This translational multi‐centre study explored early changes in serologic variables following B lymphocyte depletion by rituximab (RTX) treatment in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients and investigated in vitro effects on the activity of other immune cells and the vascular endothelium. Eighty‐five SLE patients, seventy‐five RA patients and ninety healthy donors were enrolled. Two additional cohorts of selected SLE and RA patients were treated with RTX for 3 months. Changes in circulating levels of inflammatory mediators, oxidative stress markers and NETosis‐derived bioproducts were evaluated. Serum miRNomes were identified by next‐generation sequencing, and RTX‐induced changes were delineated. Mechanistic in vitro studies were performed to assess activity profiles. Altered inflammatory, oxidative and NETosis‐derived biomolecules were found in SLE and RA patients, closely interconnected and associated to specific miRNA profiles. RTX treatment reduced SLE and RA patients' disease activity, linked to a prominent alteration in those biomolecules and the reversal of altered regulating miRNAs. In vitro studies showed inhibition of NETosis and decline of pro‐inflammatory profiles of leucocytes and human umbilical vein endothelial cells (HUVECs) after B cell depletion. This study provides evidence supporting an early RTX‐induced re‐setting of the pro‐inflammatory status in SLE and RA, involving a re‐establishment of the homeostatic equilibrium in immune system and the vascular wall. Additionally, two independent cohorts of sixteen consecutive RA patients and sixteen SLE patients treated with RTX were studied. Both cohorts attended the Reina Sofia University Hospital of Cordoba (Spain) and the St. Giovanni Bosco Hospital of Turin . All patients fulfilled the American College of Rheumatology revised criteria for SLE2 weekly for 4 weeks (in SLE patients).Patients were treated with RTX as rescue therapy because they were intolerant/refractory to standard immunosuppressants. The therapy with RTX was based on treating physicians’ judgement and agreed case‐by‐case by multidisciplinary evaluation. RTX regimens included two infusions of RTX 1 g 2 weeks apart (in RA patients), or 375 mg/m2.2Plasma and serum samples and purified leucocytes were obtained as previously reported.2.3Total RNA from lymphocytes, monocytes, neutrophils and human umbilical vein endothelial cells (HUVECs) was extracted using TRI Reagent (Sigma). Inflammatory genes were evaluated as described elsewhere.2.4. Two doses of RTX (1 and 10 μg/mL) at 2× (24 and 48 hours) were evaluated in parallel, based on previous studies.Dose‐ and time‐response experiments were carried out on lymphocytes purified from RA patientsLymphocytes purified from eight SLE and eight RA patients were incubated in vitro with 1 µg/mL of RTX for 24 hour. SLE and RA patients selected had high activity scores .The percentages of B cells before and after RTX treatment were analysed by flow cytometry on a flow cytometer FACSCalibur after incubation with PE human anti‐CD19 and FITC human anti‐CD3 (Miltenyi Biotech) as previously described.2.52 atmosphere.Human umbilical vein endothelial cells (HUVECs) were cultured in Endothelial Cell Basal Medium  with 10% FBS, 0.1% human epidermal growth factor (hEGF), 0.1% hydrocortisone, 0.1% gentamicin, amphotericin‐B (GA‐1000), 0.4% bovine brain extract, 100 U/mL penicillin, 100 mg/mL streptomycin and 250 pg/L fungizone (BioWhittaker) at 37°C in a humidified 5% COMonocytes and HUVECs were incubated for 24 hours with serum (10%) obtained from SLE and RA patients at baseline and after 3‐month RTX therapy; neutrophils were treated only for 6 hours to avoid the effect of the naturally occurring apoptotic process.2.6t test or alternatively by a non‐parametric test (Mann‐Whitney rank sum test). Paired samples within the same subjects were compared by Wilcoxon signed‐rank test. Correlations were assessed by Spearman's rank. Statistical analyses were performed with SPSS 19.0 (SPSS Inc).All data were expressed as mean ± SD or median [range] according to their distribution. Following normality and equality of variance tests, clinical characteristics were compared using paired Student's 33.1Clinical and analytical assessments after 3 months of RTX therapy revealed that 81.25% of SLE patients were early responders to the treatment (mean SLEDAI‐2K after treatment = 1.5). The mean levels of acute‐phase reactants (CRP and ESR), complement factors C3 and C4, and anti‐dsDNA levels were also significantly reversed . Mean levels of RF and acute‐phase reactants were also significantly reduced Table .3.2Patients with SLE showed increased serum levels of VEGF, IL‐2, IL‐6, IL‐8, IL‐17, IL‐23, MCP‐1 and tPA compared with HD .NETosis‐derived products, such as cell‐free elastase and cell‐free DNA levels, were strongly associated with clinical parameters in SLE patients, including high SLEDAI‐2K  and some inflammatory parameters, as well as with cell‐free DNA levels, was demonstrated , including 42 up‐regulated and 29 down‐regulated Figure A. By usiIn the miRNome of RA, the serum expression levels of 198 miRNAs were found significantly altered (cut‐off: 2‐fold change) in the exploratory phase, including 114 up‐regulated and 84 down‐regulated Figure B. The in3.5In the cohorts of SLE and RA patients treated with RTX, association studies showed that the basal levels of deregulated circulating parameters related to inflammation, oxidative stress and NETosis were interrelated and linked to a number of clinical features Figure .Circulating levels of microRNAs validated in SLE patients were found linked to levels of inflammatory parameters, as well as to clinical features, including high activity of the disease or hypocomplementemia Figure A‐B.In RA patients, similar profiles of correlation were demonstrated, involving a direct relationship between levels of the circulating miRNAs validated, inflammatory molecules and NETosis‐derived products, as well as with clinical and autoimmune parameters such as activity of the disease and positivity for RF and ACPAs Figure C‐D.Accordingly, the changes in levels of specific microRNAs after RTX treatment on each autoimmune disorder paralleled the changes in several inflammatory molecules and NETosis‐derived products, along with the normalization of clinical features  and the inflammatory status of the lymphocyte population Figure . Thus, tThe in vitro treatment of SLE and RA purified lymphocytes with 1 µg/mL of RTX for 24 hours and reduced the percentage of B lymphocytes . Besides, the treatment with the serum of those patients after RTX therapy abridged this activation status on healthy monocytes Figure A‐B.Similarly, the in vitro treatment of HUVECs with serum from SLE and RA patients before RTX therapy promoted the increased expression of inflammatory mediators such as VEGF, IL‐8, ICAM‐1 and endothelial nitric oxide synthase. The incubation with SLE and RA serum after RTX therapy significantly reduced the expression of the markers with HUVECs’ activation Figure C‐D.3.8Neutrophils from HDs, cultured in the presence of RA and SLE serum, showed a significant induction of NETosis, as well as in the levels of NETosis‐derived products released to the cell culture medium, including elastase and DNA. The NETotic process and the derived products released were reduced when the neutrophils were treated with SLE and RA serum after RTX therapy .In parallel, the serum from RA and SLE patients before RTX therapy promoted a pro‐inflammatory status in neutrophils, demonstrated by the increased expression of TF, IL‐8 and MCP‐1. The incubation with SLE and RA serum after RTX therapy mitigated this activation process Figure E and J.4In the present study, we demonstrated that RTX induced an early re‐setting of the immune and vascular system status involving a significant re‐establishment in the levels of circulating inflammatory and oxidative stress mediators, as well as a substantial reduction of NETosis‐derived products and a prominent reversal of altered miRNAs regulating those molecules. Similar profiles and responses were found in both disorders, from which clinical features most probably depend on specific autoantibodies.It has been reported that RTX administration may decrease the activated phenotype of peripheral and tissue‐resident T cells by abolishing antigen presentation by B cells and may enhance the numbers and function of regulatory T cells (Treg).Monocytes, central players in inflammation and thrombosis, have been found to be activated in SLE and RA through the release of cytokines, chemokines and pro‐thrombotic factors, and massive infiltration in inflammatory sites, such as synovial membranes, kidneys and atherosclerotic plaques.Previous studies from our group have proved the existence of an altered oxidative status in plasma and leucocytes from antiphospholipid syndrome, SLE and RA patients, further reversed by in vivo ubiquinol, statins and tocilizumab treatments, respectively.Rheumatoid arthritis ‐ and SLE‐derived neutrophils are prone to undergo NETosis. NETs themselves could contribute to the generation of autoantigens, or serve as autoantibodies targets, leading to a self‐perpetuating mechanism of disease in these autoimmune conditions.Our results are also in accordance with a very recent study by Kraaij and colleagues,It has been demonstrated that NETosis in RA and SLE consist on a “vicious cycle” on which NETs are generated through stimulation of neutrophils by autoantibodies and inflammatory mediators and, in turn, generate new autoantigens for autoantibody production, leading to a self‐perpetuating mechanism of disease in these autoimmune disorders.Furthermore, it is likely that the depletion of B cells in SLE and RA patients drives the reduction of NETosis, which, in turn, contributes to the reduction of inflammatory mediators and autoantibody production, leading to the improvement of the disease.Overall, at the molecular level, the underlying effects of RTX might involve a concomitant and interrelated reduction of NETosis, along with autoimmune, inflammatory and oxidative stress mediators. This hypothesis was supported by the direct relationship found in vivo and in vitro between the reduction of NETs extrusion products in serum from RA and SLE patients and the decline of disease activity, levels of autoantibodies, acute‐phase reactants, pro‐thrombotic and pro‐inflammatory mediators and bioproducts of oxidative stress.B cell depletion in RA setting leads to ameliorated endothelial function, along with a decrease of disease activity,A set of serum miRNAs, acting as regulators of potential key targets involved in the physiopathology of both disorders, were significantly reversed after RTX therapy. Moreover, that reversion paralleled the improvement of disease activity, as well as the changes occurred in the immune and inflammatory profiles, as well as the down‐regulation in the expression of NETs extrusion products.The miRNAs validated by RT‐PCR in the whole cohort of RA patients have been previously reported to act as relevant regulators of immune cell development, playing a crucial role in the inflammatory response, and acting as key players in the pathogenesis of chronic and autoimmune disorders, including RA.Consistent with our results, a previous study by our team found that after a 6‐month anti‐TNF‐α/DMARDs combination therapy, similar changes in three of the miRNAs found in the present RA cohort significantly increased in response to RTX .On the other hand, this is the first study that identifies the changes promoted in the serum miRNA profile of SLE patients after treatment with RTX. The selected miRNAs were validated as up‐regulated after RTX treatment included main regulators of inflammation, atherosclerosis, CV disease and nephropathy ,In our series of patients, clinical effectiveness of RTX was demonstrated in a real‐life setting, with rapid improvement in SLE and RA signs and symptoms, only after 12 weeks of therapy. Although it is well recognized that complete remission is reached only after 6‐12 months of treatment, and therefore it is not possible to conclude the precise timescale of improvement in the serological indices measured, this study allowed us to demonstrate, as previously reported,Because of the heterogeneity of clinical manifestations, add‐on therapies and applied protocols, we could not investigate the impact of different RTX regimens on immune and vascular phenotypes. In future studies, larger study populations and longer‐term time‐points may identify additional important patient‐centred outcome and molecular targets.SLE and RA patients display altered inflammatory, netotic and epigenetic profiles, which are interconnected and further related to the specific autoimmune profile and the activity of each disease.B lymphocytes play key roles in shaping pathological immune responses of SLE and RA.Associated with clinical response, RTX induced early restoration of homeostasis in immune and vascular systems.Taken together, our data demonstrated that:The authors declare no competing interests.C. P‐S, I. C, N. B, AM. P.T, M. L‐T, A. I‐C, Y. J‐G and I. A‐R developed the in vivo assays, performed the experiments and solved technical problems. L. P‐S, R. O, A. E, MC. C, M. R, MJ. C, D. R, S. S and MA. A followed up with patients and contributed useful discussion and suggestions. C.P‐S, I. C, N. B, E.C and C.L‐P formed the hypothesis, directed and coordinated the project, designed the experiments, analysed the data and wrote the manuscript. E. C, Y.J‐G, M. R, S. S and I. C performed statistical analysis and discussed results. Click here for additional data file. Click here for additional data file.


