Enchondromas (EC) and atypical cartilaginous tumours (ACT) of the knee joint represent benign/intermediate chondromatous neoplasms of the bone that are most commonly discovered incidentally. Based on small to intermediate-sized cohorts, the prevalence of cartilaginous tumours of the knee as visible in MRI is estimated at 0.2–2.9%. This study aimed at verifying/challenging these numbers via retrospective examination of a larger, uniform patient cohort.Between 01.01.2007 and 01.03.2020, 44,762 patients had received an MRI of the knee for any indication at a radiologic centre. Of these, 697 patients presented with MRI reports positive for cartilaginous lesions. In a three-step workflow, 46 patients were excluded by a trained co-author, a radiologist and an orthopaedic oncologist, as wrongly being diagnosed for a cartilage tumour.Of 44,762 patients, 651 presented with at least one EC/ACT indicating a prevalence of 1.45% for benign/intermediate cartilaginous tumours of the knee joint . As 21 patients showed 2 chondromatous lesions, altogether 672 tumours (650 ECs [96.7%] and 22 ACTs [3.3%]) could be analysed in terms of tumour characteristics: With a mean size of 1.6 ± 1.1 cm, most lesions were located in the distal femur (72.9%), in the metaphysis of the respective bone (58.9%) and centrally in the medullary canal (57.4%).This study revealed an overall prevalence of 1.45% for cartilage lesions around the knee joint. Whilst a constant increase in prevalence was found for ECs over 13.2 years, prevalence remained constant for ACTs. Enchondromas (ECs) and atypical cartilaginous tumours  are cartilaginous neoplasms located in the medullary canal . While EECs typically present clinically silent, with large lesions eventually causing pain or – especially in short bones – pathologic fractures , 5. TherAs all five studies were based on small to intermediate-sized cohorts, this study aimed at verifying these numbers via retrospective examination of a larger, uniform patient cohort with MRI scans of the knee.The current retrospective study was performed with data deriving from a private radiologic centre, performing – apart from numerous other imaging modalities – MRI scans of all body sites. The local medical ethics committee has approved the study (33–630 ex 20/21) that involved patients having received an MRI of the knee for any indication between 01.01.2007 and 01.03.2020. During that time, 44,762 patients had undergone at least one knee MRI scan. Of these, 5182 had undergone MRI scans of both knee joints, resulting in a total of 24,125 and 25,819 MRI scans of the left and right knee, respectively.All MRI reports of the knee were searched for at least one of the following terms by one of the co-authors (J.W.) who had performed an extensive study of literature and had received intensive training on imaging features of cartilage lesions before data acquisition: “Enchondrom” (enchondroma), “kartilaginäre Läsion” (cartilaginous lesion), “cartilaginäre Läsion” (cartilaginous lesion), “kartilaginärer Tumor” (cartilaginous tumour), “cartilaginärer Tumor” (cartilaginous tumour), “chondrogene Läsion” (chondrogenic lesion), “chondrogener Tumor” (chondrogenic tumour), “Chondrosarkom” (chondrosarcoma), “ACT - atypischer chondromatöser Tumor“ , “atypische chondromatöse Läsion“ .Altogether, 697 patients’ MRI reports were positive for at least one search term and were subsequently analysed in further detail Fig. . Of thesReports of the remaining 676 patients were re-examined together with related MRI scans, and a definitive radiologic diagnosis of cartilaginous tumours was confirmed in 635 patients. However, 41 patients had inconclusive reports and images. Therefore, the advice of a senior radiologist was sought for these cases. Thereafter, 20 more patients were excluded, as they did not show typical features of EC/ACT, resulting in 656 patients ultimately eligible.MRI-based differentiation into EC Fig.  and ACT Overall, 651 patients had a cartilaginous tumour, with 21 patients presenting with 2 cartilaginous lesions at the same time, resulting in 672 cases of EC/ACT.MRI was considered positive for a cartilaginous tumour on identification of a smooth or lobulated lesion that presented itself as a focal geographic area within the bone marrow, showing low signal intensity on proton-density-weighted and on T1-weighted images and high signal intensity on proton-density fat-suppressed images. Lesions positive for these characteristics but strictly subchondral in location had to be excluded, as they most likely represent different entities such as subchondral cysts, intraosseous ganglia, subchondral edema or contusion.Apart from patient gender and age, the following tumour-specific features were ascertained: lesion size , lesion site , tumour location , endosteal scalloping, perilesional edema and periosteal reaction. Furthermore, indication for MRI  and whether the patients had undergone dynamic contrast MRI was ascertained.Siemens Magnetom Skyra/Siemens Magnetom Vida; both Siemens Healthcare Diagnostics GmbH, Austria) with a 15/18-channel knee coil. Sequences acquired were (1) coronal proton density (PD) with fat suppression (FS) (field of view (VF) 160/140 mm; Matrix (M) 307 × 384/307 × 384; repetition time (TR) 3000/3200 ms; echo time (TE) 34/25 ms; slice thickness (ST) 3/3 mm; interslice gap (IG) 0.6/0.6 mm); (2) transversal PD with FS ; (3) sagittal PD with FS ; (4) coronal T1 weighted turbo spin echo (TSE)  180°/150°).MRI examinations were performed on two different 3T MRI systems (Gd-DTPA (dose 2 ml/kg body weight) via venous access was administered in 77 of 651 patients (81 of 672 tumours), followed by an MRI examination with the sequence “(4) coronal T1 weighted turbo spin echo” that was subtracted from the native T1 sequence. Additionally, T1 sequences with fat suppression in different dimensions were recorded.StataCorp, College Station, Texas, US). Patient demographics were summarized for based on the total number of patients with cartilaginous lesions. Tumour demographics were summarized from the total number of lesions found in patients. Prevalence was calculated based on the diagnosis of a benign cartilaginous lesion per patient, and not based on the number of cartilage lesions found in total. In line with this, patients without ECs/ACTs undergoing MRI scans of both knee joints during the defined period were counted once only for calculation of prevalence. Normally and non-normally distributed variables were given as means and corresponding standard deviations, as well as medians and corresponding interquartile ranges (IQR), respectively. Fisher’s exact test and t-test were used to assess differences in binary (or ordinary) and continuous variables, respectively, depending on radiological diagnosis. A p-value of < 0.05 was considered statistically significant.Statistical analyses were carried out with Stata Version 16.1 for Mac  amounted to 1.45%. In detail, the separate prevalence for ECs and ACTs was 1.4% and 0.05%, respectively. Over the years, a slight increase in prevalence was observed for ACTs and ECs, as well as ECs only, whereas the prevalence of ACTs did not equally increase  out of 44,762 patients with MRI scans of either knee joint taken from 01.01.07 to 01.03.20, a benign cartilaginous lesion was detected incidentally. In 17 of these patients, two lesions in the same knee were found. In additional 4 patients, a cartilaginous lesion was found both in the left and right knee, amounting to 672 tumours in total. Mean age of patients with EC/ACT was 52.1 ± 13.1 years, and 48.4% were males (n = 315). The reason for referral to MRI was suspected EC based on preceding imaging in 79 cases (12.2%), a suspected pathology other than EC in 463 cases (71.1%), and unknown in 109 cases (16.7%).Of the 672 lesions in total, radiological diagnosis was EC in 650 tumours (96.7%), and ACT in 22 lesions (3.3%). Mean tumour size of all cartilaginous lesions was 1.6 ± 1.1 cm. Thirty lesions (4.5%) were sized 4 cm or more, with the largest lesion measuring 9.0 cm . The most common location was the distal femur in 490 cases (72.9%), and the majority of lesions was located in the metaphysis . In relation to the medullary canal, 386 lesions were located centrally (57.4%), and the remaining 286 lesions peripherally (42.6%). Periosteal reaction was present in 2 cases (0.3%), and endosteal scalloping in 15  is an important differential diagnosis of intraosseous lesions around the knee, with a 28 times higher prevalence in comparison to their intermediate counterpart ACT (0.05%).There are some limitations that should be considered regarding the results of the current study. The herein observed prevalence of ACTs around the knee has to be interpreted with caution, given that differentiation between EC and ACT was MRI based only, and that radiological features indicative of ACT are not utterly defined. Related to this, radiological diagnosis had not been confirmed with histopathology. Given the sampling error associated with differentiation between EC and ACT upon histopathological analysis , it is bIn summary, the current study revealed an overall prevalence of 1.45% for cartilage lesions around the knee joint. Of all cartilage lesions diagnosed on MRI, 3.3% exhibited features indicative of ACT. Whilst a constant increase in prevalence was found for enchondromas over 13.2 years, prevalence remained constant for ACTs.


