Novel cell-based assays identify new metabolic targets to block Plasmodium falciparum transmission

La forma più pericolosa e mortale di malaria nell’uomo è causata dal parassita Plasmodium falciparum. Il parassita è trasmesso all’uomo dalla femmina della zanzara del genere Anopheles, che inietta gli sporozoiti nel sangue durante il suo pasto di sangue. Queste forme extracellulari migrano velocemente nel fegato attraverso il circolo ematico. Ogni sporozoita che invade un epatocita si divide mitoticamente e si differenzia in migliaia di merozoiti epatici che, rilasciati, invadono gli eritrociti, originando il ciclo asessuato intraeritrocitario. Questo ciclo, della durata di circa 48 ore, è responsabile dei sintomi clinici causati dall’infezione del parassita malarico. Nel sangue, alcune forme intra-eritrocitiche si differenziano, con un meccanismo ancora poco conosciuto, in gametociti maschili e femminili, le forme sessuali del parassita. All’interno dell’eritrocita, il parassita che ha intrapreso il differenziamento sessuale si sviluppa attraverso cinque stadi di maturazione che complessivamente durano circa 12 giorni. I gametociti maturi (stadio V), se risucchiati dalla zanzara Anopheles durante il suo pasto di sangue, si sviluppano in gameti all’interno del suo stomaco. Dalla fusione di un gamete femminile e uno maschile si forma uno zigote, il quale si differenzia in un oocinete che attraversa la parete dello stomaco e si trasforma in oocisti nell’emocele dell’insetto. Da questa oocisti sono rilasciati gli sporozoiti che migrano nelle ghiandole salivari e che verranno poi inoculati nell’ospite umano al successivo pasto di sangue. I gametociti di P. falciparum sono i responsabili della trasmissione del parassita dall’ospite umano alla zanzara. Tutti i farmaci ad oggi conosciuti sono utilizzati per curare i sintomi della malattia e quindi sono volti a eliminare le forme asessuate del parassita; l’unico farmaco ad oggi utilizzato contro i gametociti maturi di P. falciparum è la primachina. La carenza di farmaci contro i gametociti è in parte dovuta alla mancanza di un saggio cellulare veloce, quantitativo e riproducibile in grado di testare l’effetto di diverse librerie di composti sui gametociti. In parte l’assenza di tali saggi è dovuta al fatto che i gametociti maturi di P. falciparum sono stati fino ad oggi considerati cellule apparentemente quiescenti, con un metabolismo rallentato rispetto a quanto osservato per i parassiti asessuati e per i gametociti immaturi, rendendo difficile la misurazione dell’effetto di composti su questo stadio del parassita. Quest’ultima osservazione deriva dal fatto che i gametociti maturi sono spesso insensibili ai farmaci attivi contro le forme asessuate del parassita e contro i gametociti immaturi. Di conseguenza è necessario identificare vie metaboliche attive nei gametociti maturi come target per nuovi farmaci. Un’altra importante caratteristica dei gametociti di P. falciparum è la difficoltà nel valutare, tramite analisi morfologica, se siano vivi o morti dopo trattamento farmacologico in vitro. Nell’ambito di queste problematiche abbiamo sviluppato diversi saggi che abbiamo utilizzato per valutare la capacità di diversi composti di eliminare i gametociti. L'individuazione di nuovi farmaci o sinergie di farmaci capaci di agire contro i gametociti maturi di P. falciparum ha sottolineato l'esistenza di vie metaboliche attive in questi stadi, il cui studio approfondito può rappresentare un punto di partenza per lo sviluppo di nuovi trattamenti anti-trasmissione della malaria

The proposal of a clinical protocol to assess central and peripheral fatigue in myotonic dystrophy type 1

