To assess the burden of depression, anxiety and suicidality; and to determine the impact of integrated mental health and HIV services on treatment outcomes among Thai adolescents and young adults living with HIV (AYHIV).A multicentre prospective cohort study was conducted among AYHIV (15 to 25 years), and age‐ and sex‐matched HIV‐uninfected adolescents and young adults (HUAY). The Patient Health Questionnaire 9‐item (PHQ‐9) and Generalized Anxiety Disorder 7‐item scales (GAD‐7) were used as screening tools for depressive and anxiety symptoms respectively. History of lifetime and recent suicidal ideations/attempts were ascertained. Elevated mental health screening scores were defined as having either significant depressive symptoms (PHQ‐9 ≥9), significant anxiety symptoms (GAD‐7 ≥10) or suicidality . Participants meeting these criteria were referred to psychiatrists for confirmatory diagnosis and mental health services. Follow‐up assessment with PHQ‐9 and GAD‐7 was performed one year after psychiatric referral.3. At enrolment, 31 AYHIV  had elevated mental health screening scores; 17 (11%) significant depressive symptoms, 11 (7%) significant anxiety symptoms and 21 (14%) suicidality. Seven AYHIV (5%) had all three co‐existing conditions. These prevalences were not substantially different from HUAY. Urban living increased risk, whereas older age decreased risk of elevated mental health screening scores (p < 0.05). All AYHIV with elevated mental health screening scores were referred to study psychiatrists, and 19  had psychiatrist‐confirmed mental health disorders (MHDs), including adjustment disorder (n = 5), major depression (n = 4), anxiety disorders (n = 2), post‐traumatic stress disorder (n = 1) and mixed MHDs (n = 4). One year after psychiatric referral, 42% of AYHIV who received mental health services demonstrated an absence of significant mental health symptoms from the reassessments, and 26% had an improved score.From February to April 2018, 150 AYHIV and 150 HUAY were enrolled, median age was 19.0 (IQR:16.8 to 21.8) years and 56% lived in urban areas. Among AYHIV, 73% had HIV RNA <50 copies/mL, and median CD4 count was 580 (IQR:376 to 744) cells/mmWith the significant burden of MHDs among AYHIV, an integration of mental health services, including mental health screenings, and psychiatric consultation and referral, is critically needed and should be scaled up in HIV healthcare facilities. Several studies indicate elevated rates of MHDs in this population, including youth with perinatally or horizontally acquired HIV infection and adults living with HIV , 5, 6, 7Adolescents and young adults living with HIV (AYHIV) are a vulnerable population for MHDs because of the impact of HIV on physical and pubertal growth, psychological stress and HIV‐related comorbidities , 25, 26.Currently, several international guidelines recommend universal mental health screening and treatment services for people with chronic diseases , 35, 36.Given the current lack of clinical guidance on integration of mental health and HIV services for AYHIV in Asian countries, this study set out to assess the burden of depression, anxiety disorders and suicidality, and to determine the impact of integrated services on mental health and HIV treatment outcomes among Thai AYHIV.22.1We conducted a multicentre prospective cohort study among two paediatric HIV centres in Thailand, the Faculty of Medicine, Chiang Mai University (CMU) and the Faculty of Medicine, Chulalongkorn University (CU). AYHIV  who aged 15 to 25 years, were fully disclosed of HIV status, and received treatment services at both centres were enrolled. HIV‐uninfected adolescents and young adults (HUAY), including siblings, peers and neighbours of AYHIV and patients attending general paediatric clinics who had similar age and sex with AYHIV (ratio 1:1) were recruited from the same centres. Participants receiving treatments and/or interventions for psychiatric disorders, or had cognitive impairment or a condition which could compromise their ability to respond to study questionnaires and tools were excluded. This study was approved by the Research Ethics Committee, Faculty of Medicine, CMU, and the Institutional Review Board, Faculty of Medicine, CU. All participants provided written informed consent prior to study enrolment; caregiver consent was obtained for participants aged <18 years.2.2home environment, education, employment, activities, drug abuse [alcohol and illicit drugs], sexuality, suicidality and safety from injury and violence) review of systems tool : 16.8 to 21.8) years. Twenty‐four participants (8%) were LGBTQ, and 276 (92%) were heterosexual. Approximately half (54%) had biological parents  as primary caregivers, 56% lived in urban areas and 56% had household incomes ≥500 USD/month. At enrolment, 234 participants (78%) attended school, and 98 (33%) were employed in full‐ or part‐time jobs. One‐hundred and sixty‐four (55%) were current alcohol users; of whom 64 (39%) had AUDIT scores ≥8, and 16 (5%) had self‐reported illegal drug use. A comparison of sociodemographic characteristics and risk‐taking behaviours between AYHIV and HUAY is summarized in Table 3.23, and 73% were virologically suppressed (HIV RNA <50 copies/mL). Twenty‐three percent reported missed doses of cART in the past three months. Median age at disclosure of HIV status was 12.0 (IQR: 10.1 to 13.7) years. Most of AYHIV (88%) had disclosed their HIV status to other family members, but only 22% to sex partners, and 13% to other people.Of 150 AYHIV, 141 (94%) had perinatally acquired HIV infection. At enrolment, all were receiving combination antiretroviral therapy (cART); 88 (59%) were nucleoside‐reverse transcriptase inhibitor‐based, 47 (31%) protease inhibitor‐based and 15 (10%) integrase strand transfer inhibitor‐based regimens. Median CD4 T‐cell counts were 580 (IQR: 376 to 744) cells/mm3.3p < 0.05)  had experienced a relationship breakup, 44 (15%) had lost a family member and 16 (5%) had experienced a change in primary caregiver. Additionally, 15 (5%) had failed a school term/class, 8 (3%) were suspended from school and 13 (4%) were unemployed. There were also participants reporting experiencing physical abuse , forced sex , severe accidents , witnessing physical violence  and being arrested . AYHIV had greater proportion of unemployed, whereas lower proportion of family deaths and failed school term/class, compared with HUAY (3.4p = 0.66). Eleven participants (4%), seven of whom were AYHIV, had all three co‐existing conditions of significant depressive symptoms, significant anxiety symptoms and suicidality. LGBTQ participants tended to have higher prevalence of elevated mental health screening scores compared with heterosexual participants, though this comparison was not significant .Overall prevalence of elevated mental health screening scores was 20% , with no significant differences seen between AYHIV  and HUAY  (p = 0.48). Additionally, 40 participants demonstrated suicidality; 32 ever had lifetime suicidal ideations, 8 ever had lifetime suicidal attempts and 26 had a recent suicidal thoughts within two weeks, corresponding to an overall prevalence of suicidality of 13% (95%CI: 9% to 17%), which was also not different between groups . Notably, 68% of participants with elevated mental health screening scores reported suicidality.Of 300 adolescents, 34 had PHQ‐9 scores ≥9, corresponding to an overall prevalence of significant depressive symptoms of 11% (95%CI: 8% to 15%). The prevalence was the same between AYHIV and HUAY . Nineteen participants had GAD‐7 scores ≥10, corresponding to an overall prevalence of significant anxiety symptoms of 6% (95%CI: 4% to 9%), which was not significantly different between AYHIV  and HUAY  (3.5p > 0.05). For HUAY, no factors associated with elevated mental health screening scores were identified (p > 0.05).In multivariable analysis for AYHIV, residing in urban areas  increased the risk of elevated mental health screening scores, whereas older age  decreased the risk Table . Elevate3.6In this study, all 59 participants with elevated mental health screening scores (31 AYHIV and 28 HUAY) were referred to study psychiatrists (AN and PT) for confirmatory diagnosis and appropriate mental health services. Based on the DSM‐V criteria, 39 of 300 participants , including 19 AYHIV  and 20 HUAY , had at least one MHD confirmed by a psychiatrist Figure .All 39 participants with psychiatrist‐confirmed MHDs received appropriate mental health services at psychiatric clinic. Twenty‐nine (74%) received psychiatric treatment, including mental health counselling by a psychiatrist (n = 5), psychotropic medications (n = 23) and supportive psychotherapy n = 1), with regular follow‐up, whereas 10 (26%) obtained once mental health counselling without psychiatric follow‐up , and the most common psychotropic medications prescribed to participants with MDD, anxiety disorders, PDD, PTSD and mixed MHDs were selective serotonin reuptake inhibitors (SSRIs) and benzodiazepines Table . During 3.7At one‐year follow‐up, all 59 participants with elevated mental health screening scores at enrolment were re‐evaluated with PHQ‐9, GAD‐7, and recent suicidality . Thirty‐eight participants  demonstrated an absence of significant mental health symptoms from the re‐evaluations, whereas 21  had elevated scores. There were 2 of 20 participants (10%) without psychiatrist‐confirmed MHDs at enrolment demonstrating elevated scores at one‐year of follow‐up  and HIV RNA levels (40 vs. 40 copies/mL) were not different between enrolment and one‐year follow‐up (p > 0.05).Among participants with psychiatrist‐confirmed MHDs (n = 39), 20 (51%), including 8/19 AYHIV (42%) and 12/20 HUAY (60%), exhibited an absence of significant mental health symptoms from the reassessments, whereas 5/19 (26%) AYHIV and 4/20 (20%) HUAY showed an improved score at one‐year follow‐up Table . Among 14Approximately 20% of Thai AYHIV in this study demonstrated elevated mental health screening scores, of which 13% had psychiatrist‐confirmed MHDs. Younger age and urban living were associated with elevated scores. One year after appropriate mental health services, 42% of AYHIV with psychiatrist‐confirmed MHDs demonstrated an absence of significant mental health symptoms from the reassessments, and 26% showed an improved score. With the significant burden of MHDs among AYHIV, integration of mental health services into HIV healthcare settings should be implemented. Key resources include validated and culturally appropriate screening tools, well‐trained mental health counsellors, basic psychotropic medications and well‐established consultation/referral pathways between HIV and psychiatric clinics.The prevalence of significant depressive symptoms among our Thai AYHIV was lower than those reported in African and US studies , 29, 31.Likewise, the prevalence of significant anxiety symptoms among our AYHIV was much lower than the prevalence of 25% observed in South African adolescents, using the Revised Children’s Manifest