Drosophila model to test links between glycosphingolipids, neurotransmission and neurodegeneration. We found that Npc1a nulls had elevated neurotransmission at the glutamatergic neuromuscular junction (NMJ), which was phenocopied in brainiac (brn) mutants, impairing mannosyl glucosylceramide (MacCer) glycosylation. Npc1a; brn double mutants had the same elevated synaptic transmission, suggesting that Npc1a and brn function within the same pathway. Glucosylceramide (GlcCer) synthase inhibition with miglustat prevented elevated neurotransmission in Npc1a and brn mutants, further suggesting epistasis. Synaptic MacCer did not accumulate in the NPC model, but GlcCer levels were increased, suggesting that GlcCer is responsible for the elevated synaptic transmission. Null Npc1a mutants had heightened neurodegeneration, but no significant motor neuron or glial cell death, indicating that dying cells are interneurons and that elevated neurotransmission precedes neurodegeneration. Glycosphingolipid synthesis mutants also had greatly heightened neurodegeneration, with similar neurodegeneration in Npc1a; brn double mutants, again suggesting that Npc1a and brn function in the same pathway. These findings indicate causal links between glycosphingolipid-dependent neurotransmission and neurodegeneration in this NPC disease model.The lipid storage disease Niemann Pick type C (NPC) causes neurodegeneration owing primarily to loss of NPC1. Here, we employed a  Summary: Genetic analyses in a Drosophila disease model show that loss of Npc1 and glycosphingolipid pathway synthesis elevate glutamatergic neurotransmission within a common functional pathway, and this pathway links to subsequent neurodegeneration. NPC1 gene  is a well-characterized model glutamatergic synapse , the rate-limiting step in sphingolipid synthesis, exhibit elevated NMJ neurotransmission from fewer NMJ synaptic boutons and also display increased apoptosis , encoding the beta-1,3-N-acetylglucosaminyltransferase that glycosylates MacCer (egghead (egh), encoding the beta-4-mannosyltransferase that glycosylates GlcCer to MacCer  knockdown of Npc1a and brn demonstrated a presynaptic requirement. In Npc1a; brn double mutants, we found no further increase in neurotransmission, indicating that the two genes operate within the same pathway. We further found that Npc1a; egh double mutants had elevated transmission comparable to that in Npc1a nulls alone, suggesting that MacCer is not responsible for synaptic dysfunction. Consistently, the GlcCer synthase inhibitor miglustat blocked elevated neurotransmission in Npc1a and brn mutants. MacCer labeling showed no synaptic accumulation in Npc1a nulls, but maintained elevation in Npc1a; brn double mutants. Moreover, Npc1a mutants had increased GlcCer levels, suggesting that GlcCer is likely to be pathogenic. Assaying neurodegeneration with terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) labeling  as an ecdysone precursor to circumvent the early larval lethality resulting from the lack of steroid hormone signaling . The 57a; 2-286Npc1a Gal4>UAS-Npc1a::YFP line was compared to the 2-286 Gal4 driver alone control . Electrical stimulation of the wandering third-instar motor nerve with a glass suction electrode in 1.5 mM [Ca2+] was used to drive NMJ glutamatergic transmission onto ventral longitudinal muscle 6 , voltage clamped at −60 mV. To assay synaptic transmission strength, ten consecutive excitatory junction current (EJC) traces were collected (0.2 Hz) from each NMJ and then averaged for each single data point , but both were significantly increased from that of the control  closely phenocopied the 7-dehydrocholesterol-fed EJC amplitude elevation (n=18) compared to that of the 2-286 Gal4/+ control of 1.00±0.059  of beta-1,3-N-acetylglucosaminyltransferase that glycosylates MacCer  normalized EJC amplitude , fs.107brn mutants had increased synaptic transmission , which was significantly elevated , miglustat-fed Npc1a nulls had an EJC amplitude of 1.14±0.10 (n=9), which was not significantly different from that of controls  was not significantly different from that of controls  to drive both Npc1a and brn RNAi knockdown, together with TEVC recording at the NMJ to measure changes in glutamatergic neurotransmission. Compared to the transgenic driver alone control , targeted neuronal knockdown of both Npc1a and brn increased synaptic function (Npc1a RNAi elevated the EJC amplitude to 1.211±0.07 (n=9), normalized to the control 1.00±0.05 (n=14), which was a significant increase , a significant increase compared to that of control (P=0.0043), phenocopying the NPC model   , we nextt result , then GSNpc1a57a , brnfs.1 mutants , and the57a; brnfs.107Npc1a double mutants, to find the same highly elevated synaptic transmission as in each single homozygous 57aNpc1a and fs.107brn mutants alone , which was significantly increased compared to that of matched controls , which was also significantly elevated compared to that of 1118w controls , with a similar significant increase compared to that of the controls  or the fs.107brn (P=0.824) single mutant. These results indicate that brn and Npc1a function in the same pathway to increase synaptic transmission, as opposed to different pathways that would cause an additive increase in the double mutant EJC amplitude , the EJC amplitude of the 57aNpc1a single mutant was 1.408±0.09 (n=9) and that of the 57a; egh7Npc1a double mutant was 1.42±0.12 (n=9), and both were significantly increased from that of 1118w , Npc1a nulls (57aNpc1a), GSL pathway mutants  and 57a; brnfs.107Npc1a double mutants. Previous work has shown an increase in MacCer in fs.107brn/1.6P6brn mutants and decrease in MacCer in 62d18egh mutants at the Drosophila NMJ , 57aNpc1a nulls showed a mean fluorescent intensity of 0.93±0.07 (n=18), which is not different from that of controls  normalized to the 1118w genetic background control fluorescent level of 1.00±0.07 (n=10), a significant elevation in the mutant , the 57a; brnfs.107Npc1a double mutants exhibited a mean synaptic MacCer intensity of 1.44±0.15 (n=18), which is significantly increased compared to that of control  to assay the overall GlcCer levels . Levels of HexCer_NS were significantly increased in the NPC model (57aNpc1a nulls), with a measured magnitude of 23,146±1320  compared to the 1118w genetic background control with a normalized level of 14,812±1174 (n=5), which is a significant elevation  compared to 1118w controls with a normalized level of 39,561±6460 . In all four genotypes, we co-labeled for TUNEL and Brp, analyzing TUNEL-positive area in brain cerebral lobes and VNC. Representative images and data quantification are shown in To test whether GSL-mediated neurotransmission excitotoxicity is linked to neurodegeneration in our NPC disease model, we next used TUNEL labeling to analyze larval neuronal cell death . The wan1118w animals exhibit considerable normal TUNEL-labeled neuronal death in the brain cerebral lobes, but very limited neurodegeneration in the VNC , Npc1a nulls showed 50% higher TUNEL area in the brain cerebral lobes , which is a significant elevation , Npc1a nulls (57a/Npc1a1Npc1a) showed an 8-fold increase in TUNEL area in the central VNC , which is also a significant elevation  again showed essentially no TUNEL-labeled neuronal cell death along the central VNC midline and no indication of RP3 motor neuron death  showed extensive TUNEL labeling along the VNC midline, albeit with very limited overlap with RP3 motor neurons (n=11), with a trending increase in the NPC disease model , but no significant change in RP3 motor neuron TUNEL label . This trend suggests that RP3 motor neurons are in the earliest stages of neurodegeneration, but the majority of NPC model TUNEL-labeled cell death along the VNC midline clearly did not occur within these motor neurons.Given the pronounced TUNEL-labeled cell death along the VNC midline in PC model . To testP green; . We co-lcontrols . Controlon death , left. I neurons , right. 7egh and fs.107brn mutants showed increased TUNEL labeling in cerebral lobes  revealed greater TUNEL-positive area in fs.107brn mutants , 57a; brnfs.107Npc1a double mutants  and especially 7egh mutants  likewise revealed elevated TUNEL in fs.107brn  and 7egh  revealed similarly elevated TUNEL area in 57a; brnfs.107Npc1a mutants . Overall, our findings link GSLs to the maintenance of normal levels of neurotransmission and neurodegeneration.We next tested GSL pathway mutants. Both al lobes  and VNC al lobes . The Npclabeling . Quantifs or VNC . Quantifs or VNC . These rDrosophila NPC disease model. Our previous work in this model demonstrated age-dependent cholesterol accumulation and neurodegeneration in the adult brain, and shortened adult lifespan  (Npc1a NMJ synaptic transmission defect builds on our previous work showing defects in extracellular photoreceptor electroretinogram (ERG) recordings  , in contuscle 6) . Moreoveuscle 6) . Glutamauscle 6) , anotheruscle 6) . Additiouscle 6)  and couluscle 6) . The Npccordings . ERG tra defects , Npc1a mbrn mutant phenocopies the elevated synaptic transmission observed in the Npc1a mutant. These brn mutants accumulate truncated GSLs and lack elongated GSLs  and postsynaptic (DLG) membrane markers at NMJ synaptic boutons, as well as widely in the surrounding extrasynaptic muscle  or in different, parallel pathways (additive phenotypes)  . Importasmission , the synsmission ,b. In orDrosophila NPC disease model shows no increase in synaptic MacCer levels. Our work confirms previous studies that indicate that brn mutants, in contrast, display elevated MacCer at the NMJ  is causative of neuronal death. However, neurodegeneration in both Npc1a and GSL mutants is not prevented by miglustat feeding. A previous study reported that miglustat prolongs survival of Purkinje neurons in the feline NPC model . All stocks were grown on standard corn meal/molasses food at 25°C. The genetic background control was 1118w. Null Npc1a alleles (1Npc1a and 57aNpc1a) were used in both homozygous and trans-heterozygous combinations   and RP3 motor neuron driver CCAP-Gal4 . The UAS responder lines were UAS-npc::YFP , UAS-eGFP , UAS-Npc1a RNAi  and UAS-brn RNAi . Transgenes were introduced into mutants using standard recombination techniques.All mutant and transgenic lines were obtained from the Bloomington inations . Controlethality . The via (M308K) . The viaficiency . All the286-Gal4 , pan-neu2, 1.0 mM CaCl2, 70 mM sucrose and 5 mM HEPES (pH 7.2). Dissected preparations were imaged with a Zeiss Axioskop microscope using a 40× water-immersion objective. Ventral longitudinal muscle 6 in abdominal segments 3/4 was impaled by two KCl (3 M)-filled microelectrodes  with ∼15 M Ω input resistance. The cut motor nerves were sucked into a fire-polished glass suction electrode and stimulated using 0.5 ms suprathreshold voltage stimuli at 0.2 Hz (Grass S88 stimulator) to generate EJC recordings. Nerve stimulation-evoked EJC recordings were filtered at 2 kHz. To quantify EJC amplitude, ten consecutive traces were averaged to produce a mean peak value. Data were acquired with Clampex 9.0 and analyzed with Clampfit 9. Miglustat (MedChem Express) in dimethyl sulfoxide (Thermo Fisher Scientific) was fed at concentrations of 0.1-10 ng/ml (Recording was done as previously described . Briefly10 ng/ml .z-stack areas of maximum projection using the threshold and wand-tracing tools in ImageJ. All imaging setting were kept constant for comparisons within every set of experiments.Antibody labeling was done as previously described . Briefly3OH), acetonitrile (CH3CN), water (H2O), formic acid, isopropyl alcohol, chloroform (CHCl3), ammonium acetate, ammonium bicarbonate, ammonium formate (Thermo Fisher Scientific) and tert-butyl methyl ether  were used for LC-MS analyses. Global untargeted lipidomics was performed in the Center for Innovative Technology at Vanderbilt University. Five samples each of 1118w and 57aNpc1a genotypes were prepared by homogenizing whole third-instar larvae weighing 30-50 mg, rinsing with 50 mM ammonium formate and flash freezing in liquid nitrogen with storage at −80°C. Samples were thawed on ice with 0.5 ml of cold CH3OH:CH3CN:H2O  with 50 mM ammonium bicarbonate (lysis buffer), and sonicated by probe tip for 10 pulses at 50% power, followed by vortex mixing for 10 s. Normalized volumes based on the initial dry larvae weights were aliquoted and diluted to 200 µl with lysis buffer followed by an addition of 800 µl CH3OH. Samples were vortexed for 30 s and incubated at −80°C overnight for protein precipitation. Samples were then centrifuged at 4°C and 21,000 g for 15 min, and the supernatant was dried using a cold vacuum centrifuge. Dried samples were reconstituted in 100 µl H2O followed by 100 µl CH3OH and 10 µl SPLASH® LIPIDOMIX® Mass Spec Standard (Avanti Polar Lipids), and incubated for 10 min at room temperature. Next, liquid–liquid extraction was performed by adding 800 µl MTBE with 30 s of vortex mixing, followed by 10 min incubation on ice and centrifugation at 4°C and 21,000 g for 15 min. The hydrophobic supernatant (800 µl) was isolated and dried using a cold vacuum centrifuge, and extracts were stored at 4°C. Dried lipid fractions were reconstituted in 100 µl CH3OH:CHCl3  with exogenous sphingolipid standards (n=2) to assess instrument variability. Equal volumes (25 µl) of each sample were combined to create a pooled quality control (QC). Samples were analyzed using an Agilent 1290 LC system interfaced with an Agilent 6560 Ion Mobility Quadrupole Time-of-Flight . Electrospray ionization  was operated in positive polarity with sheath gas flow at 11.8 l/min, sheath gas temperature of 300°C, capillary at 3500 V, nozzle at 2000 V, octupole radio frequency at 750 peak-to-peak voltage (Vpp), drying gas temperature of 280°C, drying gas flow of 5 l/min and nebulizer at 10 psi. Agilent MassHunter Acquisition software (B.09) was used to operate the instrumentation and to acquire liquid chromatography–ion mobility–tandem mass spectrometry (LC-IM-MS/MS) data in the 75-1700 m/z range. A QC sample was injected seven times before the first sample to condition the liquid chromatography column and then injected after every five samples to assess instrument reliability through principal component analysis. Samples were injected randomly with 10% of samples reinjected for quality control purposes. Calibration solution (Agilent Tuning Mixture) was measured at the beginning and end of analysis for mass error quality assurance. Solvent blanks were used to monitor for spectral contaminants and to generate a tandem mass spectrometry (MS/MS) exclusion list. Fragmentation spectra was acquired for the pooled QC via three iterative, top two, data-dependent MS/MS acquisitions. Progenesis QI software  was used to perform data analysis including retention time alignment, peak picking and deconvolution, abundance normalization and one-factor ANOVA. Lipid annotations were performed with reference to in-house and online databases MS-DIAL  to assay neuron apoptosis as previously described . Briefly neurons , the CCAt-test or a non-parametric Mann–Whitney test. All comparisons between three or more experimental groups were analyzed with a one-way ANOVA or non-parametric Kruskal–Wallis test. All graphs were made using Prism software to show all individual data points normalized to the control. All data in this study were collected and analyzed continuously. All graphs show the mean±s.e.m., with significance indicated as *P<0.05, **P<0.01, ***P<0.001 and P>0.05 [not significant (ns)].All statistical analyses were performed using Prism software . All data were subject to D'Agostino-Pearson normality tests. If the data fell within the Gaussian distribution, a ROUT (Q=1%) outlier test was performed. All comparisons between two experimental groups were performed using a parametric unpaired two-tailed Student's 10.1242/dmm.050206_sup1Supplementary informationClick here for additional data file.