Radiation‐induced tumor immunity (RITI) influences primary tumor growth and development of metastases in preclinical cancer models with conventional radiotherapy. Antigen‐specific immune responses have also been shown for prostate cancer treated with radiotherapy. We examined whether RITI can be induced in patients with non‐small cell lung cancer (NSCLC) following proton radiotherapy.Pre‐ and post‐radiotherapy plasma samples from 26 patients with nonmetastatic NSCLC who received radiotherapy between 2010 and 2012 were evaluated by western blotting for IgG and IgM bands to assess RITI response to tumor antigens from lung cancer cell lines. Statistical analysis was used to evaluate any correlation among IgG or IgM and clinical outcomes.n = 17) or 6–12 Gy/fraction (n = 4); five received photon therapy at 2–2.5 GyRBE/fraction. Compared with the pretreatment baseline, new IgG or IgM binding was detected in 27% and 50% of patients, respectively. New IgG bands were detected in the 25–37 kD, 50–75 kD, and 75–100 kD ranges. New IgM bands were detected in the 20–25 kD, 25–37 kD, 37–50 kD, 50–75 kD, and 75–100 kD ranges. There was no difference in IgG and/or IgM RITI response in patients treated with photons versus protons, or in patients who received SBRT compared to standard fractionation (P > 0.05). There was no difference in overall survival, metastasis‐free survival, or local control based on IgG and/or IgM RITI response (P > 0.05).Twenty‐one patients received proton therapy at 2 GyRBE/fraction (RITI can be induced in patients with NSCLC through upregulated IgG and/or IgM. RITI response was not associated with proton versus photon therapy or with clinical outcomes in this small cohort and should be examined in a larger cohort in future studies. Non‐small cell lung cancer (NSCLC) is the leading cause of cancer deaths.et al. reported that external‐beam RT and brachytherapy, but not surgery, of nonmetastatic prostate cancer were associated with the development of treatment‐associated autoantibody responses in 14% and 25% of patients, respectively.Studies have demonstrated that RT‐induced tumor immunity (RITI) can affect both primary tumor growth and systemic development of metastases in murine models of breast cancer and lung cancer treated with conventional photon radiation.NSCLC lends itself to the study of anti‐tumor immunity owing to its high number of nonsynonymous mutations compared to other tumor types, due to mutagens such as tobacco smoke, thereby providing an avenue for development of anti‐tumor vaccines and immunotherapy.n = 1), concurrently (n = 15), or not at all (n = 10). Clinical outcomes recorded included death, development of metastatic disease, or local recurrence.Between 2010 and 2012, 26 patients with nonmetastatic NSCLC were enrolled prospectively on an institutional review board‐approved study at the University of Florida Proton Therapy Institute (UFPTI), which allowed collection and analysis of their blood prior to, during, and following radiation treatment to the lung with either photons or protons. Patient, tumor, and treatment information was collected from the medical record. Chemotherapy was delivered either as induction therapy before RT  supplemented with a protease inhibitor cocktail (Sigma‐Aldrich). Total lysate protein was quantified using BCA Protein Assay Kit (Thermo Fisher Scientific).Eight lung cancer cell lines were used for tumor‐associated antigens for immunoblot analysis: H460 (large cell), H23 (lung adenocarcinoma), H522 (lung adenocarcinoma), H1299 (lung carcinoma), A549 (lung carcinoma), H520 (lung squamous cell carcinoma), H2882 (lung squamous cell carcinoma), and H2170 (lung squamous cell carcinoma). This methodology has been adopted from Nesslinger Next, 600–650 μg of protein from cell lysates from the tumor cell lines were separated using 10% Bis‐Tris PAGE gel and transferred to PVDF membrane  at 90–100 V for 2 h. The membrane was blocked with Tris‐buffered saline, 0.1% Tween 20, and 5% milk for 45 min. The membrane was then cut into strips for each patient. The first antibody was patient plasma diluted 1:1000 in a diluent prepared with Tris‐buffered saline, 0.1% Tween 20, and 1% milk for each available time point. The second antibody was either horseradish peroxidase–conjugated goat anti‐human IgG  diluted 1:20 000 or anti‐human IgM  diluted 1:10 000 in the diluent and visualized by enhanced chemiluminescence . Fig. Patients were generally followed with computed tomography (CT) scans every 3 to 4 months for the first 2 years, then every 6 months. Overall survival (OS) was measured from the initiation of RT until death from any cause or last follow‐up. Metastasis‐free survival (MFS) was measured from the initiation of RT until the development of metastatic disease in the contralateral lung or distant site or last follow‐up. LC was defined as the absence of radiographic recurrence within the treatment target and measured from the initiation of RT until the development of a local recurrence or last follow‐up. Statistical analysis was performed with JMP software . The Kaplan‐Meier product limit method provided estimates of OS, LC, and MFS. The log‐rank test statistic assessed statistical significance between strata of selected prognostic factors including age, gender, histology, stage, chemotherapy, radiation modality, standard fractionation versus SBRT, IgG response, and IgM response.n = 17) or 6–12 Gy(RBE)/fraction (n = 4), and five patients received photon therapy at 2–2.5 Gy(RBE)/fraction. The median RT dose was 70 Gy in 35 fractions.Patient and tumor characteristics are summarized in Table P > 0.05).New IgG or IgM binding was detected after RT in seven patients (27%) and 13 patients (50%), respectively. IgG bands were detected in the 25–37 kD range, 50–75 kD range, and 75–100 kD range in three, one, and three patients, respectively. Each of the seven patients had only a single IgG band detected. IgM bands were detected in the 20–25 kD range (two patients), 25–37 kD range (six patients), 37–50 kD range (three patients), 50–75 kD range (five patients), and 75–100 kD range (two patients), with four patients having multiple IgM bands detected. The median time to development of new IgG bands was day 28 of RT , and IgG band persisted to 3 months in four of seven patients (57%). The median time to development of new IgM bands was day 56 of RT . At the time of last plasma assessment (3 months post‐RT), 10 of 13 patients (77%) had persistent IgM bands. Table P = 0.023). Patients with squamous cell carcinoma compared to other histologies  also experienced improved 5‐year OS . There was no difference in 5‐year OS based on IgG and/or IgM RITI response or any other examined clinical factors (P > 0.05).The median follow‐up was 36 months . For all patients, actuarial OS, MFS, and LC rates at 2 years were 62%, 49%, and 86%, respectively. The 5‐year OS, MFS, and LC rates were 27%, 34%, and 80%, respectively. As demonstrated in Table P = 0.022). There was no difference in 5‐year MFS based on IgG and/or IgM RITI response or any other examined clinical factors (P > 0.05).Table P > 0.05).As shown in Table et al., using a similar western blotting technique for IgG antibodies, has previously shown that external‐beam RT and brachytherapy for nonmetastatic prostate cancer were associated with the development of treatment‐associated autoantibody responses in 14% and 25% of patients in their study, respectively.We found that radiation does induce antihuman antibodies in patients with NSCLC treated with either photon or proton irradiation by measuring new IgG and IgM expression after radiation in response to tumor antigens from lung cancer cell lines. New IgG or IgM binding was successfully detected in 27% and 50% of patients, respectively. Nesslinger P > 0.05). In our cohort, there were only five patients who received photon RT and 21 who received protons, limiting our ability to detect a statistically significant difference in IgG and/or IgM RITI response based on RT type. Conversely, as the overall RBE of proton therapy is 1.1 and thus the biological effect is similar to that of photons, the higher LET at the end of the SOBP  may not result in more robust antigen production, supporting our finding of no difference in RITI‐response. To our knowledge, this is the first study to examine this question in patients with NSCLC treated with RT. Lupu‐Plesu et al. recently published results of an in vitro study in head and neck squamous cell carcinoma cells examining the biological effects of proton versus photon RT for genes involved in anti‐tumor autoimmunity, namely PD‐L1, and found that both proton and photon RT augment PD‐L1 mRNA expression in a dose‐dependent manner.We also aimed to examine if there was a difference in RITI response in patients treated with proton RT compared to photon RT. Although often assumed to be a low LET treatment, the LET of protons is heterogeneous, with values 10 to 100 times that of photons over the last 2 mm of the beam range (5 to 20 keV/um) at the edge of the spread‐out Bragg peak (SOBP).P > 0.05). Interestingly, in a study of patients with nonmetastatic prostate cancer who received external‐beam RT with neoadjuvant and concurrent androgen deprivation therapy, performed as a follow‐up to Nesslinger et al.,In our patient cohort, there was no statistically significant difference in OS, MFS, or LC based on IgG and/or IgM RITI response  to serve as a negative control, as demonstrated in Fig. The primary purpose of this study was to determine if radiation induces antitumor antibodies. Indeed, both new IgG and IgM were found in many patients’ blood following radiation consistent with that hypothesis. This observation confirms previous studies in prostate cancer examining for IgG. Notably, it is not possible in our study to determine if these new antibodies were therapeutic or if there was an associated antitumor cellular immune response. In future, however, it will be interesting to perform reverse genetics to discover actionable antigens, using samples from patients with unusually good outcomes. We were underpowered to test our secondary hypotheses to determine if antibody response is associated with improved outcomes or if protons induce a different response than photons.In conclusion, RITI has been consistently demonstrated across three tumor types in clinical studies: prostate cancer,BH is on the scientific advisory board of Merck & Co., Inc.