Anxiety Scale (RCMAS) , and theUnlike previous studies which commonly found the greater burden of MHDs among AYHIV than the general youth population , 44, 45,In this study, urban residence increased the risk of elevated mental health screening scores among AYHIV, whereas older age decreased the risk. A previous study in urban Uganda noted similar findings in which younger adolescents were at higher risk of psychological illnesses, evaluated by the self‐rating questionnaire 25 (SRQ‐25) . AlthougAll AYHIV with psychiatrist‐confirmed MHDs received mental health services, including mental health counselling, psychotropic medications and psychotherapy. The main prescribed psychiatric medication for MDD, anxiety disorders, PDD and PTSD were SSRIs , and benzodiazepines (e.g. lorazepam). All are listed in the Thailand National List of Essential Medicines , and canWith appropriate mental health services, 42% of our AYHIV with psychiatrist‐confirmed MHDs exhibited an absence of significant mental health symptoms from the reassessments, and 26% showed an improved score at one‐year follow‐up. This finding seems to be lower than which observed in previous studies that episodes of depression and anxiety in youth usually remit within a year after appropriate treatment , 53. OurThe strengths of this study include the availability of psychiatrist‐confirmed diagnoses in participants with elevated mental health screening scores, and a high retention of study participants. However, as MHDs are dynamic conditions with varying degrees of illnesses, a single screening assessment would not sufficiently detect the full spectrum of mental illnesses in patients. Thus, we were not able to exclude MHDs among participants who demonstrated an absence of significant mental health symptoms at enrolment since we did not refer them to a psychiatrist for confirmation. Additionally, we had limited ability to comment on the change of their mental health status over time as we did not repeat an evaluation at one‐year visit. Since diagnostic assessments were only performed on participants with elevated mental health screening scores, the sensitivity of screening measures cannot be evaluated. Furthermore, as we classified participant’s sexual orientation as heterosexual vs. LGBTQ, it might not be appropriate to make a comparison between these two groups as a person can identify as transgender and heterosexual. Recall and reporting bias were inevitable as several variables were self‐reported. In Thai culture, there is a reluctance to express true feelings to health professionals, which may have limited our ability to measure the true disease burden, especially for conditions that manifest as internalization issues more than external behavioural problems. This emphasizes the need for HIV providers to initiate conversations on mental health and actively screen for MHDs even when symptoms are not obviously apparent.5With the significant burden of MHDs among AYHIV, mental health screening with simple tools, together with provision of mental health care with well‐established psychiatric consultation and referral pathways at HIV healthcare facilities are necessary. Importantly, a collaborative integrative model of mental health and HIV services would be key to successful comprehensive care for AYHIV.All authors declare no conflict of interest related to this study.TS developed the conception of research, designed the study and developed the protocol. TS, LA, WS, TC, RN, PJ, CS and TP conducted the clinical study. AN and PT performed psychiatric evaluations, diagnosed mental health disorders and provided appropriate mental health services. TS conducted the statistical analyses and contributed to the interpretation of study results. TS wrote the first draft of manuscript. TP and WS provided revisions to the manuscript. All authors reviewed and approved the final version of the manuscript.aOR, adjusted odds ratio; AUDIT, the Alcohol Use Disorders Identification Test; AYHIV, adolescents and young adults living with HIV; BSI, the Brief Symptom Inventory (BSI); cART, combination antiretroviral treatment; CDI:S, the Children’s Depression Inventory Short Form; CDRS‐R, the Children’s Depression Rating Scale, Revised; CMU, Chiang Mai University; CU, Chulalongkorn University; DSM‐IV, the Diagnostic and Statistical Manual of Mental Disorders, 4th edition; GAD‐7, Generalized Anxiety Disorder 7‐item Scale; HUAY, HIV‐uninfected adolescents and young adults; IQR, interquartile range; LGBTQ, lesbian, gay, bisexual, transgender or queer; MDD, major depressive disorder; MHDs, Mental health disorders; MINI Kid, the Mini‐International Neuropsychiatric Interview for Children and Adolescents; OR, odds ratio; PDD, persistent depressive disorder; PHQ‐9, Patient Health Questionnaire‐9; PLHIV, people living with HIV; PTSD, post‐traumatic stress disorder; RCMAS, the Revised Children’s Manifest Anxiety Scale; SAHCS, the Southern African HIV Clinicians Society; SRQ‐25, the self‐rating questionnaire 25; SSRI, selective serotonin reuptake inhibitors; US, the United States; 95% CI, 95% confidence interval.Figure S1. The details of provided mental health services, clinical course during study follow‐up, and mental health outcomes after psychiatric referral of study participants, stratified by type of mental health disorder.Click here for additional data file.Table S1. Study participants with mental health disorders and the mental health outcomes at one year follow‐up after psychiatric referralClick here for additional data file.


We found that trophic position (TP) and resynthesis index  differ between species. The surface‐feeding M. affinis had higher TP and intermediate ∑V, both pointing to a large contribution of metazoans in its diet. P. femorata, which feeds in the subsurface layers, had lower TP and a bimodal distribution of the ∑V values, supporting previous experimental evidence of a larger feeding niche. We also evaluated whether TP and ∑V values have consequences for amphipod fecundity and embryo viability and found that embryo viability in M. affinis was negatively linked to TP. Our results indicate that the amino acid‐δ15N data paired with information about reproductive status are useful for detecting differences in the trophic ecology of sympatric amphipods.According to ecological theory, two species cannot occupy the same niche. Using nitrogen isotope analyses (δ This is a field study on niche partitioning in sympatric amphipods using the amino acid nitrogen‐isotope method. Fecundity and embryo viability were measured for each individual analyzed for isotopes, meaning that ecological and physiological causes behind observed patterns could be tested and discussed in a unique way. We demonstrate that the amino acid method was useful in detecting differences in trophic ecology between two deposit‐feeding species, but that it is crucial to include measurements of physiological status when interpreting nitrogen isotope data in amino acids of consumers. The basMonoporeia affinis, a glacial relict, and Pontoporeia femorata, of marine origin, dominate in abundance the benthic communities at soft sediment bottoms. Like all deposit‐feeders in this area, the spring phytoplankton bloom constitutes the largest annual food input  of a consumer is indicative of its trophic position, since the heavy isotope is enriched for every trophic transfer  can, however, vary considerably between ecosystems and regions  of consumers is an important component of the trophic niche; however, TP is not easily quantified in omnivorous species, such as benthic deposit‐feeders, because it is difficult to accurately sample the food items which are assimilated. The nitrogen isotope composition  has become increasingly popular for TP assessment and for encoding the baseline signal   has been suggested as a proxy for trophic transfer  more depleted δ15N values in Phe  intermediate ∑V values  content of 20 mg aliquots of oven dried (60 C°) sediment was analyzed in a Leco‐CHN analyzer (with EDTA as standard) at the accredited chemical laboratory at Department of Ecology, Environment, and Plant Sciences, Stockholm University. Acidification of Baltic sediments is not necessary as <0.1% of the carbon is inorganic .The upper 2–3 cm sediment and the amphipods living there were sampled with a bottom sled  were freeze‐dried, weighed to determine the individual dry body mass , and used for AA extraction and stable isotope analysis (hereafter AA‐δ15N). Due to the small BM , females of similar reproductive status were grouped to obtain sufficient biomass for the AA‐δ15N analysis; the target sample mass was 5 mg of dry body mass per sample. In this grouping, we considered species, station, fecundity, and percentage of the aberrant embryos. After the grouping, each sample contained between 2 and 8 females, with more individuals per sample for the smaller M. affinis. This procedure resulted in 8 (coded as M1‐M8) and 11 (coded P1‐P11) samples for AA‐δ15N analysis in M. affinis and P. femorata, respectively  for purification. To elute the amino acids fraction from columns, a 10% NH3 solution was used, and the mobile phase was evaporated until dryness and stored at −20°C. To volatilize free AAs, the derivatization step was applied according to Yarnes and Herszage , in glass vials using 6 M hydrochloric acid (HCI) for 70 min at 150°C. Thereafter, the samples were evaporated until dryness under a gentle stream of NHerszage , with sl2.415N values in the individual AAs of a single 2‐µl injection were measured on a Thermo gas chromatography/ combustion/ isotope ratio mass spectrometry (GC‐C‐IRMS) system consisting of a trace GC chromatograph, IsoLink IV combustion interface with a nickel/ copper oxide reactor, a Conflo IV unit for introduction to the Delta V Plus mass spectrometer, and a PTV injector. A SGE Analytical Science, ID: BPX70 capillary column (30 m × 0.25 mm), was used for the chromatographic separation. Standard operating conditions were applied with regard to reactor temperature , and the evolved CO2 was cryogenically removed to avoid isobaric interference by CO+ ions on mass 28. For peak integration, we used individual background type since the chromatogram peaks were within the optimal intensity range ; for the actual peak measurement, the seed oxidation method was used. Data were extracted and analyzed with the ISODAT software packages (3.0). All analyses were performed at the Department of Environmental Science and Analytical Chemistry (ACES), Stockholm University.The δ2.5P. femorata samples were found for the following trophic AAs: alanine (Ala), valine , and pyro‐glutamic acid (Glu) and for the source AA, phenylalanine (Phe) resulting in 17 samples. For eight of the samples, acceptable peaks were also found for the following trophic AAs: proline (Pro), leucine (Leu), isoleucine (Ile), and threonine (Thr), the latter which has its own category often referred to as “metabolic” AA in vertebrates  following the method of Chikaraishi et al.  and source AAs (y) in primary producers, and Δx and Δy are