The findings of this study demonstrate that early identification of MODY and diagnosis through genetic testing are critical for the treatment of the patient.Maturity-onset diabetes of the young type 3 (MODY3) is a specific type of diabetes mellitus with inherited impairment of the islet β cell function due to the mutation in the hepatocyte nuclear factor 1α (HNF1α) gene. It is a rare condition and easily misdiagnosed as T1DM or T2DM. In this study, the clinical features of two unrelated Chinese MODY3 probands were described and analyzed. Next-generation sequencing was performed to identify the mutated genes, and Sanger sequencing was employed to verify the location of the pathogenic variant in the related family members. It was found that proband 1 inherited a start codon mutation c.2T>C (p.Met1?) in exon 1 of the HNF1α gene from his affected mother, and proband 2 inherited a frameshift mutation c.1136_1137del (p.Pro379fs) in exon 6 of the HNF1α gene also from her affected mother. Proband 1 and proband 2 differed in islet dysfunction, complications, and treatments due to their different disease durations and levels of hemoglobin A To datel-pyruvate kinase, associated with insulin secretion and glucose metabolism [Among different subtypes of MODY, MODY3 was found to be associated with mutations in the hepatocyte nuclear factor 1α (HNF1α) gene. The prevalence of MODY3 in the general population ranges from 0.02 to 0.04%  [In this article, a start codon mutation and a frameshift mutation of the HNF1α gene in two unrelated Chinese families were reported. Based on the results of genetic testing, we facilitated precise diagnosis and modified appropriate treatment.22.12. No abnormalities were indicated in cardiopulmonary and abdominal examinations. Bilateral dorsalis pedis pulsed normally. The laboratory examination results were as follows: fasting blood glucose (FBG): 6.82 mmol/l; 2 h-postprandial blood glucose (PBG): 11.12 mmol/l; fasting C-peptide: 0.38 nmol/l; 2 h-postprandial C-peptide: 0.97 nmol/l; hemoglobin A1c (HbA1c): 6.80%; and urine glucose: 4+. The diabetic autoimmune antibodies were negative. Besides, no abnormal results in biochemical examination, urinary microalbumin creatinine ratio (UACR), abdominal B-ultrasound, electroneuromyography, or fundus screening were detected (c.2T>C (p.Met1?) in the start codon in exon 1 of the HNF1α gene but not his father  on July 5, 2018, due to hyperglycemia revealed by a routine examination performed 6 months earlier. He had no symptoms of diabetes such as polydipsia, polyuria, overeating, and weight loss. The proband’s mother and maternal grandmother had already been diagnosed with diabetes . Physicadetected . Based os father .For the treatment, the proband was first misdiagnosed as T2DM in the community hospital and treated with metformin. However, this therapy appeared to be useless for controlling blood glucose within the optimal range. After diagnosis in our hospital, metformin was discontinued and gliclazide sustained-release tablet, 30 mg once a day, was prescribed. During a long follow-up, the proband’s blood glucose control was satisfactory. Currently, he is still receiving gliclazide sustained-release tablet at the same dosage, and his FBG, 2 h-PBG, and HbA1c levels are controlled at 4.5–6.0 mmol/l, 6.8–7.5 mmol/l, and 6.10%, respectively. His impaired islet function shows no serious progression, and there is no occurrence of diabetes complications.2.22. No abnormalities were indicated in cardiopulmonary and abdominal examinations. Bilateral dorsalis pedis pulsed normally. The results of laboratory examination were as follows: FBG: 8.98 mmol/l; 2 h-PBG: 20.37 mmol/l; fasting C-peptide: 0.23 nmol/l; 2 h-postprandial C-peptide: 0.31 nmol/l; HbA1c: 14.4%; total cholesterol: 4.98 mmol/l; urine glucose: 4+; and urine protein: 1+. The diabetic autoimmune antibodies were negative. The UACR was 417.8 mg/g. Electroneuromyography showed severe and multiple damages in the peripheral nerve of limbs. Biochemical examination and abdominal ultrasound or fundus photography revealed no abnormalities. Color Doppler ultrasound examination showed that her lower extremity arteries were not atherosclerotic (c.1136_1137del (p.Pro379fs) in exon 6 of the HNF1α gene, but not her father  was administered by subcutaneous injection at 15 U once a day, and preprandial insulin (insulin lispro) was administered 5 min before breakfast (4 U), lunch (5 U), and dinner (5 U), respectively. According to her diabetic complication, this therapy was further supplemented with the treatment of nerve nourishing, microcirculation improvement, and urinary protein reduction. Currently, her blood glucose level fluctuates within a reasonable range, and her HbAlc level is maintained at 6.9% with insulin therapy. There is no significant progression in complications.Ethics approval and consent to participate: The present study was approved by The Affiliated Changsha Central Hospital, Hengyang Medical School, University of South China . The patients provided written informed consent for the publication of individual clinical details and all the accompanying images.3MODY3 is a monogenic disease with autosomal-dominant inheritance due to HNF1α haplo-insufficiency or dominant-negative effects. The HNF1α gene is located on chromosome 12 in the region 12q24.2 . The mutHNF1α is mainly expressed in the pancreas, kidneys, and intestine. In β-cells of the islet, HNF1α mutations can reduce β-cell proliferation and affect the biological processes including glucose transport, glucose metabolism, and glucose-stimulated ATP production, which will activate ATP-sensitive channels and attenuate depolarization of cell membranes and internal flow of calcium ion, and thus reduce insulin secretion . In the Sulfonylureas, as the preferred treatment for patients with MODY3, can specifically bind the SUR1 subunit of ATP-dependent potassium channels (KATP) and stimulate insulin secretion . SulfonyAt present, the main obstacle to the diagnosis of MODY lies in the lack of in-depth understanding and clinical suspicion, and the high cost of genetic testing. In the present study, we first detailed two heterozygous variants (c.2T>C and c.1136_1137del) in HNF1α, along with clinical manifestations, diagnosis processes, treatments, and follow-up of two patients with MODY3 in two independent Chinese families. The two patients and their families were initially misdiagnosed as T2DM and were diagnosed as MODY3 after genetic testing. These two mutations are rare, and no detailed description of patients with the same mutations has been reported until our study, which highlights our novelty. Based on these two clinical cases, we emphasized the importance of early recognition of the disease signs and symptoms, which would help improve the awareness of MODY. In clinical practice, stringent screening criteria should be established to achieve a balance between cost-effectiveness and identification. For example, the results of pancreatic autoantibodies, the family history of diabetes, the age of onset, and endogenous insulin secretion should be taken into account to determine whether genetic testing is required. Once suspicion is raised, genetic testing should be performed to support the diagnosis of MODY, allowing early treatment and accurate family screening . Clinica


JAC-Antimicrobial Resistance, Volume 5, Issue Supplement_1, February 2023, dlac133.018, https://doi.org/10.1093/jacamr/dlac133.018This is a correction to: Rob Oakley and others, P14 Improving adherence to and effectiveness of an adult critical care vancomycin continuous infusion protocol: a pilot quality improvement and administration data accuracy project, In the originally published version of this manuscript, the author and affiliations list details were incomplete.These should read:1, Ha Trinh1, Ting Yau1, Sarah Korshid1, Dagan Lonsdale1“Robert Oakley1 St George's University Hospitals NHS Foundation Trust, St George's University of London” instead of:1“Rob Oakley1 St George’s University Hospitals NHS Foundation Trust, UK”.These details have been emended in the paper online.