Congenital disorders of glycosylation type I (CDG‐I) are inborn errors of metabolism, generally characterized by multisystem clinical manifestations, including developmental delay, hepatopathy, hypotonia, and skin, skeletal, and neurological abnormalities. Among others, dolichol‐phosphate‐mannose (DPM) is the mannose donor for N‐glycosylation as well as O‐mannosylation. DOLK‐CDG, DPM1‐CDG, DPM2‐CDG, and DPM3‐CDG are defects in the DPM synthesis showing both CDG‐I abnormalities and reduced O‐mannosylation of alpha‐dystroglycan (αDG), which leads to muscular dystrophy‐dystroglycanopathy. Mannose‐phosphate‐dolichol utilization defect 1 (MPDU1) plays a role in the utilization of DPM. Here, we report two MPDU1‐CDG patients without skin involvement, but with massive dilatation of the biliary duct system and dystroglycanopathy characteristics including hypotonia, elevated creatine kinase, dilated cardiomyopathy, buphthalmos, and congenital glaucoma. Biochemical analyses revealed elevated disialotransferrin in serum, and analyses in fibroblasts showed shortened lipid linked oligosaccharides and DPM, and reduced O‐mannosylation of αDG. Thus, MPDU1‐CDG can be added to the list of disorders with overlapping biochemical and clinical abnormalities of CDG‐I and dystroglycanopathy.Mannose‐phosphate‐dolichol utilization defect 1 patients can have overlapping biochemical and clinical abnormalities of congenital disorders of glycosylation type I and dystroglycanopathy. CDG‐I leads to multiorgan phenotypes, including disorders of the brain and neuromuscular system, hepatopathy, skin, and skeletal abnormalities.The congenital disorders of glycosylation (CDG) are inborn errors of metabolism with a great genetic heterogeneity. CDG‐I defects are located in the assembly of the lipid linked oligosaccharide (LLO) glucoseGenetic defects in the biosynthesis of dolichol‐phosphate‐mannose  lead to CDG‐I profiles of transferrin. However, the clinical phenotypes associated with these defects are very different, which is partly explained by that DPM is required for N‐glycosylation, O‐mannosylation, C‐mannosylation, and GPI‐anchor synthesis. In DOLK‐CDG, DPM1‐CDG, DPM2‐CDG, and DPM3‐CDG, biochemical and phenotypic abnormalities overlap with CDG‐I and muscular dystrophy‐dystroglycanopathy, which is caused by reduced O‐mannosylation of alpha‐dystroglycan (αDG).Mannose‐phosphate‐dolichol utilization defect 1 (MPDU1) is involved in the flipping of DPM and dolichol‐phosphate‐glucose (DPG) across the ER membrane and for efficient use of DPM and DPG within the ER lumen.22.1Plasma and fibroblasts were obtained for CDG diagnostics in the Radboudumc Expertise Center for Disorders of Glycosylation in accordance with the Declaration of Helsinki. Informed consent was obtained from patients or their legal representatives. Written informed consent was obtained for inclusion of facial images.2.2Serum transferrin isoelectric focusing (TIEF) and electrospray ionization mass spectrometry (ESI‐MS) were performed as described.2.3Fibroblasts were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal calf serum (Gibco) and 1% penicillin‐streptomycin (Gibco). Fibroblast lysates were enriched for glycoproteins using agarose‐conjugated wheat germ agglutinin (Sigma). Proteins were loaded on 10% polyacrylamide gels and transferred to nitrocellulose membranes by western blotting. Membranes were used for laminin overlay (LO)33.1The two patients are brother and sister from Iraqi origin. They were the third and fourth child of consanguineous parents (first cousins), and had two healthy sisters Figure A. Table 3.1.1Patient P1, a girl, was born spontaneously in the 37th gestational week after an uneventful pregnancy. At birth, her length was 43 cm , −2.08), weight 2390 g , and head circumference 32 cm. Because of postnatal apneas and bradycardias with deep desaturations, followed by respiratory failure, she was resuscitated and referred to the neonatal intensive care unit. Slight dysmorphic features were noted including a smooth philtrum, retrognathia, low‐set, posterior‐rotated ears, and hypertelorism with megalocorneae . Brain magnetic resonance imaging (MRI) revealed enlarged outer fronto‐temporo‐parietal cerebrospinal fluid spaces. Abdominal sonography as well as magnetic resonance cholangiopancreatography showed a vast dilatation of the intrahepatic biliary ducts, especially within the left lobe of the liver . Further ophthalmologic examination revealed a buphthalmos with congenital glaucoma. The eye lenses were slightly cloudy and pupillary iris showed pigment epithelial cysts. A sensorineural hearing loss was identified with brainstem evoked response audiometry (BERA) evaluation. There were no abnormalities of the skin.Blood CK levels were elevated up to 3090 U/L without substantial elevation of aspartate amino transferases  and alanine amino transferases , biochemical signs of cholestasis or icterus. There was a marked congenital thrombocytopenia , a slightly decreased fibrinogen , and a nonmeasurable antithrombin III .Prometaphase karyotype analysis showed a normal 46,XX female karyotype and a normal array‐comparative genomic hybridization (array‐CGH), without any microdeletion or duplication. Congenital toxoplasmosis, other , rubella, cytomegalovirus, and herpes (TORCH) infections, peroxisomal diseases as well as mucopolysaccharidoses were ruled out by serology and metabolic screening. The serum amino acids, acylcarnitine profile, very long chain fatty acids, 7‐dehydrocholesterol, and urine organic acids were normal. In view of the clinical symptoms of multiorgan involvement with seizures, cardiomyopathy, eye, and liver abnormalities as well as thrombocytopenia and ATIII deficiency, a CDG was suspected and TIEF and ESI‐MS of serum transferrin were performed. These revealed reduced tetrasialotransferrin, with increased asialo‐ and disialotransferrin indicative of a CDG‐I . He was born premature per cesarean section in the 31st gestational week because of a rupture of the placental membrane and uterine bleeding. His birth length was 38 cm , his weight was 1420 g , and he had a head circumference of 28 cm. He showed a smooth philtrum, retrognathia, and low‐set, posterior‐rotated ears. He had hypertelorism with prominent appearing eyes with enlarged and cloudy corneae  at the age of 3 weeks, which developed in combination with an arterial hypertension.At the age of 6 months, he had generalized seizures accompanied by desaturations, whereas EEG showed parieto‐temporo‐occipital and multiregional spikes over both hemispheres. Sonography of the brain showed no gross abnormalities. Abdominal sonography revealed dilatation of the intrahepatic biliary duct system Figure G. KidneyThe ophthalmological examination revealed a buphthalmos with slightly opaque corneae with a diameter of 11 mm and severe congenital glaucoma, requiring prompt trabeculectomy intervention. The BERA showed a sensorineural hearing loss.Chromosomal analysis showed a normal 46,XY karyotype, and array‐CGH as well as TORCH serology were normal. He also exhibited thrombocytopenia , elevated CK levels (up to 905 U/L), and low ATIII , but transaminases, fibrinogen, thyroxin‐binding globulin, and bilirubin were normal.Like his sister, P2 hardly presented any psychomotor development, and had to be fed parenterally or by a gastric tube. He showed severe apneas and bradycardias which were not treatable, and he died at the age of 11 months from respiratory failure.3.2MPDU1): c.218G>A; p.(G73E) in both patients. This mutation has been reported in two other MPDU1‐CDG patients.Whole exome sequencing revealed a homozygous missense mutation Chr17(GRCh38): g.7585994G>A; NM_004870.3 with overlapping symptoms of CDG‐I and dystroglycanopathy. MPDU1‐CDG is the fifth disorder related to DPM biosynthesis or utilization that bridges CDG‐I and the O‐mannosylation disorders.Here, we describe two patients with mutations in So far, seven MPDU1‐CDG patients have been described. All patients showed psychomotor retardation and most patients had hypotonia, facial dysmorphism, eye defects, apnea, and skin abnormalities such as ichthyosis.Interestingly, the two siblings described here showed a very similar clinical pattern, including very similar facial features, whereas siblings with other CDG disorders do not necessarily share as many clinical characteristics, for example, in ALG3‐CDG.Unfortunately, the role of glycosylation in cardiac disease is not completely understood. Therefore, there is no clear explanation why patient 1 shows DCM, whereas patient 2 developed HCM. HCM has been reported in PMM2‐CDG, ATP6V1A‐CDG, and ATP6V1E1‐CDG.PKHD1 mutations, which are also known to cause autosomal recessive polycystic kidney disease. PKHD1 is extensively glycosylated,In addition, we reported biliary duct abnormalities and renal cysts in our MPDU1‐CDG patients. Biliary duct abnormalities have also been observed in patients with mutations in mannose‐6‐phosphate isomerase (MPI), which interconverts fructose‐6‐phosphate to mannose‐6‐phosphate.In summary, we reported on two newly identified MPDU1‐CDG patients and showed reduced N‐glycosylation and O‐mannosylation in patient material. Together with the muscular, eye, and heart abnormalities, this adds MPDU1‐CDG to the list of DPM disorders causing biochemical and clinical abnormalities overlapping CDG‐I and dystroglycanopathy. Identification of additional patients with defects in DPM availability, and extensive O‐mannosylation and N‐glycosylation analysis of patient material is required to further increase our understanding of the pathophysiology of the DPM disorders.The authors declare that they have no conflict of interest.W.V.T. has set up the study, performed laboratory experiments, acquired and interpreted data, and written the manuscript. A.A. has performed WES analysis and critically reviewed the results and revised the manuscript. E.K. has collected, analyzed, and reviewed clinical data and written the manuscript. N.A.B. has performed ESI‐MS analysis and critically reviewed the manuscript. M.A.W. has critically reviewed results and revised the manuscript. C.T. has provided LLO analysis and revised the manuscript. D.J.L. contributed to the setup of the study, has interpreted data, critically reviewed the results and the manuscript, and supervised the study.All procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in this study or from their legal representatives. Written informed consent was obtained for inclusion of facial images.


