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Abstract The Baculovirus/insect cell expression system is a powerful technology for reconstitution of eukaryotic macromolecular assemblies. Most multigene expression platforms rely on Tn7-mediated transposition for transferring the expression cassette into the baculoviral genome. This allows a rigorous characterization of recombinant bacmids but involves multiple steps, a limitation when many constructs are to be tested. For parallel expression screening and potential high throughput applications, we have established an open source multigene-expression toolbox exploiting homologous recombination, thus reducing the recombinant baculovirus generation to a single-step procedure and shortening the time from cloning to protein production to 2 weeks. The HR-bac toolbox is composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. They contain single or dual expression cassettes bearing different affinity tags and their design facilitates the mix and match utilization of expression units from Multibac constructs. The overall cost of virus generation with HR-bac toolbox is relatively low as the preparation of linearized baculoviral DNA only requires standard reagents. Various multiprotein assemblies (nuclear hormone receptor heterodimers, the P-TEFb or the ternary CAK kinase complex associated with the XPD TFIIH subunit) are used as model systems to validate the toolbox presented.

Background: Hypotension, a common intra-operative incident, bears an important potential for morbidity. It is most often manageable and sometimes preventable, which renders its study important. Therefore, we aimed at examining hospital variations in the occurrence of intra-operative hypotension and its predictors. As secondary endpoints, we determined to what extent hypotension relates to the risk of post-operative incidents and death. Methods: We used the Anaesthesia Databank Switzerland, built on routinely and prospectively collected data on all anaesthesias in 21 hospitals. The three outcomes were assessed using multi-level logistic regression models. Results: Among 147,573 anaesthesias, hypotension ranged from 0.6% to 5.2% in participating hospitals, and from 0.3% up to 12% in different surgical specialties. Most (73.4%) were minor single events. Age, ASA status, combined general and regional anaesthesia techniques, duration of surgery and hospitalization were significantly associated with hypotension. Although significantly associated, the emergency status of the surgery had a weaker effect. Hospitals' odds ratios for hypotension varied between 0.12 and 2.50 ( P≤0.001), even after adjusting for patient and anaesthesia factors, and for type of surgery. At least one post-operative incident occurred in 9.7% of the procedures, including 0.03% deaths. Intra-operative hypotension was associated with a higher risk of post-operative incidents and death. Conclusion: Wide variations remain in the occurrence of hypotension among hospitals after adjustment for risk factors. Although differential reporting from hospitals may exist, variations in anaesthesia techniques and blood pressure maintenance may also have contributed. Intra-operative hypotension is associated with morbidities and sometimes death, and constant vigilance must thus be advocated. [ABSTRACT FROM AUTHOR]

Introduction: A multidisciplinary therapeutic optimization unit (COT) was created in January 2023 at Versailles Hospital, aimed at therapeutic optimisation of patients with chronic heart failure with reduced left ventricular ejection fraction. The objective of the study was to assess the impact of the first year of COT activity on the sequential implementation and titration of heart failure treatments, the clinical evolution, and improvement of patients' quality of life., Methods: This prospective study included consecutive patients treated by the COT after hospitalisation for acute heart failure, from January to December 2023. Clinical, biological, titration, and tolerance data were analysed. Quality of life was assessed at baseline and at the end of the follow-up by COT, using standardized SF-12 and EQ-5D questionnaires., Results: We included 90 patients (men 73%, mean age 67 years). The mean left ventricular ejection fraction was 34 ± 10 %. At final visit (median number of visits 4 ; median follow-up duration 156 days), 76.7% of patients achieved optimisation with respect to maximum individually tolerated doses, but only 13.3% with respect to theoretical maximum doses for the four therapeutic classes. At 1-year follow up, total mortality was 4.4% (4/90), and 9 patients (10%) were rehospitalised unplanned for acute heart failure. COT monitoring was associated with significant improvement in NYHA class, left ventricular ejection fraction, and SF-12 and EQ-5D-5L quality of life scores., Conclusion: Although titration of heart failure treatments remained suboptimal, significant improvement was observed for NYHA class, left ventricular ejection fraction, and patient quality of life parameters., Competing Interests: Déclaration de liens d'intérêts Les auteurs déclarent n'avoir aucun lien d'intérêt en relation avec cet article., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The inhibitor of κB (IκB) kinase (IKK) is a central regulator of NF-κB signaling. All IKK complexes contain hetero- or homodimers of the catalytic IKKβ and/or IKKα subunits. Here, we identify a YDDΦxΦ motif, which is conserved in substrates of canonical (IκBα, IκBβ) and alternative (p100) NF-κB pathways, and which mediates docking to catalytic IKK dimers. We demonstrate a quantitative correlation between docking affinity and IKK activity related to IκBα phosphorylation/degradation. Furthermore, we show that phosphorylation of the motif's conserved tyrosine, an event previously reported to promote IκBα accumulation and inhibition of NF-κB gene expression, suppresses the docking interaction. Results from integrated structural analyzes indicate that the motif binds to a groove at the IKK dimer interface. Consistently, suppression of IKK dimerization also abolishes IκBα substrate binding. Finally, we show that an optimized bivalent motif peptide inhibits NF-κB signaling. This work unveils a function for IKKα/β dimerization in substrate motif recognition., (© 2024. The Author(s).)