the 15N trophic enrichment factors (TEF) for each AA(s)x and y, respectively. Values for x/yβ and TEF differ between the equations; in Equation x/y and TEF (3.4 and 7.6), (3.2 and 5.7), and (4.6 and 4.6). In Equation x/y and 5.9 for TEF.Trophic position (TP) was calculated using two alternative approaches: single pair of AAs TPxy=TPxy=15NGyΔx‐Δy+1TPxy=∑ divided by n . As trophic AAs, we used: Glu, Ala, and Val . The dece Table .2.615N of the source AA Phe and in the AA Thr, the TP values, and the ∑V values were evaluated using different methods. Phe‐δ15N, Thr‐δ15N, and TP values were compared between the species using unpaired t tests. The comparison of Thr‐δ15N between the species was conducted with a lower sample size . The ∑V values showed an approximated bimodal distribution for P. femorata and, therefore, we used Hartigan's dip‐statistics  were thereafter tested for differences in fecundity, TP and %VE using t tests and chi‐square test, respectively. To test for a station effect on the Phe‐δ15N values in amphipods and the organic carbon content in sediment, we used one‐way ANOVA followed by Tukey post hoc test. In addition, one‐way ANOVA and Kruskal–Wallis were used to evaluate station effect on the δ15N values for each of the three trophic AAs . Shapiro–Wilks test and F test were used to test for normality of the distribution and homogeneity of variance when the variables tested had 2 levels. Variables with more than two levels were visually inspected and homogeneity of variance tested using Levene's test. Tests on isotope and reproductive data were conducted both separately for each species  as described above but also tested on pooled data of amphipods  as described in the introduction .Species‐specific difference in the δ15N values against the reproductive status, TP and ∑V, were analyzed with a Spearman rank correlation. The amphipods community ∑V against the reproductive variables and against TP were analyzed with generalized additive models  regressions , differences in the reproductive variables  and biomass (BM) between the species and the species‐specific relationships of reproductive variables to BM as well as influence of station effect were evaluated. The distribution of all variables were visually explored, and when necessary, HDS was used to test whether the distribution deviated from unimodality. Species effect on fecundity and BM was tested using Mann–Whitney U test, while %VE was tested using Fisher's exact test. Relationships between BM and fecundity as well as %VE for each species were evaluated using SMA regression as described above. Since there were differences in the organic matter between the stations Figure , we usedWhen assumptions on normality or homoscedasticity for SMA regression were not met, the data were transformed. Percentage viable embryos were always Box‐Cox transformed, both for species‐specific and amphipods community data; fecundity and TP values were log‐transformed when the relationship between them was evaluated. For individual data, BM was Box‐Cox transformed when the BM‐%VE regression was evaluated. All data were analyzed using the R software environment 3.4.3 . Phe‐δ15N values (pooled for both species) differed, however, significantly among the stations . No significant differences between the stations were found for the trophic AAs . The TP calculated according to Nielsen et al., M. affinis than for P. femorata . Thr‐δ15N values differed significantly between the species . Neither TP nor % VE nor fecundity differed significantly between the two P. femorata groups (Table p < .02) for the group with ∑V < 1.The ∑V values varied between and within the species. A significant deviation from unimodality was found for ∑V values in ps Table . Howeverps Table  composed3.2M. affinis, whereas for neither of the species the TP–fecundity relationship was significant , while no significant correlation was found for fecundity or %VE  and %VE  but lower BM  than P. femorata. The median values for these traits in M. affinis versus. P. femorata were as follows: fecundity (29 vs. 15 embryos), %VE (96.4 vs. 89.8% viable embryos in the brood), and BM (1.4 vs. 2.4 mg/ind).None of the variables tested had a bimodal distribution, although it was close to significant for fecundity in M. affinis , but not significant for P. femorata  phenylalanine (Phe). However, both mean values and variation in δ15N of the trophic AAs varied significantly between the species suggesting differences in their trophic position. As hypothesized, Monoporeia affinis had higher trophic position (TP) than Pontoporeia femorata, regardless of the method for TP calculation. In addition, the difference in Thr‐δ15N values between the species supports a higher number of trophic transfers in the diet of M. affinis. P. femorata showed a greater variability in the resynthesis index (∑V) suggesting a broader feeding niche.Contrary to our expectations, the two amphipod species rely on the same primary nitrogen source as indicated by the similar δP. femorata in experiments  have been found in the guts of M. affinis  to what was found for the deposit‐feeders, including 1 Figure  could thP. femorata with high ∑V have unrealistically low TP  in both microbes  vary among species and is for both amphipods studied here higher in sediment with lower N%  and also differ in the resynthesis index (∑V), as estimated by nitrogen isotope composition of amino acids. The surface‐feeding amphipod None declared.Matias Ledesma: Conceptualization ; Data curation (lead); Formal analysis (lead); Investigation ; Methodology ; Project administration ; Validation ; Visualization (lead); Writing‐original draft (lead). Elena Gorokhova: Conceptualization ; Formal analysis (supporting); Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Visualization (supporting); Writing‐original draft (supporting); Writing‐review & editing . Henry Holmstrand: Resources ; Software (supporting); Writing‐review & editing (supporting). Andrius Garbaras: Resources ; Software (supporting); Writing‐review & editing (supporting). Agnes ML Karlson: Conceptualization ; Formal analysis (supporting); Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Validation (lead); Visualization (supporting); Writing‐original draft (supporting); Writing‐review & editing .AppendixS1Click here for additional data file.


A 51‐year‐old man attended our hospital with chief complaints of fever and diarrhoea for the past eight days. Chest computed tomography showed peripherally dominant ground‐glass opacity. Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) RNA was detected by real‐time polymerase chain reaction, and the patient was diagnosed with coronavirus disease (COVID‐19). His clinical course included respiratory failure, acute kidney injury, and paralytic ileus. Systemic management was difficult, but he recovered with high‐dose steroids, temporary haemodialysis therapy, and a nasointestinal tube, without antiviral drugs. COVID‐19 can be associated with multiple organ failure due to vascular endothelial injury. Coronavirus disease (COVID‐19) may be associated with multiple organ failure due to vascular endothelial damage. Here, we report a case of COVID‐19 in which a patient suffered from ileus complications and was difficult to manage systemically. However, he recovered with high doses of steroids, hemodialysis, and the nasal intestine without the use of antivirals. Coronavirus disease COVID‐19) can be associated with multiple organ failure due to vascular endothelial injury [9 can be A 51‐year‐old man was admitted to our hospital with a history of persistent fever and diarrhoea for the past eight days. He had a smoking history of seven pack‐years and consumed alcohol occasionally; his past and family histories were non‐contributory. Physical examination on admission revealed a fever of 39.0°C, moist skin, and coarse crackles in the lower chest bilaterally. Complete blood counts and serum chemistry suggested a serious infectious event, with a markedly elevated serum leucocyte count of 13,000/μL with a left‐shifted haemogram, C‐reactive protein of 12.7 mg/dL, elevation of lactate dehydrogenase to 430 U/L , elevation of aspartate aminotransferase to 41 U/L, elevation of alanine aminotransferase to 49 U/L, serum creatinine of 3.19 mg/ dL, serum uric acid of 110.4 mg/dL, and plasma D‐dimer of 2.5 μg/mL. A haemoglobin A1c value of 6.5% and mild glucose intolerance were found. Urinalysis showed protein 3+, uric blood −, and urine sediment of 5+ granular casts, 4+ epithelial casts, and 5+ hyaline casts. A nasopharyngeal swab was positive for severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) RNA on real‐time polymerase chain reaction (PCR). Chest X‐ray and chest computed tomography (CT) on admission demonstrated diffuse ground‐glass opacity located predominantly at the periphery of the lower lung lobes , even with 10 L/min oxygen supplied by a non‐rebreather mask, and he was intubated on a ventilator. Methylprednisolone 500 mg was intravenously administered once daily for three days. The steroid was then changed to oral prednisolone, which was given every second day at reducing doses of 60, 40, 20, 10, and 5 mg. Because bacterial pneumonia with viral pneumonia could not be ruled out, tazobactam− piperacillin 13.5 g/day and levofloxacin 500 mg/day were administered. Unfractionated heparin at 10,000 units/day was administered by subcutaneous injection to prevent thrombus formation. The patient was extubated on day 4 after intubation as his respiratory condition improved. On day 5, his serum creatinine had risen to 11.13 mg/dL and his urine volume was less than 500 mL/day, so temporary haemodialysis was performed on days 5, 6, 8, and 9. After extubation, tube feeding was continued, but vomit‐like bile was found on day 8, and an abdominal X‐ray photograph showed dilatation of the intestinal tract  receptor on cell surfaces. It was recently found that the ACE2 receptor is also expressed on vascular endothelial cells, and SARS‐CoV‐2 can infect these cells via the receptor. Vascular endothelial cell injury can lead to multiple organ damage due to microthrombus formation in the cardiovascular system [Renal injury occurs in 75.4% of COVID‐19 patients, with a median duration of 12 days; 45.7% of patients with renal injury from COVID‐19 recover completely . Owing tRapid disease exacerbation because of cytokine storm seven to 10 days after symptom onset has been suggested to occur in COVID‐19 patients . From thAlthough some success with antiviral drugs such as favipiravir has been reported, this patient had renal failure and the dosage was unknown in renal failure, so favipiravir was not used. Nevertheless, we were able to cure the patient with glucocorticosteroids.This case was complicated by a variety of organ damage caused by COVID‐19 and was difficult to manage systemically, but they recovered with supportive care and the use of systemic corticosteroids.Appropriate written informed consent was obtained for publication of this case report and accompanying images.


