PHOX2B are responsible for Congenital Central Hypoventilation Syndrome, a neurological disorder characterized by inadequate respiratory response to hypercapnia and life-threatening hypoventilation during sleep. Although no cure is currently available, it was suggested that a potent progestin drug provides partial recovery of chemoreflex response. Previous in vitro data show a direct molecular link between progestins and PHOX2B expression. However, the mechanism through which these drugs ameliorate breathing in vivo remains unknown. Here, we investigated the effects of chronic administration of the potent progestin drug Etonogestrel (ETO) on respiratory function and transcriptional activity in adult female rats. We assessed respiratory function with whole-body plethysmography and measured genomic changes in brain regions important for respiratory control. Our results show that ETO reduced metabolic activity, leading to an enhanced chemoreflex response and concurrent increased breathing cycle variability at rest. Furthermore, ETO-treated brains showed reduced mRNA and protein expression of PHOX2B and its target genes selectively in the dorsal vagal complex, while other areas were unaffected. Histological analysis suggests that changes occurred in the solitary tract nucleus (NTS). Thus, we propose that the NTS, rich in both progesterone receptors and PHOX2B, is a good candidate for ETO-induced respiratory modulation.Heterozygous mutations of the transcription factor  Congenital central hypoventilation syndrome (CCHS) is a rare complex condition, characterized by impaired chemosensitivity and autonomic disfunction, which is caused by the genetic mutation of the transcription factor paired-like homeobox 2b (PHOX2B) ,2,3,4. PA clinical feature of CCHS is sleep-related hypoventilation, with severe cases also displaying hypoventilation during wakefulness ,3,4, whi2 [2 chemosensitivity with the use of a potent progestin contraceptive, desogestrel [Respiratory stimulants commonly used in other respiratory disorders have proven ineffective in CCHS patients, and the only current options for maintaining ventilation  during sleep are mechanical ventilation or diaphragm pacing ,17,18. I2  and partogestrel .Progesterone (PG) has been proposed to act as a respiratory stimulant ,22, althPrior in vivo and in vitro studies in perinatal rodents suggest that desogestrel and its active metabolite, etonogestrel  may have acute stimulatory effects on respiratory frequency in vivo and on the response to metabolic acidosis in vitro , possiblInterestingly, both ETO and PG administration in neuroblastoma cell lines, expressing both PHOX2B and nuclear PGR, induced a direct reduction in PHOX2B expression, as well as reduced promoter activity of PHOX2B target genes . In vitr2 chemosensitivity with desogestrel in CCHS patients and the in vitro stimulatory ETO effect in perinatal rodents prompted us to investigate the effects of long-term systemic ETO administration in healthy adult female rats. We hypothesized that ETO would stimulate breathing during ventilatory challenges and based on cell culture studies, would induce changes in gene and protein targets expression that are dependent on PGR activation in the brain.The partial recovery of CO® rods  to continuously deliver ETO over a four-week period. At the end of the treatment period, brain regions of interest were isolated, and protein and gene expression was evaluated. Our results indicate that while long-term ETO administration does not alter the absolute magnitude of chemoreflex responses, it results in a mismatch between ventilation and metabolism, such that CO2-responsiveness is enhanced. We also noted increased variability in the breath cycle period of ETO-treated rats, pointing to additional changes to respiratory control. Furthermore, while expression of genes and proteins of interest was not affected by ETO treatment in the hypothalamus, locus coeruleus and the parafacial region (including the RTN), we observed a reduction in gene and protein expression of PHOX2B, PHOX2A and known PHOX2B target genes in the dorsal vagal complex (DVC). Histological analysis verified that changes in PHOX2B expression occurred specifically in the nucleus of the solitary tract.In this study, we tested baseline breathing and the chemoreflex response, following instrumentation of female rats with slices of Nexplanon® treatment results in ~200–250 pg/mL ETO in the serum for a 3-year period [® rods in pilot studies and determined ETO serum levels 4 weeks after instrumentation. We found that 1000 μm-thick Nexplanon® rods resulted in 236.8 ± 50.0 pg/mL ETO (n = 5), while thinner rods produced serum levels below our target . Thus, 1000 μm Nexplanon® rod lengths were used for the remainder of the study.In women, Nexplanonr period ,41. In oSince ETO administration in neuroblastoma cell lines affects the PHOX2B pathway through PGR activation , we invePgr mRNA expression between the control and ETO-treated groups were observed in any of the areas investigated (Phox2b mRNA showed that ETO treatment reduced Phox2b expression by 16.81 ± 0.14% selectively in the DVC (t(15) = 2.923, p = 0.01, n = 9; Phox2b in either group, as reported previously [To evaluate changes in gene expression, qPCR analysis was performed on mRNA isolated from these brain areas. No changes in stigated A. Quanti(6) = 3.464, p = 0.013, n = 3; PHOX2B mRNA levels were still reduced, but less affected [Western blot and densitometry analysis were also performed on the same tissue samples and confirmed significantly reduced PHOX2B protein levels in the DVC  and PHOX2B protein expression with immunofluorescence staining (n = 5 rats per group). Representative images of the staining in the three areas of the DVC are shown in  = 0.939, p = 0.4; −positive X motoneurons  = 0.994, p = 0.46; Phox2b mRNA did not show significant reduction in the NTS neurons  = 1.851 p = 0.09;  = 4.842, p= 0.0003, To further verify this reduction, at both mRNA and protein levels, and to investigate spatial localization within the DVC, we performed a semi-quantitative analysis of These results suggest that ETO treatment resulted in down-regulation of the PHOX2B protein in the DVC, particularly in the NTS region.+/Nmb+Phox2b RTN neurons used for the analysis are shown in +/Nmb+Phox2b cells were observed, both at the mRNA and protein levels, between SHAM- and ETO-treated rats  = 0.983, p = 0.48; Gpr4 and TASK2, two key pH sensors in RTN chemoreception, did not show significant changes between SHAM and ETO groups (Gpr4 t(20) = 1.495, p = 0.1505, n = 10; TASK2: t(20) = 0.5942, p = 0.5591, n = 10). These results suggest that ETO does not affect the expression of PHOX2B, and possibly neuronal activity through gene transcription of its target genes within the pF area.We further investigated whether ETO treatment altered the expression of PHOX2B in RTN neurons of healthy female rats . RepresePhox2a, Tyrosine hydroxylase (Th) and dopamine beta hydroxylase (Dbh) [Phox2b, a significant reduction in Phox2a  = 2.577, p = 0.02, n = 9), Th  = 2.253, p = 0.040, n = 9) and Dbh  = 2.541 p = 0.022, n = 9) mRNA levels were observed in ETO-treated rats in comparison to SHAM rats at the level of the DVC  ,45,46,47 the DVC A. Of not(4) = 15.53, p = 0.0001 n = 3; The reduction in TH expression was also confirmed at the protein level (30.45 ± 0.02% tn = 9 rats per group), there was no effect of surgery or ETO-treatment on the tidal volume (TV), respiratory frequency (f), or allometric minute ventilation (E ALLO) during room air recordings  and f  in pre-surgical recordings of ETO rats (ETO_Day 0). This led to an increased E ALLO of 127 ± 38% in 5% CO2 and 257 ± 54% in 7% CO2, as compared to restful breathing . No significant differences in TV, f or E were observed between SHAM and ETO-treated rats  = 0.788, p = 0.39; f: F = 1.38, p = 0.26; E ALLO: F = 2.17, p = 0.16).Hypercapnia produced dose-dependent increases in TV , f  and E ALLO  in ETO rat pre-surgical recordings  = 0.36, p = 0.56; f: F = 0.57, p = 0.46; E ALLO: F = 1.37, p = 0.26). The ventilatory pattern response to hypoxia was altered in both SHAM and ETO groups 28-days post-surgery: hypoxia-induced increases in TV were blunted  while f response was enhanced . However, E ALLO was unaffected .Hypoxia increased steady state Ts Day 0, A. Similap = 0.008), while resting body temperature was unchanged . Etonogestrel treatment led to significantly more weight gain as compared to SHAM rats (2) was reduced in both treatment groups 28-days post-surgery  = 11.96, p = 0.003) and this effect was more important in the ETO-treated group compared to SHAM rats  = 5.64, p = 0.03; 2 was increased following 28 days of ETO treatment  baseline  and was different from post-surgical SHAM . The absolute change vs. room air for 2 was not affected by hypercapnia exposure in either treatment group. As expected, both 2 and 2 of ETO- and SHAM-treated rats increased during 5 and 7% CO2 exposure . Interestingly, ETO treatment significantly increased 2 during 7% CO2, as compared to both pre-surgery  and SHAM rats . The increased 2 during 7% CO2 observed for ETO-treated rats may be ascribed to the significantly decreased room air 2 for this group  and slightly increased minute ventilation. These data suggest that ETO treatment enhances the hyperventilatory response to high CO2.In terms of chemoreflex responses, the absolute change in hypercapnia vs. room air for exposure D,F. The is group E, combin2 or 2, as compared to baseline; however, 2 and 2 were increased relative to room air, indicating expected increases in ventilation. No significant differences in metabolic measurements were observed between ETO-treated rats and SHAM controls in hypoxia  = 1.32, p = 0.27; 2: F = 1.41, p = 0.26).Hypoxia did not change 2, noted for room air and 7% CO2 conditions in response to ETO treatment may indicate enhanced excitability or instability in the underlying respiratory control networks [p = 0.004; vs. SHAM p = 0.028, Bonferroni) and Area . Interestingly, ETO treatment increased the convective requirement ratio during 7% CO2, suggesting a “mismatch” in respiratory control, whereby ventilatory effort was higher than predicted by metabolic rates [2 . While we cannot rule out changes in pulmonary or circulatory efficiency, the fact that ETO treatment enhanced responsiveness to high CO2, and not hypoxia, suggests changes specific to neural pathways governing the hypercapnic chemoreflex. Given that enhanced CO2 chemoreflex is associated with respiratory instability (such as periodic breathing [2 (PCO2) plays a role in restful breathing [2 sensing and integration.In contrast to perinatal data, we failed to observe any change in ew-borns , differeew-borns ,64,65, oew-borns ,66. Howeew-borns  and our ic rates . Enhanceic rates . Based oreathing ,68) and reathing , our datPhox2b and Phox2a, as shown here, or through yet-unidentified pathways. Since the functional and transcriptional role of PHOX2B on cellular function in these NTS neurons is unknown, it will be an important step forward to understand what role PHOX2B may have on the respiratory relay network, beyond its key role in the development of the autonomic nervous system and the NTS. Interestingly, recent work shows that PHOX2B neurons in the NTS are chemosensitive and partial lesioning of PHOX2B NTS neurons reduces the chemoreflex response [Our molecular results in