Although aminoglycosides are routinely used in neonates, controversy exists regarding empiric dosing regimens. The objectives were to determine gentamicin pharmacokinetics in neonates, and develop initial mg/kg dosing recommendations that optimized target peak and trough concentration attainment for conventional and extended-interval dosing (EID) regimens.Patient demographics and steady-state gentamicin concentration data were retrospectively collected for 60 neonates with no renal impairment admitted to a level III neonatal intensive care unit. Mean pharmacokinetics were calculated and multiple linear regression was performed to determine significant covariates of clearance (L/h) and volume of distribution (L). Classification and regression tree (CART) analysis identified breakpoints for significant covariates. Monte Carlo Simulation (MCS) was used to determine optimal dosing recommendations for each CART-identified sub-group.Gentamicin clearance and volume of distribution were significantly associated with weight at gentamicin initiation. CART-identified breakpoints for weight at gentamicin initiation were: ≤ 850 g, 851-1200 g, and > 1200 g. MCS identified that a conventional dose of gentamicin 3.5 mg/kg given every 48 h or an EID of 8-9 mg/kg administered every 72 h in neonates weighing ≤ 850 g, and every 24 and 48 h, respectively, in neonates weighing 851-1200 g, provided the best probability of attaining conventional (peak: 5-10 mg/L and trough: ≤ 2 mg/L) and EID targets . Insufficient sample size in the > 1200 g neonatal group precluded further investigation of this weight category.This study provides initial gentamicin dosing recommendations that optimize target attainment for conventional and EID regimens in neonates weighing ≤ 1200 g. Prospective validation and empiric dose optimization for neonates > 1200 g is needed. Although aminoglycosides are routinely used in neonates, controversy exists regarding recommended empiric dosing to optimize target attainment with either conventional dosing (peak: 5-10 mg/L and troughs ≤2 mg/L) or extended-interval dosing (higher peak and undetectable trough) –5. For tWhile data exist to support the use of EID in neonates , 11–26, The objectives of this study were to determine the pharmacokinetics of gentamicin in neonates with no clinical evidence of renal impairment in a Level III NICU, identify significant covariates of gentamicin PK parameters in neonates, and develop practical initial dosing recommendations with the highest probability of attaining target peak and trough serum concentrations currently accepted in clinical practice for both conventional dosing (trough < 2 mg/L and peak 5–10) and EID  of gentamicin.This retrospective study was conducted in the level III NICU at Sunnybrook Health Sciences Centre (SHSC) in Toronto, Ontario, Canada. SHSC is a 1325-bed tertiary care teaching hospital, with 48 NICU beds .Neonates admitted to the NICU from March 12th, 2010-November 26th, 2013 who were prescribed gentamicin to treat a documented or presumed infection and received > 48 h of gentamicin were identified from a hospital electronic database . PatientNeonates were excluded if they developed acute renal failure (urine output < 1 mL/kg/hr. or serum creatinine [sCr] > 100 μmol/L) before or during gentamicin therapy, had an increase in sCr > 25% from baseline during treatment, or had a calculated gentamicin half-life > two standard deviations (SDs) from the mean half-life observed in the study population following data analysis, without the availability of an additional set of serum concentrations to confirm the accuracy of this calculated half-life.At the time of this study, neonatal SHSC conventional gentamicin dosing recommendations aimed to target a peak and trough serum concentration of 5–10 mg/L and ≤ 2 mg/L, respectively. .e), half-life (t1/2), volume of distribution (Vd), clearance (Cl), initial estimated dose  and dosing interval for conventional (trough ≤ 2 mg/L and peak 5-10 mg/L) and EID  using an infusion time of 1 h. . Since PK parameters display a lognormal distribution, the geometric mean, 95% confidence interval (CI) and range were reported for ki.e neonates with a birth weight below the 10th percentile for neonates of the same GA] status) were input in the regression analysis. Variables that were significant (p <  0.05) with bivariate analysis and had a tolerance statistic of ≥0.4 when assessed for multicollinearity were included in a multivariable linear regression (MLR) model to identify those that remained significant using a p <  0.05. Analyses were run using SAS Version 9.4 .The data consisted of 60 neonates, of which only 4 had a second set of data with gentamicin levels. This sample size, along with the limited number of repeated measures, was insufficient to run a robust hierarchical model. To circumvent this problem, only data from the first set of gentamicin levels were included for the analyses. Clinical parameters that would have been known prior to the initiation of gentamicin, were not calculated using other parameters input into the regression analysis and were parameters with values available for > 80% of the gentamicin levels  to confirm the validity of the CART-identified breakpoints. An analysis of variance (ANOVA) with Tukey-Kramer Multiple Comparisons Test for data that passed the test for normality or a Kruskal-Wallis Test with Dunn’s Multiple Comparison Test for data that did not pass the test for normality (if > 2 sub-groups were identified) or unpaired t-test (if 2 sub-groups were identified) were used to compare the sub-groups  for differences in pharmacokinetic parameters with a p <  0.05 considered statistically significant.A Classification and Regression Tree (CART) analysis  was used to identify whether practical breakpoints existed for statistically significant MLR-identified covariates of gentamicin Cl (L/h) and Vd (L). The initial CART analyses input all statistically significant variables identified in the MLR analyses for Vd (L) and/or Cl (L/h). CART analyses for Cl and Vd were pruned to the simplest tree, utilizing forced splits to identify clinically practical breakpoints, with the lowest relative error. Forced splits were selected as practical rounded breakpoints derived from the CART identified breakpoint and which had equal or lower relative error than the CART identified breakpoint. The optimal CART model was that which allowed for the fewest sub-groups and had the lowest relative error. CART-identified breakpoints for covariates of gentamicin Vd and/or Cl were used to create patient sub-groups. Mean pharmacokinetic data were calculated for each identified sub-group and the sub-groups were compared to verify the existence of a significant difference in pharmacokinetic parameters  and would be greater than 2 SDs from the mean of any sub-group weight category. As part of each MCS, an assessment of the probability of attaining a Peak:MIC ratio of ≥8 was completed. The MIC was assumed to have a normal distribution truncated at a minimum of 0.5 mg/L and maximum of 8 mg/L  (MCS). The mean and SDs for kpatients .Of a total of 99 patients for whom there was documentation of therapeutic drug monitoring (TDM), 60 patients were eligible for study inclusion to complete the pharmacokinetic analysis Fig. . PatientEscherichia coli  and Klebsiella spp  (Table Forty-five of the 60 neonates (75%) included in this study were born at ≤28 weeks gestation. The mean (± standard deviation (SD), range) GA of neonates at birth and CGA at gentamicin initiation were 27  weeks and 28  weeks, respectively. Thirty-nine patients (65%) had a BW of < 1000 g (defined as extremely low BW ) and 55 p < 0.05) of gentamicin Vd (L) and Cl (L/h) from the bivariate screen and multivariable model are detailed in Table P < 0.0001). Covariates that remained significant following MLR for Cl (L/h) were PNA at gentamicin initiation (p = 0.0001), gender (p = 0.0447), and weight at gentamicin initiation (p < 0.0001).Significant predictors  and Cl(L/h) produced breakpoints based on the patients’ weight at gentamicin initiation, with a forced split at ≤ 850 g, > 850 g – 1200 g, and > 1200 g. These breakpoints provided the simplest trees with the lowest relative error . CART identified trees and breakpoints for other parameters in the MLR regression equations (PNA and gender) did not exist.e and Cl (L/h/kg) for neonates ≤ 850 g were significantly different from the other weight breakpoints (Table p > 0.05) , limited weight range  and wide confidence intervals of the mean calculated pharmacokinetic parameters in the > 1200 g weight sub-group caused concern regarding the robustness of any dosing recommendations derived for this weight sub-category. As a result, the > 1200 g weight sub-category of neonates was excluded from further analyses. The significant difference in both ke and Cl (L/h/kg) between the ≤850 g and 851-1200 g sub-groups (Table The mean kts Table . Mean ph5) Table . The smaMCS of weight-based dosing regimens were performed for neonates weighing ≤ 850 g Table . The MCSe and Cl (L/h/kg) between the ≤850 g and 851-1200 g sub-groups, the use of the simple weight range breakpoints of ≤850 g and 851-1200 g as the sub-groups for practical and convenient empiric gentamicin dosing calculations in neonates is rational. Dosing of 3.5 mg/kg/dose administered every 48 h for neonates weighing ≤ 850 g, and every 24 h for neonates weighing 851-1200 g provided the best probability of attaining conventional targets . Dosing of 8-9 mg/kg/dose administered every 72 h in neonates weighing ≤ 850 g and every 48 h in neonates weighing 851-1200 g provided the best probability of attaining EID targets .This retrospective pharmacokinetic study evaluated hospitalized neonates with normal renal function, and a median CGA at gentamicin initiation of < 28 weeks. Seventy-five percent of those included were born at ≤ 28 weeks gestation and 92% had a BW of < 1500 g. Gentamicin Cl (L/h) and Vd (L) were significantly associated with weight at gentamicin initiation . Since no significant difference in pharmacokinetics existed for neonates weighing > 1200 g versus 851-1200 g, due to inadequate sample size in the largest weight category, we did not explore the > 1200 g sub-group further. No CART identified trees with breakpoints for the other MLR equation covariates (PNA and gender) existed. Based on the absence of CART identified trees and breakpoints for PNA and gender and the identification of a significant difference in both kThe strengths of our study include the determination of gentamicin pharmacokinetics in a large sample of premature and low-birth weight neonates for whom data are currently lacking; the identification of significant covariates for Vd and Cl with determination of practical weight breakpoints; the utilization of MCS with 1 million iterations to develop simple initial gentamicin dosing nomograms for both conventional and EID for low-birth weight neonates with an excellent probability of target peak and trough attainment; and the provision of tables itemizing probabilities of target attainment (including Peak:MIC ratio) for a range of potential dosing options enabling institutional selection of initial dosing guidelines based on their GNB susceptibility patterns and desired target serum