Garcinia lucida is traditionally used in Cameroon for the management of cardiovascular diseases. The aim of this study was to evaluate the cardioprotective and nephroprotective effects of the aqueous extract from the stem bark of G. lucida (AEGL). The in vitro antioxidant effect of AEGL was assessed at concentrations ranging 1–300 μg/mL against DPPH, lipid peroxidation, and AAPH-induced hemolysis. The reducing power and phenolic and flavonoids contents were also determined. CKD was induced by intraperitoneal bolus injection of adenine (50 mg/kg/day) for 4 consecutive weeks to male Wistar rats. AEGL (150 and 300 mg/kg/day) or captopril (20 mg/kg/day) was concomitantly administered with adenine per os. Bodyweight and blood pressure were monitored at baseline and weekly during the test. At the end of the experiment, plasma creatinine, urea, AST, and ALT were quantified. Proteinuria, creatinine excretion, and creatinine clearance were also assessed. The effect on GSH, CAT, and SOD activity was evaluated in cardiac and renal homogenates. Sections of the heart and kidney were stained with hematoxylin and eosin. AEGL exhibited a potent in vitro antioxidant activity and was shown to possess a large amount of phenolic compounds. Adenine alone increased blood pressure, cardiac and kidney mass, proteinuria, protein to creatinine ratio, plasma creatinine, AST, and urea levels . Besides, the bodyweight and creatinine clearance were significantly reduced (p < 0.05 and p < 0.01). All these alterations were blunted by the plant extract, except the bodyweight loss. In addition, AEGL improved GSH levels and CAT and SOD activities. AEGL attenuated adenine-induced glomerular necrosis, tubular dilatation, and cardiac inflammation. AEGL exhibits cardioprotective and nephroprotective effects that may be ascribed to its antihypertensive and antioxidant activities.Chronic kidney disease (CKD) is a serious health problem with high morbidity and mortality, mainly attributable to cardiovascular risk.  In aCurrently, there is no cure for most forms of CKD, and strategies to manage CKD rely on high blood pressure control through blockade of the renin-angiotensin system . HoweverGarcinia lucida Vesque (Clusiaceae), commonly known as “essok” by the Cameroonian populations, is a small evergreen dioecious tree, reaching 25–30 cm in diameter at breast height and 12–15 m in height [G. lucida is traditionally used to prevent food poisoning, cure stomach and gynecological pains, as well as snake bites [G. lucida showed that alkaloids isolated from the stem bark exhibited trypanocidal and antileishmanial activities [G. lucida have also demonstrated cytotoxic, antimicrobial, and in vitro antioxidant activities [G. lucida prevented L-NAME-induced hypertension (unpublished data), but nothing is known about the effect of this plant on CKD. The present study was therefore undertaken to evaluate the antioxidant and the nephroprotective effects of the aqueous extract from the stem bark of G. lucida against adenine-induced chronic kidney disease in rats.n height . The steke bites . Based otivities . Extracttivities . In a prGarcinia lucida were collected in Mvengue, in the south region of Cameroon in March 2017. The plant specimen was authenticated at the National Herbarium of Cameroon in comparison with an existing voucher specimen number 57193/HNC. The fresh stem bark was shade-dried and ground into a fine powder. The aqueous extract was prepared by bringing to boil 200 g of powder in 1.5 L distilled water for an hour. The residue was again decocted in 1.2 L distilled water for 1 h. Both filtrates were mixed and evaporated in an AISET YLD-2000 temperature controlled oven at 40°C. The extraction procedure yielded 37 g of aqueous extract, corresponding to an extraction yield of 18.5%.Fresh barks of The total phenolic content was determined using the Folin–Ciocalteu method . A calibG. lucida. Briefly, 0.1 mL of aqueous extract (500 μg/mL) was mixed with 30 μL of 5% sodium nitrate and 1.4 ml distilled water and incubated at room temperature for 5 min. Then, 30 μl of 10% aluminum chloride was added followed by 200 μl of 0.1 M potassium hydroxide solution 6 min later. Optical densities were read at 510 nm. The flavonoids content was expressed as mg of quercetin per gram of sample.A modified method of Zhishen et al.  was usedMale Wistar rats aged 3-4 months and weighing 200–260 g were bred in the animal house of the Laboratory of Biology and Physiology of Animal Organism of the Faculty of Sciences, University of Douala-Cameroon, in plastic cages. Rats were maintained at a room temperature of 24 ± 2°C under natural light/dark cycle (∼12 h/12 h). They had free access to standard laboratory chow and tape water at libitum. The study was approved by the institutional ethic committee for research in human health of the University of Douala N 2043CEI-UDo/06/2019/T, and all experiments and procedures were performed in accordance with the guidelines on the protection of animals used for scientific purposes as described by the law 2010/63/EU of the European Parliament and of the Council of 22 September 2010.G. lucida was evaluated on 2,2-diphenyl-1-picrylhydrazyl (DPPH) as described by Nono et al. [μg/mL). All substances used were dissolved in methanol. The optical densities were read before (OD1) and 30 min after (OD2) adding DPPH at 515 nm using a Helios Epsilon spectrophotometer against a blank made of methanol. Experiments were performed in triplicate, and the inhibition percentage (I) was calculated as follows: I (%) = ((OD2−OD1)control−(OD2−OD1)sample) x 100/(OD2−OD1)controlThe radical scavenging ability of the aqueous extract from o et al. . AscorbiThe reducing power was performed according to a method described previously  using asμl of supernatant was incubated in the presence or absence of the plant extract. Lipid peroxidation was initiated by adding 0.2 M ferric chloride. The reaction was stopped with an ice-cold HCl (0.25 N) solution containing 15% trichloroacetic acid and 0.38% thiobarbituric acid. Optical density was read at 532 nm. Gallic acid was used as positive control, and percentage inhibition was calculated as follows: inhibition of lipid peroxidation (%) = 100 × (1–OD sample)/OD control.The effects of plant aqueous extract on lipid peroxidation were evaluated using the method described by Sawale et al.  except tG. lucida was assessed as previously reported [μL) of 5% RBC suspension, extract, and 200 mM AAPH  dihydrochloride) were mixed and incubated at 37°C for 3 h with gentle shaking. At the end of the incubation period, 4 mL of PBS was added to the reaction milieu, followed by centrifugation at 3500 rpm for 10 min. The optical density of the supernatant was read at 540 nm. To yield complete hemolysis, an aliquot of RBC suspension was treated with an equal volume of ice-cold distilled water. Control tubes were made of reaction milieu without extract or ascorbic acid, and the percentage inhibition was calculated as follows: I (%) = 100 × (1–OD sample)/OD control.The antihemolytic effect of aqueous extract from reported  with somGroup 1 served as control and received distilled water and saline (0.9 %)Group 2 received adenine (50 mg/kg) + distilled waterGroup 3 was treated with adenine (50 mg/kg) + captopril (20 mg/kg/day)Group 4 was treated with adenine (50 mg/kg) + aqueous extract (150 mg/kg/day)Group 5 was treated with adenine (50 mg/kg) + aqueous extract (300 mg/kg/days)Rats were randomly assigned into five groups of ten rats for each treatment as follows for four consecutive weeks:Chronic kidney disease was induced using a previously described method . AdenineThe doses of extract used were calculated according to the traditional healer's instructions. The amount of extract solution recommended per day was taken from this traditional healer and evaporated at 40°C in an oven. Following this procedure, 1.452 g of powder was obtained and divided by 60 kg (reference human bodyweight). This corresponds to 24.20 mg/kg human dose. The equivalent dose for rats was calculated by multiplying the human dose (24.20 mg/kg) by 6.2 [Blood pressure and heart rate were recorded at baseline and weekly using the noninvasive tail-cuff method . Bodyweight was also monitored weekly after the baseline value was taken. At the end of the treatment period, 24-hour urine samples were collected, centrifuged at 3500 rpm for 15 min at 4°C, aliquoted, and stored at −20°C for subsequent quantification of protein and creatinine. Following this, animals were anesthetized with sodium thiopental . Blood samples were collected through abdominal aorta in EDTA-coated tubes and centrifuged at 3500 rpm for 15 min at 40°C. The plasma obtained was kept at −20°C for urea, creatinine, ALT, and AST determination. The kidney and the heart were removed, freed of fats and connective tissues, washed in physiological saline, and weighed. The heart was dissected out, and pieces of each organ was fixed in 10% buffered formalin for routine hematoxylin and eosin staining technique, while the remaining part was stored at −20°C.U × V/P × 1440, where U is the urine creatinine, V is the 24 h urine volume, P is the plasma creatinine, and 1440 is time in seconds corresponding to 24 h.Urinary protein excretion was assessed using the Bradford method . Urea, c4 M−1·cm−1 as the molar extinction coefficient. Catalase (CAT) activity was evaluated according to the method of Sinha et al. [2O2) was constructed, and optical densities were read at 570 nm. One unit of catalase activity was defined as the amount of enzyme required to decompose 1 micromole of H2O2 per minute. SOD activity was measured according to the method of [The effect of the plant extract on oxidative stress parameters was determined by assaying enzymatic and nonenzymatic antioxidant status. A 15% tissue homogenate was prepared in 10 mM Tris-HCl buffer (pH 7.4) using a D‐160 handheld homogenizer . Reduced glutathione (GSH) content was determined as described by . Opticala et al. . A calibethod of .μm thickness were prepared and stained with hematoxylin and eosin. The stained sections were examined under a DN-107T light microscope for structural alterations.The kidneys and the heart from all experimental groups were fixed in 10% buffered formalin, dehydrated in graded ethanol, cleared in xylene, and then embedded in paraffin. Sections of 5 Adenine, aluminum chloride, potassium hydroxide, sodium nitrate, and Folin–Ciocalteu reagent were purchased from Carl-Roth (Germany). 2, 2-Diphenyl-1-picrylhydrazyl (DPPH),  dihydrochloride) (AAPH), gallic acid, quercetin, ascorbic acid, Tris, hydrogen peroxide, trichloroacetic acid, ferric chloride, and hydrogen chloride were purchased from Sigma–Aldrich (Germany). Captopril was purchased from Denk Pharma GmbH and Co., KG (Germany). Thiobarbituric acid was from Fluka. Sodium chloride and potassium chloride were purchased from BDH Chemicals Ltd. (England). Creatinine, urea, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) kits were purchased at SGM Italia (Roma). Sodium thiopental was from Samarth Life Sciences Pvt Ltd. (Mumbai).50 was determined using the nonlinear regression curve following logarithmic transformation. For single time point data collection , data were analyzed with one-way ANOVA followed by Tukey's post hoc test. For repeated measures (blood pressure and body gain weight), data were analyzed with two-way ANOVA with repeated measures followed by the Bonferroni post hoc test. Differences between means were considered significant at p < 0.05.Statistical analysis was performed using GraphPad Prism 5.03. All the results were expressed as mean ± standard error of the mean. ICG. lucida extract is recorded in G. lucida is high, while the total amount of flavonoids is low.The total amount of phenols and flavonoids in 50 value of aqueous extract was 14.13 μg/mL versus 3.27 μg/mL for ascorbic acid  increase in systolic and diastolic blood pressure compared to the control group. Systolic blood pressure increased from 119.00 ± 3.05 mmHg in the control group to 148.30 ± 5.75 mmHg in the adenine group at the end of the treatment period. Diastolic pressure values changed from 85.08 ± 1.84 mmHg in control rats to 112.90 ± 3.84 mmHg in animals that were chronically administered with adenine. Concomitant administration of the plant extract at all tested doses or captopril significantly (p < 0.05 and p < 0.001) prevent the rise in blood pressure elicited by adenine. Blood pressure values in these groups were similar to that of the control group at the end of the experiment.As shown in p < 0.05) effect on heart rate was observed following adenine exposure. Nevertheless, heart rate was significantly low (p < 0.05) in rats that were concomitantly treated with adenine and plant extract compared to those that received adenine alone  that was not inhibited by either captopril or the aqueous extract of G. lucida. Captopril and the extract at the dose of 150 mg/kg rather accentuated the weight loss.As observed in p < 0.001), heart (p < 0.01), and left ventricle (p < 0.001) compared to the control group. The most important organ mass increase was that of the kidney which enlarged more than double. The aqueous extract of G. lucida at the dose of 150 mg/kg completely abolished adenine-induced organ hypertrophy. Captopril acted similar to the plant extract at the dose of 150 mg/kg, except that it failed to prevent kidney hypertrophy  increased AST levels but had no effect on the ALT level (p > 0.05) compared to the control. Plasma ALT and AST levels were kept