DM1 is an autosomal-dominant disorder characterized by muscle weakness, myotonia, and multisystemic involvement. According to current literature fatigue and daytime sleepiness are among the main symptoms of DM1. Oxidative stress has been proposed to be one of the pathogenic factors of fatigue consequent to DM1. In this study, we investigated the dimensions of experienced fatigue and  physiological fatigue in a sample of 26 DM1 patients (17 males, 9 females, mean age 41.6 years, SD±12.7); experienced fatigue has been studied through Fatigue Severity Scale (FSS), and physiological fatigue was measured through an intermittent incremental exercise of the forearm muscles using a myometer; oxidative stress balance markers trend during aerobic exercise test have been collected. The occurrence of central fatigue in the sample means that central activation worsens during the motor contraction; interestingly FSS score was significantly correlated to MVC (before and after the effort, r-before=-0.583, p<0.01, r-after= -0.534, p<0.05), and to motor disability measured by MRC (r=-0.496, p<0.05); moreover we found a strong tendency towards significance in the association to lactate baseline (r=0.378, p=0.057).Results are discussed to define whether or not, based on clinical and laboratory grounds, such exercise training protocol may be suitable for proper management of DM1 patients; proper assessment of fatigue should be included in algorithms for data collection in DM1 patient registries

Detection of Plasmodium falciparum male and female gametocytes and determination of parasite sex ratio in human endemic populations by novel, cheap and robust RTqPCR assays

The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito parasite transmission. The detection of submicroscopic infections with gametocytes and the estimation of the gametocyte sex ratio are crucial to assess the human host potential ability to infect mosquitoes and transmit malaria parasites

Physical exercise and oxidative stress in muscular dystrophies: is there a good balance? Exercise and oxidative stress in MDs

The effect of oxidative stress on muscle damage inducted by physical exercise is widely debated. It is generally agreed that endurance and intense exercise can increase oxidative stress and generate changes in antioxidant power inducing muscle damage; however, regular and moderate exercise can be beneficial for the health improving the antioxidant defense mechanisms in the majority of cases. Growing evidences suggest that an increased oxidative/nitrosative stress is involved in the pathogenesis of several muscular dystrophies (MDs). Notably, physical training has been considered useful for patients with these disorders. This review will focus on the involvement of oxidative stress in MDs and on the possible effects of physical activities to decrease oxidative damage and improve motor functions in MDs patients

Muscle fatigue and exercise-related biomarkers in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder affecting motor neurons. The complex etiopathogenetic mechanism of ALS can lead to extensive alterations, including cortical changes, neuroinflammation, and changes in muscular structure. These ALS-derived alterations may contribute to fatigue, a symptom severely impacting patients’ quality of life that is commonly associated with muscular exercise. Intriguingly, muscular exercise can be at once a promoter of motor neuron degeneration in predisposed patients as well as an effective non-pharmacological treatment of ALS. To fully disclose its therapeutic potential, muscular exercise must be tailored to patients’ phenotypes, balancing potential benefits and risks that are unique to each ALS case. Biomarkers of muscular fatigue, with their potential for insight into inflammation and oxidation, can be used to ensure that the intensity of physical activity remains below the threshold level beyond which exercise might become harmful. In this review, the authors explore the concept of fatigue in ALS patients, focusing on fatigue generation, definition, detection, quantification, and treatment. The study discusses the most important fatigue biomarkers, putting them in relation to the mechanism of fatigue generation and with monitoring of muscular exercise as a possible treatment of fatigue

Real-time PCR assays for detection and quantification of early P. falciparum gametocyte stages

