Biochemical and transcript level differences between the three human phosphofructokinases show optimisation of each isoform for specific metabolic niches.

6-Phosphofructokinase-1-kinase (PFK) tetramers catalyse the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP). Vertebrates have three PFK isoforms (PFK-M, PFK-L, and PFK-P). This study is the first to compare the kinetics, structures, and transcript levels of recombinant human PFK isoforms. Under the conditions tested PFK-M has the highest affinities for F6P and ATP (K0.5ATP 152 µM; K0.5F6P 147 µM), PFK-P the lowest affinities (K0.5ATP 276 µM; K0.5F6P 1333 µM), and PFK-L demonstrates a mixed picture of high ATP affinity and low F6P affinity (K0.5ATP 160 µM; K0.5F6P 1360 µM). PFK-M is more resistant to ATP inhibition compared with PFK-L and PFK-P (respectively, 23%, 31%, 50% decreases in specificity constants). GTP is an alternate phospho donor. Interface 2, which regulates the inactive dimer to active tetramer equilibrium, differs between isoforms, resulting in varying tetrameric stability. Under the conditions tested PFK-M is less sensitive to fructose 2,6-bisphosphate (F26BP) allosteric modulation than PFK-L or PFK-P (allosteric constants [K0.5ATP+F26BP/K0.5ATP] 1.10, 0.92, 0.54, respectively). Structural analysis of two allosteric sites reveals one may be specialised for AMP/ADP and the other for smaller/flexible regulators (citrate or phosphoenolpyruvate). Correlations between PFK-L and PFK-P transcript levels indicate that simultaneous expression may expand metabolic capacity for F16BP production whilst preserving regulatory capabilities. Analysis of cancer samples reveals intriguing parallels between PFK-P and PKM2 (pyruvate kinase M2), and simultaneous increases in PFK-P and PFKFB3 (responsible for F26BP production) transcript levels, suggesting prioritisation of metabolic flexibility in cancers. Our results describe the kinetic and transcript level differences between the three PFK isoforms, explaining how each isoform may be optimised for distinct roles.

The kinetic characteristics of human and trypanosomatid phosphofructokinases for the reverse reaction.

Eukaryotic ATP-dependent phosphofructokinases (PFKs) are often considered unidirectional enzymes catalysing the transfer of a phospho moiety from ATP to fructose 6-phosphate to produce ADP and fructose 1,6-bisphosphate. The reverse reaction is not generally considered to occur under normal conditions and has never been demonstrated for any eukaryotic ATP-dependent PFKs, though it does occur in inorganic pyrophosphate-dependent PFKs and has been experimentally shown for bacterial ATP-dependent PFKs. The evidence is provided via two orthogonal assays that all three human PFK isoforms can catalyse the reverse reaction in vitro, allowing determination of kinetic properties. Additionally, the reverse reaction was shown possible for PFKs from three clinically important trypanosomatids; these enzymes are contained within glycosomes in vivo This compartmentalisation may facilitate reversal, given the potential for trypanosomatids to have an altered ATP/ADP ratio in glycosomes compared with the cytosol. The kinetic properties of each trypanosomatid PFK were determined, including the response to natural and artificial modulators of enzyme activity. The possible physiological relevance of the reverse reaction in trypanosomatid and human PFKs is discussed.

How to do it: investigate exertional rhabdomyolysis (or not).

Rhabdomyolysis is the combination of symptoms (myalgia, weakness and muscle swelling) and a substantial rise in serum creatine kinase (CK) >50 000 IU/L; there are many causes, but here we specifically address exertional rhabdomyolysis. The consequences of this condition can be severe, including acute kidney injury and requirement for higher level care with organ support. Most patients have 'physiological' exertional rhabdomyolysis with no underlying disease; they do not need investigation and should be advised to return to normal activities in a graded fashion. Rarely, exertional rhabdomyolysis may be the initial presentation of underlying muscle disease, and we review how to identify this much smaller group of patients, who do require investigation.