Background: Fibromyalgia (FM) is characterized by complex pain symptoms lacking impersonal considerations in diagnosis and treatment evaluation, which often happens in women. Chronic and persistent widespread pain is the key symptom disturbing patients with FM, leading to depression, obesity, and sleep disturbances. Toll-like receptor 4 (TLR4) activation produces a harmful sensory input involved in central pain; this is the focus of this study. Electroacupuncture (EA) has beneficial effects in reducing FM pain, but its connection with TLR4 signaling is still unknown. Methods: Intermittent cold stress significantly induced mechanical and thermal pain. EA, but not sham EA, reliably attenuated mechanical and thermal hyperalgesia. The increased inflammatory mediators in FM mice were reduced in the EA group, but not in the sham group. Results: All TLR4 and related molecule levels increased in the FM mice’s hypothalamus, periaqueductal gray (PAG), and cerebellum. These increases could be attenuated by EA but not sham stimulation. Activation of TLR4 by lipopolysaccharide (LPS) significantly induced FM and can be further reversed by a TLR4 antagonist. Conclusions: These mechanisms provide evidence that the analgesic effect of EA is related to the TLR4 pathway. In addition, we showed that inflammation can activate the TLR4 pathway and provided new possible therapeutic targets for FM pain. Fibromyalgia (FM) is accompanied by chronic general musculoskeletal pain lasting over 3 months and is highly associated with sleep disturbances, fatigue, and cognitive and somatic symptoms. These symptoms may persist over several years and cause extensive healthcare use . For cliToll-like receptor (TLR) 4 is a type I transmembrane protein that can be activated by bacterial lipopolysaccharide (LPS), further producing proinflammatory cytokines and interferons to initiate inflammatory and immune responses ,14. TLRsAcupuncture has long been used as an integrative therapy for several diseases, including pain management, with a lower risk of side effects. Traditional manual acupuncture and electroacupuncture (EA) have been reported to result in pain improvement. Acupuncture has been reported to attenuate inflammatory pain , osteoarThe current study aimed to explore the effect of EA on cold-stress-induced FM pain (CSP). Particularly, we wanted to identify whether inflammatory mediators and related signaling pathways were involved in EA analgesia in the mouse brain. We hypothesized that inflammatory mediators would increase in CSP mice and activate various receptors, including TLR4. Our data show new evidence of EA and TLR4 in the central sensitization of CSP. Furthermore, we propose that EA can attenuate CSP by regulating inflammatory factors and related molecules. Activation of TLR4 by LPS significantly induced CSP and can be further reversed by a TLR4 antagonist. These results may suggest the clinical applicability of EA for treating CSP.A total of 40 female C57BL/6 mice, which were aged 8–12 weeks old, were used in this study. After their arrival, the animals were held in a 12 h light–dark cycle and received food and water freely. The anticipated model size of 10 mice per group was counted as the amount needed for an alpha value of 0.05 and a power of 80%. The use of mice and their suffering were reduced according to animal research ethical standards. The usage of mice was allowed by the Institute of Animal Care and Use Committee of China Medical University (grant no. CMUIACUC-2019-106), Taiwan, and followed the Guide for the Use of Laboratory Animals . Mice were allocated into four main groups: normal mice ; fibromyalgia mice (Group 2: FM); 2 Hz EA group (Group 3: 2 Hz EA); and sham EA group (Group 4: sham EA).Mice were maintained in the laboratory at 24 ± 1 °C before the experiments. The intermittent FM process, which was not achieved in normal mice, required that two mice were accommodated in a flexible cage (13 cm × 18.8 cm × 29.5 cm) that was circumscribed with a stainless mesh. On day 0, the mice were placed in a cold chamber at 4 °C overnight (from 4:00 p.m. to 10:00 a.m.). The mice were subsequently moved to 24 °C for 30 min at 10:00 a.m. and further transported to the cold room for 30 min. The course continued till 4:00 p.m. The mice were then placed in the 4 °C cold room overnight. Normal mice were maintained at 24 ± 1 °C . Mouse pAll mice were anesthetized with 5% isoflurane at the beginning and 1% isoflurane for maintenance. Stainless-steel acupuncture needles  were bilaterally implanted into the ST36 acupoints of mice under anesthesia. The mouse ST36 acupoint is located ~3–4 mm deep and 1–2 mm lateral to the knee joint. In the EA group, electrical stimuli were delivered by a Trio 300 stimulator  by using 1 mA for 20 min at 2 Hz with a pulse width of 100 μs. Muscle twitching was observed during EA treatment as a de-qi sensation. Subsequent FM induction, EA management was performed from day 5 to 7. The same protocol was applied in the sham group but without electrical stimulation .After FM induction, either mechanical or thermal hyperalgesia was verified thrice from day 5 to day 7. Before behavior examinations, mice were relocated to the behavioral room and left to adjust to the room for ≥30 min. All tests were achieved at 24 ± 1 °C; the stimuli were tested when the mice were silent. Mechanical hyperalgesia was confirmed by applying force to stimulate trice the electronic von Frey filament . Furthermore, the Hargreaves’ test was applied to measure thermal hyperalgesia by analyzing the latency-to-thermal stimulation thrice by using Hargreaves’ test IITC analgesiometer . In the Hargreaves’ test, the pain threshold was measured via the latency of paw withdrawal time .3VO4, 0.02% NaN3, and 1× protease inhibitor cocktail . The proteins were further exposed to 8% Tris-Glycine-SDS glycine gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in TBS-T buffer  and incubated with a primary antibody against anti-tubulin , anti-Iba1 , anti-TLR4 , anti-MyD88 , anti-TRAF6 , anti-pERK , anti-pJNK , anti-pp38 , and anti-pNFκB  in TBS-T with 1% bovine serum albumin (BSA) for 1 h at 24 ± 1 °C. A commercial peroxidase-conjugated anti-rabbit antibody, anti-mouse antibody, or anti-goat antibody (1:5000) were used as secondary antibodies. Western blot bands were imagined through a heightened chemiluminescent substrate kit  with LAS-3000 Fujifilm . The concentration of each band was quantified by using NIH Image J 1.46 software . α-tubulin was set as the internal controller.Mice were anesthetized with 1% isoflurane and then underwent cervical dislocation. The hypothalamus, periaqueductal gray (PAG), and cerebellum VI and VII sections were removed for protein extraction. Samples were first stored at 4 °C and then kept at −80 °C, awaiting protein extraction. Sample proteins were homogenized with a radioimmunoprecipitation lysis buffer containing 50 mM Tris–HCl pH 7.4, 250 mM NaCl, 1% NP-40, 0.5% SDS, 0.5% deoxycholate, 5 mM EDTA, 50 mM NaF, 1 mM NaMice were anesthetized with 5% isoflurane and intracardially perfused with 0.9% saline and then 4% paraformaldehyde. The mouse brain was proximately separated and postfixed by 4% paraformaldehyde at 4 °C for over 3 days. The brain was then immediately stored in 30% sucrose for cryoprotection for 24 h at 4 °C. Then, the sample was quickly frozen by using liquid nitrogen and then stored at −80 °C. Frozen sections (12 images captured per group) were cut at 20 μm thickness and immediately placed on glass slides. The tissues were then fixed with 4% paraformaldehyde and incubated with a blocking solution  for 1 h at 24 ± 1 °C. The tissues were then incubated with the primary antibody against anti-Iba1 , anti-TLR4  in 1% BSA solution at 4 °C overnight. Tissues were further incubated with secondary antibodies (1: 500), 488-conjugated AffiniPure donkey anti-rabbit IgG (H + L), 594-conjugated AffiniPure donkey anti-goat IgG (H + L), and peroxidase-conjugated AffiniPure donkey anti-mouse IgG (H + L) for 2 h at 24 ± 1 °C. Tissues were detected by using an epifluorescence microscope  with a 20× numerical aperture (NA = 1.4) objective. Image colocalization was adjusted by using NIH Image J 1.46 software.Mice were anesthetized by using isoflurane with their heads fixed in a stereotaxic cannula, and the cannula was implanted at the ventricle site. The stereotaxic cannula was subsequently placed 0.5 mm in the anteroposterior axis, ±1 mm in the mediolateral axis, and −2.5 mm in the dorsoventral axis below the cortical surface. This cannula was a 23 gage, including a 2 mm stainless steel. In addition, it was immobile at the skull with a dental glue. Subsequently, the cannula was implanted and linked to a Hamilton syringe with a PE tube . Totally, 5 μL of LPS/PBS (5 μL/ventricle) was injected over a period of 5 min by using a syringe pump . After the injection, the cannula was left at the ventricle for an additional 2 min to allow the LPS to diffuse. A 25 μM TIRAP was used as an antagonist of TLR4.p < 0.05 indicated the threshold for statistical significance.Statistical analysis was performed with SPSS statistical software. All results are shown as mean ± standard error (SEM). Shapiro–Wilk examination was used to test the normal distribution of the data. Statistical differences were investigated through repeated-measures ANOVA tests. The post hoc Tukey’s test was performed after ANOVA. p < 0.05, F = 24.41, n = 10), as shown by the von Frey test. Mechanical hyperalgesia was substantially alleviated by EA, but not sham EA . This cold stress model reliably mimicked clinical symptoms, including thermal hyperalgesia. Next, we examined whether EA could alleviate thermal pain in FM mice. The Hargraves’ test showed significant thermal hyperalgesia, as measured by the paw withdrawal latency after CSP induction  = 30.02, n = 10). The hyperalgesic latency could be further reversed by EA but not by sham EA, suggesting an acupoint-specific effect .We tested the therapeutic effects of EA in a mouse FM model to address EA function in the subacute pain model to verify whether EA can reduce FM. Before FM induction, all groups showed similar mechanical thresholds. Cold stress significantly induced mechanical pain . Additionally, after a series of EA treatments, inflammatory cytokine levels were dramatically decreased , which did not occur with sham EA treatment .Since central inflammation was described in clinical patients, we next examined inflammatory mediators in FM mice via Bio-Plex ELISA. In normal mice, several inflammatory cytokines were recorded at a low level in basal conditions. Furthermore, we found that IL-1β, IL-2, IL-5, IL-6, IL-9, IL-12, IL-17A, TNF-α, IFN-γ, and MCP-1 all increased in FM mice 7 days after induction  = 20.27 and F = 22.88, n = 6, respectively). EA at 2 Hz reliably reduced Iba1 and TLR4 overexpression  but not sham EA . Furthermore, we measured MyD88 and TRAF6 levels as TLR4-associated downstream proteins and found that both increased in the FM group  = 20.26 and F = 18.41, n = 6, respectively) and were decreased by EA treatment  but not sham EA . Furthermore, we verified that all downstream molecules, including pERK, pp38, and pJNK, were potentiated in the FM mouse hypothalamus  = 11.08, F = 10.02, and F = 23.17, n = 6, respectively). These increased levels were further reversed in EA mice, but not in sham EA mice . Moreover, we measured the levels of the transcription factor pNFκB in the mouse hypothalamus. We observed that pNFκB increased in both with FM  = 13.31, n = 6) and was reversed by EA but not sham EA . Similar results were observed in the mouse PAG . These results indicate the contribution of inflammatory factors and TLR4 pathways to the FM phenotype. Moreover, these changes in TRPV1 and related molecules were reversed by EA treatment. We used immunostaining for TLR4 localization on the hypothalamus and PAG, a brain area involved in pain modulation. We observed very low levels of TLR4 in normal mice and increased levels in the FM mouse hypothalamus and PAG (n = 4). EA, but not sham EA, reliably alleviated this overexpression found in the CSP model (n = 4). Furthermore, a similar tendency showed increased Iba1, which was further attenuated in the EA group but not in the sham group (n = 4). Additionally, TLR4 and Iba1 colocalization was detected in the mouse hypothalamus and PAG (n = 4). This pattern of localization was further abrogated by EA but not sham treatment.Western blotting served to explore the Iba1–TLR4 signaling pathway in the mouse hypothalamus and PAG. Iba1 and TLR4 expression was detected in normal mice; in FM mouse hypothalamus, their expression levels increased  = 15.46 and F = 30.24, n = 6). EA significantly decreased Iba1 and TLR4 increased , but this was not the case in sham mice . MyD88 and TRAF6 protein levels in the CVI served to characterize TLR4 downstream effectors. Similar to TLR4, MyD88 and TRAF6 levels simultaneously increased after FM induction  = 21.47 and F = 20.16, n = 6). EA caused a significant