near to the normal value following treatment with the plant extract. Captopril significantly (p > 0.05) rose ALT but lowered (p < 0.05) adenine-evoked AST increase (Long-term administration of adenine significantly (increase .p < 0.05 and p < 0.001) reduced in the kidneys following chronic adenine administration. In the cardiac tissue, adenine also induced a decrease in these parameters, but only the reduction in SOD activity reached statistical significance (p < 0.05). The plant extract regardless of the dose used failed to inhibit the drop in renal GSH, eventhough this decrease was a little bit delay with the dose of 150 mg/kg  were significantly  in adenine-treated rats, while urinary creatinine excretion and creatinine clearance declined significantly (p < 0.05) in the same animals. Creatinine clearance dropped from 0.07 ± 0.01 mL/min in control rats to 0.02 ± 0.01 mL/min in the adenine group . This experimental model was chosen in this study because it exhibits structural and functional alterations observed in CKD patients [The present study was designed to evaluate the nephroprotective and cardioprotective effects of the aqueous extract from the stem bark of patients , 30.G. lucida was evaluated in vitro and in vivo. Results from in vitro antioxidant tests showed that G. lucida extract exhibited a strong radical scavenging activity against DPPH. In alcoholic solution, DPPH forms a stable free radical, and the effect of antioxidant substances on this radical is thought to result from their hydrogen and/or their electron donating ability [Many reports have evidenced the role of oxidative stress in the pathogenesis and progression of adenine-induced CKD , 31. The ability , 33.G. lucida extract could be attributed to the presence of phenolic compounds.It is well known that phenolic compounds and mostly flavonoids possess an ideal structure for free radical scavenging activity. In fact, the antioxidant properties of phenols arise from their high reactivity as hydrogen or electron donors , 35. In G. lucida strongly inhibited the peroxidation of brain's lipids. It is well known that radical scavenging activity and reduction of ferric ions are among the mechanisms by which antioxidants inhibit lipid [G. lucida inhibit lipid peroxidation by one of the above mechanisms.One of the main mechanisms by which free radicals exert their deleterious effects on cells and organs is through lipid peroxidation of biological membranes. Since the extract used in this study showed interesting antiradical and reducing power, we tested their ability to inhibit lipid peroxidation initiated by ferric chloride through OH radical generation by Fenton's reaction. It was noticed that the aqueous extract from the stem bark of it lipid , 36. ThuG. lucida extracts on a living organism, they were tested on AAPH-induced red blood cells hemolysis. The results showed that aqueous extract exhibited a good antihemolytic effect, although these activities were observed at high extract concentrations (100 and 300 μg/ml). AAPH induces red blood cells hemolysis by two mechanisms, namely, lipid peroxidation and protein oxidation [G. lucida extract matches well with the above demonstrated antilipid peroxidation activity.Red blood cells are more prone to lipid peroxidation . In an axidation . TherefoG. lucida was evaluated on a model of CKD using adenine. The results showed that adenine administration was associated with a reduction in GSH, CAT, and SOD supporting the findings of many other authors [The in vivo antioxidant effect of  authors , 31, 39.G. lucida may possess a slimming effect.It was observed in this study that animals treated only with adenine progressively lost weight compared to healthy controls. This result is in accordance with previous works , 40, 41.G. lucida possesses metabolites with the nephroprotective effect.It has been observed in this study that intraperitoneal injection of adenine alone increased proteinuria, protein to creatinine ratio, plasma creatinine, and serum urea but decreased creatinine excretion and creatinine clearance. These alterations are typical signs of CKD , 3 and cG. lucida on the cardiovascular system was evaluated. The results showed that chronic adenine injection was associated with increased systolic and diastolic blood pressure, cardiac hypertrophy, cardiac inflammation, and increased cardiomyocyte intercellular space. Nguy et al. [G. lucida successfully prevented cardiac remodeling. This protective effect of the plant extract on cardiac architecture could be explained by its antihypertensive effect. Captopril also exhibited a cardioprotective effect suggesting that adenine-induced cardiovascular damages might be mediated by the renin-angiotensin system.Cardiovascular disease is one of the major causes of mortality and morbidity in patients with CKD, rather than from end-stage renal disease , 44. Thuy et al.  and Kashy et al.  found thHistological analysis of sections from the renal tissue showed adenine-induced glomerular necrosis. This observation is supported by the high level of plasma AST. ALT levels did not change in the adenine group. However, Kim et al.  and Al ZG. lucida exhibits in vitro and in vivo antioxidant activities. This antioxidant activity could be ascribed to its content in phenolic compounds. Besides, the cardioprotective and nephroprotective effects of G. lucida may be ascribed to its antihypertensive and antioxidant activities.In the light of what precedes, it can be concluded that the aqueous extract from the stem bark of


Unloading/disuse induces skeletal muscle atrophy in bedridden patients and aged people, who cannot prevent it by means of exercise. Because interventions against known atrophy initiators, such as oxidative stress and neuronal NO synthase (nNOS) redistribution, are only partially effective, we investigated the involvement of melusin, a muscle‐specific integrin‐associated protein and a recognized regulator of protein kinases and mechanotransduction in cardiomyocytes.in vivo melusin replacement by cDNA electroporation, and muscle force, myofiber size, and atrogene expression after adeno‐associated virus infection. In vivo interference of exogenous melusin with dominant‐negative kinases and other atrophy attenuators  on size of unloaded rat myofibers was also explored.Muscle atrophy was induced in the rat soleus by tail suspension and in the human vastus lateralis by bed rest. Melusin expression was investigated at the protein and transcript level and after treatment of tail‐suspended rats with atrophy initiator inhibitors. Myofiber size, sarcolemmal nNOS activity, FoxO3 myonuclear localization, and myofiber carbonylation of the unloaded rat soleus were studied after P < 0.001), and to about 35% after 8 day bed rest in humans (P < 0.05). In the unloaded rat, melusin loss occurred despite of the maintenance of β1D integrin levels and was not abolished by treatments inhibiting mitochondrial oxidative stress, or nNOS activity and redistribution. Expression of exogenous melusin by cDNA transfection attenuated atrophy of 7 day unloaded rat myofibers (−31%), compared with controls , without hampering the decrease in sarcolemmal nNOS activity and the increase in myonuclear FoxO3 and carbonylated myofibers. Infection with melusin‐expressing adeno‐associated virus ameliorated contractile properties of 7 day unloaded muscles (P ≤ 0.05) and relieved myofiber atrophy (−33%) by reducing Atrogin‐1 and MurF‐1 transcripts (P ≤ 0.002), despite of a two‐fold increase in FoxO3 protein levels (P = 0.03). Atrophy attenuation by exogenous melusin did not result from rescue of Akt, ERK, or focal adhesion kinase activity, because it persisted after co‐transfection with dominant‐negative kinase forms (P < 0.01). Conversely, melusin cDNA transfection, combined with 7‐nitroindazole treatment or with cDNA transfection of the nNOS‐interacting chaperone Grp94, abolished 7 day unloaded myofiber atrophy.Unloading/disuse reduced muscle melusin protein levels to about 50%, already after 6 h in the tail‐suspended rat (Disuse/unloading‐induced loss of melusin is an early event in muscle atrophy which occurs independently from mitochondrial oxidative stress, nNOS redistribution, and FoxO3 activation. Only preservation of melusin levels and sarcolemmal nNOS localization fully prevented muscle mass loss, demonstrating that both of them act as independent, but complementary, master switches of muscle disuse atrophy. Therefore, the aim of the present study was to investigate the signal transduction pathway bound to the widespread mechanosensitive receptor integrins, focusing on the muscle‐specific transducer melusin.2+‐binding domain, rich in aspartic and glutamic acid residues.Melusin is a chaperone protein expressed specifically in cardiac and skeletal muscles. It consists of two N‐terminal cysteine‐rich and histidine‐rich domains: a CS domain (peculiar of cysteine‐rich and histidine‐rich domains and Sgt1 proteins)Melusin is also expressed in skeletal muscles; however, its function in this context has been poorly investigated so far. Early studies indicated that melusin expression is regulated during skeletal muscle development, becoming detectable in embryo limbs at 15 days of gestation and reaching a peak in newborn muscles. In accordance with a possible role in myogenesis, melusin expression is upregulated during muscle regeneration after trauma.in vivo transfection and viral infection. In vivo transfections allowed to compare melusin‐transfected myofibers with untransfected ones in the same muscle section concerning myofiber cross‐sectional area (CSA), sarcolemmal nNOS activity by means of histochemistry for NADPH‐diaphorase, FoxO3 nuclear localization, and myofiber carbonylation by means of DNPH adducts. In vivo melusin cDNA transfection was further used during unloading in combination with 7‐NI treatments or other constructs, such as dominant‐negative (DN) forms of the melusin‐interacting kinases, or other pro‐trophic players, such as the Grp94/gp96 chaperone. Infections with adeno‐associated virus (AAV) coding for melusin allowed to investigate whether unloaded muscles preserved contractility, in addition to myofiber CSA, composition in fiber types, myosin heavy chains, levels of atrogene transcripts, and FoxO3 and kinase proteins.By investigating rat and human skeletal muscles after short and long periods of unloading/bed rest, here we identify melusin as a new and early disuse target. The rat tail suspension model was further used to monitor melusin expression after exposure to muscle atrophy attenuators such as 7‐NI,The combination of these approaches demonstrates here that melusin is required to maintain mass and force of unloaded muscle. Interestingly, unloading‐induced melusin loss is not rescued by known atrophy attenuators, and melusin replacement appears to counteract atrophy independently from both integrin‐downstream signalling pathways and the preservation of nNOS activity and subcellular distribution.22.1Exogenous melusin expression was achieved by expressing human melusin cDNA added with a c‐myc tag at the amino‐terminus, either subcloned in a pCEP plasmid or in the AAV 9 (AAV‐MEL).Plasmids for DN non‐receptor kinases were provided from Addgene: (i) HA‐Akt‐DN (plasmid 16243) codifies for a mouse Akt variant, which is catalytically inactive due to K179M substitution at the ATP‐binding site and was effective with both rat and human cellsPlasmid pT94, a bicistronic vector containing rabbit Grp94 and GFP cDNAs,2.2n = 8–9 in total) and were randomly selected for standard caging (n = 2–3) or hindlimb unloading (n = 6) using the tail suspension model.About 136 six week old female Wistar rats were used in the study. Rats entered in each experimental protocol in small groups n = 8– in totalin vivo gene transfer, that is, direct injection in the soleus muscle of plasmids or AAV particles, or both pharmacological treatments and plasmid injection.Several different experimental protocols combined standard caging and hindlimb unloading to either pharmacological treatments or TableData concerning number of animals and soleus muscles, together with body and absolute and normalized muscle weight, were reported in Supporting Information, 2.2.1Plasmid transfection was performed as previously describedn = 5); (b) freely ambulating animals transfected with melusin cDNA (n = 5); (c) hindlimb‐unloaded animals transfected with EV (n = 11); and (d) hindlimb‐unloaded animals transfected with melusin cDNA (n = 11); (ii) about 50 μg of each plasmid codifying for DN forms of Akt or ERK2 or FAK were injected in one soleus and in combination with 50 μg melusin cDNA in the contralateral one in 15 rats (five rats for each DN‐kinase plasmid), which were tail‐suspended for 7 days; (iii) about 50 μg of plasmids codifying for melusin combined either with 50 μg of plasmid pT94 or with pT one were injected in each soleus of the same rat. This procedure involved eight rats, which were then tail‐suspended for 7 days.The following constructs were used for soleus muscle transfections: (i) about 50 μg of melusin cDNA or EV were co‐injected with 30 μg of pT bilaterally in the soleus muscles of 32 rats, which were then subdivided in four different groups corresponding to (a) freely ambulating animals transfected with EV  were used to determine melusin protein levels. Data concerning these experimental groups were already published .Still available frozen ambulatory and unloaded muscles obtained from rats treated with curcumin or MitoTEMPO , and details on ethical