Epeleuton, a novel synthetic ω-3 fatty acid, reduces hypoxia/ reperfusion stress in a mouse model of sickle cell disease.

Inflammatory vasculopathy is critical in sickle cell disease (SCD)-associated organ damage. An imbalance between pro-inflammatory and pro-resolving mechanisms in response to different triggers such as hypoxia/reoxygenation or infections has been proposed to contribute to the progression of SCD. Administration of specialized pro-resolving lipid mediators may provide an effective therapeutic strategy to target inflammatory vasculopathy and to modulate inflammatory response. Epeleuton (15 hydroxy eicosapentaenoic acid ethyl ester) is a novel, orally administered, second-generation ω-3 fatty acid with a favorable clinical safety profile. In this study we show that epeleuton re-programs the lipidomic pattern of target organs for SCD towards a pro-resolving pattern. This protects against systemic and local inflammatory responses and improves red cell features, resulting in reduced hemolysis and sickling compared with that in vehicle-treated SCD mice. In addition, epeleuton prevents hypoxia/reoxygenation-induced activation of nuclear factor-κB with downregulation of the NLRP3 inflammasome in lung, kidney, and liver. This was associated with downregulation of markers of vascular activation in epeleuton-treated SCD mice when compared to vehicle-treated animals. Collectively our data support the potential therapeutic utility of epeleuton and provide the rationale for the design of clinical trials to evaluate the efficacy of epeleuton in patients with SCD.

Maternal, newborn and breast milk concentrations of elexacaftor/tezacaftor/ivacaftor in a F508del heterozygous woman with cystic fibrosis following successful pregnancy.

With the introduction of elexacaftor/tezacaftor/ivacaftor (ETI), more women with cystic fibrosis (CF) are likely to grow families. Hence, an understanding long-term safety and effects of CFTR modulators on fertile women and children while monitoring their concentrations is crucial. Here, we report on the development of an improved LC-MS/MS methodology to measure ETI concentrations in maternal and child blood and breastmilk, applied in one case of successful pregnancy of a 30-year-old woman with CF (F508del/R334W). We observed that ETI remains stable in breastmilk, is absorbed by the infant and can be detected in child plasma. Our results confirm accumulating evidence of a successful pregnancy in women treated with CFTR modulators without significant side effects on the child and provide valuable analytical procedures suitable for the post-marketing evaluation of CFTR modulators in pregnant and lactating women, as well as in their infants.

Fluid shear stress triggers cholesterol biosynthesis and uptake in inner medullary collecting duct cells, independently of nephrocystin-1 and nephrocystin-4.

Renal epithelial cells are subjected to fluid shear stress of urine flow. Several cellular structures act as mechanosensors-the primary cilium, microvilli and cell adhesion complexes-that directly relay signals to the cytoskeleton to regulate various processes including cell differentiation and renal cell functions. Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy leading to end-stage kidney failure before adulthood. NPHP1 and NPHP4 are the major genes which code for proteins that form a complex at the transition zone of the primary cilium, a crucial region required for the maintenance of the ciliary composition integrity. These two proteins also interact with signaling components and proteins associated with the actin cytoskeleton at cell junctions. Due to their specific subcellular localization, we wondered whether NPHP1 and NPHP4 could ensure mechanosensory functions. Using a microfluidic set up, we showed that murine inner medullary collecting ductal cells invalidated for Nphp1 or Nphp4 are more responsive to immediate shear exposure with a fast calcium influx, and upon a prolonged shear condition, an inability to properly regulate cilium length and actin cytoskeleton remodeling. Following a transcriptomic study highlighting shear stress-induced gene expression changes, we showed that prolonged shear triggers both cholesterol biosynthesis pathway and uptake, processes that do not seem to involve neither NPHP1 nor NPHP4. To conclude, our study allowed us to determine a moderate role of NPHP1 and NPHP4 in flow sensation, and to highlight a new signaling pathway induced by shear stress, the cholesterol biosynthesis and uptake pathways, which would allow cells to cope with mechanical stress by strengthening their plasma membrane through the supply of cholesterol.