decrease in these levels  compared to sham EA . Increased pERK, pp38, and pJNK were also detected in FM mice  = 17.43, F = 7.93, and F = 25.03, n = 6, respectively). However, their levels significantly decreased in mice treated with EA  but not with sham EA . With respect to pNFκB, the protein levels of pNFκB increased in FM mice, an increase that was further reversed with EA treatment  = 22.96, n = 6). The aforementioned tendency, as a TLR4 signaling pathway, was also obtained in CVII, suggesting its crucial role in FM and EA treatment effects .Because the cerebellum plays a critical role in FM pain, we evaluated Iba1 and TLR4 signaling pathways in the mouse cerebellum after FM. Our data showed that Iba1 and TLR4 expression significantly increased after FM induction in the cerebellum lobule VI (n = 4) and were then reversed by EA, but not sham EA (n = 4). Similarly, Iba1 increased with FM and was attenuated in EA, but not in sham EA (n = 4). Moreover, double-staining of TLR4 and Iba1 were observed in the mice CVI and VII (n = 4); a pattern abrogated by EA but not sham treatment.Furthermore, we tried to identify whether TLR4 and Iba1 levels were altered and distributed in the mouse cerebellum, a brain area involved in patients with FM. Our data indicated that TLR4 was distributed in the CVI and VII in normal mice. TLR4 levels further increased after FM induction in the mouse CVI and VII . Furthermore, co-administration of LPS and toll-interleukin 1 receptor domain-containing adapter protein (TIRAP) reliably reversed this phenomenon . A similar tendency was also observed in thermal hyperalgesia . By using Western blot, we indicated that Iba1 and TLR4 level were significantly augmented after LPS injection in the mouse hypothalamus . TIRAP meaningfully diminished Iba1 and TLR4 rises . MyD88 and TRAF6 levels served to describe TLR4 downstream effectors. Similarly, MyD88 and TRAF6 proteins were concurrently augmented after FM induction . TIRAP produced a significant reduction in these levels . Amplified pERK, pp38, and pJNK were also identified in the LPS group. However, their expressions dramatically declined in TIRAP-treated mice . The expression of pNFκB was increased in LPS mice and then reversed with TIRAP treatment .Recent articles showed that alterations in higher brain areas are essential for developing chronic pain ,28. TherFM is a complicated symptom with chronic pain, fatigue, sleep disturbance, obesity, and depression affecting the whole body. FM is often resistant to opioid drugs or nonsteroidal anti-inflammatory drugs . AdditioTLR4 was reported to be expressed in both neuronal and glial cells in the CNS responding to inflammatory mediators. In inflammation or nerve injury, TLR4 was suggested to activate microglia and astrocytes to induce the release of inflammatory cytokines at the spinal cord level to develop inflammatory pain and neuropathic pain ,33,34. MThe study results proved that several inflammatory mediators increased in the circulating blood of FM mice and can be attenuated by EA, suggesting an anti-inflammatory effect. In the neuropathic pain model, mechanical or thermal hyperalgesia was abrogated after inhibition of TLR4. Ma and colleagues suggested that TLR4 increased in the neuropathic pain model . InhibitIn conclusion, the main outcomes in this study are that FM induction significantly activates pain, inflammation, and central sensitization in a mouse central pain model. Mechanical and thermal hyperalgesia were observed in the FM mice. Inflammation contains increased IL-1β, IL-2, IL-5, IL-6, IL-9, IL-12, IL-17A, TNF-α, IFN-γ, and MCP-1 in the mice plasma. Furthermore, we have confirmed that TLR4 and related molecules increased in the mice’s hypothalamus, PAG, and cerebellum. Particularly, we reported TLR4 is an inflammation detector for FM. Additionally, inflammatory modulators were released to activate TLR4 for pain processing. All signals further activate the aforementioned brain regions for the pain signaling pathway, including MYD88 and TRAF6. EA potently suppressed these complicated molecular pathways in the FM mice brain .


Bradyrhizobium japonicum strains. Soybean inoculated with USDA123 has a lower yield than strains known to have high nitrogen fixation efficiency, such as USDA110. In the main soybean-producing area in the Midwest of the United States, USDA123 has a high nodule incidence in field-grown soybean and is competitive but inefficient in nitrogen fixation. In this study, a high-throughput system was developed to characterize nodule number among 1,321 Glycine max and 69 Glycine soja accessions single inoculated with USDA110 and USDA123.Symbiotic nitrogen fixation differs among G. max accessions with significantly different nodule number of USDA110 and USDA123 were identified. After double inoculating 35 of the 73 accessions, it was observed that PI189939, PI317335, PI324187B, PI548461, PI562373, and PI628961 were occupied by USDA110 and double-strain nodules but not by USDA123 nodules alone. PI567624 was only occupied by USDA110 nodules, and PI507429 restricted all strains. Analysis showed that 35 loci were associated with nodule number in G. max when inoculated with strain USDA110 and 35 loci with USDA123. Twenty-three loci were identified in G. soja when inoculated with strain USDA110 and 34 with USDA123. Only four loci were common across two treatments, and each locus could only explain 0.8 to 1.5% of phenotypic variation.Seventy-three High-throughput phenotyping systems to characterize nodule number and occupancy were developed, and soybean germplasm restricting rhizobium strain USDA123 but preferring USDA110 was identified. The larger number of minor effects and a small few common loci controlling the nodule number indicated trait genetic complexity and strain-dependent nodulation restriction. The information from the present study will add to the development of cultivars that limit USDA123, thereby increasing nitrogen fixation efficiency and productivity.The online version contains supplementary material available at 10.1186/s12864-023-09627-4. Glycine max (L.) Merr.] is one of the most economically important crops in the world due to its rich protein and oil content, about 42 and 20%, respectively [Soybean [ectively , 2. Protectively , 4. It iectively , 6.B. japonicum has been observed. For instance, several authors have reported that the seed yield of soybeans grown in rhizobia-free soil inoculated with B. japonicum USDA123 was lower than in plants inoculated with strains known to have high nitrogen fixation efficiency, such as USDA110 [B. japonicum strain USDA123 in field-grown soybean nodules is high [Variability in symbiotic nitrogen fixation among different strains of  USDA110 –9. In th is high , and the is high , 17.Since the early days, many attempts have been made to increase soybean nitrogen fixation efficiency through the use of superior inoculant strains like USDA110 , 18, impRj2 restricts nodulation of B. japonicum strain USDA122 [Rj3 locus restricts nodulation of B. elkanii strain USDA33, the Rj4 locus restricts B. elkanii strain USDA61 [Rfg1 restricts nodulation of Ensifer/Sinorhizobium fredii strain USDA257 [rj1, rj5, and rj6 alleles restrict all rhizobium strains [The Soybean Germplasm Collection of the United States Department of Agriculture includes 18,480 cultivated and 1,168 wild soybean accessions introduced from 84 countries or developed in the United States . This co USDA122 , 30, then USDA61 , 32, and strains . NeverthB. japonicum strains USDA110 and USDA123, to identify new germplasm that may restrict nodulation of USDA123 but prefer USDA110 strain by characterizing nodule occupancy in soybean double inoculated with USDA110 and USDA123, and to determine genetic complexity of the NN trait in soybean accessions.Therefore, the objectives of the present study were to determine the genetic variation of nodule number (NN) among sets of diverse cultivated and wild soybean accessions after single inoculation with G. max after single inoculation with B. japonicum strains USDA110 and USDA123, respectively (Table G. max accessions did not nodulate with any of the two strains tested. They were grown in field soil and confirmed no nodules on their roots. The non-nodulated accessions tested were PI548193 (T201), a well-known accession with genotype rj1 non-nodulating, and PI507429 (Tousan 89) introduced to the U.S. from Japan in 1987 (www.ars-grin.gov) which has not been previously reported with restricted nodulation.The NN was counted on 1,321 accessions of ly Table . Two of G. max accessions was 35.3 nodules per plant and ranged from 0 to 75.3 when inoculated with USDA110, and the average was 33.8 nodules per plant, ranging from 0 to 75 when inoculated with USDA123. The average number of nodules of G. soja was 12.9 and 13.8 per plant, ranging from 5.3 to 23.5 and 2.3 to 27.8 when inoculated with USDA110 and USDA123, respectively. The NN in both cultivated and wild soybean were normally distributed with Skewness between − 2 to + 2 and Kurtosis between − 7 to + 7  and USDA123 (0.74). The phenotypic variation of NN in G. max and G. soja was mainly attributed to genetic effects, and the trait was less affected by the environment, i.e., relatively high consistency of NN among replicates in the experiment.The average NN of the 1,321 P < 0.05) was performed to determine the statistical difference in NN of USDA110 vs. USDA123 in 1,319 cultivated, and 69 wild soybean accessions single inoculated with USDA110 and USDA123, respectively. The non-nodulated accessions were not included in this analysis. Forty-two cultivated soybean accessions showed significantly higher NN of USDA110 than USDA123, and 31 had a lower NN of USDA110 than USDA123 , and three with high NN of USDA110  in the same trial Fig. .Out of the 35 accessions, 27 formed three different types of nodules, which consisted of USDA110, USDA123, and mixed nodules (USDA110 + USDA123 nodules). The occupancy preference of nodules in 16 of these accessions was USDA110 > mixed nodules > USDA123; seven genotypes had two types of nodules: USDA110 and mixed nodules, with a preference for USDA110, and one accession had all nodules occupied by strain USDA110  > 3) when G. max accessions were single inoculated with USDA110 (NNmax110), 50 SNPs when the same set was inoculated with USDA123 (NNmax123), and 32 and 51 in G. soja single inoculated with USDA110 (NNsoja110) and USDA123 (NNsoja123), respectively  haploblock Gm14_Hap123 in the euchromatic region spanning 0.43 Mb (from 9 to 9.5 Mb). In NNmax123, 11 of the 50 SNPs were identified in the euchromatic region on chromosome 11, spanning 1.4 Mb (from 0.1 to 1.5 Mb) and positioned in five different haploblocks with one to four SNPs in each block. Only four SNPs were in common between NNmax110 and NNmax123, i.e., SNPs ss715584397 at position 11.5 Mb on chromosome 3, haploblock Gm03_Hap5; ss715593108 at position 13.8 Mb on chromosome 6; ss715620123 at 9.4 Mb on chromosome 14, haploblock Gm14_Hap123; and ss715624623 at position 33.7 Mb on chromosome 16.In NNsoja110, six over 32 significant SNPs were located in the heterochromatic region on chromosome 11. Five of the six SNPs were located in haploblock Gs11_Hap28 (25.116–25.123 Mb), plus one SNP located 0.9 Mb downstream. For NNsoja123, 21 over 51 SNPs were found on chromosome 17, six in haploblock Gs17_Hap5 spanning 4.2 Mb (22.7–23.7 Mb) in the heterochromatic region, and 15 SNPs were located in blocks with one to six significant SNPs, or not in haploblocks. No significant SNPs in common were found between treatments NNsoja110 and NNsoja123 or interspecies.In the final count of significant loci for NN, we kept the significant leading SNP to represent each haploblock, as well as the SNPs outside the haploblocks. As a result, 35, 35, 23, and 34 loci were detected from NNmax110, NNmax123, NNsoja110, and NNsoja123, respectively Table 2. For experiments with a large number of samples, such as in this study, establishing an efficient system of phenotyping that maximizes space is fundamental to maintaining similar growth conditions. Therefore, Cone-tainers of 164 ml (3.8 cm diameter x 21 cm depth)  with the same advantages but smaller than the Deepots and Leonard Jars were used to evaluate soybean nodules. Cone-tainers were distributed in sets of 45 units/tray entailing an area of approximately 22 plants/m2, which increased the area by 83% compared with the Deepots and Leonard Jar systems  [Conventionally, scientists have used Leonard Jars as a growth system for the study of nodulation in legumes –40. Lateabaceae) , this isLikewise, developing methods to determine nodule occupancy in a large number of accessions was critical for this nodulation competition study. The rapid DNA extraction and high-throughput Kompetitive Allele Specific (KASP) genotyping method established in this study allowed us to accuraterly differentiate the types of nodules, which was verified by Sanger sequencing of nodules and USDA110 and USDA123 strain controls. In initial