permission have been already published.2.4Whole homogenates were prepared by dissolving muscle cryosections as previously described .2.5TableTotal RNA from muscles was isolated using Trizol Reagent (Thermo Fisher Scientific), following the manufacturer's instructions. RNA was reverse transcribed by using the high capacity cDNA reverse transcription kit (Applied Biosystem). Gene expression was evaluated using specific primers designed for Itgb1bp2, Atrogin, MuRF‐1, FoxO1, Foxo3a, and Gapdh as endogenous control  and/or with mouse anti‐myc mAb clone 9E10 or with anti‐FoxO3 rabbit pAb (Sigma). In this last case, to distinguish myofiber nuclei from interstitial ones, sections were doubly labelled with anti‐α sarcoglycan mouse mAb (Monosan) and nuclei counterstained with DAPI, as previously described.2.6.2Consecutive transverse cryosections from ambulatory and 7 days tail‐suspended rats, transfected or co‐transfected with melusin cDNA or EV, and DN‐kinase constructs, or pTGrp94 or pT, or infected with AAV‐MEL or AAV‐EV, were processed for indirect immunoperoxidase using antibodies specific for c‐myc and HA tags, or EGFP and GFP proteins (Invitrogen) as primary antibodies. Bound antibodies were revealed as previously described .2.Myofiber size was evaluated by measuring CSA. In some cases, measurements were repeated using minimal Feret's diameter to exclude overestimation secondary to sample misorientation.2.6.3Histochemistry for NADPH‐diaphorase to reveal distribution of active nNOS molecules was performed on paraformaldehyde‐fixed cryosections following the previously described protocol.2.6.4Demonstration of myofiber carbonylation by formation of DNPH adducts was performed on freshly cut cryosections as previously described.Slides were observed with a Leitz Axioplan optic microscope. All morphometric analyses were performed on micrographs, obtained the same day from cryosections of different samples stained on the same glass slide together with sections from a control sample. All measurements were performed independently by two investigators using ImageJ software.2.7in vitro as previously described,2, 3.15 mM MgCl2, 1.3 mM NaH2PO4, 25 mM NaHCO3, 11 mM glucose, 30 μM d‐tubocurarine, and pH 7.2–7.4, 30 °C, bubbled with 95% O2–5% CO2. Muscles were stretched to the optimal length  and electrically stimulated, by two parallel electrodes, with supramaximal pulses (0.5 ms duration) delivered by a Grass S44 electronic stimulator through a stimulus isolation unit (Grass SIU5). Muscle response was recorded through an isometric force transducer (Harvard) connected to an AT‐MIO 16AD acquisition card , and data were analysed by a specific module of the National Instruments LabVIEW software. Twitch and tetanic tensions were normalized to the muscle wet weight . Tetanic stimulation was obtained by applying trains of supramaximal stimuli at 80 Hz frequency. Maximum rate of rise of tetanus was also measured (Vmax) (N/s).The experiments were performed 2.8Analysis of My heavy chain isoforms was performed by the SDS‐PAGE method previously described, which allows separation of slow from fast My isoforms.2.9The experimental unit corresponded to the ambulatory/unloaded muscle. Data were expressed as mean, median, and 5th–95th percentiles with outliers, when using box plots representation, and as mean + SEM, when using histograms.t‐test. Paired Student's t‐test analysis was used when comparing average CSA or minimal Feret's diameter values or percentage of FoxO3‐positive myonuclei between transfected and untransfected myofibers of the same muscles. One‐way repeated measure analysis of variance (ANOVA) or within‐subject ANOVA, and Tukey or Bonferroni test for post hoc analysis, respectively, were used for multiple comparison. In the case of within‐subject ANOVA, average values of transfected and untransfected myofibers were compared with values obtained from transfected muscles obtained from animals of the same body weight (matched participants). Human data were analysed using within‐subject ANOVA with time as repeated factor. Post hoc Bonferroni tests were performed to assess specific differences between times.Statistical analysis between two groups was performed utilizing Student's 0.05 was set as the limit for significance.Graphs were prepared using the SigmaPlot version 2.0 software .33.1Tableet al. Our experimental model of rat tail suspension induced a statistically significant decrease in soleus muscle mass normalized to body weight (MW/BW), which reached −28% and −40% after 7 and 15 days of unloading, respectively, as previously describedFigureFigureP < 0.0001 and <0.005, after SA and GAPDH normalization, respectively) and again at U15. Quantification of melusin transcripts by quantitative real time PCR showed a significant, transient decrease in mRNA accumulation in U4 muscles compared with ambulatory and U1 muscles and a significant increase compared with both ambulatory and U1 levels at U7 , as demonstrated by western blotting analyses and normalization with two different loading controls, such as SA and GAPDH . Such a result was confirmed by confocal microscopy studies   were 0.77 ± 0.11, 0.80 ± 0.13, and 0.77 ± 0.15, respectively. Infection with AAV‐MEL apparently involved every muscle fiber and significantly increased MW/BW , without apparently affecting the relative percentage of fast and slow fiber populations and My heavy chains  of U7 soleus muscle. In fact, Vmax of ambulatory soleus was 3.65 ± 0.29 N/s and significantly decreased to 1.43 ± 0.15 N/s (P < 0.001) in U7 and recovered to 2.56 ± 0.20 N/s (P < 0.001) in AAV‐MEL treated U7 muscles.Mechanical studies performed in unloaded muscles showed that infection with AAV‐MEL significantly ameliorated force development, because higher twitch and tetanic tensions were observed after normalization with MW . The possibility that DN‐kinase forms did not blunt exogenous melusin effects on myofiber CSA, because of increased kinase protein levels secondary to melusin expression, was excluded by comparing protein kinase total amounts among sham‐infected U7 muscles, and AAV‐MEL‐infected and AAV‐EV‐infected ones. No difference was detectable . It is likely that such a further reduction in sarcolemmal nNOS activity reflects the increase in myofiber cross‐sectional sarcolemma consequent to the pro‐trophic effect of exogenus melusin expression.We therefore investigated whether exogenous melusin expression counteracted the effects of other recognized pro‐atrophic drivers, such as the loss of nNOS sarcolemmal activity and localization, and atrogene activation by nuclear FoxO3 translocation FiguresA and 6B.Figuren = 5 muscles examined; more than 100 myonuclei investigated for muscle, P = 0.33, paired Student's t‐test).The possible interference of melusin transfection with the unloading‐induced activation of the FoxO3 transcription factor was addressed by measuring the percentage of FoxO3‐positive myonuclei relative to total myonuclei of melusin‐transfected myofibers and untransfected or EV transfected ones FigureB. ResultFiguresP ≤ 0.002, Supporting Information, FigureP ≤ 0.002, FigureFigureFigureWe then analysed whether and how exogenous melusin affected the atrogene expression FiguresC and 6D.Figuret‐test, P = 0.03).Western botting analyses were then performed to clarify whether the perturbation of FoxO3 transcript downregulation in unloaded AAV‐MEL muscles corresponded to increased protein levels FigureD. ResultFigureP = 0.01), that is, despite of the attenuation of muscle atrophy consequent to each treatment . On the other hand, mitochondrial oxidative stress induced by a 6 h unloading bout did not appear to be responsible for the early decrease in melusin protein amount  and untreated muscles (n = 4), respectively, Student's t‐test, P = 0.41).Because we demonstrated that unloading‐induced activation of the nNOS/FoxO3 pathway requires a mitochondrial‐derived increase in oxidative stress,t FigureD. In fac4This study demonstrates that (i) melusin is early involved in the unloading‐induced loss of muscle mass and force and (ii) maintenance of the protein at physiological levels is sufficient to partially counteract disuse muscle atrophy. Indeed, here we show that unloading‐induced muscle atrophy results from the early deregulation of at least two distinct, independent pathways, that is, one leading to melusin loss and the other raising mitochondrial oxidative stress and nNOS subcellular redistribution. Only interventions aimed to prevent both of them fully counteracted the development of muscle disuse atrophy.et al.Our work on unloaded, yet not atrophic, soleus muscle showed that mitochondrial‐derived oxidative stress represents a required signal driving the myoplasmic redistribution of active nNOS sarcolemmal molecules, which are determinant for FoxO3 nuclear traslocation.Here, we identify melusin, the muscle‐specific integrin‐associated protein, as a novel relevant regulator of skeletal muscle mass and force. At variance with nNOS and Grp94, melusin protein levels appear already significantly reduced after a 6 h unloading bout. This event occurs independently from mitochondrial‐derived oxidative stress and appears to be post‐transcriptionally regulated, because downregulation of melusin gene transcription becomes detectable later on. We also show here that the unloading‐induced decrease in melusin protein levels is changed neither after inhibition of mitochondrial derived oxidative stress with MitoTEMPO, nor of nNOS activity by means of 7‐NI, nor after Grp94 upregulation by curcumin administration. Because all these treatments were shown to blunt FoxO3 nuclear translocation/activity,Furthermore, melusin, but not β1D integrin, is lost from costameres, the macromolecular complexes aligned between the sarcolemma and underlying sarcomeric myofibrils coincident with Z‐discs.Indeed, prevention of melusin loss by means of exogenous melusin expression had a beneficial effect in the rat model of muscle disuse/unloading, on both the degree of myofiber atrophy and force development. The pro‐trophic role of melusin has been extensively investigated for cardiac myocytes, where melusin absence accelerates eccentric left ventricle remodelling and transition toward heart failure, whereas melusin overexpression leads to compensatory hypertrophy with increased pump function and protects from dilation.In addition to contractility, exogenous melusin attenuated the unloading‐induced loss in myofiber size. Melusin pro‐trophic effect occurred acting on ubiquitin‐ligase expression, that is, exogenous melusin expression blunted unloading‐induced Atrogin‐1 transcript accumulation and reduced, without silencing, MurRF‐1 one. Strikingly, such a downregulation occurred in the presence of deregulation of their major inductor FoxO3,Consistent with such a hypothesis is the evidence that melusin transfection combined with interventions aimed to block nNOS/FoxO3/Grp94 deregulation, such as treatment with the NOS inhibitor 7‐NI or co‐transfection with Grp94, fully counteracted myofiber atrophy in U7 soleus muscle, implying that both nNOS/Grp94 and melusin are required, because they are independently involved in the maintenance of myofiber size challenged by muscle unloading/disuse.The possibility that the full pro‐trophic effect resulted from downstream complementation between the two pathways has to be considered too. Increased Grp94 levels may enhance folding and maturation of IGFI and II,2Na‐K‐ATPase pump.Although both nNOS and melusin are costamere proteins, unloading‐induced, mitochondrial‐derived oxidative stress initiates nNOS redistribution in the sarcoplasm,In conclusion, this study shows that disuse muscle atrophy results from the deregulation of at least two master switches, melusin and nNOS, only the latter being oxidative stress dependent. In both humans and experimental laboratory animals, the loss of active nNOS molecules from sarcolemma and the decreased melusin amount represent very early events in the development of disuse muscle atrophy. In spite of the desire to uncover a single master regulatory switch, here we demonstrate that unloading‐induced muscle atrophy can be abolished only by addressing simultaneously melusin and nNOS deregulations.M.V., M.S., D.D.B., M.B., G.T., and L.G. conceived the experiments and analysed the data; M.V., E.P., M.S., E.T., E.G. carried out experiments. All the authors, except G.T., were involved in writing the paper and had final approval of the submitted version.All authors declare that they have no conflict of interest.Figure S1. A) Representative Western blot of whole homogenates from the midbelly region of ambulatory soleus muscle (A) and unloaded (U) ones for 4 days (left panels) and 7 days (right panels), stained with anti‐melusin (mel) antibodies. Red Ponceau staining of serum albumin (SA) and Western blot for GAPDH are shown as loading reference. B) Box plots with mean (dotted) and median (solid) values of normalized melusin protein levels to GAPDH. n indicates the number of animals examined. Single asterisk indicates significant difference to A values; double asterisk indicates significant difference to A and U6h. (P < 0.005 ANOVA).Click here for additional data file.Figure S2. Transverse consecutive cryosections from ambulatory rat soleus muscle stained for immunoperoxidase with anti‐melusin mAb (mel) and anti‐slow myosin (slow‐My). Labelling for melusin is detectable in every myofiber with a slight heterogeneity, involving mostly fast myofibers (thin arrow) in addition to slow myofibers (thick arrow). Bar: 50 μmClick here for additional data file.Figure S3. A) Histograms showing mean and SEM of the percentage of fast fibers in sham‐infected ambulatory (A) and 7‐day unloaded (U) muscles and in AAV‐infected 7‐day U muscles with melusin (U MEL) or empty virus (U EV). N indicates the number of muscles examined. More than 200 fibers were evaluated for muscle. ANOVA P=ns B) Upper panel shows the Coomassie blue staining of a representative gel electrophoresis showing separation of myosin heavy chains (My). Slow My migrates faster than fast My. Lower panels show histograms of mean and SD values of the relative percentage of fast My densitometric values on total ones. ANOVA P = nsClick here for additional data file.Figure S4. A) Representative Western blots of different whole homogenates from 7‐days unloaded soleus muscles after infection with AAV (U7 + AAV) expressing melusin (MEL) or empty vector (EV) labelled for total Akt and ERK1/2. Parallel staining with anti‐GAPDH antibodies and Red Ponceau staining of serum albumin (SA) is shown as loading reference. B) Left and right panels illustrate histograms of mean and SEM values of normalized total Akt protein levels with SA and GAPDH, respectively. n indicates the number of examined muscles. ANOVA P=ns C) Left and right panels illustrate histograms of mean and SEM values of normalized total ERK1/2 protein levels with SA and GAPDH, respectively. n indicates the number of examined muscles. ANOVA P=nsClick here for additional data file.