the NTS are particularly interesting, as this area has an important role in relaying multiple autonomic functions and integration of chemosensory stimuli ,12, someresponse ,72, whilPHOX2B gene, resulting in aberrant protein production [It is well known that CCHS is caused by mutations in the oduction ,74,75,76oduction . Howeveroduction ,79. Givead libitum. All handling and experimental procedures were approved by the Health Science Animal Policy and Welfare Committee at the University of Alberta (AUP#461) and performed in accordance with guidelines established by the Canadian Council on Animal Care.Experiments were performed using female Sprague–Dawley rats  bred born to pregnant dams obtained from Charles River and housed at the University of Alberta Health Sciences Animal Housing Facility to . Animals were maintained on a 12-h dark/light cycle and received food and water Vaginal smears by lavage were obtained daily to monitor changes in vaginal cytology resulting from fluctuations in circulating hormones over the oestrous cycle. Smears were obtained using a plastic pipette filled with ~100 µL distilled water and examined under a light microscope . Proestrus was defined by the disappearance of leukocytes from the preceding day with a dominance of nucleated epithelial cells either appearing in clumps or with mucous, as described by . Only ra® rods  were implanted subcutaneously to deliver Etonogestrel (ETO). In an initial pilot study, 14 female rats (200–250 g) were implanted with either 250, 500 or 1000 μm long sections of Nexplanon® rods to determine the appropriate length to achieve comparable ETO serum levels as observed in women instrumented with Nexplanon® rods for contraception  and a 0.5 cm skin incision was made below the shoulder blades (using aseptic techniques) to permit implantation of the Nexplanon® rod . Bupivacaine  was administered post-surgically. Four weeks following implantation, rats were anesthetized with urethane  and ~2 to 2.5 mL blood samples were collected intracardially. After 30 min at room temperature, samples were centrifuged at 2000× g (15 min) and the serum stored at −20 °C. Serum ETO concentration was determined by liquid–liquid extraction methods performed by Q2 lab solution . Both serum and the internal standards underwent reverse phase high-performance liquid chromatography and tandem mass spectrometric detection  utilizing a turbo ion spray interface in the positive ion mode.Nexplanon00 pg/mL ,41). Ana2, and 10% O2 (8–15 min). Oxygen consumption and CO2 production were determined by recording the% composition of gasses flowing into, and out of, the recording chamber . Estrous testing conducted post-experimentally in ETO-treated rats verified a drug-induced cessation of oestrous cycling, resulting in maintenance of the dioestrus and/or proestrus stages. SHAM rats were recorded only in the proestrus stage to limit confounding effects of cycling sex hormones on breathing.Rats were habituated to the whole-body plethysmography chambers  on two occasions (~1.5 h) 3–4 days before baseline recordings. On the day of the experiment, rats were placed in the chamber and testing commenced once the animals were quiet but awake (30 min–1 h). Various gas mixtures were produced by a GSM−3  and delivered continuously at 1.5 L/min. Ventilatory reflexes were measured during exposure to 5% and 7% COV) and breathing frequency . The amplitude of the pressure signal was converted to TV (ml·kg−1) using the equations of [f = 50, 75, 100, 150 beats·min−1). Chamber temperature and humidity were continuously monitored using a RH-300 water vapour analyzer  plus HPR Plus Handheld PIT Tag reader  or digital thermometer/hygrometer sensor (Wifehelper), depending on the setup configuration. Rectal temperature was read using a Homeothermic Monitor with Probe . Minute ventilation (E) was calculated as f × TV and expressed as ml·min−1·kg−1, and allometrically corrected for weight gain according to [E ALLO. Metabolic parameters 2, 2, E/2 and E/2 were calculated by pull-mode indirect calorimetry [t-test (weight gain) or Mann–Whitney test  described by ,82. Brietions of  and calirding to  resultinorimetry . Brieflyorimetry ,85. Metaorimetry ,87. BrieRats were anesthetized with 3% isoflurane (nose cone) and blood samples collected intracardially and the serum stored at −80 °C until further use. Under isoflurane anaesthesia, rats were then decapitated, and the brain was quickly removed and placed on an ice-cold tissue chopper (Mc Ilwain Tissue Chopper). The brain was sliced into 1–2 mm sections and regions of interest were collected using a 2 mm diameter tissue punch according to gross anatomical coordinates: medial hypothalamus , locus coeruleus . The parafacial region (PF), including the retrotrapezoid nucleus and midline structures, was collected as the 1 mm ventral margin of the brainstem in a 1–1.5 mm slice caudal to the one used for LC. The dorsal vagal complex (DVC), including NTS, AP and X, was collected from the dorsomedial brainstem in a 2-mm thick section collected around the obex. Fresh resected tissues were immediately frozen in dry ice and stored at −80 °C for RNA and protein extraction.To confirm the anatomical boundaries and reduce the variability among samples, the leftover tissues were processed for thionin counterstaining. After dissection, tissue was fixed in PFA, cryoprotected in 30% sucrose and frozen in Tissue-Tek O.C.T compound at −80 °C, and sectioned on a cryostat . Fifty µm thick brain sections were washed in phosphate buffer saline , mounted sequentially on microscope slides, and dried overnight. Slides were then rehydrated in increasingly graded ethanol and reacted with thionin for 30 s, dehydrated, and coverslipped with CytoSeal 60 mounting medium (Electron Microscopy Sciences) and observed under Zeiss Primo Star binocular microscope.g). The RNA in the upper phase was collected and purified using the RNeasy Mini kit Qiagen), according to the manufacturer’s instructions.The total RNA and proteins were isolated from the same tissue simultaneously. Tissue samples were thawed, disrupted and homogenised in QIAzol Lysis Reagent (Qiagen). After the addition of chloroform, the homogenate was separated into aqueous and organic phases by centrifugation  buffer and stored at −80 °C; the total protein concentration was evaluated using BCA Protein Assay .Proteins in the lower organic phase were precipitated by adding 4 volumes of ice-cold acetone and incubated for 30 min on ice. After centrifugation at 1200× Phox2b and its target genes mRNA expression level, one µg of RNA was reverse transcribed using the GoScript™ Reverse Transcriptase kit  according to the manufacturer’s instruction manual, and the cDNAs of interest were then amplified and quantitatively analysed using a StepOnePlus™ Real-Time PCR System . The TaqMan® primer and probe assays  used in the study were the following: rat Phox2b (ID #Rn01413076_mH), rat Phox2a (ID #Rn00587898_m1), rat Pgr (ID #Rn01448227_m1), rat Th(ID #Rn00562500_m1), rat Dbh (ID #Rn00565819_m1) and the endogenous control rat Actb (ID #Rn00667869_m1). Each sample was run in duplicate, and the results were calculated using the 2−ΔCt and the 2−ΔΔCt methods to allow for normalization of each sample to Actb [t-test) using GraphPad Prism 5 Software . p values < 0.05 were considered significant.To study the effect of ETO on  to Actb . Results2O, saturated with blocking buffer  for at least 1 h and incubated over night at 4 °C with the appropriate antibody diluted in blocking buffer. The primary antibodies used in the study were: polyclonal rabbit anti-PHOX2B , monoclonal rabbit anti-beta Tubulin SR25-04  and polyclonal anti-TH . On the following day, the membrane was washed in blocking buffer (3 × 5 min), incubated with the secondary antibody, and diluted in blocking solution for 1 h. The excess antibody was then removed by multiple washes with blocking buffer; TS 1X buffer  + Tween®20 0.1%; three washes in TS 1X + Tween®20 0.3% and three final washes in TS 1X. Primary antibodies were revealed by infrared-conjugated anti-rabbit IgG IRDye®  and scanned with the Odyssey Infrared Imaging System . Densitometric analysis of the obtained signals was carried out using Image Studio software , and the results are shown as the mean ± SD of at least three independent experiments. Statistical analysis was performed to assess differences, within each brain area, between SHAM and ETO groups . p values of < 0.05 were considered significant.In order to evaluate the effect of ETO on PHOX2B and its target gene protein levels, 80 μg of protein extract from the areas of interest was analysed by means of Western blot as previously described . Protein® in situ hybridization  and immunofluorescence detection to quantify Phox2b mRNA and protein expression within the RTN and the DVC according to our previous protocols [Following the plethysmography recordings (4-weeks post-Nexplanon implantation), rats were transcardially perfused in 4% paraformaldehyde (PFA) and the brains were post-fixed in 4% PFA and cryoprotected in 30% sucrose in 1× PBS frozen in O.C.T and sectioned on a cryostat . Thirty µm thick sections were mounted on slides for combined RNAScoperotocols . In ordePhox2b  and Nmb  for 2 h at 40 °C. In parallel, two sections/rat were treated with positive (low-copy housekeeping gene) and negative  control probes provided by ACDBio. Finally, slides were processed using the RNAScope Multiplex Fluorescent Assay kit V2 (ACDBio) according to the manufacturer’s instructions. Phox2b and Nmb probes were visualized using Opal 570 and Opal 690 reagent, respectively .For the RNAScope assay, slides were processed as detailed in  and incuImmediately following the RNAScope assay, immunofluorescence staining was performed using commercial mouse monoclonal antibody against PHOX2B  and the goat polyclonal antibody against ChAT  followed by the secondary antibodies as follows: donkey CY5-conjugated anti-mouse IgG and donkey Cy2-conjugated donkey anti-goat IgG . Slides were then cover-slipped with Fluorosave mounting media  and fluorescence labelling was observed and acquired under a Leica TCS SP8 Laser Scanning Confocal microscope .Pho2xb mRNA and protein expression in serial section, the opening of the central canal was set as our reference point  in 1 mm caudo-rostral distribution from the opening of the central canal and encompassing the facial motor nucleus. The mRNA expression for Nmb was used as marker of RTN CO2-sensing neurons [Phox2b expression within the DVC we selected 4 sections surrounding the opening of the central canal , and ChAT staining was used as anatomical marker to distinguish between PHOX2B-positive cells in the X (ChAT+/PHOX2B+) and the NTS (ChAT−/PHOX2B+).For quantification of rdinates ). To ide neurons . To invep values of < 0.05 were considered significant.Under the Leica TCS SP8 Laser Scanning Confocal microscope, exposure time and acquisition parameters were set for the SHAM group and kept unchanged for the entire dataset acquisition. The collected images were than analysed using FIJI software  by selec