Resolvin D1 improves airway inflammation and exercise capacity in cystic fibrosis lung disease.

Mucus plugging and non-resolving inflammation are inherent features of cystic fibrosis (CF) that may lead to progressive lung disease and exercise intolerance, which are the main causes of morbidity and mortality for people with CF. Therefore, understanding the influence of mucus on basic mechanisms underlying the inflammatory response and identifying strategies to resolve mucus-driven airway inflammation and consequent morbidity in CF are of wide interest. Here, we investigated the effects of the proresolving lipid mediator resolvin (Rv) D1 on mucus-related inflammation as a proof-of-concept to alleviate the burden of lung disease and restore exercise intolerance in CF. We tested the effects of RvD1 on inflammatory responses of human organotypic airways and leukocytes to CF mucus and of humanized mice expressing the epithelial Na + channel (ßENaC-Tg) having CF-like mucus obstruction, lung disease, and physical exercise intolerance. RvD1 reduced pathogenic phenotypes of CF-airway supernatant (ASN)-stimulated human neutrophils, including loss of L-selectin shedding and CD16. RNASeq analysis identified select transcripts and pathways regulated by RvD1 in ASN-stimulated CF bronchial epithelial cells that are involved in sugar metabolism, NF-κB activation and inflammation, and response to stress. In in vivo inflammation using ßENaC TG mice, RvD1 reduced total leukocytes, PMN, and interstitial Siglec-MΦ when given at 6-8 weeks of age, and in older mice at 10-12 weeks of age, along with the decrease of pro-inflammatory chemokines and increase of anti-inflammatory IL-10. Furthermore, RvD1 treatment promoted the resolution of pulmonary exacerbation caused by Pseudomonas aeruginosa infection and significantly enhanced physical activity and energy expenditure associated with mucus obstruction, which was impaired in ßENaC-Tg mice compared with wild-type. These results demonstrate that RvD1 can rectify features of CF and offer proof-of-concept for its therapeutic application in this and other muco-obstructive lung diseases.

Mitapivat reprograms the RBC metabolome and improves anemia in a mouse model of hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common, nonimmune, hereditary, chronic hemolytic anemia after hemoglobinopathies. The genetic defects in membrane function causing HS lead to perturbation of the RBC metabolome, with altered glycolysis. In mice genetically lacking protein 4.2 (4.2-/-; Epb42), a murine model of HS, we showed increased expression of pyruvate kinase (PK) isoforms in whole and fractioned RBCs in conjunction with abnormalities in the glycolytic pathway and in the glutathione (GSH) system. Mitapivat, a PK activator, metabolically reprogrammed 4.2-/- mouse RBCs with amelioration of glycolysis and the GSH cycle. This resulted in improved osmotic fragility, reduced phosphatidylserine positivity, amelioration of RBC cation content, reduction of Na/K/Cl cotransport and Na/H-exchange overactivation, and decrease in erythroid vesicles release in vitro. Mitapivat treatment significantly decreased erythrophagocytosis and beneficially affected iron homeostasis. In mild-to-moderate HS, the beneficial effect of splenectomy is still controversial. Here, we showed that splenectomy improves anemia in 4.2-/- mice and that mitapivat is noninferior to splenectomy. An additional benefit of mitapivat treatment was lower expression of markers of inflammatory vasculopathy in 4.2-/- mice with or without splenectomy, indicating a multisystemic action of mitapivat. These findings support the notion that mitapivat treatment should be considered for symptomatic HS.

Specialized pro-resolving lipid mediators and resolution of viral diseases.

The COVID-19 pandemics has made sparkly evident the importance of acute inflammation and its timely resolution to protect humans from pathogenic viruses while sparing them from collateral damages due to an uncontrolled immune response. It is clear now that resolution of inflammation is an active process regulated by endogenous specialized proresolving lipid mediators (SPM) biosynthesized from essential polyunsaturated fatty acids. Accruing evidence indicates that SPM are produced during viral infections and play key roles in controlling the magnitude and duration of the inflammatory response and in regulating adaptive immunity. Here, we reviewed biosynthesis and bioactions of SPM in virus-mediated human diseases. Harnessing SPM and their proresolutive actions can help in providing new therapeutic approaches to current and future human viral diseases by controlling infection, stimulating host immunity, and protecting from organ damage.