< 0.05, **p < 0.01; ***p < 0.001).Figure S5. Dot plots showing normalized values of four atrogene transcript amount detected in ambulatory (A) soleus muscle and after 1, 4 and 7 days of unloading (U). FoxO1 , Atrogin  and MufF1  transcript were all significantly upregulated at U7. Bars in graphs represent standard errors and asterisks indicate the presence of significant difference (*p Click here for additional data file.Table S1. Body and soleus muscle weights of ambulatory and tail‐suspended ratsClick here for additional data file.Table S2. Primer sets used for qPCRClick here for additional data file.


Fasting plasma glucose (FPG) variability is a significant predictor of mortality, especially in patients with poor glycemic control. This study aimed to explore the temporal age- and sex-specific profiles of temporal FPG variability in a Chinese population undergoing routine health screening and to guide the development of targeted public health interventions for the prevention and control of diabetes.In this cross-sectional study, we used a general linear model to compare differences in temporal FPG values between sexes and across age groups in 101,886 Nanjing residents who underwent a routine physical health examination at the Health Management Center, the First Affiliated Hospital of Nanjing Medical University, in 2018. The variability of FPG as a function of time, age, and sex, independently and in combination, was analyzed.P < 0.05), with an excellent fitting effect (Eta-squared =0.15). The variability of FPG showed sex differences in the percentage difference of the coefficient of variation, which was 34.1% higher in males than females. There were significant interaction effects for month*age*sex and day*age*sex.The participants included 57,455 (56.4%) males and 44,431 (43.6%) females, with a mean ± SD age of 42.8 ± 15.0 years. The average ± SD FPG level was 5.5 ± 1.1 mmol/L. The monthly variation contributed to 22% of the overall FPG variability. A significant main effect for the age group was observed (F = 7.39, Temporal variability in FPG is evident in the general Chinese population and is affected by both age and sex. To avoid complications associated with FPG variability, interventions should be directed at females and males at specific ages for optimal control of FPG variability and to reduce the risk of diabetes and cardiovascular events.The online version contains supplementary material available at 10.1186/s12889-021-10367-x. Human health outcomes are unequally distributed throughout populations . Due to A fasting plasma glucose (FPG) test is usually used in the diagnosis of diabetes. Plasma glucose levels are controlled by the release of insulin to ensure a dynamic equilibrium (homeostasis). Glycemic or glucose variability makes it difficult to achieve good glycemic control. Generally, blood sugar levels between 70 mg/dL 3.9 mmol/l) and 100 mg/dL l 5.6 mmol/) are considered normal. It is evident that glucose variability (GV) plays a role in the development of diabetic complications, particularly cardiovascular events , 5. FPG .6 mmol/  mmol/l aTime-dependent FPG variation, as represented by the coefficient of variation (CV), could predict mortality in subsequent all-cause, expanded or nonexpanded cardiovascular disease-related mortality independent of mean FPG, renal function, mean glycated hemoglobin A1C (HbA1C), HbA1C variation, and other risk factors in patients with type 2 diabetes, suggesting that GV may be a valuable clinical biomarker in the management of patients , 14. DayTo modify risk factors related to impaired glycemic control and diabetes, there is a need to identify populations at risk of developing impaired fasting glucose (IFG) and diabetes and to find measures to curb the progression. An uncontrolled and progressive increase in FPG can predict impending IFG and diabetes. In the current study, we explored the variability of FPG among a general population in eastern China to reveal the blood glucose distribution by age and sex for use in a targeted public health intervention.This cross-sectional study involved 101,886 Nanjing residents who took part in a routine health screening at the Health Management Center of the First Affiliated Hospital of Nanjing Medical University in 2018. The participants took part in the routine physical examination once a year. A simple questionnaire was used to collect demographic characteristics and behavior factors. Abdominal venous blood was collected by vacuum anticoagulant tube or nonanticoagulant tube according to the physical examination items. Blood samples were stored at a suitable temperature and sent to the laboratory for testing within an hour. The FPG test was performed using a glucometer after an overnight or at least 8 to 12 h fast in all participants. We extracted demographic characteristics, behavioral factors, body mass index (BMI), blood glucose values, and blood pressure of the subjects from the database. To avoid a potential source of selection bias, we included all people (diagnosed with or treated for diabetes) attending the routine health screening program for the whole year.A fasting blood sugar level between 70 mg/dL (3.9 mmol/l) and 100 mg/dL (5.6 mmol/l) is considered normal, from 100 to 125 mg/dL (5.6 to 6.9 mmol/L) is considered prediabetes or IFG, and over 126 mg/dL (7 mmol/L) is considered diabetes [A univariate generalized linear model (GLM) approach was used for regression analysis and analysis of variance for FPG and FPG categories . We tested null hypotheses about the effects of various age groups and different sexes on the FPG category and investigated interactions between temporal factors and personal effects. Tukey’s test was applied for post hoc analysis if statistical significance was observed for the F test to evaluate differences among specific means. Estimated marginal means gave estimates of the predicted mean values for the cells in the model, and profile plots were used to visualize the interaction of these means. The process of model construction and analysis is described in Supplementary file P-value < 0.05 was considered statistically significant.Data are given as the mean and standard deviation (SD), and cross-tabulation of FPG was estimated by age, sex, height, weight, SBP, DBP, and BMI. The observed trends of FPG over days, months, and seasons were explored for interindividual variability with respect to age and sex. All analyses were performed with SPSS 25 , and a Variability measures, such as variance, SD, and the coefficient of variation, have different strengths and applications. Although SD is generally preferred over variance because of its straightforward interpretation, the coefficient of variation has the power to compare data from different populations .This study was approved by the ethics committee of Nanjing Medical University. The data used in this study were anonymized and irreversibly deidentified to protect participants, health care professionals, and hospital privacy.The participants included 57,455 (56.4%) males and 44,431 (43.6%) females. Their mean ± SD age was 42.8 ± 15 years (range 12–100 years). The overall mean ± SD FPG was 5.59 ± 1.1 mmol/L, with an interquartile range of 5.0–5.7 mmol/L. The overall CV of FPG was 0.2, which implied that the SD was 20% of its mean value , with an interquartile range of 5.0–5.7 mmol/L. The seasonal FPG variation also showed a mean value of 5.2 ± 1.23 mmol/L (range 4.3–6.7), with an interquartile range of 4.8–5.5 mmol/L. FPG values for males and females were 5.7 ± 1.3 mmol/L and 5.3 ± 0.9 mmol/L, respectively. Overall, the monthly variation contributed 22% of the overall FPG variability by sex and age group  =1571.5, P < 0.001). A Tukey honest significant difference (HSD) test revealed that all nine means were significantly different from each other, although there was a slight deviation from this pattern for males aged 70–79 years and a sharp deviation for those older than 90 years in both sexes. The sex*sex mixed model revealed that the main effect of sex was significant  =83.461, P < 0.05). Thus, there was an overall significant difference in FPG means between males and females. A significant main effect for age group was obtained  =7.39, P < 0.05), with a good fitting effect (Eta-squared =0.15). FPG variability was higher in males than in females. The variability of FPG showed sex differences in the CV percentage difference (PD): PD =100/CVfemale, specifically the sex variability ratio = 34.1% more in males than females  and day*age*sex  =1.45, P < 0.05). However, there were no significant interaction effects for season*age*sex  =1.50, P = 0.10) and half-year*age*sex  =1.41, P = 0.19). Thus, the month and day were significantly attributed to the variability in FPG, whereas season and half-year FPG were not.We observed significant interactions for month*age*sex  =1.89, P = 0.37).The amplitude percent mean (A % M = (highest FPG − lowest FPG)/(mean FPG) ∗ 100) for males and females was 24.7 and 24.5%, respectively. The SD percent mean, /(mean FPG) ∗ 100), was higher in males (22.8%) than in females (17.0%). Examination of the multiple comparisons by age group and month was significant from January to December, except for in September . Women aged 30–39 years and men aged 40–49 years had an increased risk of type 2 diabetes  and the United Kingdom Prospective Diabetes Study (UKPDS), it should be without frequent, prolonged, or severe hypoglycemia.A follow-up of blood glucose distribution characteristics in a health examination population in Chengdu, China, from 2010 to 2016 showed higher glucose levels with increasing age, and males had higher glucose levels than females [The Taichung Diabetes Study measured glycemic variability by computing the annual CV of all FPG measurements in a given year and showed that the annual CV of FPG was independently associated with all-cause mortality in patients with type 2 diabetes aged 30 years and over. Participants classified based on BMI category showed that as BMI increased, FPG increased according. Patients with type 2 diabetes are generally associated with inactivity and overweight . The VerA better understanding of these differences among the population is critical for implementing effective preventive measures. Participants classified based on FPG category revealed that the risks of IFG or prediabetes and diabetes were associated with maximal swings in the SD and CV. The most straightforward and easy way to measure interday variability is to calculate the SD of fasting blood glucose concentrations . SiegelaThe population density is relatively high in eastern China, and knowing the demographic transition within and across age groups and sex is key for developing public health interventions to improve overall health. To control FPG and avoid complications associated with FPG variability, such as hypoglycemia, there is a need for treatment strategies to control fluctuations in FPG variability to reduce the risk of diabetes and cardiovascular events.This study has several limitations. First, the variability of FPG may be underestimated due to the short length of the study. Second, this study examined age- and sex-specific profile of FPG over time, but there could be other factors that might be attributed to FPG variability. Third, models other than GLM could be used in future studies. Thus, generalizing the study results should be done cautiously and in consideration of the characteristics of different populations.Temporal variability in FPG is evident in the general Chinese population and is affected by both age and sex. Interventions for optimal control of FPG variability should be directed at females and males aged 30–39 years and 40–49 years, respectively. Additionally, people should be encouraged to perform self-monitoring of fasting blood glucose and report unusual changes in blood sugar patterns for early treatment and to avoid the risk of diabetes and cardiovascular diseases.Additional file 1. Supplementary file 1. Description of statistical modeling.


