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BackgroundPegunigalsidase alfa is a PEGylated α-galactosidase A enzyme replacement therapy. BALANCE (NCT02795676) assessed non-inferiority of pegunigalsidase alfa versus agalsidase beta in adults with Fabry disease with an annualised estimated glomerular filtration rate (eGFR) slope more negative than −2 mL/min/1.73 m2/year who had received agalsidase beta for ≥1 year.MethodsPatients were randomly assigned 2:1 to receive 1 mg/kg pegunigalsidase alfa or agalsidase beta every 2 weeks for 2 years. The primary efficacy analysis assessed non-inferiority based on median annualised eGFR slope differences between treatment arms.ResultsSeventy-seven patients received either pegunigalsidase alfa (n=52) or agalsidase beta (n=25). At baseline, mean (range) age was 44 (18–60) years, 47 (61%) patients were male, median eGFR was 74.5 mL/min/1.73 m2 and median (range) eGFR slope was −7.3 (−30.5, 6.3) mL/min/1.73 m2/year. At 2 years, the difference between median eGFR slopes was −0.36 mL/min/1.73 m2/year, meeting the prespecified non-inferiority margin. Minimal changes were observed in lyso-Gb3 concentrations in both treatment arms at 2 years. Proportions of patients experiencing treatment-related adverse events and mild or moderate infusion-related reactions were similar in both groups, yet exposure-adjusted rates were 3.6-fold and 7.8-fold higher, respectively, with agalsidase beta than pegunigalsidase alfa. At the end of the study, neutralising antibodies were detected in 7 out of 47 (15%) pegunigalsidase alfa-treated patients and 6 out of 23 (26%) agalsidase beta-treated patients. There were no deaths.ConclusionsBased on rate of eGFR decline over 2 years, pegunigalsidase alfa was non-inferior to agalsidase beta. Pegunigalsidase alfa had lower rates of treatment-emergent adverse events and mild or moderate infusion-related reactions.Trial registration number NCT02795676.

BackgroundPatients with Fabry disease (FD) on reduced dose of agalsidase-beta or after switch to agalsidase-alfa show a decline in chronic kidney disease epidemiology collaboration-based estimated glomerular filtration rate (eGFR) and a worsened plasma lyso-Gb3 decrease. Hence, the most effective dose is still a matter of debate.MethodsIn this prospective observational study, we assessed end-organ damage and clinical symptoms in 78 patients who had received agalsidase-beta (1.0 mg/kg) for >1 year, which were assigned to continue this treatment (agalsidase-beta, regular-dose group, n=17); received a reduced dose of agalsidase-beta and subsequent switch to agalsidase-alfa (0.2 mg/kg) or a direct switch to 0.2 mg/kg agalsidase-alfa (switch group, n=22); or were re-switched to agalsidase-beta after receiving agalsidase-alfa for 12 months (re-switch group, n=39) with a follow-up of 88±25 months.ResultsNo differences for clinical events were observed for all groups. Patients within the re-switch group started with the worst eGFR values at baseline (p=0.0217). Overall, eGFR values remained stable in the regular-dose group (p=0.1052) and decreased significantly in the re-switch and switch groups (p<0.0001 and p=0.0052, respectively). However, in all groups males presented with an annual loss of eGFR by –2.9, –2.5 and −3.9 mL/min/1.73 m² (regular-dose, re-switch, switch groups, all p<0.05). In females, eGFR decreased significantly only in the re-switch group by −2.9 mL/min/1.73 m² per year (p<0.01). Lyso-Gb3 decreased in the re-switch group after a change back to agalsidase-beta (p<0.05).ConclusionsOur data suggest that a re-switch to high dosage of agalsidase results in a better biochemical response, but not in a significant renal amelioration especially in classical males.