WDR5 is required for DUX4 expression and its pathological effects in FSHD muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.

Neurorehabilitation of severe acquired brain injury in the time of COVID-19: impact of the absence of caregivers.

INTRODUCTION: During the COVID-19 pandemic, several restrictions were imposed to limit the circulation of the infection within communities. Hospitals denied access to the family and friends of inpatients, and thus to caregivers. This observational study evaluated the impact of the physical absence of caregivers during the lockdown period due to the COVID-19 emergency on the rehabilitation of inpatients with severe acquired brain injury (sABI). METHODS: The functional outcome at discharge was measured in 25 inpatients with sABI through the Disability Rating Scale (DRS), Glasgow Outcome Scale (GOS), and Levels of Cognitive Functioning scale (LCF) after neuropsychological rehabilitation in an Adult Inpatient Neurorehabilitation Unit for Patients with sABI. Fourteen patients were directly assisted by their informal caregivers physically present in the neurorehabilitation ward. Eleven patients were indirectly supported via remote connection because during the lockdown period (from March to July 2020) caregivers could not be admitted to the rehabilitation hospital. The Caregiving Impact on Neuro-Rehabilitation Scale (CINRS) was also used to evaluate both the change since the admission and the impact of the caregiver from the perspective of the cognitive therapist. Demographic characteristics, time since injury, injury severity (duration of impaired consciousness measured by the time to follow commands), level of functioning at the beginning of the rehabilitation, and duration of the rehabilitation treatment were comparable between the groups. RESULTS: Both groups improved after the treatment; however, the improvement was consistently greater in the group directly assisted by the caregivers. The results showed that although the caregivers ensured their virtual presence at distance, their physical absence played a role in hindering the functional outcome of the patients. CONCLUSIONS: The role of the caregiver of patients with sABI is underlined in being not only a person handing out generic aid, cares, and affection, but also an integral part of the rehabilitation process.

Self-Awareness Multilevel Assessment Scale (SAMAS): psychometric analysis of inter-rater reliability.

BACKGROUND: Severe acquired brain injury (sABI) frequently causes impairment in self-awareness (ISA), leading to reduced patients' compliance to treatment, worse functional outcome, and high caregiver distress. Self-awareness (SA) is a multilevel and complex function that, as such, requires a specific and effective assessment. To date, many tools are available to evaluate the declarative, but not emergent and anticipatory levels of awareness, therefore the Self-Awareness Multilevel Assessment Scale (SAMAS) was recently proposed. The new tool proved to be useful to assess SA at different levels across all domains of functioning (motor, cognitive, psycho-behavioural, etc.) because it measures not only the declarative SA, but also emergent and anticipatory levels of SA, thus overcoming some important limits of other current assessment methods. AIM: This study evaluated the inter-rater reliability (IRR) of the SAMAS. METHODS: Four professionals blind to each other evaluated 12 patients with sABI. Each patient was rated by two professionals. RESULTS: Inter-rater reliability was moderate-to-excellent, adding evidence in support of the use of SAMAS to specifically diagnose ISA after sABI. CONCLUSIONS: The SAMAS can help to better address neurorehabilitation, as it allows assessing ISA as early as possible, at all possible levels of awareness and functional domains.

Gene Expression of the D-Series Resolvin Pathway Predicts Activation of Anti-Tumor Immunity and Clinical Outcomes in Head and Neck Cancer.

In human medicine, the progression from early neoplasia development to either complete resolution of tumorigenesis and associated inflammation or chronicity and fatal outcomes remain difficult to predict. Resolution of inflammation is an active process that stimulates the termination of the inflammatory response and promotes return to homeostasis, while failure in resolution contributes to the development of a number of diseases. To understand how resolution pathways contribute to tumorigenesis, we defined and employed a cumulative score based on the expression level of genes involved in synthesis, signaling, and metabolism of the D-series resolvin (RvD). This score was used for comparative analyses of clinical, cellular, and molecular features of tumors, based on RNA-sequencing (RNA-seq) datasets collected within The Cancer Genome Atlas (TCGA) program. Our results indicate that higher RvD scores are associated with better clinical outcome of patients with head and neck squamous cell carcinoma (HNSC), and with molecular and cellular signatures indicative of enhanced anti-tumor immunity and better response to immune-checkpoint inhibitors (ICI), also in human papilloma virus (HPV) negative HNSC subtypes. Thus, higher activity of the RvD pathway identifies patients with improved resolution and a more efficient immune reaction against cancer.