concentrations. In addition, our rigorous study design which limited the inclusion of gentamicin levels to those with a confirmed time for dose administration and serum sampling increases the validity of our results.The weaknesses of our study include its retrospective design and associated risk of unrecognized confounders; the inability to generalize our results to neonates > 1200 g and SGA infants; and the risk of incomplete gentamicin distribution at time of sampling for peak concentrations. However, since our mean pharmacokinetic parameters were comparable to those reported in other studies , our samSimilar to other pharmacokinetic studies, our multivariable analysis indicated that the Vd of gentamicin in neonates is associated with body weight , 28, 29.Our multivariable analysis indicated that gentamicin clearance in neonates is associated with PNA, as well as bodyweight, and gender. The correlation between PNA and gentamicin elimination has been previously reported in the literature , 28, 29,CART analysis confirmed breakpoints for weight at gentamicin initiation for both Vd and Cl and demonstrated that neonates had altered Vd (L) and Cl (L/h) based on these weight breakpoints. This allowed the use of the CART derived weight breakpoints (≤850 g and 851-1200 g) to divide our data into homogenous patient sub-groups for practical empiric gentamicin dosing recommendations and provides a new and convenient nomogram for gentamicin dosing  with a MCS demonstrated high probability of target attainment. The mean gentamicin Vd  and Cl  identified in this study are comparable to those reported in a study of infants born at less than 28 weeks gestation (Vd = 0.50 L/kg and Cl = 0.032 L/h/kg) .E coli, are emerging as the leading cause of systemic infections in neonates. Recent microbiological reports of E coli isolates from Canadian pediatric patients report a mean MIC90 of 2 mg/L for gentamicin [Our study confirms previous reports , 27, 38 ntamicin . Therefontamicin . In thisntamicin . Our MCSntamicin . HoweverE. coli.Although the study by Lanao et al  was publThe study contributes new data based gentamicin dosing guidelines for both initial conventional and EID in neonates ≤ 1200 g, a patient population under-represented in neonatal studies and for whom limited data exists for gentamicin dosing. Our results provide clinicians with practical and simple initial dosing recommendations based on weight at time of gentamicin initiation with a high probability of target peak and trough attainment. Confirmatory gentamicin levels  are recommended to further refine dosing. If more prolonged therapy is needed, then repeat levels are recommended to identify changes in the neonate’s gentamicin pharmacokinetics with PNA and weight. The gentamicin levels that were targeted in this study reflect accepted safe and effective levels for gentamicin in neonates , 11–26.


Coptis chinensis (CC) with a history of using for thousands of years. This work aimed at summarizing coptisine's activity and providing advice for its clinical use. We analysed the online papers in the database of SciFinder, Web of Science, PubMed, Google scholar and CNKI by setting keywords as ‘coptisine’ in combination of ‘each pivotal pathway target’. Based on the existing literatures, we find (a) coptisine exerted potential to be an anti‐cancer, anti‐inflammatory, CAD ameliorating or anti‐bacterial drug through regulating the signalling transduction of pathways such as NF‐κB, MAPK, PI3K/Akt, NLRP3 inflammasome, RANKL/RANK and Beclin 1/Sirt1. However, we also (b) observe that the plasma concentration of coptisine demonstrates obvious non‐liner relationship with dosage, and even the highest dosage used in animal study actually cannot reach the minimum concentration level used in cell experiments owing to the poor absorption and low availability of coptisine. We conclude (a) further investigations can focus on coptisine's effect on caspase‐1‐involved inflammasome assembling and pyroptosis activation, as well as autophagy. (b) Under circumstance of promoting coptisine availability by pursuing nano‐ or microrods strategies or applying salt‐forming process to coptisine, can it be introduced to clinical trial.Coptisine is a natural small‐molecular compound extracted from  A study by Su et al employed rat model with two administration routes, authors reported the oral bioavailability and tIt is worth mentioning that the plasma concentration of coptisine and other CC isoquinoline alkaloids displayed explicit non‐liner relationship with the oral dosage, it exerted an obvious process of limitation, and thus, the bioavailability decreased along with the elevated dosage. More details are depicted in Table 3Numerous researches have verified the vital role of MMPs, MMP‐2 and MMP‐9 in particular, in promoting the membrane‐basement invasion of tumour cells.Similar anti‐cancer property of coptisine was observed in liver HepG2, breast MDA‐MB‐231, pancreas PANC‐1 cell and lung A549 cancer cells. To be specific, coptisine  functioned as reducing viability and growth of HepG2 through diminishing miR‐122 expression. Meanwhile, it induced apoptosis by enhancing transduction of 67kD laminin receptor/cGMP pathway in multiple human hepatoma cells.Several studies have established that coptisine exerts ameliorative effect on cancer in in vivo model. Cao et al reported that coptisine  could prevent tumour development by inhibiting MFG‐E8, MMP‐2/9, N‐cadherin, vimentin and Snail expressions and increase E‐cadherin expression in HCT116‐challenged nude mouse.4Involved in the pathogenesis of many types of cancer, inflammation is one part of protective biological response to harmful stimuli . Inflammatory transductions depend on cellular pathways, among which NF‐κB, MAPK and PI3K/AKT attract most attention in resent research.Accumulative studies have been designed to assess coptisine's anti‐inflammatory effect in vitro Figure . It was Similarly, inhibition of inflammatory process was also observed in animal models including xylene‐induced ear oedema, carrageenan‐elicited paw oedema, LPS‐induced shock, OVA‐induced allergic rhinitis and MSU‐elicited gouty after coptisine administration.5The typical cardiovascular diseases (CVDs) includes heart attack , atherosclerosis, heart failure and stroke.2O4‐stimulated H9c2 cardiomyocyte, and the underlying mechanism was the suppressing of autophagy and apoptosis markers.2+ influx.Coptisine exerted ameliorative effects on CVDs both in vitro and in vivo. A study by Wang et al reported the cardiovascular protection property of coptisine  in NaS‐/‐ C57BL/6J mice demonstrated coptisine exerted lipid‐lowering property through regulating biomarkers in serum. Coptisine reduced mRNA level of p65, VCAM‐1, ICAM‐1 and IL‐6/1β in aorta and liver, and it also prevented the phosphorylation of both p38 and JNK1/2.A recent in vivo study which conducted to examine how coptisine  affects ApoE6bifdobacterium adolescentis property was methylenedioxy at C2 or C3.The study screened the five protoberberine alkaloids by microcalorimetry and confirmed berberine and coptisine were more appropriate candidates as new anti‐infective drug.71‐42 in microglia cells. Coptisine was also reported to down‐regulate TXNIP protein concentration in SH‐SY5Y cells, which checked oxidative damage.A study designed to evaluate the neuroprotection effect of coptisine discovered that coptisine  reversed the enhanced IDO activity in AβPPswe/PS1ΔE9 mice, and it as well prevented AD pathogenesis by means of blocking microglia and astrocyte activation through inactivating CD11b and GFAP, respectively.Interestingly, the effect of coptisine  on MAPK subfamilies ERK, JNK and p38 demonstrated to be reversed in oxidative injury model of AAPH‐challenged adult zebrafish.Pharmacological research in current years indicates coptisine is a multi‐targeting isoquinoline alkaloid Tables  and 3, w8Coptisine is a typical quaternary proberberine alkaloid (QPA) with a parent nucleus of isoquinoline Figure , which p9Documented in ancient pharmaceutical books, CC has been used since 2000 years ago in the treatment for ocular inflammation, diarrhoea and damp‐heat‐caused abdomen disorders, which are also the common indications of CC’s clinical use by now. Since CC exerts effect of clearing away damp‐induced heat, we can also be informed from some Chinese plant medicine books of its anti‐tumour action.On the other hand, dose‐effect relationship determines how well the drug works. In clinical, owing to the affecting factors such as age, disease location, administration route and processing method, CC exhibits a very wide daily single dose range of 1.5‐40 g.50 value of CC in mice was 4.89 g/kg,50 value of berberine was, respectively, 329 , 9.0386 (i.v) and 57.6103(i.p) mg/kg, and coptisine's LD50 value was 880.10 mg/kg,50 study is probably due to the gastrointestinal toxicity, rather than systemic toxicity.When evaluating drug efficacy, toxicity and safety should be firstly taken into consideration. It has been reported that the oral LD10Epimedium koreanum Nakai, emodin from Rheum palmatum L, quercetin from Hypericun ascyron L and curcumin from Curcuma louga L are all reported to modulate various chronic diseases within safe dose range.Chemically modified drug plays a dominant role in clinical because it exerts potent therapeutical effect by affecting accurate and specific target.The main anti‐cancer approaches include (1) invasion and metastasis prevention, (2) apoptosis or (3) autophagy induction, among which (1) and (2) are reported to be successfully achieved by coptisine treatment in many cancer cell lines to date. Apart from mechanisms mentioned above, cellular senescence is another promising approach in anti‐cancer therapy.Cardiovascular diseases such as hyperlipidaemia and hypertension are global health issue and closely related to metabolic disorder which is accompanied by low‐grade chronic inflammation.Autophagy is involved in the progression of multiple disorders including cancer and CADs.As mentioned in previous chapters, the plasma concentration of coptisine and other CC isoquinoline alkaloids demonstrates obvious non‐liner relationship with dosage, and the low dissolution in intestinal fluid dominantly limits the absorption amount. In recent years, pharmaceutics methods like nano strategies and microrods have been employed to promote the intestinal dissolution of berberine. Given the similar parent structure shared by berberine and coptisine, further study is advised to focus on new pharmaceutics strategy which is able to improve coptisine's dissolve rate.To sum up, coptisine is a promising drug with multiple targets, while there is still a knowledge gap before coptisine meets the requirements to be introduced to clinic and further applied to the prevention and therapy of diseases such as cancer, inflammation, anti‐bacteria and CVDs. Besides, more investigation is needed to promote coptisine's bioavailability and meanwhile reach the balance between toxicological safety and therapeutic efficacy.The authors declare no conflicts of interest.JS.W and Y.L wrote the draft; JS. W, S.L and SY.S analysed the data; DH.D, L.X and YF.H searched the database and extracted literatures; JS.W and DH.D prepared all the figures; and P.W and XL.M supervised the work.