In this study, we observed that two rice recombinant inbred lines, H138 and H217 (RILF11 plants from Sasanishiki × IRAT10), have higher net CO2 assimilation (An) than their parent Sasanishiki due mainly to the improvement of leaf anatomy. Our results showed that An positively correlated with anatomy traits’ mesophyll cell number per cross-sectional area (NO.mescell/Acros) and mesophyll area (Ames). NO.mescell/Acros exert direct and indirect effects on An. Compared to Sasanishiki flag leaves, IRAT10, H138, and H217 have higher mesophyll cell numbers. Simultaneously, higher chlorophyll content and expression of genes encoding the light-harvesting protein of PSII and PSI  were recorded in IRAT10, H138, and H217, which facilitates light use efficiency. Higher electron transport rate and RuBP concentration were recorded in IRAT10, H138, and H217 flag leaves. Retinoblastoma-related gene (OsRBR1), exerting effects on mesophyll cell density, can be used to modify leaf anatomy for improving leaf photosynthesis. Additionally, higher stomatal conductance and mesophyll conductance were also recorded in H138 and H217 than in Sasanishiki. Furthermore, we modeled mesophyll conductance through anatomical traits, and the results revealed that chloroplast thickness was the dominant factor restricting CO2 diffusion within mesophyll cells rather than cell wall thickness. Higher RuBP content accompanied by higher CO2 concentration within the carboxylation set in H138 and H217 flag leaves contributed to higher CO2 assimilation.Leaf anatomy determining the light distribution within the leaf and exerting influence on CO Mffusion) . Howeveroperties . Among lximum gm . The celthan Tcw . High SCat level . Howeverme cases . Moreovee tissue . Furtherr stress . This vi2 diffusion or biochemical factors mediated by leaf anatomy traits. In addition, we modeled mesophyll conductance from anatomical traits to reveal the dominant components that restrict CO2 diffusion and make up for the lack of application of the methods in rice.Under the field condition, we found that IRAT10 with thicker leaves has a higher photosynthetic rate than Sasanishiki, which may be benefited from leaf anatomy traits. Additionally, H138 and H217 from the recombinant inbred line of IRAT10 × Sasanishiki have higher photosynthetic rates than their parent Sasanishiki. We suspect that the increase in photosynthetic efficiency of H138 and H217 might be related to the leaf anatomical traits inherited from their parent IRAT10. This has great significance for rice breeding. It makes sense to investigate the mechanism of photosynthetic improvements benefited from COOryza sativa japonica subspecies) and IRAT10 , as well as two recombinant inbred lines, H138 and H217 (RILF11 plants from Sasanishiki × IRAT10), were used in this study. The genetic map of tested materials is shown in Sasanishiki (–1) = 13.95 (A665) – 6.88 (A649), chlorophyll b (μg mL–1) = 24.96 (A649) – 7.32 (A665). The total chlorophyll content is the sum content of chlorophyll a and chlorophyll b. For each genotype, six biological replicates were measured.Chlorophyll was extracted from flag leaves in 20 ml of 95% alcohol for 48 h at room temperature under a dark environment according to the method described by –2 s–1 provided by a LED source with a light ratio of red: green: blue: white 90: 0: 5: 5%. CO2 concentration in the leaf chamber was maintained at 400 μmol mol–1 provided by a CO2 injector system. The leaf temperature in the chamber was controlled at 25°C, and the flow rate was controlled at 200 μmol s–1. Net CO2 assimilation , internal CO2 concentration , and stomatal conductance  were recorded simultaneously when the system stabilized to a steady-state. Then, a fluorometric procedure was performed to measure the steady-state fluorescence (FS) and maximum fluorescence (Fm′). The saturating light pulse was 8,000 μmol m–2 s–1. The actual photochemical efficiency of photosystem II (ΦPSII) was calculated with the following equations:A portable infrared gas analyzer  equipped with an integrated leaf fluorometer head  was used to measure gas exchange and chlorophyll fluorescence parameters simultaneously on a sunny day. About nine flag leaves on the main stem of each genotype were selected to exert gas exchange measurement. The photon flux density (PPFD) in the leaf chamber was maintained at 1,200 μmol mThe electron transport rate (J) was calculated as:–2 s–1 at low O2 concentrations (<2%). The slope of the relationship between ΦPSII and 4ΦCO2 (the quantum efficiency of CO2 uptake) was taken as the value of α⋅β  and PSII. Previously, α was assumed as 0.85, and β was assumed as 0.5 between PSI and PSII . In thise of α⋅β .2 (gm) was determined as the following equation described by The mesophyll conductance of CO2 compensation point in the absence of photorespiration, and Rd is the respiration in the daytime. Γ* and Rd were estimated from A/Ci curves, and CO2 concentrations in the leaf chamber were set in a series of 200, 150, 100, 75, and 50 μmol mol–1, under three light intensities  and low O2 concentrations (<2%) (Rd and Γ*(2 leakage was measured to make the gas exchange measurement more accurate (where Γ* refers to the COns (<2%)  (Pons etns (<2%) . The cooRd and Γ*. CO2 leaaccurate .Nine flag leaves on the main stem of each genotype were graphed to measure leaf length, leaf width, and leaf area using Image J software . Dry weights were determined when the flag leaves were dried to a constant weight at 80°C for 48 h. Leaf mass per area (LMA) is the ratio of dry weight and leaf area. Then the dried samples were used for nitrogen concentration determination through a carbon and nitrogen analyzer .For light microscopy, leaf sections (approximately 1 cm in length) from the middle of leaves were collected with the formalin acetic-alcohol solution. Samples were dehydrated with ethanol series  and then, were embedded with paraffin (melting point 56–58 degrees). A sliding microtome (Leica RM2255) was used to cut the block into transverse sections 7 μm in thickness. Light sections were stained using 1% toluidine blue, then the sections were scanned under a digital whole slide scanning. Anatomical characteristics were determined using CaseViewer.leaf/Tmes, μm), the cross-sectional area of each mesophyll cell , the ratio of mesophyll cell (Ames), bulfom cell (Abf), main vein (Amain vein), major vein (Amaj vein), and minor vein (Amin vein) area to cross-sectional area (Acros), and the lengths between two neighboring major/minor vascular bundle , were determined. Mesophyll cell number per cross-sectional area  was calculated as a quotient of dividing Ames by Acell. The cross-sectional areas of >30 mesophyll cells were measured between the vascular bundles. For each genotype, at least six leaf sections from flag leaves of different main stems were used for anatomical character measurement.The thickness of leaf/mesophyll (Tchl), chloroplast thickness (Tchl), and the cross-sectional area of chloroplast (Achl) were measured at 1,200–1,500× magnification. Mesophyll cell wall thickness (Tcw) and cytoplasm thickness (Tcyt) were measured at 12,000× magnification. For each genotype, six-leaf sections from different plants were used. For each section, three different view fields were measured.Leaf pieces between the first and the second major vein (approximately 4 mm × 1 mm) from the same leaves used for light microscopy were cut and immersed in 0.1 M phosphate buffer (pH 7.2) with 2.5% glutaric aldehyde and 3% paraformaldehyde under vacuum for 48 h. Then, the leaf pieces were further fixed in 1% osmium tetroxide for another 2 h, followed by washing three times with phosphate buffer. Then, an ethanol series was used to dehydrate leaf pieces. After dehydration, the leaf samples were embedded in resin for ultrathin sections. Ultrathin sections were further stained with 7.7% uranyl acetate (30 min) and lead citrate (4 min) for photographing under an electron microscope . Chloroplast length (Lfias) was determined as  were calculated as follows (The surface area of mesophyll and chloroplasts exposed to the intercellular airspaces (S follows :m and Lc refer to the length of mesophyll and chloroplast exposed to the intercellular airspace (μm). F is the curvature correction factor and was taken as 1.55 in rice (The L in rice .m ana)  ,b. In thgias is the gas phase conductance that links to CO2 moving from substomatal internal cavities to the outer surface of the mesophyll cell wall, and gliq is the liquid phase conductance that links to CO2 moving from the outer surface of cell walls to the stroma. Gas constant , Henry’s law constant , and absolute temperature  were used to convert gliq to gas equivalent conductance (where ductance .ias) is determined as (The gas-phase conductance (gmined as :–1); and Da, the diffusion coefficiency for CO2 in the gas phase, was assumed as 1.51 × 10–5 (m2 s–1 at 25°C); ΔLias, the average gas-phase thickness, was assumed as half of the mesophyll thickness between epidermal layers.where ς, the diffusion path tortuosity, was assumed as 1.57 (mmgcw), plasmalemma (gpl), cytosol (gcyt), chloroplast envelope (gen), and chloroplast stroma (gst).The total liquid phase conductance is the sum conductance of each component including cell wall  at 25°C. ΔLi (m) is the corresponding thickness of each component in the CO2 diffusion pathways. The effective porosity  in cytosol and chloroplast was taken as 1. Cell wall porosity (pcw) was calculated from Tcw (pcw = -0.3733 × Tcw+.3378) (pl and gen were assumed as 0.0035 ms–1 (–1 were converted into molar units according to the equation g[mol m–2 s–1] = g[ms–1] × 44.6 × 273.15/(273.15 + TemL) × (P/101.325), where TemL is the leaf temperature (°C) and P (Pa) is the air pressure (where dimensionless factor w+.3378) . Both gp035 ms–1 ,b. Condupressure .2 diffusion pathways were calculated as  under dark for 48 h. Then, the leaf samples were washed three times with phosphate buffer. After an ethanol series and a tertButanol series, the samples were freeze-dried. Images were photographed using a scanning electron microscope (HITACHI Regulus 8100). At least three views on abaxial and adaxial sides were photographed from one section, and at least six leaves from different plants were used for each genotype. Stomatal density , stomatal length , and stomatal width  on each leaf side was measured using ImageJ. The stomatal size was determined by the maximum area of the open stomatal pore  calculated as described in Another two leaf sections (approximately 5 mm × 5 mm) from the same leaf used for anatomical measurements were collected with the fixative 2.5% glutaric aldehyde in 0.1 mol LS max) can be estimated according to,Maximum theoretical stomatal diffusive conductance , v is the molar volume of air , and l is the stomatal pore depth for fully open stomata, which assumed to be equal to the width of stomatal guard cell. The gS max is the sum of abaxial and adaxial conductance.where After the gas exchange measurement flag leaves on the main stem were collected and stored at –80°C until to determine the biochemical indices. For each genotype, six biological replicates were measured. The contents of RuBP, Rubisco, PGA (3-phosphoglycerate), and the activity of Rubisco were measured using commercial ELISA kits following the manufacturer’s instructions .