Key Points Dynamin, the founding member of a family of dynamin-like proteins (DLPs) implicated in membrane remodelling, has a critical role in endocytic membrane fission events. The use of complementary approaches, including live-cell imaging, cell-free studies, X-ray crystallography and genetic studies in mice, has greatly advanced our understanding of the mechanisms by which dynamin acts. The mechanisms by which dynamin drives membrane fission have been the subject of intense debate. Recent crystallographic and cryo-electron microscopy studies of dynamin and DLPs support a model in which dynamin polymerization serves to bring two GTPase domains together, which allows GTP hydrolysis and the conformational changes in dynamin that are necessary for helix constriction and membrane fission. The role of dynamin is best defined during clathrin-dependent endocytosis and is essential only for a late step when membrane fission occurs. Gene-knockout studies in mice and the cells derived from them have provided numerous insights into dynamin function and the specific roles of the three dynamin isoforms. Dynamin 2 is ubiquitously expressed and has a housekeeping role in membrane dynamics. By contrast, dynamin 1 and dynamin 3 are specific to the nervous system and, although neither is essential for supporting a specific form of endocytosis at synapses, they may be important for allowing clathrin-mediated endocytosis to function over a very broad range of neuronal activities. Roles of abnormal dynamin function in genetic disease have begun to emerge. Whereas mutations in dynamin 2 show links to tissue-specific diseases, mutations in dynamin 1 specifically affect the nervous system. The dynamin GTPase mediates membrane remodelling during endocytosis. Through complementary approaches, including structural and genetic studies, the mechanisms by which dynamin regulates membrane fission events, and the unique physiological roles of its three isoforms, are becoming clear. Dynamin, the founding member of a family of dynamin-like proteins (DLPs) implicated in membrane remodelling, has a critical role in endocytic membrane fission events. The use of complementary approaches, including live-cell imaging, cell-free studies, X-ray crystallography and genetic studies in mice, has greatly advanced our understanding of the mechanisms by which dynamin acts, its essential roles in cell physiology and the specific function of different dynamin isoforms. In addition, several connections between dynamin and human disease have also emerged, highlighting specific contributions of this GTPase to the physiology of different tissues.

A novel ALK transcript expressed in a subset of human cancers, arising from a de novo alternative transcription initiation site within the ALK gene, is described; the ALK transcript encodes three protein isoforms that stimulate tumorigenesis in vivo in mouse models; resultant tumours are sensitive to treatments with ALK inhibitors, indicating a possible therapeutic avenue for patients expressing these isoforms. A novel oncogene activation mechanism Oncogenes are usually activated by genetic abberations. Ping Chi and colleagues have identified a novel isoform of the anaplastic lymphoma kinase (ALK) in a subset of human cancers, arising independently of genomic aberrations at the ALK locus through alternative transcription initiation in ALK intron 19. Tumours driven by the transcript, termed ALK ATI , are sensitive to ALK inhibitors, suggesting ALK inhibitors as possible therapeutics in patients expressing these isoforms. Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK ATI . In ALK ATI -expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites 1 . ALK ATI is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK ATI transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK ATI stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK ATI , suggesting that patients with ALK ATI -expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, transcriptionally independent truncated isoform of ACE2 , which we designate as deltaACE2 ( dACE2 ). We demonstrate that dACE2 , but not ACE2 , is an ISG. In The Cancer Genome Atlas, the expression of dACE2 was enriched in squamous tumors of the respiratory, gastrointestinal and urogenital tracts. In vitro, dACE2, which lacks 356 amino-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, an inflammatory tumor microenvironment or viral co-infections is unlikely to increase the cellular entry of SARS-CoV-2 and promote infection. dACE2 is a newly identified isoform of ACE2 that is unable to bind the SARS-CoV-2 spike protein. Truncated dACE2, but not full-length ACE2, is induced by interferons and viruses, thus suggesting that such conditions are unlikely to increase cellular entry of SARS-CoV-2.

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis; using in vivo RNA interference and mathematical modelling it is shown here that the endolysosomal system is resilient to loss of Rab5 until its concentration drops below a critical level, at which point endosomes are lost, leading to increased serum low-density lipoprotein levels, alterations in metabolism and hepatocellular polarity. Rab5 is endosome organizer in vivo Regulation of organelle size and number is a fundamental question in biology. The small GTPase Rab5 has been proposed as a master regulator of the biogenesis of endosomes: membrane-associated vacuoles involved in endocytosis. In this study, Marino Zerial and colleagues use a combination of mathematical modelling and in vivo RNA interference analysis to reduce the levels of Rab5 in mouse liver. They demonstrate that Rab5 is a principal component of endosome biogenesis in vivo . Consistent with the loss of endosomes following reduction in Rab5 levels below a key point, animals had elevated levels of serum low-density lipoprotein as a result of decreased endocytosis.

