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.

Inhaled Nitric Oxide in Fibrotic Lung Disease: A Randomized, Double-Blind, Placebo-Controlled Trial.

RATIONALE: Inhaled nitric oxide (iNO) has been shown to result in benefits in moderate to vigorous physical activity (MVPA) in patients with fibrotic interstitial lung disease (if-ILD) on supplemental oxygen in two independent trials. OBJECTIVE: This phase 3 randomized double-blind placebo-controlled study sought to validate the benefit of ambulatory iNO in patients with f-ILD requiring supplemental oxygen. METHODS: Patients with f-ILD on supplemental long-term oxygen were randomized in a 1:1 fashion to inhaled nitric oxide at 45 µg/kg ideal body weight/hour or placebo for 16 weeks. The primary outcome was the change from baseline to week 16 in MVPA assessed by accelerometry. Secondary outcomes included overall activity, six-minute walk distance and patient reported outcomes. MEASUREMENT AND MAIN RESULTS: 145 patients were enrolled; 75 were assigned to receive iNO and 70 to placebo. The change from baseline in MVPA at 16 weeks was -9.2 minute/day (SE 3.51) in the iNO45 group versus -3.7 (3.76) minute/day in the placebo group (difference, 5.5; P=0.265). No statistically significant differences between the two treatment arms were found for any of the secondary outcomes. A subgroup analysis of patients with intermediate or high probability of pulmonary hypertension on echocardiography did not demonstrate any benefit. The most common adverse events reported were respiratory tract infections, but the therapy was generally very well tolerated. CONCLUSIONS: There was no demonstrable benefit to iNO in patients with f-ILD on supplemental oxygen in daily physical activity assessed by actigraphy, a potential novel clinical trial endpoint. Clinical trial registration available at www. CLINICALTRIALS: gov, ID: NCT03267108.

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.

Risk factors for intracranial hemorrhage in acute ischemic stroke patients treated with recombinant tissue plasminogen activator: a systematic review and meta-analysis of 55 studies.

BACKGROUND AND PURPOSE: Recombinant tissue plasminogen activator (rtPA) is an effective treatment for acute ischemic stroke but is associated with an increased risk of intracranial hemorrhage (ICH). We sought to identify the risk factors for ICH with a systematic review of the published literature. METHODS: We searched for studies of rtPA-treated stroke patients that reported an association between a variable measured before rtPA infusion and clinically important ICH (parenchymal ICH or ICH associated with clinical deterioration). We calculated associations between baseline variables and ICH with random-effect meta-analyses. RESULTS: We identified 55 studies that measured 43 baseline variables in 65 264 acute ischemic stroke patients. Post-rtPA ICH was associated with higher age (odds ratio, 1.03 per year; 95% confidence interval, 1.01-1.04), higher stroke severity (odds ratio, 1.08 per National Institutes of Health Stroke Scale point; 95% confidence interval, 1.06-1.11), and higher glucose (odds ratio, 1.10 per mmol/L; 95% confidence interval, 1.05-1.14). There was approximately a doubling of the odds of ICH with the presence of atrial fibrillation, congestive heart failure, renal impairment, previous antiplatelet agents, leukoaraiosis, and a visible acute cerebral ischemic lesion on pretreatment brain imaging. Little of the variation in the sizes of the associations among different studies was explained by the source of the cohort, definition of ICH, or degree of adjustment for confounding variables. CONCLUSIONS: Individual baseline variables were modestly associated with post-rtPA ICH. Prediction of post-rtPA ICH therefore is likely to be difficult if based on single clinical or imaging factors alone. These observational data do not provide a reliable method for the individualization of treatment according to predicted ICH risk.

Pulmonary hypertension in interstitial lung disease: Clinical trial design and endpoints: A consensus statement from the Pulmonary Vascular Research Institute's Innovative Drug Development Initiative-Group 3 Pulmonary Hypertension.