® Premix Ex Taq™ II (Tli RNaseH Plus) . Genes related to photosynthesis were checked, and the sequences of primers are shown in Flag leaves of tested varieties  were used for total RNA isolation using MiniBEST Plant RNA Extraction Kit . Then, total RNA was used for complementary DNA (cDNA) library construction. Real-time PCR was performed on an Applied Biosystems QuantStudio 3 Real-Time PCR System  using TB GreenO. sativa ssp. japonica1 by DESeq2  were used for total RNA isolation on the same day after gas exchange measurements. Total RNA was isolated for cDNA library construction. The cDNA libraries were sequenced using Illumina HiSeq™ 2500 sequencing platform  by MetWare . After filtrating the adapter and low-quality reads, clean reads were aligned to the reference genome Nipponbare y DESeq2 , and they DESeq2 . Genes wOne-way analysis of variance (ANOVA) was used to test the statistically significant differences among tested varieties, which was performed through SPSS 22.0 software (IBM Inc.). Multivariate correlation analysis was used to reveal the relationship between leaf anatomical traits and photosynthetic parameters. Structural equation modeling (SEM) was performed to determine the causal relationships between leaf anatomy and photosynthetic capacity, which was exerted with AMOS 22.0.0 software.2, 6.76 mg cm–2). Much longer distances between two neighboring major veins or minor veins (Lmajor vein/Lminor vein) were observed in H217  flag leaves. The cross-area of a single mesophyll cell varied from 157 to 249 μm2, with IRAT10 having the largest and H138 having the smallest. Leaf thickness and the mesophyll thickness of IRAT10 , H138 , and H217  flag leaves were significantly thicker than that of Sasanishiki . The cross-sectional area, the proportion of mesophyll cell area accounts for the cross-sectional area, and mesophyll cell number per cross-sectional area of flag leaves in IRAT10, H138, and H217 were significantly higher than that in Sasanishiki (Sasanishiki flag leaves have the longest leaf length (21.08 cm) and H217 has the shortest (29.69 cm) . Contraranishiki .chl) and chloroplast thickness (Tchl) in Sasanishiki flag leaves  were significantly higher than that of IRAT10, H138, and H217 (chl) in flag leaves of IRAT10, Sasanishiki, and H138 was 13.8, 10.9, and 12.8% larger than H217 (4.69 μm), respectively. The significantly bigger Achl of Sasanishiki flag leaves was mainly due to the thicker chloroplast thickness (Tchl), and shorter Lchl contributed to a smaller Achl in H217 flag leaves. The number of chloroplast per mesophyll cell (NOchl/Cell) has no difference among tested varieties. Cytoplasm thickness (Tcyt) of IRAT10 (0.161 μm) and H217 (0.178 μm) were significantly higher than Sasanishiki (0.110 μm) and H138 (0.134 μm). Compared to Sasanishiki (0.224 μm) and H217 (0.238 μm), IRAT10 (0.189 μm) and H138 (0.216 μm) have a thinner cell wall thickness (Tcw). The surface area of mesophyll and chloroplasts exposed to the intercellular airspaces (Sm/S and SC/S) and the fraction of intercellular airspace of Sasanishiki, H138, and H217 were significantly higher than that of IRAT10 .Chloroplast cross-sectional area (Aand H217 . Chloropanam ), modeled from anatomical traits, was estimated according to the component diffusion conductance of the corresponding diffusion pathway, and the limitation of each component was calculated. The ganam  in flag leaves of IRAT10, H138, and H217 was 33.3, 16.9, and 28.4%, respectively, was remarkably higher than that of Sasanishiki (.081 mol m–2 s–1) (ias), ranging from 15.7 to 42.4%, was lower than the total limitation from liquid phase components in all tested varieties (st) were the main limitations among all components in all cultivars in the liquid phase. The Lmst in IRAT10 (38.6%) flag leaves were significantly lower than that of Sasanishiki (62.5%), H138 (51.8%), and H217 (49.4%). Despite the value of gm being dramatically higher than gmana, a significant positive correlation  was observed between gmana and gm (mana and Tleaf (r = 0.72) and Tmes (r = 0.7). However, Tchl, Sm/S, and Sc/S negative correlate with gm ana.Mesophyll conductance (gm–2 s–1) . The limarieties . A signia and gm . Also, smax) of IRAT10 flag leaves on the adaxial side and the abaxial side was the largest , and H138 having the smallest  (–2) was significantly higher than that of IRAT10, Sasanishiki, and H217. The variation of stomatal density on the adaxial side ranged from 611 (H217) to 778 NO.mm–2 (IRAT10). Maximum theoretical stomatal diffusive conductance (gmaxS) was calculated from stomatal density and stomatal area, and the total gSmax was the sum of the adaxial and abaxial sides. The total gSmax in the flag leaves of IRAT10, H138, and H217 was higher than that of Sasanishiki (1.33 mol m–2 s–1).Scanning electron microscope results showed that the maximum area of the open stomatal pore (a5.3 μm2) . The var–2 s–1), IRAT10, H138, and H217 have higher CO2 assimilation (An) of 29.1, 27.5, and 29.1 μmol CO2 m–2 s–1, respectively, at the heading stage (S), estimated from the gas exchange, in IRAT10, H138, and H217 flag leaves, were 99, 30, and 26%, respectively, significantly higher than Sasanishiki (554 mmol m–2 s–1). Intercellular CO2 concentration (Ci) in flag leaves of IRAT10, H138, and H217 were 7, 6.9, and 5.9%, respectively, significantly higher than Sasanishiki (309 μmol mol–2). Mesophyll conductance (gm) was estimated from the gas exchange and fluorescence methods to show the CO2 diffusion within the mesophyll cell level. Mesophyll conductance in flag leaves of H217, IRAT10, and H138 were 92, 88, and 58%, respectively, significantly higher than Sasanishiki (0.26 mol m–2 s–1). Hence, CO2 concentration within the chloroplast (Cc) in flag leaves of IRAT10 (269 μmol mol–2), H138 (263 μmol mol–2), and H217 (268 μmol mol–2) were remarkably higher than Sasanishiki (221 μmol mol–2). A significant higher electron transport rate (J) was observed in IRAT10 (190 μmol m–2 s–1), H138 (185 μmol m–2 s–1), and H217 (189 μmol m–2 s–1) flag leaves than in Sasanishiki (173 μmol m–2 s–1).Relative to Sasanishiki flag leaves , H138 , and H217  flag leaves compared to Sasanishiki  (–1), H138 (921 ng g–1), and H217 (908 ng g–1) flag leaves were significantly higher than in Sasanishiki (773 ng g–1).Significant higher chlorophyll content, N content, and RuBP content were observed in IRAT10 (3.3 mg g ng g–1) . However2 diffusion traits were selected to explore the relationship between them and An. Significant higher positive correlations were observed for An with J (r = 0.85), chlorophyll content (r = 0.79), gm (r = 0.74), NO.mescell/Acros (r = 0.80), Ames (r = 0.72), N (r = 0.75), Cc (r = 0.70), ganam  (r = 0.66), RuBP (r = 0.63), Across (r = 0.62), Ci (r = 0.52), and gS (r = 0.45)  . Signifies on An . AnotherLOC_Os02g04080 , LOC_Os08g41070 , LOC_Os09g27700 , LOC_Os11g44014 , LOC_Os12g20324  , LOC_Os05g10580 , LOC_Os06g44040 , LOC_Os07g10070 , and LOC_Os11g47410  were recorded in comparisons IRAT10 vs. Sasanishiki, IRAT10 vs. H138, and IRAT10 vs. H217. Cyclins and cyclin-dependent kinases (CDKs) are positive regulators of cell proliferation  were lowly expressed in H138 and H217. The LOC_Os08g42600 (OsRBR1) in H138 and H217 were inherited from their parent IRAT10 and were lowly expressed, which may contribute to small mesophyll cells and larger mesophyll cell numbers in H138 and H217 flag leaves.The KOG enrichment analysis was performed on the DEGs obtained from five pairwise comparisons of transcriptomes . Genes rpressed) . Additioferation . LOC_Os1anishiki . HoweverLeaves exposed to complex environmental conditions of different temperatures and different irradiations inside the canopy often show dramatically different photosynthetic properties . AdditioS) and mesophyll conductance (gm) in flag leaves were observed in IRAT10, H138, and H217 compared to Sasanishiki, though differences existed in achieving higher gS and gm among tested varieties. This significantly facilitates higher CO2 concentration in intercellular spaces, as well as at the carboxylation site, higher Ci and Cc were observed in IRAT10, H138, and H217  were recorded in IRAT10, H138, and H217, which facilitates light use efficiency. Better light use efficiency may facilitate RuBP regeneration and promotes CO2 assimilation. Fortunately, cell density can be manipulated by modifying cell cycle-associated genes. Retinoblastoma-related gene, RBR1, plays a central role in regulating gene expression, endoreduplication, the number, size, and death  in H138 and H217 were inherited from IRAT10 and were lowly expressed than IRAT10 , together with higher mesophyll conductance (mediated by chloroplast thickness), contributed to higher An in flag leaves of H138 and H217 at the heading stage. Chloroplast thickness was the dominant factor restricting CO2 diffusion within mesophyll cells rather than cell wall thickness. Mesophyll cell density will be a good target to improve photosynthesis in engineering leaf anatomy.In summary, we analyzed the photosynthetic capacity and corresponding constraints on COPRJNA834750.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI; DM and JW designed experiments. FZ, LZ, and YL performed experiments. FZ, LZ, and GW analyzed data and compiled figures. FZ and JW wrote the manuscript. DM edited the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The study aimed to estimate the prevalence and associated factors of undiagnosed type 2 diabetes (T2D) among adults in Morocco. Cross-sectional data were analyzed from 4779 people  who participated in the Morocco STEPS nationally representative survey in 2017 and had completed fasting blood glucose measurement. The results indicate that the prevalence of undiagnosed T2D was 5.9% , diagnosed T2D 7.3% and total T2D 13.2%. In the adjusted multinomial logistic regression analysis, older age (≥ 50 years), receipt of health care advice, and obesity were positively associated with undiagnosed T2D. Older age (≥ 50 years), urban residence, receipt of health care advice, ever cholesterol screening, moderate sedentary behaviour, obesity, hypertension, and elevated total cholesterol were positively associated with diagnosed T2D. In adjusted logistic regression analysis, older age (≥ 50 years), receipt of health care advice and cholesterol screening were negatively associated with undiagnosed T2D versus diagnosed T2D. A significant proportion of adults in Morocco had undiagnosed T2D and several associated factors were identified that can help guide interventions. Untreated undiagnosed type 2 diabetes (T2D) may have serious consequences, including microvascular and macrovascular complications3 and an increased risk of mortality5, highlighting the crucial importance of early diagnosis. In 2021, worldwide 44.7% of adults with T2D did not know they had T2D6. In an earlier review of studies in 29 low- and middle-income countries (LMICs), the prevalence of undiagnosed T2D was 4.9%7, and in a review of 55 studies in the eastern Mediterranean region, the pooled prevalence of undiagnosed T2D was 5.45%, for example, in Tunisia 7.7% and in Iraq 6.2%8. In a small sample of urban Sahraoui women in South Morocco, the prevalence of undiagnosed T2D was 6.4% in 2001/20029. Due to 44% of the diabetes population being undiagnosed in Morocco10, the Moroccan Ministry of Health launched a public awareness campaign in 2015 on the different types of diabetes, its risk factors, warning signs and complications, the importance of screening and the adoption of a healthy lifestyle, involving health professionals and other lay persons in awareness and health education and involving the media in information and awareness on diabetes and its complications11. There is a lack of national data on undiagnosed T2D and its correlates in