Background: Acid sphingomyelinase deficiency (ASMD) or Niemann-Pick disease types A, A/B, and B is a progressive, life-limiting, autosomal recessive disorder caused by sphingomyelin phosphodiesterase 1 (SMPD1) gene mutations. There is a need to increase the understanding of morbidity and mortality across children to adults diagnosed with ASMD., Methods: This observational retrospective survey analysed medical records of patients with ASMD with retrievable data from 27 hospitals in France, diagnosed/followed up between 1 st January 1990 and 31 st December 2020. Eligible records were abstracted to collect demographic, medical/developmental history, and mortality data. Survival outcomes were estimated from birth until death using Kaplan-Meier survival analyses; standardised mortality ratio (SMR) was also explored., Results: A total of 118 medical records of patients with ASMD (type B [n = 94], type A [n = 15], and type A/B [n = 9]) were assessed. The majority of patients were males (63.6%); the median [range] age at diagnosis was 8.0 [1.0-18.0] months (type A), 1.0 [0-3] year (type A/B), and 5.5 [0-73] years (type B). Overall, 30 patients were deceased at the study completion date; the median [range] age at death for patients with ASMD type A (n = 14) was 1 [0-3.6] year, type A/B (n = 6) was 8.5 [3.0-30.9] years, and type B (n = 10) was 57.6 [3.4-74.1] years. The median [95% confidence interval (CI)] survival age from birth in patients with ASMD type A and type A/B was 2.0 [1.8-2.7] years and 11.4 [5.5-18.5] years, respectively. Survival analysis in ASMD type B was explored using SMR [95% CI] analysis (3.5 [1.6-5.9]), which showed that age-specific deaths in the ASMD type B population were 3.5 times more frequent than those in the general French population. The causes of death were mostly severe progressive neurodegeneration (type A: 16.7%), cancer (type B: 16.7%), or unspecified (across groups: 33.3%)., Conclusions: This study illustrated a substantial burden of illness with high mortality rates in patients with ASMD, including adults with ASMD type B, in France., (© 2024. The Author(s).)

A number of studies have shown the effect of cereals and sourdough on bread nutritional and organoleptic quality, but the impact of the milling technique remains little studied. There are two main types of milling technic depending on the bread-making food chain. Industrial bakeries mainly use roller mills while artisanal bakeries may also use stone mill. We set up a participatory experiment with six millers and four bakers to study the impact of these two milling techniques on the quality of flours, sourdough microbiota and the quality of breads. Millers made twenty-two different flours from four different wheat grain varieties using either roller or stone mills. Each baker initiated and maintained sourdoughs with three roller-milled and three stone-milled flours during at least 32 backsloppings and then made bread. The analysis of flours revealed a typical granulometry profile linked to wheat hardness with higher particle sizes for stone-milled flours. Stone-milled flours also had a higher maltose content. However, the milling technic did not drive the composition of the sourdough microbiota. Moreover, the analysis of bread revealed that variation in bread protein fractions and in bread aroma compounds were more related to the specific baker microbial community than to the milling technique. Carbohydrate contents were clearly linked to the main LAB species metabolism. These results revealed that the sourdough microbial community shapes the organoleptic and nutritional quality of bread more than milling techniques., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The baculovirus expression vector system (BEVS) is recognized as a powerful platform for producing challenging proteins and multiprotein complexes both in academia and industry. Since a baculovirus was first used to produce heterologous human IFN-β protein in insect cells, the BEVS has continuously been developed and its applications expanded. We have recently established a multigene expression toolbox (HR-bac) composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. Unlike platforms that rely on Tn7-medidated transposition for the construction of baculoviruses, HR-bac relies on homologous recombination, which allows to evaluate expression constructs in 2 weeks and is thus perfectly adapted to parallel expression screening. In this chapter, we detail our standard operating procedures for the preparation of the reagents, the construction and evaluation of baculoviruses, and the optimization of protein production for both intracellularly expressed and secreted proteins., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Classical infantile-onset Pompe disease (IOPD) is the most severe form of Pompe disease. Enzyme replacement therapy (ERT) has significantly increased survival but only a few studies have reported long-term outcomes., Methods: We retrospectively analyzed the outcomes of classical