Pulmonary hypertension (PH) associated with interstitial lung disease (ILD) is an attractive target for clinical trials of PH medications. There are many factors that need to be considered to prime such studies for success. The patient phenotype most likely to respond to the intervention requires weighing the extent of the parenchymal lung disease against the severity of the hemodynamic impairment. The inclusion criteria should not be too restrictive, thus enabling recruitment. The trial should be of sufficient duration to meet the chosen endpoint which should reflect how the patient feels, functions, or survives. This paper summarizes prior studies in PH-ILD and provides a framework of the type of studies to be considered. Inclusion criteria, clinical trial endpoints, and pharmacovigilance in the context of PH-ILD trials are also addressed. Through lessons learnt from prior studies, suggestions and guidance for future clinical trials in PH-ILD are also provided.

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.

Clinical significance of pulmonary hypertension in interstitial lung disease: A consensus statement from the Pulmonary Vascular Research Institute's innovative drug development initiative-Group 3 pulmonary hypertension.

Pulmonary hypertension (PH) has been linked to worse outcomes in chronic lung diseases. The presence of PH in the setting of underlying Interstitial Lung Disease (ILD) is strongly associated with decreased exercise and functional capacity, an increased risk of hospitalizations and death. Examining the scope of this issue and its impact on patients is the first step in trying to define a roadmap to facilitate and encourage future research in this area. The aim of our working group is to strengthen the communities understanding of PH due to lung diseases and to improve the care and quality of life of affected patients. This introductory statement provides a broad overview and lays the foundation for further in-depth papers on specific topics pertaining to PH-ILD.

A Phase-2 Exploratory Randomized Controlled Trial of INOpulse in Patients with Fibrotic Interstitial Lung Disease Requiring Oxygen.

Rationale: Patients with fibrotic interstitial lung disease often progress to the point of requiring supplemental oxygen. This is invariably accompanied by an impaired quality of life and limitations on activities of daily living. Objectives: This study aimed to assess the improvement in physical activity in patients with interstitial lung disease requiring supplemental oxygen treated with pulsed inhaled nitric oxide via INOpulse (Bellerophon Therapeutics). In addition, it sought to explore the safety and clinical benefits of INOpulse on multiple patient-reported outcomes. Methods: Ambulatory patients with fibrotic lung disease on supplemental oxygen were randomized in a 2:1 ratio to inhaled nitric oxide at 45 µg/kg ideal body weight/h (iNO45) or placebo for 4 months (3 months after baseline) of blinded treatment. The study assessed multiple exploratory efficacy endpoints, including moderate to vigorous physical activity as measured by actigraphy and patient-reported outcomes using the University of California San Diego shortness of breath questionnaire and the St. George's Respiratory Questionnaire (SGRQ). Results: A total of 44 patients (30 iNO45 and 14 placebo) were enrolled. A placebo-corrected clinical benefit of 12.3 min/d increase in MVPA was observed in the iNO45 group. Clinically meaningful beneficial trends were observed for the University of California San Diego shortness of breath questionnaire (6.05 points) and the SGRQ total (3.75) scores, as well as the SGRQ activity (5.84), and SGRQ impact (6.30) domains. Conclusions: INOpulse was well tolerated and associated with maintenance of physical activity and improved symptomatology in patients with interstitial lung disease who require supplemental oxygen. Further validation of this beneficial effect warrants further study in a phase-3 trial that is currently underway.

Poor quality vital anti-malarials in Africa - an urgent neglected public health priority.

BACKGROUND: Plasmodium falciparum malaria remains a major public health problem. A vital component of malaria control rests on the availability of good quality artemisinin-derivative based combination therapy (ACT) at the correct dose. However, there are increasing reports of poor quality anti-malarials in Africa. METHODS: Seven collections of artemisinin derivative monotherapies, ACT and halofantrine anti-malarials of suspicious quality were collected in 2002/10 in eleven African countries and in Asia en route to Africa. Packaging, chemical composition (high performance liquid chromatography, direct ionization mass spectrometry, X-ray diffractometry, stable isotope analysis) and botanical investigations were performed. RESULTS: Counterfeit artesunate containing chloroquine, counterfeit dihydroartemisinin (DHA) containing paracetamol (acetaminophen), counterfeit DHA-piperaquine containing sildenafil, counterfeit artemether-lumefantrine containing pyrimethamine, counterfeit halofantrine containing artemisinin, and substandard/counterfeit or degraded artesunate and artesunate+amodiaquine in eight countries are described. Pollen analysis was consistent with manufacture of counterfeits in eastern Asia. These data do not allow estimation of the frequency of poor quality anti-malarials in Africa. CONCLUSIONS: Criminals are producing diverse harmful anti-malarial counterfeits with important public health consequences. The presence of artesunate monotherapy, substandard and/or degraded and counterfeit medicines containing sub-therapeutic amounts of unexpected anti-malarials will engender drug resistance. With the threatening spread of artemisinin resistance to Africa, much greater investment is required to ensure the quality of ACTs and removal of artemisinin monotherapies. The International Health Regulations may need to be invoked to counter these serious public health problems.