experiments, isolation of DNA was performed with PrepMan Ultra (Applied Biosystems); however, DNA amplification with the KASP protocol did not amplify as expected, affecting allelic discrimination, probably due to interaction between the nodule’s polysaccharides and PCR chemical reagents. It is well known that polysaccharides could inhibit PCR , 50.Identifying accessions with preference for nodulation with one strain over the other was the first step in selecting genotypes of interest for the strain competition experiment. In this study, 11 of 30 accessions selected in the first screening of single inoculation with a high NN of USDA110 versus USDA123 showed the same significant preference for USDA110 nodulation compared to USDA123 in competition experiments, while two of five accessions initially selected with a high NN of USDA123 versus USDA110, had the same preference in the competition experiment as single inoculation, and the remaining three accessions preferred USDA110 to USDA123, suggesting the complexity of the strain competition mechanisms. In the competition study, the incidence of mixed nodules ranged from 0 to 67% under controlled conditions, a wide variation among the genotypes. Nodule mixed infections in soybean, and other legumes species are common and have been previously reported , 51–55.In the early days, Lindemann et al.  found thB. japonicum and inoculated with USDA123 and either USDA110, USDA122, or USDA138. In each competition treatment, greater than 75% of the nodules of ‘Williams’ were occupied by USDA123, whereas in all cases, less than 10% of the nodules of the PI371607 genotypes contained USDA123. The accessions genotypes excluded strain USDA123 in favor of the inoculant strain. In the present experiment, PI371607 did not significantly differ when inoculated with USDA110 (35 nodules/plant) or USDA123 (37.3 nodules/plant). Consequently, it was not included in further competition experiments in this study. Nevertheless, when PI548461 (‘Improved Pelican’), classified with restricted nodulation to USDA123 by Cregan and Keyser [P < 0.05) was found.With the intent to find genotypes with restricted nodulation to the strain USDA123, Cregan and Keyser  screenedd Keyser  was testUnder a different approach, Cregan et al.  identifiIdentifying accessions with potential nodulation restriction for USDA123 strain and specificity for superior rhizobium strain would facilitate the development of soybean cultivars with improved symbiosis through hybridization and introgression of genes from the accessions. Several USDA123-restricting and USDA110-preferring accessions were identified from this study could be a valuable source for further genetic research on this trait and soybean improvement.Bradyrhizobium or Esinfer, efforts to find genes that limit USDA123 nodulation have not yet been reported [Although several articles have reported in soybean restricted nodulation to specific strains of reported –26, 59.G. max and G. soja were single-inoculated with B. japonicum strains USDA110 and USDA123, 35 loci were identified in treatment NNmax110 and the same number of loci in NNmax123. Moreover, 23 loci were identified in NNsoja110 and 34 in NNsoja123. From the total of 127 loci, 107 unique significant loci were identified, and 18 have been previously reported for NN or nodule-related traits in SoyBase (http://soybase.org). The significant SNP ss715590625 in NNsoja123 located at 31.4 Mb on chromosome 5 was adjacent to the gene Glyma.05G121600 (GmVTL1a/nodulin-21), which is a vacuolar iron transporter-like gene indispensable for symbiotic nitrogen-fixation in nodules, annotated as a transporter of ferrous iron from the infected root cell cytosol to the symbiosome [In the present study, when accessions of The region on chromosome 6 where two loci were found, ss715595435  in NNmax110 and the common SNP ss715593108  in NNmax110 and NNmax123, have been previously described by Yang et al.  in the iOn chromosome 10 SNPs ss715607620 in haploblock Gm10_Hap93  in NNmax110 and ss715607699 at 47,626,066 bp in NNmax123, were found in a similar region  previously reported by Yang et al.  for six On chromosome 11, Yang et al.  and SantGlyma.16G175600 (GmUGT1), Glyma.16G175900 (GmUGT7), and Glyma.16G176000 (GmIF7GT5), are related to symbiosis and are involved in the isoflavonoid pathway coding for isoflavone 7-O-glucosyltransferase, which plays important roles in plant-microbe interaction and is essential for establishing symbiosis with B. japonicum [nod gene expression in B. japonicum [SNP ss715624623 on chromosome 16 was among the four significantly associated common markers in NNmax110 and NNmax123, where seven putative genes reside. Three of them, aponicum . Moreoveaponicum –72, howeOn chromosome 19, the SNP ss715635907 at 49,239,109 bp was significant in NNmax110, and the region from 49,210,133 to 49,243,536 was also described by Nicolás et al. (2006) for nodule dry weight. On chromosome 20, two of the significant SNPs found in NNsoja123, ss715638590  in haploblock Gs20_Hap55 , and ss715638603 , were previously described by Hwang et al. , and oneThe detection of multiple QTLs associated with NN, the small number of common or overlapped QTLs between inoculation treatments, and the small effects at each locus implied the complexity of the trait and complex signal exchange between rhizobia and the host to establish symbiosis. This study also supported the evidence that host-controlled restriction or nodulation preference was strain dependent.G. max and 1168 G. soja accessions were provided by the USDA Soybean Germplasm Collection located at Urbana-Champaign, Illinois, USA (https://www.ars-grin.gov/) and previously genotyped with the SoySNP50K assay, containing more than 50,000 SNPs [G. max (23%) and 362 G. soja (30%) accessions were at least 99.9% identical to another accession in the Collection. From the remaining 14,186 cultivated and 806 wild soybean accessions, diverse sets of 1,417 cultivated and 81 wild soybean accessions were selected using the following procedures: pairwise distances among 14,186 cultivated and among 806 wild soybean accessions were estimated by calculating the ratio of the number of SNPs with different alleles to the total number of loci between paired accessions. The cultivated and wild soybeans were clustered into 1,418 and 82 clusters, respectively, equal to approximately 10% of the cultivated and wild soybean accessions in the Collection. From each cluster, one accession with the largest average distance to other accessions was chosen. Further analysis showed that the two sets could capture > 90% of the genetic diversity in cultivated and wild soybean collections based on the ratio of the number of polymorphic SNPs in the selected accessions versus the total number of polymorphic SNPs among all cultivated and wild soybean accessions. However, due to the lack or deficiency of seeds for some accessions, only 1,321 accessions of the 1,418 G. max (landrace and modern cultivars) and 69 accessions of the 81 G. soja were used in this study   were used to grow plants. A medium cotton ball was placed on the bottom of each Cone-tainer and filled with prewashed sand. Cone-tainers were supported by seedling flats of 162 cells (9 × 18), distributed in sets of 45 units leaving an empty cell between each Cone-tainer (5 columns x 9 rows). Seedling flats and Cone-tainers were placed in trays filled with 3 inches of tap water. Sand and cotton balls were autoclaved, and Cone-tainers, flats, and trays were disinfected for 5 min in a 1:4 bleach solution, then rinsed three times with water. For disinfection and sowing, a seedling flat of 162 cells was modified into a compartmentalized strainer. First, the size was reduced to 45 cells (5 × 9 cells), and on the bottom of each cell, a plastic mesh # 10 was glued. Next, seeds and plastic labels of 45 accessions were distributed over the seed strainer, then disinfection took place by sequentially immersing the seed strainer, first in the tray with 70% ethanol for 30 s, then rinsed in water, washed in 1:4 bleach solution for 4 min in another tray, and finally, rinsed four times with water by transferring the seed strainer tray by tray. The seed strainer had the same distribution as the experimental unit of the Cone-tainers. Therefore, four seed strainers were used at a time per disinfection. With this method, seeds of 180 accessions were disinfected in 6 min.G. max and 69 G. soja accessions in four replicates. For each replicate, four seeds were sowed per Cone-tainer, and after four days, seedlings were thinned to one and wetted with Nitrogen-free Hoagland-Arnon plant nutrient solution [8 CFU/ml-1) of B. japonicum USDA110 and USDA123 strains, respectively. Water in trays was changed weekly. After 30 days, roots were harvested, washed, dried, and frozen, and then nodules were removed and counted. Thirty-six un-inoculated ‘Williams 82’ plants were included as controls distributed in four units along the benches, and no nodules were found on them when phenotyped.The experiment was conducted in a randomized complete block design with 1,321 solution . Five-da8 CFU/ml− 1) and volume (1:1) of USDA110 and USDA123. After 30 days, roots were harvested, washed, and disinfected with alcohol 75%, dried up for 20 min at 28 °C, then frozen for further analysis. After that, nodules were detached from roots and placed in a sterile 96-well plate (0.8 ml), one nodule per well, then lyophilized overnight for DNA extraction. A single steel bead of 2.4 mm was placed in each well, and the plate was sealed with thermo-aluminum film. Nodules were ground at 30 Hz for 30 s in a Retsch MM400 mixer mill. Plates were centrifuged at 5,200 rpm for 5 min, and 500 µl of distilled water was added to each sample. Plates were sealed, vortexed, spun down and sat for 20 min. From the supernatant, 100 µl was transferred to a new 96-well PCR plate, sealed, and centrifuged at 5,200 rpm for 10 min; the supernatant was removed without disturbing the pellet, then 50 µl of distilled water was added to the pellet, the plate was briefly vortexed, then heated at 100 °C for 10 min, centrifuged at 3,000 rpm for 5 min, and finally, 20 µl of the supernatant was taken and transferred into a new 96-well plate. DNA was diluted to 10% for the PCR reaction.A completely randomized design experiment with four replicates was performed on 35 accessions that showed a significant difference in NN after single inoculation with USDA110 and USDA123, respectively. Five-day-old plants were double inoculated with a 2 ml blend suspension of equal concentration (10https://bioinfo.biotec.or.th/WASP) [https://www.ncbi.nlm.nih.gov) and USDA123 (http://genome.jgipsf.org). Among these, two markers, rhi_snp2 , and rhi_snp24 , had the best amplification and allelic discrimination. Thus, both markers were chosen to test nodule occupancy on 35 G. max accessions. Nodules were genotyped by KASP markers in a final reaction volume of 5 µL with 2.5 µL of 2x KASP MasterMix Low Rox 5000 V4.0 TF (LGC Genomics), 0.07 µl of the primers mix containing 12 µM of each forward primer plus 30 µM of the reverse primer, and 5–20 ng of genomic DNA. QuantStudio 6 Flex  was used for PCR under the following cycling conditions, activation for 15 min at 94 °C; 10 cycles of 20 s at 94 °C and 60 s at 61–55 °C ; 26 cycles of 20 s at 94 °C and 60 s at 55 °C, followed by 15 cycles of 20 s at 94 °C and 60 s at 57 °C. Fluorescence detection was performed at the end of the run for 30 s at 35 °C, and data were analyzed with Quant Studio Real-Time PCR software . For the validation of the phenotyping results, 12, 13, and 18 bacteroids from 3 mixed nodules were used for Sanger-sequencing at seven loci defined in the Bradyrhizobium Multilocus Sequence Analysis [To determine nodule occupancy of USDA110 and USDA123 in soybean after double inoculation of USDA110 and USDA123, a set of 23 Kompetitive Allele Specific PCR (KASP) markers were designed using WASP Web-based Allele Specific Primer Design Tool (th/WASP) , based oAnalysis . A t-tesG. max single inoculated with B. japonicum strains USDA110 (NNmax110) and USDA123 (NNmax123) and in G. soja single inoculated with USDA110 (NNsoja110) and USDA123 (NNsoja123). To determine the complexity of NN, the number of loci, and their effects on the trait were analyzed through genome-wide association studies (GWAS). Population structure was analyzed by ADMIXTURE v1.22 [K from 2 to 10 for G. max and 2 to 5 for G. soja, and a 10-fold cross-validation (CV) procedure was performed with 30, 100, 500, 1000, and 2000 seeds for each K-value. The most likely K-value was determined using the CV values. Consequently, marker and NN associations were tested in Tassel [P < 0.001 or -Log10 (P) > 3. The haplotype blocks of the soybean genome were determined in Haploview 4.2 [http://soybase.org) and previous reports. When a given QTL was outside the previously described interval position, we considered it as a novel QTL. Genes located in the significant regions were searched for expression abundance in nodule-related traits in Phytozome (https://phytozome-next.jgi.doe.gov/) [http://systemsbiology.cau.edu.cn/agriGOv2) [Analysis of variance and heritability were performed for the NN in RE v1.22  with K fn Tassel  using thview 4.2 , and sigoe.gov/) , genes igriGOv2) . In caseBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5