Morocco12. These data may help to assess public health interventions related to the screening and diagnosis of T2D in Morocco.According to the World Health Organization15, older adults19, male sex17, living alone 20, and family history of diabetes22. Enabling/disabling factors associated with undiagnosed T2D include rural residence16, in some studies lower economic status23, food insecurity24, and lower education25, and in other studies higher economic status17, and higher education23. Other enabling/disabling factors associated with undiagnosed T2D include knowing symptoms of diabetes21, no health care visit in the past 12 months15, health insurance status , obesity31, low HDL-C32, high triglycerides32, dyslipidaemia22, cardiovascular disease status , and perceived poor health16. The study aimed to estimate the prevalence and associated factors of undiagnosed T2D among people 18 years and older in Morocco.The need factors associated with undiagnosed T2D include other chronic diseases33 with complete measurements of fasting blood glucose were analyzed; the overall response rate was 89.0%34. STEPS focus is on “obtaining population-based data on the established risk factors that determine the major disease burden on a regular basis”33. Participants were randomly selected from the target population (18 years and older), using a multi-stage stratified sampling procedures34. Following the STEP-wise survey procedures, in Step 1 behavioural and sociodemographic data were collected. In Step 2, physical measurements and blood pressure were assessed, and in Step 3. biochemical measurements were collected to assess blood glucose and cholesterol33. Blood glucose, total cholesterol and triglycerides were measured in peripheral (capillary) blood at the data collection site; equipment used was the Cardiochek® PA  with a Chip MeMo, Blood Glucose Strips and lipids34.Secondary data from the ‘STEPwise approach to NCD risk factor Surveillance’ (STEPS) cross-sectional survey in Morocco in 2017Ethics approval was provided by the Biomedical Research Ethics Committee, Faculty of Medicine and Pharmacy of Rabat, Morocco, and participants provided written informed consent. All methods were performed in accordance with the relevant guidelines and regulations.35.Undiagnosed T2D was defined as fasting plasma glucose level ≥ 126 mg/dL among people who responded “no” to the question “Have you ever been told by a doctor or other health worker that you have raised blood sugar or diabetes?” Diagnosed T2D was defined as those who answered “yes” to the question of ever having been told by a health care worker that they had diabetes, and total T2D included those with undiagnosed and diagnosed T2DPredisposing factors included age, sex, and marital status.Enabling or disabling factors included residence status, receipt of health care advice, ever screening for cholesterol, smoking tobacco history, physical activity, and sedentary behaviour. The receipt of health care advice was assessed with the question, “During the past three years, has a doctor or other health worker advised you to maintain a healthy body weight or lose weight?” (yes/no). Smoking history was assessed with two questions, “Do you currently smoke any tobacco products, such as cigarettes, cigars or pipes?”  and “In the past, did you ever smoke any tobacco products?”34. Self-reported physical activity and sedentary behaviour were assessed with the Global Physical Activity Questionnaire (GPAQ) and categorized by the median metabolic equivalent (METs) of performed activities as low, moderate, and high36, and sedentary behaviour defined as low (< 4 h), moderate (4 to < 8 h) and high (≥ 8 h sitting/day)37.The need factors included BMI, hypertension, heart attack or stroke, and elevated total cholesterol. Body mass index (BMI) was classified as “underweight (< 18.5 kg/m2), normal weight (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2), and obesity (≥ 30.0 kg/m2)”33. Hypertension was defined as “systolic BP ≥ 140 mmHg and/or diastolic BP ≥ 90 mmHg and/or previous or current treatment with antihypertensive drugs”38. History of heart attack or stroke included self-reported “Have you ever had a heart attack or chest pain from heart disease (angina) or a stroke (cerebrovascular accident or incident)? ”34. Elevated total cholesterol was classified39 as: “being on antilipidemic medication or having elevated total cholesterol (TC): ≥ 5.17 mmol/l (200 mg/dl)”.8. We calculated the sample size with Epi-Info with population 100,000, expected frequency 5.45, acceptable margin of error 5% confidence level 99.99, minimum sample size is 317. In our study we calculated that based on the existing data set of 4799 people, the sample size is sufficient.Sample size calculation. This data set had an overall N = 5429 and 4779 (88%) persons from the total sample had completed fasting blood glucose measurement. From a review in the study region, the pooled prevalence of undiagnosed T2D was 5.45%33 Descriptive statistics are used to describe the sample. Pearson chi-square statistics were used to calculate differences in proportion. Multinomial logistic regression was used to estimate factors associated with undiagnosed T2D and diagnosed T2D (with not having T2D as reference category). Logistic regressions were used to assess the associations with undiagnosed T2D versus diagnosed T2D. Covariates in the logistic regression models included predisposing factors , enabling, or disabling factors  and need factors . Variables significant in univariable analyses were subsequently included in the multivariable models. To account for the multi-stage sample design, Taylor linearization methods were utilized. P-values < 0.05 were considered significant and missing values were discarded.All statistical analyses were conducted with STATA software version 14.0 . Analysis weights were calculated by taking the inverse of the probability of selection of each participant adjusted for differences in the age-sex composition of the sample population compared to the target population.The sample with complete fasting blood glucose measurement included 4779 persons ≥ 18 years), with a mean age of 41.7 years (SD = 16.5 years) in 2017. The prevalence of undiagnosed T2D was 5.9% , diagnosed T2D 7.3%, and total T2D 13.2%. Further sociodemographic and health characteristics of the sample by T2D status are described in Table 8 years, In the adjusted multinomial logistic regression analysis, older age (≥ 50 years) , receipt of health care advice , and obesity  were positively associated with undiagnosed T2D. Older age (≥ 50 years) , urban residence , receipt of health care advice , ever cholesterol screening , moderate sedentary behaviour , obesity , hypertension , and elevated total cholesterol  were positively associated with diagnosed T2D. In addition, in unadjusted analyses, male sex, higher education, and high physical activity were negatively associated, and hypertension and elevated total cholesterol were positively associated with undiagnosed T2D  , receipt of health care advice , and ever cholesterol screening  were negatively associated with undiagnosed T2D versus diagnosed T2D. Furthermore, in the unadjusted analysis, urban residence, sedentary behaviour, obesity, hypertension, and heart attack or stroke were negatively associated with undiagnosed T2D versus diagnosed T2D 7, higher than in a review of studies in the Eastern Mediterranean region (5.45%)8, and lower than in a local study among women in Morocco (6.4%)9. People with undiagnosed T2D versus diagnosed T2D showed fewer diabetes-related risk factors, such as younger age, no obesity, and no hypertension than those with diagnosed T2D. This may be explained by people with undiagnosed T2D being generally younger and healthier than those with diagnosed T2D, mostly at an earlier stage of T2D40.The study found a national prevalence of undiagnosed T2D , which is similar to recent global Figs. 19, some predisposing factors (older age) were associated with undiagnosed T2D versus no T2D. According to some previous studies26, enabling / disabling factors associated with undiagnosed T2D versus diagnosed T2D included no receipt of health care advice (to lose weight) in the past three years, and had never been screened for cholesterol. People with receipt of health care advice and cholesterol screening are more likely to use health services and may consequently reduce the odds of undiagnosed T2D16. Following the T2D management guidelines in Morocco, patients with diabetes are expected to attend health care services more often41, which may explain that people with diagnosed T2D visit health care providers more often than people with undiagnosed T2D15. While some studies16 found an association between rural residence and undiagnosed T2D, we only found this association in unadjusted analysis. Previous studies25 found mixed results regarding the association between educational status and undiagnosed T2D, while we found no significant association.Consistent with some previous research28, need factors associated with undiagnosed T2D included other chronic diseases, such as obesity. Obesity is a known risk factor for diabetes21. Consistent with some research15, in unadjusted analysis, we found a negative association between hypertension, cardiovascular disease, and undiagnosed T2D versus diagnosed T2D. Unlike some previous studies32, we did not find an association between elevated total cholesterol and undiagnosed T2D. However, we found a statistically significant positive association between elevated total cholesterol and diagnosed T2D. This may be explained by people with diagnosed T2D who are generally older and less healthy, mostly at a later stage of T2D than those with undiagnosed T2D40.Consistent with previous research42. However, Priya et al.43 showed that capillary blood glucose is a feasible alternative for screening for diabetes in epidemiological studies in developing countries where obtaining venous samples may be difficult. Some variables were evaluated by self-report, which may have biased responses, and the cross-sectional design precludes causative conclusions between the evaluated variables. The sample only included those persons who were non-institutionalized, while the inclusion of institutionalized persons would have given different estimates. Furthermore, certain variables, such as knowledge of diabetes symptoms and a family history of diabetes, were not evaluated and should be included in future research.The study strengths include the use of nationally representative adult sample of all ages and standardized STEPS methodology and measures. The limitation of the study is using peripheral (capillary) blood instead venous plasma glucose44.Policy implications are that increased public awareness campaigns, and screening of T2D are needed to reduce undiagnosed T2D in Morocco. The Morocco national NCD programme includes community awareness campaigns on diabetes, screening/early detection, and integrated care for diabetesA significant proportion of adults in Morocco had undiagnosed T2D. Predisposing factors (older age), enabling factors  and need factors (obesity) were identified as associated with undiagnosed T2D versus no T2D, and predisposing factors (younger age), and enabling / enabling factors  were identified as associated with undiagnosed T2D versus diagnosed T2D, which can be targeted in interventions.