IOPD patients diagnosed in France between 2004 and 2020., Results: Sixty-four patients were identified. At diagnosis (median age 4 months) all patients had cardiomyopathy and most had severe hypotonia (57 of 62 patients, 92%). ERT was initiated in 50 (78%) patients and stopped later due to being ineffective in 10 (21%). Thirty-seven (58%) patients died during follow-up, including all untreated and discontinued ERT patients, and 13 additional patients. Mortality was higher during the first 3 years of life and after the age of 12 years. Persistence of cardiomyopathy during follow-up and/or the presence of heart failure were highly associated with an increased risk of death. In contrast, cross-reactive immunologic material (CRIM)-negative status (n = 16, 26%) was unrelated to increased mortality, presumably because immunomodulation protocols prevent the emergence of high antibody titers to ERT. Besides survival, decreased ERT efficacy appeared after the age of 6 years, with a progressive decline in motor and pulmonary functions for most survivors., Conclusions: This study reports the long-term follow-up of one of the largest cohorts of classical IOPD patients and demonstrates high long-term mortality and morbidity rates with a secondary decline in muscular and respiratory functions. This decreased efficacy seems to be multifactorial, highlighting the importance of developing new therapeutic approaches targeting various aspects of pathogenesis., (© 2023 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

GM1 gangliosidosis is a rare lysosomal storage disorder associated with β-galactosidase enzyme deficiency. There are three types of GM1 gangliosidosis based on age of symptom onset, which correlate with disease severity. In 2019, we performed a retrospective multicentric study including all patients diagnosed with GM1 gangliosidosis in France since 1998. We had access to data for 61 of the 88 patients diagnosed between 1998 and 2019. There were 41 patients with type 1 (symptom onset ≤6 months), 11 with type 2a (symptom onset from 7 months to 2 years), 5 with type 2b (symptom onset from 2 to 3 years), and 4 with type 3 (symptom onset >3 years). The estimated incidence in France was 1/210000. In patients with type 1, the first symptoms were hypotonia (26/41, 63%), dyspnea (7/41, 17%), and nystagmus (6/41, 15%), whereas in patients with type 2a, these were psychomotor regression (9/11, 82%) and seizures (3/11, 27%). In types 2b and 3, the initial symptoms were mild, such as speech difficulties, school difficulties, and progressive psychomotor regression. Hypotonia was observed in all patients, except type 3. The mean overall survival was 23 months (95% confidence interval [CI]: 7, 39) for type 1 and 9.1 years (95% CI: 4.5, 13.5) for type 2a. To the best of our knowledge, this is one of the largest historical cohorts reported, which provides important information on the evolution of all types of GM1 gangliosidosis. These data could be used as a historical cohort in studies assessing potential therapies for this rare genetic disease., (© 2023 SSIEM.)

In Infantile Onset Pompe Disease (IOPD), enzyme replacement therapy (ERT) may improve survival, cardiac function, and motor development. However, even with early enzyme replacement therapy, some patients experienced poor response to ERT and abnormal motor milestones that could be due to motor neuron involvement. In this long-term retrospective study, we analyzed concomitant clinical motor outcomes and electroneuromyography (ENMG) findings in patients with IOPD and Juvenile Onset Pompe Disease (JOPD). Twenty-nine pediatric patients were included and 20 surviving were analyzed for neuromotor studies: 12 had IOPD (group 1), 4 had JOPD (group 2) and 4 (group 3) received ERT in the first month of age. Motor nerve conduction studies were mostly normal. Needle EMG performed at diagnosis always indicated the existence of myopathy that responded to ERT. Two IOPD patients (group 1) presenting with mixed motor neuropathy and myopathy displayed a poor outcome and never walked. Two patients became non-walkers (one IOPD patient and one patient of group 3) at respectively 9 and 3 years of age. One JOPD patient is about to lose walking ability. This motor deterioration was associated with the development of a motor neuropathy. Patients older than 10 years of age develop a motor neuropathy. Initial or secondary motor neuron involvement seems to be associated with a poor motor outcome showing that ERT may fail to prevent the accumulation of glycogen in motor neuron. Neurophysiological findings are important to assess severity of motor neuron damage in all Pompe pediatric patients and should be systematically performed., Competing Interests: Declaration of Competing Interest Authors have no conflict of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Niemann-Pick disease type C (NP-C) is a rare neurovisceral lysosomal lipid storage disease characterized by progressive neurodegeneration and premature death. While