 = sometimes, 3 = often and 4 = very often/always. For each coping strategy, mean scores for respective items were computed, where higher scores implied higher level of use. The descriptive statistics for the coping strategies are shown in The associations between coping strategies used and level of anxiety were tested using Analysis of variance (ANOVA) procedures. In the analyses, the variances were similar. The results are shown in Out of the four, only “Seek social support” and “Humanitarian” were significantly associated with level of anxiety. The usage of “Seek social support” coping strategy was higher in the “Severe to Extreme” anxiety group compared to the “Minimal to Moderate” and “Normal” groups. The usage of “Humanitarian” coping strategy was higher in the “Severe to Extreme” anxiety group compared to the “Normal” groups.The following table is the summary from the post-host test. The pairwise differences are indicators using superscripts of a and b in The differences in the usage of the four coping strategies were tested using Multivariate analysis of variance (MANOVA) procedure. The results are tabulated in The results in This study examined the coping strategies adopted by university students in Pakistan due to psychological impacts of the COVID-19 pandemic. Findings indicate that approximately 41% of students are experiencing some level of anxiety. From these students, 16% are having severe to extreme anxiety levels, which is in line with earlier studies about students’ anxiety during the pandemic in other countries ,25,26.Four types of coping strategies i.e., acceptance, humanitarian, mental disengagement, and seeking social support were examined. Salin et al.  mention The results of this study also indicated that the coping action involving seeking social support seemed to be the least preferred coping strategy (Mean 1.98 ± 0.77). American Psychological Association  defines While others are also going through pandemic, this may be the underlying reason for this strategy adopted by the students to be the least preferred. Findings also show that female students seek social support , humanitarian , and acceptance  coping strategies more than males. Earlier studies have shown that females prefer maladaptive coping more than males . This apAge-wise findings show that students of age bracket over 25 adopted more acceptance, humanitarian, and mental disengagement strategies than students of other age brackets . For seeking social support strategy, all the age groups had almost same mean values. Mental disengagement strategy, according to Traeger , is “empWhile many studies have explored the psychological effects of the pandemic to the society in general, limited studies focused on the mental health of the youth, particularly, the university students in Pakistan. This study examined how university students in Pakistan cope with the psychological impact of the COVID-19 pandemic. Based on Zung’s self-rating anxiety scale (SAS), 41% of the students are experiencing some level of anxiety, including 16% experiencing severe to extreme anxiety levels. Students’ coping mechanisms were evaluated based on four strategies, notably, seeking social support, humanitarian, acceptance, and mental disengagement. Seeking social support appears to be the least preferred coping method among the students. However, further analysis indicates that students who are experiencing severe to extreme level of anxiety sought social support more than others. Our findings also indicate that coping strategies vary based on students’ gender. Female students significantly seek social support, humanitarian, and acceptance coping strategies more than male students. Similarly, age, ethnicity, level, and type of study, and living arrangement of the students were also associated with usage of coping strategies.This study can help inform the government and policy makers about the youths’ preferred method of coping during a stressful situation like a pandemic. Students are young adults and at the vulnerable stage who get easily cajoled into opinions and views from the social media , which mUniversities and higher learning institutions shall conduct periodic assessment on students’ physical and mental health. While physical illness can be easily identified, mental health issues are more difficult to assess and resolve, particularly, in an environment where the students generally do not prefer seeking social support. Thus, learning institutions should assign trained mentors or advisors to review students’ emotional state during these critical times. Peer-to-peer counseling and chatgroups, moderated by a psychologist and an academic expert, can also help students to freely express their concerns and issues about their mental health and its impacts on their learning.Our study, which is based on cross-sectional design, is not intended to test for any causal relationships between students’ level of anxiety, coping strategies, and the overall psychological impact to the students. We suggest that future studies shall employ longitudinal design to test for these effects. Next, this study that uses self-reported questionnaire is subject to subjectivity and reliability issues. Finally, the limited sample size and convenience sampling approach may not reflect the bigger population of university students in Pakistan, which poses as another limitation to the study.