Introduction The use of reverse transcription, quantitative qRT-PCR assays for detection and quantification of late gametocyte stages has revealed the high transmission capacity of the human malaria parasite, Plasmodium falciparum. A full understanding how the parasite adjusts its transmission in response to varying in-host environmental conditions during natural infections requires simultaneous quantification of early and late gametocytes. Here, we describe qRT-PCR assays that are specific for detection and quantification of early-stage gametocytes of P. falciparum. Methods The assays are based on expression of known early gametocyte genes (pfpeg4, pfg27, pfge1, pfge3 and pfgexp5). The specificity of the qRT-PCR assays was tested using purified stage II and stage V gametocytes. These validated assays were used with qRT-PCR assays targeting late stage (pfs25) and all-stage (pfs16) gametocyte-specific transcripts to quantify gametocytes in natural P. falciparum infections and in a controlled human clinical infection study. Results The relative expression of pfpeg4, pfg27 and pfge3, but not of pfge1 and pfgexp5, was significantly higher in purified stage II compared to stage V gametocytes, indicating early gametocyte specificity. In natural infections, 71.2% of individuals had both early and late gametocyte transcripts (pfpeg4/pfg27 plus pfs25), 12.6% harboured only early gametocytes transcripts (pfpeg4/pfg27), and 15.2% had only late gametocytes transcripts (pfs25). In natural infections, the limit of detection was equivalent to 190 and 390 gametocytes/mL blood for pfpeg4 and pfg27, respectively. In infected volunteers, transcripts of pfpeg4 and pfg27 were detected shortly after the onset of blood stage infection, demonstrating the specificity of the assays. Conclusion The pfpeg4 and pfg27 qRT-PCR assays can be used specifically to quantify circulating immature gametocytes. Quantification of early gametocytes will improve understanding of epidemiological processes that modulate P. falciparum transmission and enhance the evaluation of transmission blocking interventions

Clinical expression of facioscapulohumeral muscular dystrophy in carriers of 1-3 D4Z4 reduced alleles: Experience of the FSHD Italian National Registry

OBJECTIVES: Facioscapulohumeral muscular dystrophy type 1 (FSHD1) has been genetically linked to reduced numbers ( 64 8) of D4Z4 repeats at 4q35. Particularly severe FSHD cases, characterised by an infantile onset and presence of additional extra-muscular features, have been associated with the shortest D4Z4 reduced alleles with 1-3 repeats (1-3 DRA). We searched for signs of perinatal onset and evaluated disease outcome through the systematic collection of clinical and anamnestic records of de novo and familial index cases and their relatives, carrying 1-3 DRA. SETTING: Italy. PARTICIPANTS: 66 index cases and 33 relatives carrying 1-3 DRA. OUTCOMES: The clinical examination was performed using the standardised FSHD evaluation form with validated inter-rater reliability. To investigate the earliest signs of disease, we designed the Infantile Anamnestic Questionnaire (IAQ). Comparison of age at onset was performed using the non-parametric Wilcoxon rank-sum or Kruskal-Wallis test. Comparison of the FSHD score was performed using a general linear model and Wald test. Kaplan-Meier survival analysis was used to estimate the age-specific cumulative motor impairment risk. RESULTS: No patients had perinatal onset. Among index cases, 36 (54.5%) showed the first signs by 10 years of age. The large majority of patients with early disease onset (26 out of 36, 72.2%) were de novo; whereas the majority of patients with disease onset after 10 years of age were familial (16, 53.3%). Comparison of the disease severity outcome between index cases with age at onset before and over 10 years of age, failed to detect statistical significance (Wald test p value=0.064). Of 61 index cases, only 17 (27.9%) presented extra-muscular conditions. Relatives carrying 1-3 DRA showed a large clinical variability ranging from healthy subjects, to patients with severe motor impairment. CONCLUSIONS: The size of the D4Z4 allele is not always predictive of severe clinical outcome. The high degree of clinical variability suggests that additional factors contribute to the phenotype complexity

Non-neural phenotype of spinal and bulbar muscular atrophy: Results from a large cohort of Italian patients