Mesona procumbens Hemsl) on human HSCs, the expression levels of cleaved caspase‐3, p38, and c‐Jun N‐terminal kinase (JNK) were assessed using Western blotting, and the caspase‐3 activity was measured using caspase‐3/CPP32 colorimetric assay kit. Hsian‐tsao extract (HTE) increased the activity of caspase‐3 and the level of activated caspase‐3, indicating the activation of apoptosis. The intracellular reactive oxygen species (ROS) level increased in a dose‐dependent manner. This increase was prevented by an antioxidant, suggesting that HTE induces ROS accumulation. In addition, we found that HTE induced the phosphorylation of the mitogen‐activated protein kinases JNK and p38. These collective data indicate that HTE induces apoptosis via ROS production through the p38, JNK, and caspase‐3‐dependent pathways. HTE may decrease HSC activation in liver fibrosis and may have a therapeutic potential.The activation of hepatic stellate cells (HSCs) is an important step in the progress of liver fibrosis. Fibrosis can be impeded by HSC reversion to a quiescent state or HSC clearance through apoptosis. To investigate the apoptotic effects of hsian‐tsao ( Sigma‐Aldrich was the manufacturer of all other chemicals.2.3Human primary hepatic stellate cells (hHSCs) were obtained from ScienCell Research Laboratories and were cultured according to the manufacturer's instructions. Briefly, the cells were seeded into poly‐l‐lysine‐coated T‐25 flasks in Stellate Cell Medium (ScienCell Research Laboratories) containing 2% fetal calf serum (FCS) and stellate cell growth supplement (ScienCell Research Laboratories).2.4We used a WST‐1 cell proliferation assay kit and caspase‐3/CPP32 colorimetric assay kit to detect the cell viability and caspase‐3 activity in this study, respectively. The protocols were supplied by the manufacturer and were modified according to our previous study  was used to detect the relative ROS levels after the cells were stained with the reagent 2′,7′‐dichlorofluorescein diacetate .2.6The cell was harvested and lyzed according to a protocol from our previous study . Additionally, the Bonferroni post hoc test was used in this study. A 33.1The cell viability was inhibited in a dose‐dependent manner with HTE treatment (10–100 μg/ml) Figure a–c. We h3.2DCF‐DA staining showed that HTE 100 μg/ml) significantly induced ROS production Figure a. Furthe00 μg/ml 3.3The JNK and p38 pathways have been implicated in cell apoptosis  were extracted from hsian‐tsao (Yeh, Huang, & Yen, p‐Hydrobenzoic acid has antimicrobial, antialgal, antimutagenic, antiestrogenic, hypoglycemic, anti‐inflammatory, antiplatelet aggregating, nematicidal, antiviral, and antioxidant functions; in addition, it is widely used in cosmetic products (Azam, Dharanya, Mehta, & Sachdeva, Currently, phenolic compounds (kaempferol, apigenin, caffeic acid, protocatechuic acid, syringic acid, vanillic acid, and Herein, JNK and p38 were activated significantly after HTE treatment Figure . Consist5We have shown a significant induction of apoptosis in HSCs by hsian‐tsao treatment. These apoptotic effects are mediated through multiple mechanisms: the increased accumulation of ROS and the activation of JNK and p38. The structures of the bioactive compounds in HTE remain to be elucidated. In conclusion, our study makes a significant contribution toward the identification of pathways which can be pharmaceutical targets for the development of novel therapeutic approaches in the treatment of liver fibrosis.None declared.This study does not involve any human or animal experiments.


Defining hemostatic profile for preterm infants is a challenge when severe bleedings are frequent.The aim was to define the hemostatic profile at birth of infants with spontaneous prematurity and to evaluate whether characteristic profiles can predict the development of intraventricular hemorrhage (IVH) in prematures.5/7 gestational age (GA)  and median weight of 1145 g . Levels of fibrinogen, factor II (FII) and factor V (FV) rose with GA . In the group of 230/7 – 286/7 GA, the 5th percentile was defined as 0.6 g/L for fibrinogen, 15 IU/dL for FII and 16 IU/dL for factor V (n = 30). In the group of 290/7–326/7 GA, the 5th percentile was defined as 1.0 g/L for fibrinogen, 24 IU/dL for FII and 41 IU/dL for factor V (n = 46). In the group of 330/7–366/7 GA, the 5th percentile was defined as 1.0 g/L for fibrinogen, 24 IU/dL for FII and 30 IU/dL for factor V (n = 46). Level of fibrinogen was higher in case of vaginal delivery and lower in case of IUGR. Only lower level of FV at birth was significantly associated with IVH  with an unadjusted OR per SD increase in FV of 0.57 . After adjustment for age, the association between FV level and IVH was slightly attenuated  but remained not significant (p = 0.22).There was no correlation with FII and fibrinogen.We included 122 newborns with a median age of 31We can define hemostastic profile of prematures and corroborate references ranges for studied parameters. Further large studies are still called for, to correlate the grade of hemorrhage and the factor V level at birth. The coagulation system of the newborn differs from that of adults, and among full-term newborn, premature newborn and fetus, the hemostatic balance is different. This is the developmental hemostasis concept –5.The premature hemostatic balance is an evolving process depending on fetal hepatic maturation  and the maturation inherent to birth  . Indeed,Most data on coagulation factors in the preterm neonate’s population have been obtained from fetuses at the occasion of prenatal diagnosis. However, data from fetus are considering only fetal hepatic maturation whereas it was demonstrated that there is an effect of labour and birth itself on the coagulation system in the neonate , 7.Thanks to advances in neonatal resuscitation, more extremely premature infants can survive and there is a crucial need for consensus in interpreting coagulation profile in preterm neonates . Indeed,Among bleeding complications, intraventricular hemorrhage (IVH) is associated with an increased risk of mortality and long-term morbidity. Indeed, seizures, mental retardation and cerebral palsy have been reported in 45 to 86% of preterm neonates with IVH and even a low grades of IVH can adversely influence long-term neurodevelopmental outcomes in extremely preterm infants . The potThe aim of this study was to define the hemostatic profile  of premature infants and to evaluate whether characteristic profiles can predict the development of IVH in premature infant. Secondary objectives were to correlate the fibrinogen, factor V and factor II values with antenatal corticosteroids, delivery, gestational age (GA), weight, intrauterine growth restriction (IUGR), biological data and the newborn outcome.This retrospective observational cohort study was conducted in the neonatal intensive care unit (NICU) of the Lille University Hospital (France). Infants born between 23 and 36 weeks of gestation who had coagulation testing were eligible if diagnosed with spontaneous prematurity  between January 2016 and December 2017. Exclusion criteria were preeclampsia or perinatal asphyxia context.According to our department protocol, coagulation tests were performed within the first hour of life before vitamin K administration as part of routine laboratory testing. Coagulation testing was not exhaustive, but performed according to clinician appreciation. Coagulation tests included fibrinogen and factors II and V in order to cover the main coagulation pathway.9/L platelets in neonates < 28 weeks and < 140 × 109/L platelets in neonates > 28 weeks.The following characteristics were collected in infants: gestational age, birth weight, administration of antenatal steroids, mode of delivery, birth fibrinogen, birth factor II, birth factor V, presence and grade of IVH. GA at delivery was determined by routine first trimester ultrasound examination. Cranial ultrasound was performed by a trained neonatologist within the first 24 h of life and control between day 3 and day 5. Cranial ultrasound was repeated every week the first month of life, and then once a month until term, if normal. Cranial ultrasounds were conducted more frequently in case of IVH grade 3 or 4 (frequency depending on evolution and complications).The time of the diagnosis of IVH was not recorded. The presence and grade of IVH were determined as part of clinical care of the patients, using the Papile classification . ThromboData from patients were retrospectively collected. In this context, we obtained a CNIL  authorization (CNIL DEC18–342).Blood samples were obtained by umbilical vein catheter at routine immediately after birth. Blood was collected into citrated (0.109 M) tubes with a final 9:1 ratio of blood to anticoagulant followed by gentle mixing. Platelet poor plasma (PPP) was prepared by centrifugation of citrated blood at 3000 x g for 10 min at room temperature.Factor II, factor V and fibrinogen were measured in platelet poor plasma (PPP) using the fully automated STA R Max2  coagulometer with Neoplastin R and Stago factor specific deficient plasma and STA liquid fibrinogen . All testing were performed in accordance with manufacturer’s instructions.0/7–28 6/7 GA, 29 0/7–32 6/7 GA and 33 0/7–36 6/7 GA), we calculated the reference ranges defined as 5 to 95th percentiles for fibrinogen, factor II and factor V.Categorical variables were expressed as frequency (percentage) and continuous variables as mean ± standard deviation (SD) or median [interquartile range] in case of non-Gaussian distribution. Normality of distribution was checked graphically and by using the Shapiro–Wilk test. For each GA subgroups ) using Student t or Mann-Whitney U tests , with 3-levels categorical variable  using Kruskall-Wallis tests, and with continuous variables  IUGR, biological data and with the newborn outcome in univariate analyses. The Mann-Whitney U test was used for binary categorical variables, a Kruskall-Wallis test for 3-levels categorical variable and using Spearman’s rank coefficient correlation for continuous variables.Finally, the association between of thrombocytopenia and intraventricular hemorrhage was studied using the Chi-square test. Given that age is a main confounder in association between factor V and intraventricular hemorrhage, we performed a multivariable logistic regression model to adjust this association for age; odds ratio (OR) of intraventricular hemorrhage per 1 SD increase in factor V was calculated as effect size.Among 142 eligible premature newborns, 122 were included .5/7 GA  and median weight was 1145 g . Among premature, 42.6% (n = 52) did not have