Meanwhile, the capacity retention rate remains 73.8% after 10 000 charge and discharge cycles. In particular, this cathode material can be used at low temperatures (up to −30 °C) with 60% capacity remained, which enlarges the application temperature range of ZHSCs. These results of this study can help understand new properties of organic–inorganic hybrid materials.Organic and inorganic materials have their own advantages and limitations, and new properties can be displayed in organic–inorganic hybrid materials by uniformly combining the two categories of materials at small scale. The objective of this study is to hybridize activated carbon (AC) with ferrocene to obtain a new material, ferrocene/AC, as the cathode for Zn-ion hybrid supercapacitors (ZHSCs). The optimized ferrocene/AC material owns fast charge transfer kinetics and can obtain pseudo-capacitance through redox reaction. Due to the introduction of ferrocene/AC, the ZHSCs exhibit remarkable electrochemical performances relative to that using ferrocene cathode, including high discharge specific capacity of 125.1 F g Organic and inorganic materials have their own advantages and limitations, and new properties can be displayed in organic–inorganic hybrid materials by uniformly combining the two categories of materials at small scale. In this context, aqueous Zn-ion hybrid supercapacitors (ZHSCs) have emerged as a promising cost-efficient technique for large scale energy storage applications.6,7 As an energy storage device, ZHSCs is composed of a capacitor-type cathode, a battery-type metal zinc anode, and aqueous electrolyte containing Zn2+.8 During the charging and discharging process, Zn2+ deposits/dissolves on the Zn anode rapidly, which ensures a high energy density, and the simultaneous reversible adsorption/desorption process of anions on the cathode material ensures an excellent power density.9 Besides the benefits from Zn anodes, aqueous ZHSCs also take the advantages of supercapacitors (SCs), i.e., fast charge/discharge rate, high power density, good cycling stability and environmental friendliness.10,11Zn anodes have long been used in electrochemical energy storage devices, especially in primary and secondary aqueous batteries,12,13 most of which are inorganic materials. These inorganic cathodes mainly consist of transition metal oxides and Prussian blue analogs, and manganese oxides (commonly MnO2) and vanadium oxides (commonly V2O5) are representative transition metal oxides.14,15 Ma et al. reported the aqueous ZHSCs using V2O5 as the cathode, showing an operating voltage range of 0–2 V, with the maximum specific capacity and energy density of 57.4 mA h g−1 and 34.6 Wh kg−1, respectively.16 MnO2 nanorods were used as the cathode in ZHSCs, showing the maximum specific capacity of 54.1 mA h g−1 and maximum energy density of 34.8 Wh kg−1, and good cycling stability with 93.4% capacity retention over 5000 charge/discharge cycles.17 However, manganese oxides are plagued by the issues of dissolution in aqueous electrolytes and their low electrical conductivity.18,19 The crystal structure of vanadium oxides is unstable,20,21 and the capacity of the corresponding aqueous ZHSCs devices decreases rapidly during Zn2+ intercalation and deintercalation cycling.22,23Studies of Zn-ion energy storage devices mainly focus on the development of cathode materials,24,25 and exhibited unique advantages, such as fast reaction kinetics, rich abundance of composition elements, high sustainability, facile structural design, and high capacity generated by multi-electron redox reactions.5,26 However, organic materials usually have the problem of inherent low electrical conductivity, which greatly increases the internal resistance, reduces the areal capacity and volume energy density.5 Since organic and inorganic cathode materials have their own limitations, new properties can be displayed in organic–inorganic hybrid materials by uniformly combining the two categories of materials at small scale.27 Xin et al. reported the aqueous Zn-ion hybrid energy storage device using poly-modified activated carbon (AC) as the cathode.10 The application of this hybrid cathode not only widens the voltage window from 0.2–1.8 V to 0.1–1.9 V, but also maintains the capacitance retention rate of 71% after 2000 charge–discharge cycles. Du et al. reported a novel organic–inorganic hybrid V2O5@polyaniline as the cathode for aqueous Zn ion batteries, showing a high specific capacitance of 61 mA h g−1 at 0.1 A g−1.28On the other hand, compared with inorganic materials, organic cathode materials have also been used in aqueous energy storage devices to couple with Zn anodes,29,30 Specifically, it possesses remarkable electrochemical performances, including the high discharge specific capacity of 125.1 F g−1, high energy density (up to 44.8 Wh kg−1 at 0.1 A g−1) and large power density (up to 1839 W kg−1 at 5 A g−1). The capacity retention rate can maintain 73.8% after 10 000 cycles. This cathode material can be used at low temperatures (up to −30 °C) with 60% capacity remained, which enlarges temperature application field of ZHSCs.In this work, ferrocene and AC are combined by a hybrid method to obtain a new organic–inorganic material ferrocene/AC. This hybridization allows fast charge transfer kinetics of the resulting cathode material, and can obtain pseudo-capacitance through redox reactions.i.e., 3 : 1, 5 : 1 and 7 : 1) were also prepared using the same method. The active material, conductive additive (Super P) and polyvinylidene fluoride (PVDF) in a mass ratio of 8 : 1 : 1 were mixed, and then appropriate amount of N-methyl pyrrolidone (NMP) was added to form a homogeneous slurry. The resulting slurry was coated on stainless steel mesh (with a thickness of 244 μm) and dried in a vacuum oven at 70 °C for 12 h as cathode.Ferrocene was purchased from Adamas and used directly without any purification, and AC powder was obtained from Kejing (Hefei) and was sintered in argon at 480 °C for 3 h before use. These two materials were ground and mixed with a pestle and mortar in a mass ratio of 1 : 1 as the active material (denoted as ferrocene/AC). For comparison, cathodes of ferrocene, AC powder, separately, and mixtures of ferrocene and AC powder with different mass ratios other than 1 : 1  was used to observe the microstructure and morphologies of electrode. The X-ray photoelectron spectroscopy  was used to obtain the information of the chemical bonding states. The specific surface area was measured by a Brunauer–Emmett–Teller analyzer . The corresponding surface areas of micropores and mesopores were calculated by t-plot method.X-ray diffraction  was used to obtain the information of the diffraction pattern and the composition of materials. The scan rate was set as 5° min4 electrolyte and fiber glass separator. Zn foils were polished by abrasive papers before use. The electrochemical tests were performed on a CHI660E electrochemical workstation and a LAND CT2001A battery-testing instrument. The electrochemical tests include cyclic voltammetry (CV), galvanostatic charge–discharge (GCD), electrochemical impedance spectroscopy (EIS), long cycling stability, and rate performance. The voltage range of all tests were 0.2–1.8 V. The frequency of EIS was ranging from 10 mHz to 100 kHz with 5 mV AC  amplitude.ZHSCs were assembled as CR2032 coin cells using the hybrid cathode , Zn anode, 2 M ZnSOCs, F g−1), energy density  and power density  were calculated by the following 31,32I, (V2 − V1), v, m and Δt represent the discharge current, voltage window of the discharge process, scan rate, mass loading of the active materials based on the cathode, and discharge time, respectively. Is the area under the curve I/V.The specific capacitance , in the same conditions, ferrocene/AC shows comparable specific capacitance with AC (125.1 vs. 107.9 F g−1). Given that ferrocene/AC comprises of ferrocene and AC in the mass ratio of 1 : 1, the faradaic reaction of ferrocene must contribute to the capacitance of this hybrid material. −1 to 1 mV s−1, with voltage window in between 0.2–1.8 V. All the curves show asymmetric deviation from the standard rectangular shapes of ideal SCs, suggesting that faradaic reactions take place with the hybrid material.38 In addition, the reduction and oxidation peaks appear at around 0.8 V and 1.1 V, respectively. As a comparison, the CV curves of pure ferrocene in Fig. S1a (in ESI)17,39,40 and the constant peak position without varying with the scan rate indicates the fast charge transfer kinetics in these processes. The CV curves of pure AC in Fig. S3a41−1 at the scan rates of 10, 20, 50, 100 and 200 mV s−1, respectively. The ZHSCs can achieve high energy and power densities up to 44.8 Wh kg−1 at 0.1 A g−1 and 1839 W kg−1 at 5 A g−1, respectively. The electrochemical performance of other samples, with different mass ratios between ferrocene and AC, were also tested using the same methods, and the results are shown in Fig. S4–S6.−1 for ferrocene  of ferrocene/AC (with 1 : 1 mass ratio of ferrocene to AC) are much higher than the sum of the two component materials (4.8/2 F g−1 + 107.9/2 F g−1 = 56.4 F g−1). The faradaic reaction of ferrocene is responsible for the increased capacitance. Because pure ferrocene as the cathode in ZHSCs only show very small amount of capacitance, the capacitance contribution of ferrocene is probably due to the synergistic effect by combination of the two types of materials, in which AC provides pores to distribute ferrocene, and improve the charge transfer rate for the organic molecule.From i, mA) of CV curve and scan rates  have a relationship as below:a and b are adjustable parameters, and the value of b expresses the charge storage mechanism: b close to 0.5 represents a traditional battery behavior, and energy storage in the ZHSC relies on the process of ionic diffusion control; b close to 1 represents a traditional pseudo-capacitor behavior; b in the range of 0.5–1 corresponds to devices that exhibit both battery and pseudo-capacitance properties. According to the slope of log(i) and log(v) plots for all peaks in b of oxidation peak 1 and reduction peak 2 are fitted to be 0.60 ± 0.05 and 0.63 ± 0.03, respectively. The two values indicate that the charge and discharge process are controlled by both ionic diffusion and pseudo-capacitance. As shown in −1, 38% of the total stored charge is contributed by the capacitive-controlled process (the red region). Other capacitive contribution curves at different scan rates are shown in Fig. S7.