Taguchi analysis for sliding wear characteristics of carbon nanotube-flyash reinforced aluminium nanocomposites.

Dry sliding wear characteristics of aluminium nano-composites reinforced with different wt. % of multiwall carbon nanotubes (0.25, 0.5, 0.75 wt. %) and fly ash (4, 8, 16 wt. %) produced by powder metallurgy were investigated. ANOVA and Taguchi methods of design of experiment technique were successfully used to determine the predominant factors and optimisation of the testing parameters on wear. MWCNT (wt. %) and FA (wt. %) was found to be the predominant parameter affecting wear loss with percentage contribution of 43.71% and 30.78%. The results of Taguchi indicate the optimized values of wear parameters were 0.25wt. % MWCNT, 8 wt. % FA, 2 h ball milling, 6 h sintering, 10 N applied load, 200 rpm sliding speed and 500 m sliding distance. The microstructure of composites exhibited well dispersion of the reinforcements in the aluminium matrix. The study of worn surfaces revealed minor grooves and delamination wear due to abrasive and adhesive wear mechanisms.

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.

Clinical trial design in phase 2 and 3 trials for pulmonary hypertension.

This article on clinical trial design incorporates the broad experience of members of the Pulmonary Vascular Research Institute's (PVRI) Innovative Drug Development Initiative (IDDI) as an open debate platform for academia, the pharmaceutical industry and regulatory experts surrounding the future design of clinical trials in pulmonary hypertension. It is increasingly clear that the design of phase 2 and 3 trials in pulmonary hypertension will have to diversify from the traditional randomised double-blind design, given the anticipated need to trial novel therapeutic approaches in the immediate future. This article reviews a wide range of differing approaches and places these into context within the field of pulmonary hypertension.

A Randomized, Double-Blind, Placebo-Controlled Study of Pulsed, Inhaled Nitric Oxide in Subjects at Risk of Pulmonary Hypertension Associated With Pulmonary Fibrosis.

BACKGROUND: The interstitial lung diseases include a variety of disorders, many of which are characterized by fibrotic changes (fILD). Of the fILDs, Idiopathic pulmonary fibrosis is the most common. Pulmonary hypertension (PH) frequently complicates fILD and is associated with impaired functional capability, lower physical activity, and significantly reduced life expectancy. There is no proven treatment for patients with fILD-PH. We report results from the first cohort of a phase 2b/3 trial with pulsed inhaled nitric oxide (iNO) in patients with fILD-PH. METHODS: Subjects in cohort 1 were randomized to iNO 30 µg/kg ideal body weight/h (iNO30) or placebo for 8 weeks of blinded treatment; subjects then transitioned to open-label extension (OLE) on iNO30 followed by dose escalation to iNO45 then iNO75. Activity monitoring was used to assess changes in daily activity. Safety and efficacy were evaluated. RESULTS: Twenty-three patients were randomized to iNO30 and 18 to placebo. During blinded treatment, iNO30 subjects showed an average improvement in moderate/vigorous physical activity (MVPA) and remained stable in overall activity. Placebo subjects showed an average drop of 26% in MVPA and a 12% drop in overall activity. The iNO group had an improvement in oxygen saturation. During OLE, subjects maintained their activity levels including placebo subjects who transitioned from a decline to a maintenance in all activity parameters. Inhaled nitric oxide at all doses (30, 45, and 75) was safe and well tolerated. CONCLUSIONS: Treatment with iNO30 demonstrated clinically and statistically significant benefit in MVPA and clinically significant benefit in overall activity. In the OLE, higher doses of iNO were also safe and well tolerated while showing maintenance in activity parameters.