miglustat can stabilize neurological manifestations in later onset forms of NP-C, its efficacy in the early-infantile neurological form has not been demonstrated. In this observational retrospective study, we compared long-term neurodevelopmental outcome and survival between an untreated and a treated group of early infantile NP-C patients., Methods: Data available on all NP-C patients with early infantile neurological onset diagnosed in France between 1990 and 2013 were compiled. Patients with incomplete data or who had died from a systemic perinatal, rapidly fatal form were excluded., Results: Ten patients were included in the treated group (year of birth: 2006-2012), and 16 patients in the untreated group [born 1987-2005 (n = 15), 2012 (n = 1)]. The median age at neurological onset was 9 months (5-18) in the treated group, and 12 months (3-18) in the untreated group (p = 0.22). Miglustat therapy was started at a median age of 24.5 months (9-29) and median duration was 30 months (11-56). Gastrointestinal adverse events were reported in 7/10 patients on miglustat. All patients developed loss of psychomotor acquisitions or additional neurological symptoms despite miglustat therapy. The ages of developmental milestones and neurological involvement did not significantly differ between the two groups. Four patients in the untreated group were lost to follow up. The 22 remaining patients had died by the end of the study and no patient survived beyond the age of 7.4 years. The median survival age was 4.42 years in the untreated group and 5.56 years in the treated group; the Kaplan-Meier survival curves were not significantly different (log-rank test: p = 0.11)., Conclusions: Miglustat allowed no significant long-term neurodevelopmental improvement nor significant increase of survival in patients with early infantile NP-C., (© 2023. The Author(s).)

Mucopolysaccharidosis type I-H (MPS I-H) is a rare lysosomal storage disorder caused by α-L-Iduronidase deficiency. Early haematopoietic stem cell transplantation (HSCT) is the sole available therapeutic option to preserve neurocognitive functions. We report long-term follow-up (median 9 years, interquartile range 8-16.5) for 51 MPS I-H patients who underwent HSCT between 1986 and 2018 in France. 4 patients died from complications of HSCT and one from disease progression. Complete chimerism and normal α-L-Iduronidase activity were obtained in 84% and 71% of patients respectively. No difference of outcomes was observed between bone marrow and cord blood stem cell sources. All patients acquired independent walking and 91% and 78% acquired intelligible language or reading and writing. Intelligence Quotient evaluation (n = 23) showed that 69% had IQ ≥ 70 at last follow-up. 58% of patients had normal or remedial schooling and 62% of the 13 adults had good socio-professional insertion. Skeletal dysplasia as well as vision and hearing impairments progressed despite HSCT, with significant disability. These results provide a long-term assessment of HSCT efficacy in MPS I-H and could be useful in the evaluation of novel promising treatments such as gene therapy., (© 2022. The Author(s).)

Objective: The objective of this study was to compare the quality of life (QoL) for parents of children with inborn errors of metabolism (IEMs) requiring a restricted diet with French population norms and investigate parental QoL determinants., Study Design: This cross-sectional study included mothers and/or fathers of children < 18 years of age affected by IEMs requiring a restricted diet (except phenylketonuria) from January 2015 to December 2017. Parents' QoL was assessed using the World Health Organization Quality of Life BREF questionnaire and compared with age- and sex-matched reference values from the French general population. Linear mixed models were used to examine the effects of demographic, socioeconomic, disease-related, and psychocognitive factors on parental QoL, according to a 2-level regression model considering individuals (parents) nested within families., Results: Of the 1156 parents invited to participate, 785 (68%) were included. Compared with the general population, parents of children with IEMs requiring a restricted diet reported a lower QoL in physical and social relationship domains but a higher QoL in the psychological domain. In the multivariate analysis, characteristics associated with poorer parental QoL included both parent-related factors (being a father, older age, more educated parent, nonworking parent, greater anxiety, seeking more social support, and using less positive thinking and problem-solving coping strategies) and family-related factors (disease complications, increased number of hospital medical providers, child's younger age, single-parent family, and lower family material wealth)., Conclusion: Parents of children with IEMs requiring a restricted diet reported poorer QoL in physical and social relationship domains than population norms. Psychocognitive factors, beyond disease-specific and