Strokes: mimics and chameleons.

Diagnosing stroke is not always straightforward. Stroke mimics such as Todd's paresis or hemiplegic migraine account for between a fifth and a quarter of suspected strokes (depending on the setting in which they are assessed). Stroke chameleons can arise when the tempo of symptom onset is not apoplectic or if the loss of function is not clearly consistent with a deficit within an arterial territory. Thrombolysis and secondary prevention have much to offer patients with stroke chameleons, though those with stroke mimics may be harmed by these treatments and have more to gain from other therapies.

Guillain-Barré syndrome following SARS-CoV-2 vaccination in the UK: a prospective surveillance study.

Objective: To investigate features of Guillain-Barré syndrome (GBS) following SARS-CoV-2 vaccines and evaluate for a causal link between the two. Methods: We captured cases of GBS after SARS-CoV-2 vaccination through a national, open-access, online surveillance system. For each case, the certainty of GBS was graded using the Brighton criteria, and the relationship to the vaccine was examined using modified WHO Causality Assessment criteria. We compared age distribution of cases with that of prepandemic GBS cases and clinical features with the International GBS Outcome Study (IGOS). Results: Between 1 January and 30 June 2021, we received 67 reports of GBS following the ChAdOx1 vaccine (65 first doses) and three reports following the BNT162b2 vaccine (all first doses). The causal association with the vaccine was classified as probable for 56 (80%, all ChAdOx1), possible for 12 (17%, 10 ChAdOx1) and unlikely for two (3%, 1 ChAdOx1). A greater proportion of cases occurred in the 50-59 age group in comparison with prepandemic GBS. Most common clinical variants were sensorimotor GBS (n=55; 79%) and facial diplegia with paraesthesias (n=10; 14%). 10% (n=7/69) of patients reported an antecedent infection, compared with 77% (n=502/652) of the IGOS cohort (p<0.00001). Facial weakness (63% (n=44/70) vs 36% (n=220/620); p<0.00001) and sensory dysfunction (93% (n=63/68) vs 69% (n=408/588); p=0.00005) were more common but disease severity and outcomes were similar to the IGOS study. Interpretation: Most reports of GBS followed the first dose of ChAdOx1 vaccine. While our study cannot confirm or refute causation, this observation, together with the absence of alternative aetiologies, different than expected age distribution and the presence of unusual clinical features support a causal link. Clinicians and surveillance bodies should remain vigilant to the possibility of this very rare adverse event and its atypical variants.

Fast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice.

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.

Novel treatments for progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy is a rare demyelinating disorder of the CNS, caused by John Cunningham virus, that occurs in those with impaired immune systems. Existing treatment options are ineffective or unproven. This article reviews research into novel therapies: immune checkpoint-blocking antibodies (nivolumab and pembrolizumab), allogenic BK virus-specific T cell treatment and filgrastim. Results for these therapies in small clinical trials are promising, but further research is required to assess efficacy fully.

Progressive multifocal leukoencephalopathy (PML) following autologous peripheral blood stem cell transplantation for multiple myeloma.

PML should be considered in patients with neurological symptoms following MM and in those who are immunosuppressed. Symptoms are diverse and often rapidly progressing. Prompt referral and early involvement of the multidisciplinary team are crucial.

Kinetic and structural studies of Trypanosoma and Leishmania phosphofructokinases show evolutionary divergence and identify AMP as a switch regulating glycolysis versus gluconeogenesis.