Mass fractal dimension from 2D microscopy images via an aggregation model with variable compactness.

Microscopy-image analysis provides precious information on size and structure of colloidal aggregates and agglomerates. The structure of colloids is often characterized using the mass fractal dimension d f $d_f$ , which is different from the two-dimensional (2D) fractal dimension d p $d_p$ that can be computed from microscopy images. In this work, we propose to use a recent morphological aggregation model to find a relationship between 2D image fractal dimension and 3D mass fractal dimension of aggregates and agglomerates. Our case study is represented by scanning transmission electron microscopy images of boehmite colloidal suspensions. The behaviour of the computed d f $d_f$ at different acid and base concentration shows a fair agreement with the results of small angle x-ray scattering and with the literature, enabling to use the d f $d_f$ versus d p $d_p$ relationship to study the impact of the composition of the colloidal suspension on the density of colloidal aggregates and agglomerates.

Boehmite powder is often employed for the manufacturing of γ $\gamma$ -alumina catalyst carriers and absorbents. This process involves boehmite colloidal particles coagulation, the size and shape of the particles affect the porosity of the final solid. As there is a high interest in tuning the porosity via the synthesis parameters, this work is aimed at the use of microscopy analysis to evaluate the mass fractal dimension of boehmite aggregates and agglomerates formed under controlled physical chemical conditions (acid and alkaline content and Brownian motion). Since the mass fractal dimension cannot be directly computed on binary two-dimensional images, a recently developed morphological model has been used to generate three-dimensional clusters characterized by a certain mass fractal dimension d f $d_f$ , the projection of the clusters leads to the estimation on the projected fractal dimension d p $d_p$ , being related to the slope on a bi-logarithmic plot perimeter versus area. According to the morphological model, the higher the d f $d_f$ , the lower the d p $d_p$ . The relationship between d f $d_f$ and d p $d_p$ has been used to estimate the mass fractal dimension from STEM images of boehmite suspensions. The trend of d f $d_f$ with respect to the acid and alkaline content in the suspensions agrees with small angle X-ray scattering measurement and with the literature.

The renal inflammatory network of nephronophthisis.

Renal ciliopathies are the leading cause of inherited kidney failure. In autosomal dominant polycystic kidney disease (ADPKD), mutations in the ciliary gene PKD1 lead to the induction of CCL2, which promotes macrophage infiltration in the kidney. Whether or not mutations in genes involved in other renal ciliopathies also lead to immune cells recruitment is controversial. Through the parallel analysis of patients' derived material and murine models, we investigated the inflammatory components of nephronophthisis (NPH), a rare renal ciliopathy affecting children and adults. Our results show that NPH mutations lead to kidney infiltration by neutrophils, macrophages and T cells. Contrary to ADPKD, this immune cell recruitment does not rely on the induction of CCL2 in mutated cells, which is dispensable for disease progression. Through an unbiased approach, we identified a set of inflammatory cytokines that are upregulated precociously and independently of CCL2 in murine models of NPH. The majority of these transcripts is also upregulated in NPH patient renal cells at a level exceeding those found in common non-immune chronic kidney diseases. This study reveals that inflammation is a central aspect in NPH and delineates a specific set of inflammatory mediators that likely regulates immune cell recruitment in response to NPH genes mutations.

Xeroderma Pigmentosum: General Aspects and Management.