family-related characteristics, were the most important determinants influencing parental QoL and may represent essential aspects for interventions., Clinical Trial Registration: ClinicalTrials.gov: NCT02552784., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: Mini-COMET (NCT03019406; Sanofi) is a phase 2, open-label, ascending-dose, 3-cohort study, evaluating avalglucosidase alfa safety, pharmacokinetics, and efficacy in individuals with infantile-onset Pompe disease aged <18 years who previously received alglucosidase alfa and showed clinical decline (cohorts 1 and 2) or suboptimal response (cohort 3)., Methods: During a 25-week primary analysis period, cohorts 1 and 2 received avalglucosidase alfa 20 and 40 mg/kg every other week, respectively, for 6 months, whereas cohort 3 individuals were randomized (1:1) to receive avalglucosidase alfa 40 mg/kg every other week or alglucosidase alfa (current stable dose) for 6 months., Results: In total, 22 individuals were enrolled (cohort 1 [n = 6], cohort 2 [n = 5], cohort 3-avalglucosidase alfa [n = 5], and cohort 3-alglucosidase alfa [n = 6]). Median treatment compliance was 100%. None of the individuals discontinued treatment or died. Percentages of individuals with treatment-emergent adverse events were similar across dose and treatment groups. No serious or severe treatment-related treatment-emergent adverse events occurred. Trends for better motor function from baseline to week 25 were observed for 40 mg/kg every other week avalglucosidase alfa compared with either 20 mg/kg every other week avalglucosidase alfa or alglucosidase alfa up to 40 mg/kg weekly., Conclusion: These data support the positive clinical effect of avalglucosidase alfa in patients with infantile-onset Pompe disease previously declining on alglucosidase alfa., Competing Interests: Conflict of Interest P.S.K. reports advisory board participation for Amicus Therapeutics, Baebies, and Sanofi; receiving consulting fees from Amicus Therapeutics, Asklepios Biopharmaceutical Inc (AskBio), JCR Pharma Co Ltd, Maze Therapeutics, and Sanofi; performing contracted research for Amicus Therapeutics and Sanofi; receiving honoraria from Amicus Therapeutics, AskBio, Maze Therapeutics, and Sanofi; having ownership interest of <5% (stocks, stock options, or other ownership interest excluding diversified mutual funds) in AskBio and Maze Therapeutics; and receiving travel expenses from Amicus Therapeutics and Sanofi. D.K. reports advisory board participation for AskBio and Sanofi; performing contracted research for Sanofi; serving on the Speaker’s bureau for Sanofi; and receiving travel expenses from AskBio and Sanofi. A.Bra. reports advisory board participation for Alexion, Sanofi, and Shire; receiving consulting fees from Alexion; and receiving travel expenses from Alexion, BioMarin, Orphan Europe, Sanofi, and Shire. A.Bro. reports advisory board participation for Sanofi and receiving honoraria from Orchard Therapeutics and Sanofi. J.D. reports advisory board participation for Sanofi, Recordati RRD, and Orchard Therapeutics; receiving consulting fees from bluebird bio Inc; and serving on the Speaker’s bureau for Sanofi, Recordati RRD, and FYMCA Medical Ltd. S.H.H. reports advisory board participation for Alexion Pharmaceutical Inc; performing contracted research for Sanofi; receiving honoraria from Alexion Pharmaceutical Inc; membership of the Seattle Children’s Hospital workforce; serving as temporary CEO of KEY PROTEO, Inc; being an inventor of intellectual property that has been licensed to KEY PROTEO, Inc; being a founder of KEY PROTEO, Inc and having ownership equity interests in the company; and receiving royalties from UpToDate. S.K., F.L., and H.O. declare no conflicts of interest. S.P. reports receiving travel expenses from Sanofi. S.G.P. reports performing contracted research for Sanofi and receiving consulting fees from Sanofi. K.A.H. and her spouse are employees of Sanofi and both have ownership interest of <5% (stocks, stock options, or other ownership interest excluding diversified mutual funds) in Sanofi. B.K. is an employee of Sanofi and has ownership interest of <5% (stocks, stock options, or other ownership interest excluding diversified mutual funds) in Sanofi. X.M. is an employee of TechData Service LLC (contracted to Sanofi). S.S. is an employee of Sanofi and has ownership interest of <5% (stocks, stock options, or other ownership interest excluding diversified mutual funds) in Sanofi. C.W. reports receiving consulting fees from Sanofi and Regenxbio. A.Z. is an employee of Sanofi and has ownership interest of <5% (stocks, stock options, or other ownership interest excluding diversified mutual funds) in Sanofi Canada. Y.-H.C. reports advisory board participation for Amicus Therapeutics and Sanofi; receiving consulting fees from Amicus Therapeutics and Sanofi; performing contracted research for Sanofi; and receiving honoraria from Biogen, Novartis, Sanofi, and Takeda., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)