−1, respectively. The contribution of capacitive contribution ratio increases with the scan rate, indicating the capacitive-dominant nature and fast kinetics of ferrocene/AC at high scan rates.To understand the electron transfer kinetics of ZHSCs using ferrocene/AC cathode, CVs were investigated at various scan rates . The peaZn2+), respectively. The diffusion coefficients of Zn2+ are in the range of 1.11 × 10−9 ∼ 1.12 × 10−8 cm2 S−1, higher than those of 8.08 × 10−10 ∼ 1.07 × 10−8 cm2 S−1 for the AC cathode ,44–46 ethylene glycol (EG) was added to water as the co-solvent, in varying volume ratios of 0% (EG0) to 20% (EG20), 40% (EG40) and 60% (EG60). As shown in Fig. S9,2+ with EG, the hydrogen bond between EG and H2O is strengthened, while the hydrogen bond between H2O and H2O is weakened, leading to the higher ionic conductivity of the mixed electrolyte.42 EG40 can still maintain a ionic conductivity of 1.95 mS cm−1 at −20 °C, higher than EG20 and EG60. Hence, EG40 was chosen as the low-temperature electrolyte in this study.To further explore the electrochemical performance of ZHSCs using ferrocene/AC at low temperature, 2 M of ZnSO47 As the cycle progresses, there is no obvious plateau at the 25th cycle. For comparison, the GCD curves for −30 °C does not show the plateau of redox reaction of ferrocene at all cycles, and the curves at the 2nd, 250th and 500th cycles overlap well, suggesting the long-term stability of the capacitance. −1 at −20 °C and 70 F g−1 at −30 °C. The chelation between Zn2+ and EG weakens the solvation interaction of Zn2+ with H2O, and also enhances the hydrogen bonding between EG and H2O. EG40 shows a high Zn2+ conductivity and reversibility for energy storage, even at temperature as low as −30 °C, and thereby enables ZHSCs to operate well in a wide range of temperatures from 25 °C to −30 °C.42,43 Fig. S11−1 and 62 F g−1 at −20 °C and −30 °C, respectively.−1. Both curves show CE remains 100% after 500 cycles, reflecting the extremely cycling durability. These results confirm the excellent electrochemical performance of ZHSCs using ferrocene/AC cathode, at low temperature up to −30 °C. The rate performance of ZHSCs using ferrocene/AC cathode at −40 °C is shown in Fig. S10.−1, probably due to the low ionic conductivity of EG40 at −40 °C.39 sheet-like structures are evolved after cycling  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O and sp2-C respectively. Apparently, more oxygen-containing functional groups have been formed on ferrocene/AC cathode after cycling, which may provide additional pseudo-capacitance through faradaic reactions and improve the electrochemical performances of ZHSCs.53–55 This phenomenon indicates the nature of synergistic effect of the ferrocene/AC hybrid material.The chemical composite of AC and ferrocene/AC cathode is analyzed by XPS. The C 1s spectrum of AC cathode before cycling shows three strong peaks at 284.8, 286.6 and 291.7 eV that can be assigned to the binding energy of C–C, C–O–C, and π–π*, respectively.3/2 and Fe 2p1/2 are 710.4 eV and 723.5 eV, respectively. The satellite peak obtains at about 716.5 eV,56 with about 6.1 eV difference from the Fe 2p3/2 peak. Another satellite peak appearing at 735.5 eV might be the satellite peak for Fe 2p1/2. The binding energies of Fe 2p3/2, Fe 2p1/2 and satellite peaks after cycling, at 709.6, 722.6 eV and 715.9 eV, respectively, are all very close to those before cycling, indicating the electrochemical cycling process is reversible for ferrocene.−1, high energy density up to 44.8 Wh kg−1 at 0.1 A g−1, and large power density up to 1839 W kg−1 at 5 A g−1. The capacity retention rate remains 73.8% after 10 000 charge and discharge cycles. In particular, this cathode material can be used at low temperatures (up to −30 °C) with 60% capacity remained, which enlarges the application temperature range of ZHSCs. The findings of this study can help explore new application of organic–inorganic hybrid materials in the field of energy storage materials.In summary, we developed a ferrocene/AC hybrid cathode material for aqueous ZHSCs, showing high safety, wide operation temperature and long cycle life. This cathode material combines the advantages of inorganic and organic materials, in which a rapid charge transfer of ferrocene undergoes at the interface on the carbon surface of AC. Meanwhile, the large contact area between the finely dispersed ferrocene molecules and AC surface enables fast redox reactions of ferrocene, resulting in high power densities. Namely, AC matric provides abundant electroactive sites for insulate ferrocene organic material, which inherently has limited active sites for redox reaction. The ZHSCs using ferrocene/AC cathodes possess remarkably improved electrochemical performance related to that using ferrocene, including the high discharge specific capacitance of 125.1 F gThere are no conflicts to declare.RA-012-D2RA02907C-s001


Lupus nephritis (LN) is an inflammatory disease of the kidneys affecting patients with systemic lupus erythematosus. Current immunosuppressive and cytotoxic therapies are associated with serious side effects and fail to protect 20–40% of LN patients from end-stage renal disease. In this study, we investigated whether a small heat shock protein, HSPB5, can reduce kidney inflammation and the clinical manifestations of the disease in NZB/W F1 mice. Furthermore, we investigated whether HSPB5 can enhance the effects of methylprednisolone, a standard-of-care drug in LN, in an endotoxemia mouse model.NZB/W F1 mice were treated with HSPB5, methylprednisolone, or vehicle from 23 to 38 weeks of age. Disease progression was evaluated by weekly proteinuria scores. At the end of the study, the blood, urine, spleens, and kidneys were collected for the assessment of proteinuria, blood urea nitrogen, kidney histology, serum IL-6 and anti-dsDNA levels, immune cell populations, and their phenotypes, as well as the transcript levels of proinflammatory chemokine/cytokines in the kidneys. HSPB5 was also evaluated in combination with methylprednisolone in a lipopolysaccharide-induced endotoxemia mouse model; serum IL-6 levels were measured at 24 h post-endotoxemia induction.HSPB5 significantly reduced terminal proteinuria and BUN and substantially improved kidney pathology. Similar trends, although to a lower extent, were observed with methylprednisolone treatment. Serum IL-6 levels and kidney expression of BAFF, IL-6, IFNγ, MCP-1 (CCL2), and KIM-1 were reduced, whereas nephrin expression was significantly preserved compared to vehicle-treated mice. Lastly, splenic Tregs and Bregs were significantly induced with HSPB5 treatment. HSPB5 in combination with methylprednisolone also significantly reduced serum IL-6 levels in endotoxemia mice.HSPB5 treatment reduces kidney inflammation and injury, providing therapeutic benefits in NZB/W F1 mice. Given that HSPB5 enhances the anti-inflammatory effects of methylprednisolone, there is a strong interest to develop HSBP5 as a therapeutic for the treatment of LN.The online version contains supplementary material available at 10.1186/s13075-022-02958-9. Lupus nephritis (LN) affects nearly 50% of adults and 80% of children diagnosed with systemic lupus erythematosus (SLE), a chronic autoimmune and inflammatory disease . The curHeat shock proteins (HSP) perform pleiotropic functions. Intracellularly, HSPs function as molecular chaperones to prevent protein aggregation during cellular stress –7. HSPs Of particular interest is the small HSP ɑB-crystallin (HSPB5), which was originally described as a major component of the eye protecting against lens protein aggregation  but has Macrophages are key regulators of inflammation, immunity, and tissue homeostasis with a wide variety of activities and presence in a broad range of tissues. Upon inflammatory injury, resident and infiltrating macrophages may be differentiated and activated and often take on pathogenic roles in autoimmune settings. Macrophages are dysfunctional in SLE and LN in a number of ways, including deficient clearance of apoptotic cells, abnormal polarization, secretion of inflammatory mediators, and tissue destruction through the expression of proteolytic enzymes . FurtherBecause of the potent anti-inflammatory effects of HSPB5 that are mediated through macrophages and the involvement of these cells in SLE/LN pathology, we hypothesized that HSPB5 could provide a therapeutic effect in SLE by reducing the aberrant activation and inflammatory signaling of macrophages. We have previously demonstrated the protective effects of HSPB5 treatment in the MRL/lpr mouse model of SLE and in particular showed its ability to reduce the extent of LN by limiting renal damage . In thisRecombinant HSPB5 was manufactured by Delta Crystallon, and negligible endotoxin levels (< 0.05 EU/mg) were confirmed as previously described . MethylpMice were purchased from the Jackson Laboratories and housed under pathogen-free conditions at the animal research facility of the University of Waterloo . All procedures involving mice were approved by the Office of Research Ethics and animal care committee at the University of Waterloo (AUPs 41798 and 43991). Mice were acclimated for at least 4 weeks prior to the study start.At 23 weeks of age, female NZB/W F1 mice (strain #100008) were evenly distributed by body weight and proteinuria score into three groups with 14–15 mice per group. Mice were treated three