Objective: To carry out a deep characterisation of the main androgen-responsive tissues involved in spinal and bulbar muscular atrophy (SBMA). Methods: 73 consecutive Italian patients underwent a full clinical protocol including biochemical and hormonal analyses, genitourinary examination, bone metabolism and densitometry, cardiological evaluation and muscle pathology. Results: Creatine kinase levels were slightly to markedly elevated in almost all cases (68 of the 73; 94%). 30 (41%) patients had fasting glucose above the reference limit, and many patients had total cholesterol (40; 54.7%), low-density lipoproteins cholesterol (29; 39.7%) and triglyceride (35; 48%) levels above the recommended values. Although testosterone, luteinising hormone and follicle-stimulating hormone values were generally normal, in one-third of cases we calculated an increased Androgen Sensitivity Index reflecting the presence of androgen resistance in these patients. According to the International Prostate Symptom Score (IPSS), 7/70 (10%) patients reported severe lower urinal tract symptoms (IPSS score >19), and 21/73 (30%) patients were moderately symptomatic (IPSS score from 8 to 19). In addition, 3 patients were carriers of an indwelling bladder catheter. Videourodynamic evaluation indicated that 4 of the 7 patients reporting severe urinary symptoms had an overt prostate-unrelated bladder outlet obstruction. Dual-energy X-ray absorptiometry scan data were consistent with low bone mass in 25/61 (41%) patients. Low bone mass was more frequent at the femoral than at the lumbar level. Skeletal muscle biopsy was carried out in 20 patients and myogenic changes in addition to the neurogenic atrophy were mostly observed. Conclusions: Our study provides evidence of a wide non-neural clinical phenotype in SBMA, suggesting the need for comprehensive multidisciplinary protocols for these patients. \ua9 2016 Published by the BMJ Publishing Group Limited

Fusion Hindrance and Pauli Blocking in 58Ni + 64Ni

58Ni +64Ni is the first case where the influence of positive Q-value transfer channels on sub-barrier fusion was evidenced, in a very well known experiment by Beckerman et al., by comparing with the two systems 58Ni + 58Ni and 64Ni+64Ni. Subsequent measurements on 64Ni + 64Ni showed that fusion hindrance is clearly present in this case. On the other hand, no indication of hindrance can be observed for 58Ni + 64Ni down to the measured level of 0.1 mb. In the present experiment the excitation function has been extended by two orders of magnitude downward. The cross sections for 58Ni + 64Ni continue decreasing very smoothly below the barrier, down to '1 µb. The logarithmic slope of the excitation function increases slowly, showing a tendency to saturate at the lowest energies. No maximum of the astrophysical S -factor is observed. Coupled-channels (CC) calculations using a Woods-Saxon potential and includinginelastic excitations only, underestimate the sub-barrier cross sections by a large amount. Good agreement is found by adding two-neutron transfer couplings to a schematical level. This behaviour is quite different from what already observed for 64Ni+ 64Ni (no positive Q-value transfer channels available), where a clear low-energy maximum of the S -factorappears, and whose excitation function is overestimated by a standard Woods-Saxon CC calculation. No hindrance effect is observed in 58Ni+ 64Ni in the measured energy range. This trend at deep sub-barrier energies reinforces the recent suggestion that the availability of several states following transfer with Q>0, effectively counterbalances the Pauli repulsion that, in general, is predicted to reduce tunneling probability inside the Coulomb barrier

A Molecular Assay to Quantify Male and Female Plasmodium falciparum Gametocytes: Results From 2 Randomized Controlled Trials Using Primaquine for Gametocyte Clearance.

Background: Single low-dose primaquine (PQ) reduces Plasmodium falciparum infectivity before it impacts gametocyte density. Here, we examined the effect of PQ on gametocyte sex ratio as a possible explanation for this early sterilizing effect. Methods: Quantitative reverse-transcription polymerase chain reaction assays were developed to quantify female gametocytes (targeting Pfs25 messenger RNA [mRNA]) and male gametocytes (targeting Pf3D7_1469900 mRNA) in 2 randomized trials in Kenya and Mali, comparing dihydroartemisinin-piperaquine (DP) alone to DP with PQ. Gametocyte sex ratio was examined in relation to time since treatment and infectivity to mosquitoes. Results: In Kenya, the median proportion of male gametocytes was 0.33 at baseline. Seven days after treatment, gametocyte density was significantly reduced in the DP-PQ arm relative to the DP arm (females: 0.05% [interquartile range {IQR}, 0.0-0.7%] of baseline; males: 3.4% [IQR, 0.4%-32.9%] of baseline; P 0.125 mg/kg) 48 hours after treatment, and gametocyte sex ratio was not associated with mosquito infection rates. Conclusions: The early sterilizing effects of PQ may not be explained by the preferential clearance of male gametocytes and may be due to an effect on gametocyte fitness