Type 2 diabetes (T2D) is expected to worsen the prognosis of inpatients with heart failure (HF) but the evidence from observational studies is inconsistent. We aimed to compare mortality outcomes and life expectancy among inpatients with HF with or without T2D and explored whether chronic kidney disease (CKD) influenced these associations.We collected hospital and civil registry records of consecutive inpatients from a tertiary hospital in Switzerland with a diagnosis of HF from the year 2015 to 2019. We evaluated the association of T2D with mortality risk using Cox regression and adjusted for confounders.Our final cohort consisted of 10,532 patients with HF of whom 27% had T2D. The median age (interquartile range [IQR]) was 75 [68 to 82] and 78 [68 to 86] for the diabetes and non-diabetes groups, respectively. Over a median follow-up [IQR] of 4.5 years [3.3 to 5.6], 5,347 (51%) of patients died. T2D patients had higher risk of all-cause mortality . Compared to control (i.e. no T2D nor CKD), average life expectancy (95% CI) among T2D patients, CKD, or both was shorter by 5.4 months (95% CI 1.1 to 9.7), 9.0 months (95% CI 8.4 to 9.6), or 14.8 months (95% CI 12.4 to 17.2), respectively. No difference by sex or ejection fraction category was observed.T2D is associated with a significantly higher risk of all-cause mortality and shorter life expectancy compared to those without among middle-aged and elderly inpatients with HF; presence of CKD may further increase these risks.The online version contains supplementary material available at 10.1186/s12933-023-01903-7. Global trends on the burden of diabetes and heart failure (HF) continue to increase, thus highlighting the need for more effective preventive strategies , 2. TypeHospitalised patients with HF represent advanced stages of disease with poor prognosis: high risk of in-hospital mortality from 4 to 7%, rehospitalisation from 25 to 30%, and mortality from 7 to 11%, with shorter life expectancy , 7, and Studies that have explored the association between diabetes and mortality in HF patients did not estimate life expectancy of inpatients with HF, nor compare life expectancy between those with or without diabetes . PreviouTherefore, by using data of inpatients with HF at the largest tertiary cardiovascular referral hospital in Switzerland, we compared mortality outcomes among patients with or without T2D and estimated the differences in life expectancy between groups. Additionally, we explored whether co-existence of T2D and CKD was associated with worse prognosis.All patients with heart failure who were admitted at Bern University Hospital , Switzerland—a large tertiary cardiology center—between January 2015 and December 2019, were followed-up for the present study. Participants were excluded if they are 40 years old and younger, had type 1 diabetes, or developed diabetes after hospitalisation. The clinical data warehouse at the Inselspital contains administrative and medical data of all patients from any department including the Department of Cardiology. Information about diagnoses were obtained based on the International Statistical Classification of Diseases and Related Health Problems 10th version (ICD-10). In this study, we identified eligible participants based on relevant ICD-10 codes for heart failure . Among other data, demographic and clinical characteristics, information on hospitalizations, comorbidities, implantable cardiac devices, heart failure and antidiabetic medications, laboratory data, and survival status were obtained. Demographic and clinical characteristics included age, sex, body mass index (BMI) and left ventricular ejection fraction (EF) measured using ultrasound. Patients were categorized as having heart failure with preserved ejection fraction  or reduced ejection fraction . Comorbidities included were T2D, CKD, hypertension, and atherosclerotic cardiovascular disease , and chronic obstructive pulmonary disease (COPD). Medication information was obtained using Anatomical Therapeutic Chemical Classification System (ATC) codes. Survival status was assessed by linking with the national mortality record. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines .Data were summarized as mean  or median [interquartile range] for continuous variables and as counts (proportions in %) for categorical variables.Survival by T2D status and by sex for the overall inpatient population was presented using Kaplan–Meier (KM) curves. We estimated the association of T2D status (yes vs no) with the risk of all-cause mortality using Cox proportional hazards regression. In Model 1, we adjusted for age and sex . In Model 2, we additionally adjusted for CKD, ASCVD, EF category, hypertension, atrial fibrillation, COPD, and dyslipidemia. In Model 3, we included interaction terms between T2D status, CKD status, and EF category. We tested for proportionality of hazards (PH) assumption using p > 0.05 as cut-off for Schoenfeld test and plotting Schoenfeld residuals against follow-up time. When PH assumption was not met, we stratified our analyses by time period: < 30 days (Period 1), 30 days to 1 year (Period 2), 1 to 2 years (Period 3), and > 2 years (Period 4), and reported hazard ratios (HRs) per period. Furthermore, we estimated whether the association between T2D and mortality differed by sex, EF category, or presence of CKD; for the last analysis we compared four patient groups (i) without T2D nor CKD (T2D-/CKD-); (ii) without T2D, with CKD (T2D-/CKD +); (iii) with T2D, without CKD (T2D + /CKD-); and (iv) with T2D and CKD (T2D + /CKD +). We compared life expectancy  between (i) patients with and without T2D and (ii) T2D and CKD groups, by fitting a survival curve using a non-parametric step function . We usedWe performed sensitivity analysis by i) adjusting for, or ii) excluding patients who have died during that episode of hospital admission, herein defined as in-hospital mortality.A total of 12,190 inpatients with an ICD-10 diagnosis of HF  from January 2015 to December 2019 were included in this study. Of these patients, 1658 were excluded because they did not meet eligibility criteria: developed T2D after hospitalization (n = 1423), < 40 years old (n = 181), with type 1 diabetes (n = 45), or erroneous civil registry data (n = 9). Our eligible study population was 10,532 patients with 4218 females  and 6314 males   and illustrated on Schoenfeld residuals plot on Additional file  and illuIn the four exposure group analysis (i.e. by T2D and CKD status), T2D was not associated with an increased mortality risk in Periods 1–2 (i.e. when observed within 1 year), but was statistically significantly associated with increased risk of all-cause mortality in Periods 3–4 (when observed beyond 1 year) Fig. . MeanwhiOverall, patients with T2D had on average shorter life expectancy compared to those without. Furthermore, those with both T2D and CKD had even shorter life expectancy than those with T2D alone. Bootstrapped estimates showed that compared to control, average life expectancy (95% CI) among inpatients with T2D, CKD, or both was 5.4 months (1.1 to 9.7) less, 9.0 months 8.4 to 9.6) less, or 14.8 months (12.4 to 17.2) less, respectively, with no sex differences   on its association with all-cause mortality. Stratified analyses by sex or EF category showed results were similar to overall  among inpatients with T2D, CKD, or both was 2.6 months (0.3 to 4.9) less, 9.1 months (7.0 to 11.2) less, or 15.1 months  less, respectively  with longer follow-up periods up to 15 years [Our study population is consistent with a large multinational study describing a contemporary population of patients with heart failure, where ASCVD such as ischemic heart disease and CKD are found to be common comorbidities . Moreove15 years . A rando15 years . Moreove15 years . NeverthIn our analysis that stratified patients according to EF category, we did not observe a statistically significant interaction between T2D and EF category for all-cause mortality. This is in line with findings from the CHARM program, in that the magnitude of all-cause mortality did not differ between EF categories . They alWe observed that the association of T2D on HF could be time-dependent, with similar risk of mortality in the acute term (< 30 days follow-up), but an increasing risk of mortality with longer follow-up even after accounting for age. This could partly be explained by the delayed effects of diabetes on HF, and the episode of hospitalization accelerated the longstanding maladaptive alterations, structural changes, cardiovascular dysfunction that T2D already imparts before hospitalization . MoreoveOverall, we observed that T2D and CKD were associated with higher risk of mortality among HF inpatients in the long term (i.e. beyond acute period). We extended evidence by translating relative risks into absolute measures more interpretable to the patients, clinicians, and policymakers. Inpatients with heart failure and T2D had shorter survival than those without T2D, those with CKD had shorter survival than those with T2D whilst those with both T2D and CKD had shorter survival than those with either T2D or CKD alone. These estimates constitute a substantial life expectancy reduction, considering that our patient population is relatively older and represents patient populations that already have a very high mortality risk. Moreover, healthcare costs at this age and disease severity are expected to increase, and particularly among patients with CKD who incur much higher hospital care costs, this could mean even much greater societal costs .The observed association of diabetes on long-term survival among HF inpatients could be explained by ischemic and non-ischemic mechanisms , 18: insTo our knowledge, this is the first study that reported relative (i.e. HR) and absolute estimates  on the association of T2D with life expectancy among inpatients with HF, increasing bedside interpretability. We posit that after hospital admission with advanced stages of disease, life expectancy is a more useful information that could improve risk communication from clinicans to patients. Another strength of our study relies on its large, real - world hospital setting of inpatients with HF patients in Switzerland, exceeding the size of some multinational registries of HF in Europe –28. ThisOur findings should be interpreted against several considerable limitations. First, the data we analysed were based on medical records at the hospital. Thus, we lacked information on routine subclinical measures that stratify risk in patients with T2D or CKD: glycosylated hemoglobin and estimated glomerular filtration rate, that could have provided important insights clinical risk stratification. Further, data on cause of death were not available, which precluded us from exploring whether our findings were driven by CV deaths. This could have allowed us to compare results with other similar studies. Second, our study lacked information on durations of T2D, HF, and other comorbidities such as CKD. Our reliance on ICD-10 codes to identify most T2D and CKD cases could also have led to misclassification of non-T2D and CKD cases if the ICD code was not encoded, hence in the non-T2D group some cases of T2D maybe present. This is a pattern that is in general observed when using medical record data . HoweverIn conclusion, we showed that T2D is associated with a significantly higher risk of all-cause mortality with significant reduction in life expectancy among patients with HF, even when clinical risk factors and potential confounders such as age, sex, hypertension, EF, CKD, ASCVD, and other comorbidities were taken into account. Among those with T2D, CKD was associated with further reduction in life expectancy.Additional file 1: Table S1. Baseline characteristics of patients hospitalised for heart failure at Inselspital, 2015-2019, by sex. Figure S1. Schoenfeld residuals plotted against time. Table S2a. Time-partitioned Cox proportional hazards regression models* assessing the risk of mortality according to diabetes status, adjusting for in-hospital mortality. Table S2b. Time-partitioned Cox proportional hazards regression models* assessing the risk of mortality according to diabetes status, excluding patients that had in-hospital mortality. Figure S2. Cox proportional hazards regression models* assessing the risk of mortality according to T2D status, stratified by ejection fraction. Figure S3. Time-partitioned Cox proportional hazards regression models* assessing the risk of mortality according to T2D status, stratified by ejection fraction. Figure S4. Estimated life expectancy according to diabetes and CKD status, stratified by EF.