Osimertinib is recommended for T790M mutation‐positive advanced non‐small cell lung cancer (NSCLC) resistant to first‐ and second‐generation epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitors (TKIs). Recently, some reports exist on the real‐world use of osimertinib; however, reports involving third/later‐line use are few. Hence, this study was conducted to evaluate the efficacy and safety of osimertinib used in various treatment lines for T790M‐positive NSCLC patients.This retrospective, observational, multicenter study included T790M‐positive advanced/recurrent NSCLC patients treated with osimertinib from May 2016 to March 2018. The clinical characteristics, efficacy, and adverse events were retrospectively investigated. The Kaplan‐Meier method was used to analyze progression‐free survival (PFS) and overall survival (OS). PFS‐associated clinical characteristics were evaluated using the Cox proportional hazards model.P = 0.327). Analysis using the Cox proportional hazards model for clinical features affecting PFS also revealed no significant factors. Adverse events of grade ≥ 3 were reported in 15 patients (26.8%); the most common were anemia (n = 3) and cutaneous toxicity (n = 3). Grade 4 neutropenia was observed in one patient; any‐grade pneumonitis was observed in six patients (10.7%), including one with grade 3 pneumonitis.The objective response rate (ORR) and disease control rate (DCR) were 60.7% and 91.1%, respectively; the median PFS was 11.0 months. There were no significant differences in the median PFS for patients treated with osimertinib as second‐line and third−/later‐line (14.5 vs. 11.0 months respectively, Osimertinib demonstrated efficacy even when administered as third−/later‐line treatment to NSCLC patients. Osimertinib‐related pneumonitis was observed more frequently than previously reported.Osimertinib shows efficacy even as later‐line treatment in T790M mutation‐positive NSCLC patients previously treated with EGFR‐TKIs. However, the incidence of ≥ grade 3 adverse events, especially pneumonitis, was higher than that previously reported by other studies.Osimertinib was approved for previously EGFR‐TKI‐treated EGFR T790M‐positive NSCLC. With the increasing frequency of its use as first‐line treatment, this study provides valuable evidence for the efficacy and safety of osimertinib for previously EGFR‐TKI‐treated NSCLC. EGFR mutation‐positive, advanced NSCLC.EGFR mutations respond to first‐ or second‐generation EGFR‐TKIs, such as gefitinib, erlotinib, and afatinib.EGFR mutant.EGFR T790M mutation, osimertinib treatment is recommended as second‐line treatment for T790 mutation‐positive patients. More recently, in a phase III trial (FLAURA),The 2018 National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines for Non‐Small Cell Lung Cancer (NSCLC) recommend epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitors (TKIs) as first‐line treatment for unresectable, EGFR mutations at diagnosis, testing method to confirm T790M mutations before osimertinib treatment, number of previous treatments before osimertinib, initial dose of osimertinib, subsequent dose reduction and discontinuation, the duration of administration, efficacy data of osimertinib, and adverse events.A total of 56 patients with EGFR T790M‐positive advanced or recurrent NSCLC (stage IIIB or IV), who had received osimertinib as second−/later‐line treatment in one of four hospitals belonging to the Tochigi‐kitakan Thoracic Oncology Research Organization (TOTORO) from 1 May 2016 to 31 March 2018, were included in this study. The following characteristics of the 56 patients were collected from medical reports obtained from the hospitals: demographic information, smoking status, renal function, Eastern Cooperative Oncology Group performance status (ECOG‐PS), tumor histology, clinical stage, the presence/absence of central nervous system (CNS) metastasis, Objective response rate (ORR) and disease control rate (DCR) were assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 to examine the clinical efficacy of osimertinib.Osimertinib‐related adverse events were also evaluated according to the Common Terminology Criteria for Adverse Events version 4.0 (CTCAE v4.0).This retrospective study was approved by the Institutional Review Board of the Tochigi Cancer Center (No. A‐458). Owing to the retrospective nature of the study, the requirement for informed consent was waived according to the Japanese ethical guidelines for clinical research.All statistical analyses were performed with EZR , which is a graphical user interface for R ; moreover, it is a modified version of R Commander designed to add statistical functions that are frequently used in biostatistics.EGFR mutations at initial diagnosis were as follows: exon 19 deletion‐positive  and L858R point mutation‐positive . Two of the L858R point mutation‐positive patients were de novo T790M mutation‐positive. The following methods were used to detect the T790M mutation prior to osimertinib treatment: rebiopsy specimens from diseased tissue , cytological specimens such as pleural effusion , plasma specimens , and both tissue and plasma specimens (n = 2).In total, 56 patients who received osimertinib treatment were registered at the four hospitals. Patient demographics are listed in Table n = 38, 67.9%), erlotinib , and afatinib . A total of 23 patients (41.1%) were treated with two or more EGFR‐TKIs, and three patients (5.4%) were rechallenged with the same type of TKI. Moreover, 35 patients (62.5%) received treatment with cytotoxic agents. A total of 33 patients (58.9%) received platinum combination therapy, and one patient (1.8%) received antiprogrammed cell death protein‐1 (PD‐1) antibody. The osimertinib treatment line was as follows: second‐line , third‐line , and fourth−/later‐line . The median number of previous regimens was two . Osimertinib (80 mg/day) was orally administered to all patients.The treatments administered before and after osimertinib are shown in Table n = 2, 3.6%), partial response , stable disease , progressive disease , and not evaluable . ORR was 60.7% , and DCR was 91.1% (95% CI 80.4–97.0). The median PFS was 11.0 months (95% CI 7.6–13.8), and the median OS was 24.1 months  (Table P = 0.327) between the groups of patients classified by treatment line   . At the time of final analysis, 17 patients 30.4%) were still receiving osimertinib. A total of 24 patients (42.9%) died during the follow‐up period; of these, except for one death caused by another disease, all other patients died of the primary disease. The median duration of osimertinib treatment was 10.8 months . The best outcomes determined according to the RECIST guideline were as follows: complete response  received some treatment after osimertinib (Table n = 3), cutaneous toxicity (n = 3), stomatitis (n = 2), leukopenia (n = 2), neutropenia (n = 2), and aspartate aminotransferase/alanine aminotransferase elevation (n = 2). A total of 20 patients (35.7%) underwent dose reduction or a transient washout due to adverse events, and 10 patients (17.9%) discontinued treatment due to adverse events. Any‐grade pneumonitis was observed in six patients (10.7%), including one with grade 3 pneumonitis, and osimertinib treatment was discontinued in all patients. There was no treatment‐related death. Multivariate logistic regression analysis was performed to assess the factors associated with severe adverse events. The incidence tended to be high in patients aged ≥ 75 years; however, no statistically significant differences were observed  reported at least one adverse event, and 15 patients 26.8%) reported all‐causality ≥ grade 3 adverse events. Grade 4 neutropenia was observed in one patient (Table .8% reporEGFR mutation‐positive advanced NSCLC.EGFR‐TKIs are the standard treatment for n = 19) and the third−/later‐line group (n = 37), including 22 patients (39.3%) who received osimertinib as fourth−/later‐line treatment. Additionally, in this study, two cases of complete response (CR) were observed: one patient received osimertinib as second‐line treatment and the other as third‐line. These results suggest that osimertinib shows clinical efficacy even when administered to patients as a subsequent treatment.In this retrospective study, ORR and median PFS were 60.7% and 11.0 months, respectively. This study included nine patients with PS 2/3 and many elderly patients; moreover, although osimertinib was administered as second‐line treatment to only 19 patients (33.9%), its efficacy was similar to that observed in the AURA3 trial (ORR 71% and median PFS 10.1 months).P = 0.096); no association with other significant factors was observed. Moreover, in this population, six patients (10.7%) had any‐grade pneumonitis, including one patient with grade 3 pneumonitis, which was a higher frequency than that observed in previous studies.A total of 15 patients (26.8%) had ≥ grade 3 adverse events, most of which were hematologic toxicity . Although the adverse event profile was similar to that seen in previous studies, the incidence was more frequent.Recently, several similar retrospective studies of osimertinib in a real world data for treated NSCLC have been reported.However, toxicity such as pneumonitis was observed more frequently, necessitating the earlier discontinuation of osimertinib treatment.There are few reports of clinical data on such late‐line osimertinib treatment, making the design of prospective studies considering treatment sequence and long‐term prognosis difficult. Therefore, the investigation of the efficacy and safety of osimertinib as a late‐line treatment for previously treated EGFR‐TKI‐treated NSCLC patients depends on retrospective data. To the best of our knowledge, our study provides invaluable evidence for the efficacy and safety of osimertinib in the real world. However, the small sample size and short observation period are limitations of this study. Hence, a higher number of cases and long‐term data follow‐up are desirable. In conclusion, our study shows that osimertinib may improve PFS and OS even in a population that includes many patients who received osimertinib as a third−/later‐line treatment. This real‐world evidence may help answer the clinical question of the optimal treatment sequence of osimertinib.The authors declare they have no conflict of interest.