IUGR. Regarding the mode of delivery, 27.0% (n = 33) were vaginally delivered and 73.0% (n = 89) were caesarean delivery (Table Median age was 31n = 16) did not received prenatal corticosteroids, 67.2% (n = 82) were exposed to a partial course of antenatal corticosteroids and 19.7% (n = 24) were exposed to a complete course of antenatal corticosteroids. None of the mother received medications like anticoagulants or antiepleptics and none of the mothers presented with thrombocytopenia related with immune disorder.Among mothers, 13.1% (n = 42) of newborns presented with hypophosphatemia and 29.5% (n = 36) with thrombocytopenia. Mean number of platelets was 181 × 109/L (77.5).Regarding biological data, 34.4% (n = 28) and classified as grade I (n = 8), grade II (n = 15), grade III (n = 3) and grade IV (n = 2).IVH was diagnosed in 23.0% of premature (n = 44) received one or more red blood cell (RBC) transfusions (maximum 9) and 10.7% (n = 13) received fresh frozen plasma (15 ml/kg). Four premature (3.3%) died in the NICU .Levels of fibrinogen, factor II and factor V did not differ significantly according to the use of antenatal corticosteroids (Fibrinogen p = 0.027). There was no difference regarding factors II and V.Level of fibrinogen was higher in case of vaginal delivery compare to caesarean  and with increasing birth weight . In the group of 23 0/7–28 6/7 GA, the 5th percentile was defined as 0.6 g/L for fibrinogen, 15 IU/dL for FII and 16 IU/dL for factor V (n = 30). In the group of 29 0/7–32 6/7 GA, the 5th percentile was defined as 1.0 g/L for fibrinogen, 24 IU/dL for FII and 41 IU/dL for factor V (n = 46). In the group of 330/7–366/7 GA, the 5th percentile was defined as 1.0 g/L for fibrinogen, 24 IU/dL for FII and 30 IU/dL for factor V (n = 46) . There was no difference regarding factors II and V.Level of fibrinogen (g/L) was lower in case of IUGR .Levels of fibrinogen (g/L), factor II (%) and factor V (%) were lower in case of thrombocytopenia . There was no correlation between hypophosphatemia and fibrinogen and factor II .Level of factor V (%) was lower in case of hypophosphatemia . Factor II and fibrinogen at birth were also not associated with Intraventricular hemorrhage (Table p = 0.026) (Table p = 0.22).There was no correlation with factor II and fibrinogen (Table There was no association between birth platelets count and intraventricular hemorrhage development (ge Table . Only fa6) Table  with an en Table .Table 3Correlations between coagulation factor’s levels and death could not be assessed given the small number of deaths.Preterm birth is defined by the World Health Organization (WHO) as delivery before 37 completed weeks of gestation. Each year, 15 million babies are born preterm, which is estimated to be about 11% of all deliveries . Among bn = 20), 0.65–1.65 for fetuses of 24–29 GA (n = 22) and 1.25–1.65 for fetuses of 30–38 GA (n = 22). Regarding factor II and factor V, the lower and upper boundaries including 95% of the population were respectively as follows (%): 10–24 and 21–44 for fetuses of 19–23 GA (n = 20), 11–30 and 25–50 for fetuses of 24–29 GA (n = 22) and 15–50 and 23–70 for fetuses of 30–38 GA (n = 22). In each group, the lower boundary seems to be lower than in our study [In 1996, Reverdiau-Moalic et al. evaluated blood coagulation parameters in healthy human fetuses between 19 and 38 weeks’ gestation. The lower and upper boundaries including 95% of the population for fibrinogen (g/L) were as follows: 0.57–1.50 for fetuses of 19–23 GA , 5th percentile was 0.71 g/L for fibrinogen versus 0.87 g/L in the group 28–34 [Few studies have examined the levels of coagulation factors in premature infants shortly after birth. First reference ranges were published by Andrew in 1988 among 137 infants but were limited to healthy premature aged from 30 to 36 GA. The lower boundary was 1.5 g/L for fibrinogen, 20% for factor II and 41% for factor V . In 2014up 28–34 . These dWe found in this study that levels of fibrinogen, factor II and factor V rose with increasing gestational age. This data seems consistent with the literature. Early in the 70’s Bleyer et al. described developmental hemostasis concept. They reported for the first time that the premature infant is born with low levels of nearly all the clotting factors known compared to the full term newborn . It is nWe found that preterm neonates with IUGR had lower fibrinogen level at birth in consistence with previous studies , 18.Franzoi et al. also reported that fibrinogen was more elevated in vaginally delivered neonates in comparison to those delivered by caesarean section but the difference was not statistically significant .We found that factor V level at birth was correlated with presence of IVH. To the best of our knowledge, this is the first report of an association between factor V level at birth and IVH occurrence. However, in our study, the strength of association was slightly attenuated compared to unadjusted OR but remained not significant and a larger study is needed to establish if the factor V was an independent risk factor of IVH. Previous studies on the topic mainly focused on global coagulation tests. In 1982, Setzer reported that among premature the IVH group had a mean bleeding time which was significantly prolonged compared to that of the group without IVH . Same, iThe overall prevalence of thrombocytopenia in neonates admitted to NICUs ranges from 22 to 35%. In our study there was no significant association between thrombocytopenia and IVH. Conversely, in some studies thrombocytopenia was independently associated with IVH or IVH severity , 24.The place of fresh frozen plasma in the prevention of IVH is still unclear. The only randomized control trial assessing the effect of prophylactic fresh frozen plasma in 776 preterm babies reported no effect on early mortality and morbidity . HoweverSome authors have even suggested that the expansion of blood volume could increase cerebral blood flow and cerebral capillary pressure, enough to rupture subependymal vessels . Recent Our study has limitations inherent to the method and strengths. Limitations included the retrospective design of the study and the lack of exhaustive coagulation screening. Another limitation of the study is that prematurity is an independent risk factor for IVH even in newborns with normal coagulation factor levels. The major strength of our study is the important effective of premature and the homogeneity of its population .In this study, we can define hemostatic profile among 122 newborns with spontaneous prematurity, corroborating references ranges for studied parameters. One of the important feature of this study was that factor V level at birth was correlated with IVH development.Further large and prospective studies are still called for, to increase the number of patients and in particular in order to correlate the grade of hemorrhage and the factor V level at birth.


PDP1 gene, c.575dupT (p.L192FfsX5), with absent PDP1 product in fibroblasts. Unexpectedly, the patient also had low branched‐chain 2‐ketoacid dehydrogenase (BCKDH) activity in fibroblasts with slight elevation of branched‐chain amino acids in plasma and ketoacids in urine but with no pathogenic mutations in the enzymes of BCKDH, which could suggest shared regulatory function of PDC and BCKDH in fibroblasts, potentially in other tissues or cell types as well, but this remains to be determined. The clinical presentation of this patient overlaps that of other patients with primary‐specific PDC deficiency, with neonatal/infantile and childhood lactic acidosis, normal lactate to pyruvate ratio, elevated plasma alanine, delayed psychomotor development, epileptic encephalopathy, feeding difficulties, and hypotonia. This patient exhibited marked improvement of overall development following initiation of ketogenic diet at 31 months of age. To the best of our knowledge, this is the fourth case of functional PDC deficiency with a defined mutation in PDP1.Congenital lactic acidosis due to pyruvate dehydrogenase phosphatase (PDP) deficiency is very rare. PDP regulates pyruvate dehydrogenase complex (PDC) and defective PDP leads to PDC deficiency. We report a case with functional PDC deficiency with low activated (+dichloroacetate) and inactivated (+fluoride) PDC activities in lymphocytes and fibroblasts, normal activity of other mitochondrial enzymes in fibroblasts, and novel biallelic frameshift mutation in the PDP1 mutations leads to PDC deficiency and congenital lactic acidosis.Pyruvate dehydrogenase phosphatase (PDP) regulates pyruvate dehydrogenase complex (PDC) and defective PDP due to  BCKDHbranched‐chain 2‐ketoacid dehydrogenaseDCAdichloroacetateKDketogenic dietKDC2‐ketoglutarate dehydrogenase complexPDCpyruvate dehydrogenase complexPDPpyruvate dehydrogenase phosphatase1PDHA1, PDHB, DLAT, and DLD genes, respectively) and a structural protein (E3BP/PDHX). E3 is also a part of other 2‐keto acid dehydrogenases  as are the three cofactors .The mitochondrial multienzyme pyruvate dehydrogenase complex (PDC) irreversibly catalyzes oxidative decarboxylation of pyruvate into acetyl‐CoA as the primary substrate from carbohydrate for the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Defective complex function results in increased cellular pyruvate levels which leads to elevated lactate and alanine levels. PDC is comprised of four core catalytic subunits , while activation of PDC is achieved by removal of these phosphates from E1α by two pyruvate dehydrogenase phosphatases (PDPs: PDP1 and PDP2).PDP1) and a larger regulatory FAD‐associated subunit . PDPr regulates the activity of PDP1c by blocking and distorting its active site. The other isoform consists of only the phosphatase catalytic subunit encoded by PDP2.PDP1 and PDP2, respectively).Two isoforms of PDP are known.PDP1, PDP2, and PDPR) explaining the functional PDC deficiency.Several cases with “atypical” PDC deficiency attributed to a defect in phosphatase activation of the PDC complex have been reported, without identifying pathogenic variants in genes associated with phosphatase deficiency  not surprisingly overlaps that of PDC deficiencies, with neonatal/infantile and childhood lactic acidosis, encephalopathy, brain abnormalities, seizures, feeding difficulties, hypotonia, cardiomyopathy, corneal clouding, and delayed psychomotor development reported to date, although cardiomyopathy and corneal clouding are not typical of primary PDC deficiency.PDP1 presenting with lactic acidosis, delayed psychomotor development, and epilepsy with review of literature of cases with confirmed PDP1 mutations.Consequently, the reported cases of PDC