Trypanosomatids possess glycosome organelles that contain much of the glycolytic machinery, including phosphofructokinase (PFK). We present kinetic and structural data for PFK from three human pathogenic trypanosomatids, illustrating intriguing differences that may reflect evolutionary adaptations to differing ecological niches. The activity of Leishmania PFK - to a much larger extent than Trypanosoma PFK - is reliant on AMP for activity regulation, with 1 mm AMP increasing the L. infantum PFK (LiPFK) kcat/K0.5F6P value by 10-fold, compared to only a 1.3- and 1.4-fold increase for T. cruzi and T. brucei PFK, respectively. We also show that Leishmania PFK melts at a significantly lower (> 15 °C) temperature than Trypanosoma PFKs and that addition of either AMP or ATP results in a marked stabilization of the protein. Sequence comparisons of Trypanosoma spp. and Leishmania spp. show that divergence of the two genera involved amino acid substitutions that occur in the enzyme's 'reaching arms' and 'embracing arms' that determine tetramer stability. The dramatic effects of AMP on Leishmania activity compared with the Trypanosoma PFKs may be explained by differences between the T-to-R equilibria for the two families, with the low-melting Leishmania PFK favouring the flexible inactive T-state in the absence of AMP. Sequence comparisons along with the enzymatic and structural data presented here also suggest there was a loss of AMP-dependent regulation in Trypanosoma species rather than gain of this characteristic in Leishmania species and that AMP acts as a key regulator in Leishmania governing the balance between glycolysis and gluconeogenesis.

Impact of the first COVID-19 pandemic wave on the Scottish Multiple Sclerosis Register population.

Background: The impact of the coronavirus disease 2019 (COVID-19) pandemic on people with multiple sclerosis (MS) is a major current concern, in particular the risk of death. Here we describe the impact of the first wave of COVID-19 infections (Mar 2020-July 2020) on the Scottish MS Register (SMSR) population, a cohort of 4702 individuals with MS, all newly diagnosed in the past decade. Methods: We established a clinician alert system, linking the SMSR with the Electronic Communication of Surveillance in Scotland (ECOSS). This allows identification of patients within this cohort who had a positive SARS-CoV-2 PCR test. The SMSR was also linked to death records from National Records Scotland. Results: Of 4702 people with MS, 246 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR tests were performed, of which 17 were positive. The proportion of positive tests were similar to the general Scotland population (Observed PCR confirmed cases = 17, expected = 17.5, O/E = 0.97, 95% CI: 0.60 - 1.56, p=.90). Between 1 st March - 31 st July 2020 12 individuals on the SMSR died, 5 of which were linked to COVID-19 (1 PCR confirmed, 4 clinical diagnoses without PCR confirmation). This number of COVID-19-related deaths was higher than expected (observed deaths = 5, expected deaths = 1.2, O/E = 4.03, 95% CI = 1.48 - 8.94, p=.01). All COVID-19-related deaths in the SMSR occurred in individuals with advanced disability (Expanded Disability Status Scale ≥7), and no deaths occurred in patients receiving disease modifying therapy (DMT) therapies. Conclusion: In this nationally comprehensive cohort of MS patients diagnosed in Scotland within the past 10 years, we observed similar rates of PCR-confirmed SARS-CoV-2 infection compared to the general Scottish population, but a small number of excess COVID-19 related deaths. These deaths occurred in individuals with advanced disability who were not receiving DMTs.

Visual disturbance with systemic symptoms: old lessons revisited.

We describe a retired physician who presented with visual disturbance and systemic symptoms. The presence of general malaise, headache and scalp tenderness, with raised inflammatory markers, suggested that giant cell arteritis (GCA) was the likely diagnosis. Rapid response to initial steroid therapy and histological evidence of inflammation in the temporal artery supported this diagnosis. The character of these visual symptoms was, however, atypical for GCA. The patient, who had heart valve disease, subsequently deteriorated and developed further symptoms warranting investigation of bacterial endocarditis. Retinal emboli are a recognised complication of endocarditis, which could account for these visual symptoms. Moreover, interpretation of the temporal artery biopsy is limited in the context of existing steroid therapy. Our patient was consequently diagnosed with bacterial endocarditis. This case reminds us to consider the wider differential diagnoses for headache, visual disturbance and systemic symptoms, where echocardiogram and blood cultures may be crucial to reach the diagnosis.