Dictyostelium discoideum cells secrete a chemorepellent protein called AprA. Examining existing knockout strains, we previously identified proteins required for AprA-induced chemorepulsion, and a genetic screen suggested that the enzyme phosphatidylinositol phosphate kinase A  might also be needed for chemorepulsion. Here, we show that cells lacking PIPkinA are not repelled by AprA, and that this phenotype is rescued by expression of PIPkinA. To bias cell movement, AprA inhibits Ras activation at the side of the cell closest to the source of AprA, and we find that PIPkinA is required for AprA to inhibit Ras activation. PIPkinA decreases levels of phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol -trisphosphate [PIP3], and possibly because of these effects, potentiates phagocytosis and inhibits cell proliferation. Cells lacking PIPkinA show normal AprA binding, suggesting that PIPkinA regulates chemorepulsion at a step between the AprA receptor and AprA inhibition of Ras activation.During developmental and immune responses, cells move towards or away from some signals. Although much is known about chemoattraction, chemorepulsion (the movement of cells away from a stimulus) remains poorly understood. Proliferating  Highlighted Article: This study expands upon the role of Dictyostelium PIPkinA, furthering our understanding of the underlying mechanism of chemorepulsion and informing development of therapeutics for acute respiratory distress syndrome. Dictyostelium discoideum. During development, D. discoideum cells aggregate using chemoattraction to relayed pulses of cyclic adenosine monophosphate (cAMP) (carA), including the mammalian target of rapamycin complex 2 (mTORC2), guanylyl cyclase, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)–phosphatidylinositol -trisphosphate [PIP3] and phospholipase A (PlA) pathways, appear to act synergistically to drive chemoattraction towards cAMP  . Five mards cAMP .Dictyostelium cells called autocrine proliferation repressor protein A (AprA)  . In a coA (AprA) .Dictyostelium and neutrophils move by extending a pseudopod, attaching the pseudopod to the substrate and then essentially squeezing the cell into the pseudopod P2 -trisphosphate (IP3), diacylglycerol (DAG) or PIP3 P2 . PIPkinAIP2 . Both PI,5)P2 3,4,5)P3 , that re3,4,5)P3 , 2011.pipkinA gene (referred to in this report as pipkinAins cells) do not have the ability to develop, fail to form aggregates even after 48 h (pipkinAins cells (Saccharomyces cerevisiae Mss4 (type I), rat type Iβ and human type IIβ PI(4)P 5-kinases (PIP5Ks), and cells expressing these chimeric proteins showed normal development, indicating that Dictyostelium PIPkinA is homologous to these other PI(4)P kinases P and PIP3 without significantly affecting levels of PIP2, suggesting the existence of a complex mechanism regulating phosphatidylinositol phosphate levels, and that PIPkinA inhibits cell proliferation and potentiates phagocytosis.In this report, we made a gene replacement knockout of pipkinA open reading frame. Blasticidin-resistant clones were screened for homologous recombination by PCR using a primer in the pipkinA 5′ flanking region and a primer in the coding region that gave a 1.77 kb product from wild-type (WT) DNA . One of the transformants lacking the pipkinA open reading frame was chosen for further analysis and was designated −pipkinA. We then expressed PIPkinA as a fusion with a green fluorescent protein (GFP) in −pipkinA cells to make the strain pipkinA−/pipkinA-GFP. Fluorescence microscopy confirmed that, as previously observed  DNA . Of eigh(WT) DNA , unlike ter 48 h . This phobserved , expressobserved B. As prens cells , expressA− cells . CompareA− cells C,D.2 and PIP3 at the plasma membrane play a major role in cell migration P2 P, PIP2 and PIP3 levels were measured in WT and −pipkinA cells. Compared to WT cells, −pipkinA cells showed significantly higher basal levels of PI(4)P and PIP3 P2 through other pathways. The addition of recombinant AprA P3 levels at 10 and 20 min P2 levels, albeit with a different time course P and PIP3 levels as well as the timing of the effect of AprA on PIP2 levels.The localizations of PIPigration . The kinIP2 . To dete3,4,5)P3 A,C. TherA− cells B, indicaA rAprA;  to WT ceA rAprA; D but incd 20 min E,F. Whene course H. UnlikeA− cells G and inc3 levels I. These −grlH cells nor −pipkinA cells moved away from the source of rAprA  that have defective chemorepulsion from AprA, and some mutants (such as cells lacking AprA) that show chemorepulsion from exogenous AprA . To deteof rAprA A. Similaby rAprA A. As preH− cells   or increase roundness (Movies 1–4) in response to rAprA, and this effect was rescued by expression of PIPkinA–GFP (Movies 5 and 6). For these assays, cells were in submerged culture exposed to air, whereas for the chemorepulsion assays the cells were in a narrow space in an Insall chamber. The different conditions might account for the slower speed of cells in the Insall chamber compared to that of cells in submerged culture , which preferentially binds GTP-bound Ras P3 is fundamental for vesicular trafficking Pfficking . The lococytosis . PIfficking  and playfficking . To detearticles A,C but darticles B,C. We hn viable . To detebacteria . The losand 48 h D,E. Thes−aprA, −pipkinA and −pipkinA/pipkinA-GFP cells but showed reduced binding to −grlH cells  and for actin as a loading control P3, and PIP3 can enhance the activity of Ras (PIPkinA converts PI(4)P to PIP2 . Althougactivity . Conversivity P3. As we expected, we observed that cells lacking PIPkinA had a high basal level of PI(4)P. However, −pipkinA cells showed no change in the basal level of PIP2 and a high basal level of PIP3. This suggests that additional pathways not involving PIPkinA regulate basal PIP2 and PIP3 levels.In cells lacking PIPkinA, we would expect a high basal level of PI(4)P, low basal level of PIP2 to generate PIP3, and activation of Ras, can induce phagocytosis P3 also generate a phagocytic cup P3.In mammalian cells, phosphorylation of PIPocytosis . In D. dytic cup , and Rasytic cup . In mammocytosis . Since P−pipkinA cells, AprA increased PI(4)P levels. This suggests that PIPkinA counteracts some AprA-induced activity that increases PI(4)P levels. In both WT and −pipkinA cells, AprA increased PIP2 levels, albeit with different time courses. This suggests that AprA might increase CnrN and PTEN activity to convert PIP3 to PIP2 P2. The regulation of PIP2 and PIP3 levels involve complex pathways P2, other pathways are likely to be involved in the AprA-induced increase in PIP2 levels. In WT cells, AprA decreased PIP3 levels, whereas in −pipkinA cells, AprA increased PIP3 levels. PIPkinA inhibits Ras activation, and Ras activates PI3K P3 levels in −pipkinA cells suggests that AprA activates an additional pathway that, in the absence of PIPkinA, increases PIP3 levels. Taken together, these observations suggest that PIPkinA is part of a complex network that regulates levels of phosphatidylinositol phosphates, and that PIPkinA mediates chemorepulsion and inhibits proliferation by inhibiting Ras.In WT cells, AprA caused no significant change in PI(4)P levels, whereas in IP2 ,b, and tpathways . Since ttes PI3K . The resD. discoideum strains were purchased from the Dictyostelium stock center (−grlH (DBS0350226) (−aprA (DBS0235509)  and then electroporated into WT Ax2 cells  plate for single clone isolation. After 4 days, cells in plaques on the lawns were picked and cultured in HL5  with 10 µg/ml blasticidin. The genomic DNA from each clone was extracted and purified using a Quick DNA Miniprep kit , then underwent PCR to verify the correct orientation of the insert using the primers listed in k center . The str0350226)  and aprA0235509) . pipkinAct15/Bsr . The 5′ x2 cells . After 1−pipkinA cells were transformed with the D. discoideum plasmid PKG3016  by electroporation (act15) promoter driving the expression of the pipkinA cDNA open reading frame (encoding residues 31 to 4767) fused in frame to a C-terminal GFP tag  and incubated on ice for 5 min. Phosphatidylinositol extractions and ELISAs were performed using phosphatidylinositol 4-phosphate [PI(4)P] Mass ELISA kits , phosphatidylinositol 4,5-bisphosphate [PIP2] Mass ELISA kits  and phosphatidylinositol 3,4,5-trisphosphate [PIP3] Mass ELISA kits . Vacuum-dried samples were stored at −20°C and analyzed by ELISA within 6 months of collection. Test samples were reconstituted with PBS-Tween-20/0.25% Protein Stabilizer solution, included with the kit, and assayed in duplicate.For the phosphatidylinositol extractions, 1.25×10E. coli K-12 survival in D. discoideum and phagocytosis of Alexa Fluor 594-conjugated zymosan A bioparticles  by D. discoideum cells were assayed as described previously (Recombinant AprA (rAprA) was expressed and purified as previously described , then coeviously .D. discoideum cells were grown to 1.0×106 cells/ml in HL5 in shaken suspension culture (160 rpm) and collected by centrifugation at 500 g for 3 min. Cells were resuspended in HL5 and washed twice more by centrifugation and resuspension. The cells were then resuspended in HL5 to 1.0×105 cells/ml, and 300 µl of the cell suspension was placed in a chamber of a type 354118 8-chamber tissue culture treated glass slide . Cells were then allowed to adhere for 30 min at room temperature. From a 250–300 ng/µl Myc-tagged rAprA stock in 20 mM NaPO4 pH 7.4, ∼0.5–0.8 µl was added gently to each well to produce a uniform concentration as described previously  and allowed to adhere for 30 min. rAprA was added to a final concentration of 300 ng/ml, or an equal volume of sodium phosphate buffer was added. Cells were lysed and fractionated by centrifugation at 14,000 g for 1 h at 4°C into supernatant and pellet as described previously  was used for 10.1242/joces.260541_sup1Supplementary informationClick here for additional data file.