deficiency due to confirmed mutations in either 21P0‐1 mother by C‐section due to prolonged labor, failure to progress, and fetal distress. Pregnancy was complicated by vaginal bleeding requiring bed rest. Perinatal course was notable for a 5‐day NICU stay for oxygen support. He was noted to be hypotonic from birth. Subsequently, there was failure to thrive. He did not sit until a year of age, was not walking at 20 months of age, and early on had limited physical (motor) endurance. Seizures began at 8 months of age with noted pallor, rigidity, bruxism, drooling and perioral cyanosis, and recurred with addition of eyelid closure, and enhanced hypotonia. He would be unresponsive during the episodes, which usually lasted between 5 and 10 minutes. His seizures ceased after initiating Keppra, with last seizure being at 14 months of age. At 15 months of age, brain MRI suggested a subtle region of increased FLAIR/T2 signal in the right frontal subcortical white matter, a small transmantle cortical dysplasia could not be ruled out. There were prominent CSF spaces around the cerebellum suggestive of cerebellar volume loss. He later developed reactive airways disease requiring Pulmicort and then Flovent. He also was noted to have an everted and pronated left foot and everted right foot.This male proband was born full term, 3.4‐kg birth weight to a GAt 25 months of age, his weight, length, and head circumference were 10.8 kg (5th percentile), 81.9 cm (5th percentile), and 48 cm (28th percentile), respectively. Metabolic testing showed lactic acidosis, elevated pyruvate with normal lactate to pyruvate ratio of 12, and abnormal findings on urine organic acids that included large amounts of 2‐keto‐isocaproic and 2‐keto‐isovaleric acids and small amounts of 2‐keto‐3‐methylvaleric and 2‐hydroxyisovaleric acids. Levels of methylmalonic acid in serum and creatine in plasma were normal. A chromosomal microarray analysis revealed a loss of heterozygosity (LOH) of about 16.3 Mb involving 10p11.2‐q11.23 . At 28 months of age, a repeat brain MRI Figure  showed aHe was started on an oral 3:1 (grams of fats to grams of carbohydrates plus proteins) ketogenic diet (KD) at 31 months of age once PDC deficiency was diagnosed and ketosis was adequately maintained (plasma beta‐hydroxybutyrate between 4.5 and 5.5 mM). His physical endurance markedly improved while on KD. However, due to continued feeding difficulty and food refusal, he would exhibit neurological dysfunction such as tremor, increased ataxia, and decreased alertness when unable to maintain adequate ketosis. About 15 months after KD initiation, a gastrostomy tube was placed to provide 75% to 100% of energy needs by ketogenic formula, resulting in the ability to maintain a more consistent level of ketosis. He required physical, occupational and speech therapies with placement in special education classes at school. His growth and development significantly improved while on KD, and KD stringency was reduced to 2.5:1 with advanced age. Although he exhibited speech delay early in development, by 7 years of age he spoke fluent English and Spanish, and attended class for children with special needs. Although he missed motor milestones and exhibited motor delay, at 7 years of age he was able to jump and attempted to climb stairs, but continued to need scheduled occupational, physical, and speech therapies. Despite developmental improvement, he continues to have poor oral intake and food refusal. When on KD, plasma alanine and proline were within their respective reference ranges, but the branched‐chain amino acids remained slightly above their respective reference ranges, but allo‐isoleucine was not noted on plasma amino acids. The highest levels of plasma branched‐chain amino acids noted at 3.5 years of age were valine 569 μM (RR 105‐407), isoleucine 206 μM (RR 24‐124) and leucine 331 μM (RR 56‐228). He also was maintained on thiamine, L‐carnitine, calcium and vitamin D3, polycitra‐K or baking soda, multivitamin, and Keppra. Family history was non‐contributory with no reported consanguinity. Parents were of Hispanic origin.33.1BCKDHA, BCKDHB, DBT, and PPM1K genes using NGS technology were performed at Invitae .Coding exons and immediate flanking intronic regions of 23 genes associated with pyruvate metabolism3.214CO2 from 14C‐leucine decarboxylation was performed at Emory Genetics Laboratory .Assays of PDC, both activated‐dephosphorylated (+DCA) and inactivated phosphorylated (+fluoride), 2‐ketoglutarate dehydrogenase complex (KDC), and dihydrolipoamide dehydrogenase (E3) activities in disrupted blood lymphocytes and cultured skin fibroblasts were measured as previously described.3.32. The cells were grown to confluence in a 75 cm3 dish. Harvested cells were lysed via two freeze‐thaw cycles and suspended in PBS with 10 μL of 0.1 M PMSF and 0.2 M Leupeptin. For each sample, 50 μg/mL of lysate protein were resolved by SDS‐PAGE and transferred overnight to a PVDF membrane. The PVDF membrane was blocked in 5% milk for 1.5 hours at room temperature (RT) then incubated with primary antibody against PDP1 (492 636 700 Sigma) at 1:600 dilution and against GADPH (TAB1001 Thermo Fisher) at 1:2000 dilution for 1.5 hours at RT. After washing off the primary antibody, the membrane was incubated with 1:1500 dilution of secondary antibody, goat anti‐rabbit IgG HRP (32460 Thermo Scientific), for 1.5 hours at RT. Signals were detected with HyBlot CL autoradiography film (E3018 Denville Scientific Inc.) after applying the SuperSignal West Pico Plus chemiluminescent substrate (34577 Thermo Scientific) to the blot.Fibroblasts were grown in Advanced DMEM medium (12491‐015 Gibco) with 16% fetal bovine serum (F2442 Sigma) supplemented with 82 U/mL penicillin and 82 μg/mL streptomycin (15140‐122 Gibco), 20 μg/mL uridine (194 763 MP Bio), and 8 ng/mL fibroblast growth factor (F3133 Sigma) in a 37°C incubator with humidified atmosphere of 10% CO3.4The proband was enrolled in the IRB‐approved Manton Center for Orphan Disease Research Gene Discovery Core at Boston Children's Hospital. Informed consent was also obtained from the parents/guardians for additional investigative studies by inclusion in the University Hospitals Cleveland Medical Center IRB‐approved Disorders of Pyruvate Metabolism: Phenotype Genotype Study as well as the IRB‐approved NIH‐funded North American Mitochondrial Disease Consortium research project titled “Natural History and Advanced Genetic Study of PDC Deficiencies,” for additional functional and/or molecular analyses. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from the patient for being included in the study.44.1PDP1 variant in the proband, which was subsequently confirmed by Sanger sequencing and predicted to produce a premature stop codon  8  inhibits the pyruvate dehydrogenase kinases and is often used to identify PDC activation deficiency. Fluoride (F) is used to inhibit pyruvate dehydrogenase phosphatase. Assay of PDC in blood lymphocytes and cultured skin fibroblasts from patient showed decreased activated (+DCA) and inactivated (+F) activity with low PDC/E3 ratio consistent with PDC deficiency Table . In blooAnalysis of integrated oxidative phosphorylation in digitonin‐permeabilized proband fibroblasts  showed normal oxygen consumption in the presence of pyruvate, malate and adenosine diphosphate (ADP) as substrates as well as long‐chain acylcarnitine as substrate Table . Changes5PDHA1, PDHB, DLAT, PDHX, PDP1, PDP2, and PDPR, constitute 70% to 90% of PDC activity deficiencies, with those due to X‐linked PDHA1 mutations representing >80% of genetically defined primary‐specific deficiencies.The clinical presentation of PDC deficiency is highly variable and ranges from fatal congenital lactic acidosis and congenital brain abnormalities including corpus callosum abnormalities 15%‐55%), ventriculomegaly (35%‐85%), and Leigh syndrome (12%‐25%), to relatively mild ataxia or neuropathy with normal cognitive function and long survival.%‐55%, ve5.1PDP1 mutations have been placed on a ketogenic diet. The patient reported here and others with PDP deficiency due to PDP1 mutations have shown good response to KD with qualitative improvement of psychomotor development and increased survival.Life‐long ketogenic diet (KD) use (and avoidance of high carbohydrate diets) is currently the main therapeutic intervention for primary‐specific PDC deficiency with positive outcomes noted in the areas of epilepsy, ataxia, sleep disturbance, speech/language development, social functioning, and frequency of hospitalizations,PDP1 , the most common gene mutated in maple syrup urine disease, as well as BCKDHB (on Chr 6), DBT (on Chr 1), and PPM1K (on Chr 4) were all normal (data not shown). Similar to PDC regulation, BCKDH activity is regulated posttranscriptionally by phosphorylation (inactivation) and dephosphorylation (activation).PPM1K is a manganese (Mn+2)‐dependent BCKDH complex phosphatase and not a magnesium‐ or calcium‐dependent enzyme.PDP1 mutations or in the canine model of PDP deficiency due to PDP1 mutations. Therefore, the suggested potentially shared regulatory function of PDC and BCKDH activities by PDP1 is speculative and remains to be determined.The regulatory enzymes pyruvate dehydrogenase E1) kinase and phosphatase are believed to be specific for PDC, and to the best of our knowledge, there is not an analogous regulatory function for KDC. This is supported by the observation of normal KDC activity in this proband in fibroblasts with biallelic pathogenic  kinase ans Table .9 InterePDP1 variant (p.E93X) showed decreased ETC complex I + III activity relative to citrate synthase in skeletal muscle (Table PDP1 genotype, and/or other modifying genes, which remains to be investigated.Finally, the case with the biallelic pathogenic J. B., G. G., C. H., and D. K. were involved in the biochemical/functional analyses of this patient. L. H., S. T., A. B., E. Y., and G. B. were involved in clinical management of the patient. D. D. assisted with compilation and analysis of clinical data. S. Z. and H. K. S. performed mutational and expression analyses, respectively. J. B. and S. D. were involved in enrolling this patient in the IRB‐approved studies. J. B. wrote the first draft, reviewed, and revised the manuscript, and approved the final version as submitted. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. All authors confirm the absence of previous similar or simultaneous publications.The authors declare that they have no conflict of interest.Supplementary Figure 1 Supplementary FileClick here for additional data file.Supplementary Table 1 Supplementary FileClick here for additional data file.