times weekly for 15 weeks by intraperitoneal injection (IP) with HSPB5 (0.3 mg/kg), methylprednisolone (3 mg/kg), or PBS as a vehicle control, at a volume of 50 µL per 10 g body weight. Disease progression was monitored by body weight and condition, behavior, and weekly measurements of urinary protein content (proteinuria). Treatments and scoring were performed by animal health technicians (AHTs) blinded to the experimental groups. Mice were terminated upon reaching 38 weeks of age or upon the recommendation of AHTs and the consulting veterinarian after achieving humane endpoints , and tissues  were collected for further analysis.At 11 weeks of age, male BALB/c mice (strain #00065) were evenly distributed by body weight into 4 groups with 11 mice per group. Endotoxemia was induced by IP injection of 6 mg/kg LPS . Mice were treated 30 min before and 6 h after the LPS challenge, by IP injection with HSPB5 (0.3 mg/kg), methylprednisolone (1.5 mg/kg), HSPB5 (0.3 mg/kg) + methylprednisolone (1.5 mg/kg), or PBS as a vehicle control at a volume of 80 µL per 10 g body weight. Mice were euthanized 24 h post-LPS injection, and the serum was collected for further analysis.Proteinuria was measured using a commercial dipstick test  and scored semi-quantitatively as follows: 0 (no/trace protein), 1 (30 mg/dL), 2 (100 mg/dL), 3 (300 mg/dL), and 4 (≥ 2000 mg/dL). Half scores were assigned when the color was intermediate between two whole values. Scoring was performed by AHTs blinded to the study. Blood urea nitrogen (BUN) was measured from terminal serum samples using a commercial kit (Thermo Fisher) as per the manufacturer’s instructions.The whole kidneys and spleen portions were fixed in 10% formalin and embedded in paraffin. Two sections (top and middle) were cut for staining with hematoxylin and eosin (H&E). The kidneys were additionally cut for periodic acid–Schiff (PAS) staining. Slides were scanned using TissueScope LE120 digital slide scanner  at 20 × and evaluated by an experienced veterinary pathologist blinded to the study. The severity of glomerular deposits, mesangial expansion, endocapillary proliferation, extracapillary proliferation, tubular atrophy, and interstitial infiltrates was graded semi-quantitatively: 0 (no change), 1 (mild), 2 (moderate), and 3 (severe). The total glomerular lesions score represents the sum of individual scores for glomerular deposits, mesangial expansion, endocapillary proliferation, and extracapillary proliferation. The total kidney lesion score represents the sum of all individual scores .IL-6 was measured from terminal serum samples using a commercial ELISA kit  as per the manufacturer’s instructions. Anti-dsDNA antibody titers were measured from terminal serum samples using a commercial ELISA kit  as per the manufacturer’s instructions.−ΔΔCT method as previously described [Kidney portions were stored in RNAlater™ (Thermo Fisher) until analysis. RNA was isolated using a commercial kit  as per the manufacturer’s instructions and reverse transcribed with GoScript™ Reverse Transcription System kit using random primers (Promega). Reactions were run on 10-ng RNA equivalents in triplicate with Fast Advanced MasterMix (Thermo Fisher) and TaqMan™ probe and primer sets (Thermo Fisher): CCL2 (Mm00441242_m1), IFNγ (Mm01168134_m1), IL-6 (Mm00446190_m1), IL-10 (Mm01288386_m1), TNF (Mm00443258_m1), Nphs1 (Mm01176615_g1), Havcr1 (Mm00506686_m1), BAFF (Mm00446347_m1), and TATA-Binding Protein  as an endogenous control. Probes against genes of interest were labeled with FAM, whereas the TBP probe was labeled with VIC. Data were analyzed using the 2escribed . Statistl-glutamine, 50 µM β-mercaptoethanol (Thermo Fisher), 25 mM HEPES (Thermo Fisher), 1% penicillin–streptomycin (Sigma Aldrich), 1 mM sodium pyruvate (Thermo Fisher), and 1 × MEM non-essential amino acids (Thermo Fisher)) with 1 mg/mL collagenase IV (Thermo Fisher) for 30 min at 37 °C. Kidney pieces were further mechanically disrupted by sequential pipetting using 25 mL, 10 mL, and 5 mL serological pipettes, then passed through the 40-µm strainer as described for the spleens. Both spleen and kidney single-cell suspensions were incubated with RBC lysis buffer (Millipore Sigma) for 3 min, then prepared for flow cytometric analysis. Briefly, cells were incubated with Fc block  for 10 min, followed by a 20-min staining with fluorochrome-conjugated antibodies: CD45-PE , CD11b-FITC , F4/80-PerCP-Cy5.5 , Ly6G-PE-Cy7 , Ly6C-APC , CD11c-APC-R700 , CD86-PE-Cy7 , CD206-AlexaFluor700 , Siglec-1-APCeFluor780 , CD3-FITC , CD4-PE , CD25-PerCP-Cy5.5 , CD19-PE-Cy7 , and CD5-APC-R700 . For intracellular staining, cells were fixed and permeabilized using eBioscience Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher) followed by a 30-min staining with anti-Arg1-APC  and anti-FoxP3-APC antibodies . Events were acquired using Attune NxT cytometer (Thermo Fisher) and analyzed using FlowJo v.10.8 (BD Biosciences).A portion of the spleen and kidney was processed to single cells for flow cytometric analysis. Spleen fragments were dissociated using a plunger from a sterile 1-mL syringe (BD) and a 40-µm strainer (Bio Basic). Kidney pieces were digested in complete DMEM media (2 mM t-test (2 groups). Data that did not follow a normal distribution were analyzed by the non-parametric Kruskal–Wallis test and corrected for multiple testing using Dunn’s test (> 2 groups) or the Mann–Whitney test (2 groups). The Pearson correlation coefficient was used to determine the relationship between kidney function  and pathology scores . Data were analyzed in GraphPad Prism 9.0.0. The results were considered statistically significant when p-values were less than 0.05.Data are presented as means ± standard error of the mean (SEM) unless otherwise indicated in the figure legends. All data were assessed for normality by the Shapiro–Wilk test. Data following a normal distribution were analyzed by ordinary one-way ANOVA and corrected for multiple comparisons using Dunnett’s test (> 2 groups) or by Student’s Given that our previous study with HSPB5 demonstrated attenuated LN in the MRL/lpr lupus mouse model , we wereProgression of proteinuria in NZB/W F1 mice and disease activity in SLE patients are associated with elevated IL-6 levels . Thus, wThe observed improvement in renal function prompted us to examine whether HSPB5 played an immunoregulatory role in the spleen, where pathogenic lymphocytes are activated and expanded by APCs presenting autoantigens. To assess the effect of HSPB5 treatment on APC and lymphocyte activation in the spleen, we performed flow cytometry. HSPB5 had no effect on the distribution of T cells CD3 +), B cells (CD19 +), myeloid cells (CD11b +), neutrophils (CD11b + Ly6G +), or mDCs (CD11b + CD11c +) (data not shown). Myeloid cells were further evaluated for the expression of activation markers, CD86, and Siglec-1. Compared to the vehicle group, mice treated with HSPB5 had decreased expression of CD86 and Siglec-1 on splenic monocytes/macrophages (CD11b + F4/80lo)  and reduced total kidney lesions  in the HSPB5 group. Similarly, the decrease in BUN correlated with improved total glomerular lesion scores  and total kidney lesion scores  in HSPB5-treated mice. Treatment with methylprednisolone also alleviated kidney pathology, but the extent of the improvement was greater in HSPB5-treated mice . Importantly, IFNγ, IL-6, and BAFF were significantly reduced in HSPB5-treated mice , and BAFF. No major changes were observed in the expression of TNF and IL-10 between the vehicle and HSPB5 groups (Fig. The side effects from chronic glucocorticoid use contribute significantly to morbidity in LN . In thisA therapeutic approach that combines immunomodulation with immunosuppression represents an attractive strategy for the treatment of LN. This approach becomes even more attractive if it can reduce the use of glucocorticoids, as their side effects contribute to morbidity. In this report, we demonstrate that HSPB5 can induce regulatory lymphocytes in the spleen, reduce activation of splenic monocytes/macrophages, reduce systemic IL-6, reduce renal infiltration by inflammatory macrophages/monocytes, and ameliorate renal pathology, thus improving the renal function of NZB/W F1 mice. Importantly, the effects of HSPB5 were comparable and, in some instances, superior to methylprednisolone. The results from the LPS-induced endotoxemia study demonstrate that the co-formulation of HSPB5 with methylprednisolone can provide enhanced anti-inflammatory effects, even in the face of a potent and acute inflammatory insult. Taken together, these results suggest that HSPB5 treatment could reduce disease activity, prevent kidney injury, and be steroid-sparing in patients with LN.HSPB5-treated mice showed substantial improvements in renal pathology and outperformed methylprednisolone in reducing total glomerular and kidney lesions. These results are consistent with our previous study demonstrating improved kidney pathology in MRL/lpr mice treated with HSBP5 . FurtherImmune cell infiltration is a hallmark of LN and the nephritic kidney of NZB/W F1 mice. Monocytes are the most abundant infiltrating immune cell in LN patients and the most elevated in NZB/W F1 mice , 36. HSPKidney inflammation in LN is initiated by the deposition of immune complexes composed of autoantibodies; macrophages play an essential role in propagating this inflammation and causing renal damage , 43. PerHSPB5 treatment also had a profound effect on the induction of Tregs and Bregs in the spleen of NZB/W F1 mice. Tregs limit T effector cell proliferation by consuming local IL-2, secreting suppressive mediators such as IDO, IL-10, and TGFβ; by direct killing through granzyme B secretion; and by inhibiting the stimulatory capacity of APCs –51. BregWhen LN becomes clinically evident due to intrarenal inflammation and cellular injury, the autoimmune processes leading to these events have already been active for some time , 60. HigAdditional file 1.