In muscle, digoxin inhibits Na+,K+‐ATPase (NKA) whereas acute exercise can increase NKA gene expression, consistent with training‐induced increased NKA content. We investigated whether oral digoxin increased NKA isoform mRNA expression (qPCR) in muscle at rest, during and post‐exercise in 10 healthy adults, who received digoxin (DIG, 0.25 mg per day) or placebo (CON) for 14 days, in a randomised, double‐blind and cross‐over design. Muscle was biopsied at rest, after cycling 20 min (10 min each at 33%, then 67% V̇O2peak ${{\\dot{V}}_{{{\\mathrm{O}}}_2}{\\mathrm{peak}}}$ ), then to fatigue at 90% V̇O2peak ${{\\dot{V}}_{{{\\mathrm{O}}}_2}{\\mathrm{peak}}}$and 3 h post‐exercise. No differences were found between DIG and CON for NKA α1–3 or β1–3 isoform mRNA. Both α1 (354%, P = 0.001) and β3 mRNA (P = 0.008) were increased 3 h post‐exercise, with α2 and β1–2 mRNA unchanged, whilst α3 mRNA declined at fatigue (−43%, P = 0.045). In resting muscle, total β mRNA (∑(β1+β2+β3)) increased in DIG (60%, P = 0.025) and also when transcripts for each isoform were normalised to CON then either summed (P = 0.030) or pooled (n = 30, P = 0.034). In contrast, total α mRNA (∑(α1+α2+α3), P = 0.348), normalised then summed (P = 0.332), or pooled transcripts (n = 30, P = 0.717) did not differ with DIG. At rest, NKA α1–2 and β1–2 protein abundances were unchanged by DIG. Post‐exercise, α1 and β1–2 proteins were unchanged, but α2 declined at 3 h (19%, P = 0.020). In conclusion, digoxin did not modify gene expression of individual NKA isoforms at rest or with exercise, indicating NKA gene expression was maintained consistent with protein abundances. However, elevated resting muscle total β mRNA with digoxin suggests a possible underlying β gene‐stimulatory effect. Highlights What is the central question of this study? Na+,K+‐ATPase (NKA) in muscle is important for Na+/K+ homeostasis. We investigated whether the NKA‐inhibitor digoxin stimulates increased NKA gene expression in muscle and exacerbates NKA gene responses to exercise in healthy adults. What is the main finding and its importance? Digoxin did not modify exercise effects on muscle NKA α1–3 and β1–3 gene transcripts, which comprised increased post‐exercise α1 and β3 mRNA and reduced α3 mRNA during exercise. However, in resting muscle, digoxin increased NKA total β isoform mRNA expression. Despite inhibitory‐digoxin or acute exercise stressors, NKA gene regulation in muscle is consistent with the maintenance of NKA protein contents.

A metabolomics quantification of NADPH production and consumption fluxes in proliferating mammalian cells reveals that, in addition to canonical pathways such as the oxidative pentose phosphate pathway, NADPH can also be produced by a folate metabolism pathway, a discovery providing new insights into the metabolism of cell growth. Folate generates the reducing agent NADPH NADPH is a coenzyme that is involved in many redox processes in cells, including lipogenesis, oxidative stress and tumour growth. The most direct route for the production of NADPH from glucose is via the oxidative pentose phosphate pathway. In this manuscript, the authors use various metabolomics methodologies to quantify NADPH production and consumption fluxes in proliferating mammalian cells, and show that NADPH can also be produced when methylene tetrahydrofolate is oxidized to 10-formyl-tetrahydrofolate. This is unexpected — folate metabolism was not previously recognized as an important source of NADPH — and is particularly interesting in light of the importance of serine and glycine, the major carbon sources of this folate-dependent pathway, in cancer growth. ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis 1 . The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important 2 , 3 . Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography–mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP + to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase ( MTHFD ) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP + and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

Metacaspases are cysteine-dependent proteases found in protozoa, fungi and plants and are distantly related to metazoan caspases. Although metacaspases share structural properties with those of caspases, they lack Asp specificity and cleave their targets after Arg or Lys residues. Studies performed over the past 10 years have demonstrated that metacaspases are multifunctional proteases essential for normal physiology of non-metazoan organisms. This article provides a comprehensive overview of the metacaspase function and molecular regulation during programmed cell death, stress and cell proliferation, as well as an analysis of the first metacaspase-mediated proteolytic pathway. To prevent further misapplication of caspase-specific molecular probes for measuring and inhibiting metacaspase activity, we provide a list of probes suitable for metacaspases.