Xeroderma Pigmentosum (XP) is a rare genetic syndrome with a defective DNA nucleotide excision repair. It is characterized by (i) an extreme sensitivity to ultraviolet (UV)-induced damages in the skin and eyes; (ii) high risk to develop multiple skin tumours; and (iii) neurologic alterations in the most severe form. To date, the management of XP patients consists of (i) early diagnosis; (ii) a long-life protection from ultraviolet radiation, including avoidance of unnecessary UV exposure, wearing UV blocking clothing, and use of topical sunscreens; and (iii) surgical resections of skin cancers. No curative treatment is available at present. Thus, in the last decade, in order to prevent or delay the progression of the clinical signs of XP, numerous strategies have been proposed and tested, in some cases, with adverse effects. The present review provides an overview of the molecular mechanisms featuring the development of XP and highlights both advantages and disadvantages of the clinical approaches developed throughout the years. The intention of the authors is to sensitize scientists to the crucial aspects of the pathology that could be differently targeted. In this context, the exploration of the process underlining the conception of liposomal nanocarriers is reported to focus the attention on the potentialities of liposomal technology to optimize the administration of chemoprotective agents in XP patients.

Home fetal heart rate monitoring in anti Ro/SSA positive pregnancies: Literature review and case report.

Anti-Ro/SSA antibodies are associated with a risk of 1-2 % to develop complete atrioventricular block (AVB) in fetuses of positive mothers. Complete AVB is irreversible, but studies suggest that anti-inflammatory treatment during the transition period from a normal fetal heart rate (FHR) to an AVB might stop this progression and restore sinus rhythm. The most efficient method for diagnostic evaluation of this arrhythmia is the pulsed-Doppler fetal echocardiography. However, weekly or bi-weekly recommended fetal echocardiographic surveillance can rarely identify an AVB in time for treatment success, also because the transition from a normal rhythm to a third degree AVB is very fast. Daily FHR monitoring in a medical facility could increase the chances of identifying the AVB onset but is difficult to realize. For this reason, an alternative method of FHR monitoring, performed directly by mothers in their home context, has been recently proposed. We present a case report utilizing this approach and review the current evidence about this condition.

Laser capture microdissection on formalin-fixed and paraffin-embedded renal transplanted biopsies: Technical perspectives for clinical practice application.

Renal biopsy (RBx) is an essential tool in the diagnostic and therapeutic process of most native kidney diseases and in the renal transplanted graft. Laser capture microdissection (LCM), combined with molecular biology, might improve the diagnostic power of RBx. However, the limited amount of available renal tissue is often an obstacle for achieving a satisfactory qualitative and quantitative analysis. In our work we present a method which allows us to obtain good quality and quantity of RNA from formalin-fixed and paraffin-embedded (FFPE) renal tissue derived from RBx performed in transplanted patients. Histology, immunohistochemistry, LCM, pre-amplify system and qRT-PCR of biomarkers related to tubular damage, inflammation and fibrosis on FFPE RBx were performed. Glomeruli, tubules and interstitium of three RBx (RB-A: no alteration; RB-B and -C: the progressive rise of creatinine) were compared. The method proposed, could well be useful in future clinical practice. It is quick, easy to perform and allows the analyses of many biomarkers. In addition, it could be extended to all types of RBx without any limitation on the sample amount. Nevertheless, the need for a higher number of well-trained technicians might represent some limitation, counterbalanced by the opportunity to elaborate more accurate diagnosis and, consequently, more targeted therapies.

The Self-Awareness Multilevel Assessment Scale, a New Tool for the Assessment of Self-Awareness After Severe Acquired Brain Injury: Preliminary Findings.

Self-awareness (SA) is frequently impaired after severe acquired brain injury (sABI) and may lead to reduced subject's compliance to treatment, worse functional outcome, and high caregiver distress. Considering the multifaceted nature of SA, a specific and effective assessment is crucial to address treatment of impairment of SA (ISA). Many tools can currently assess ISA; however, they have some important limits. In the present study, we proposed the Self-Awareness Multilevel Assessment Scale (SAMAS), a new scale for assessment of SA at different levels (i.e., declarative, emergent, and anticipatory) across all domains of functioning. The SAMAS has been designed to be administered by the cognitive/behavioral therapist with the involvement of a patient's relative. Findings showed that the SAMAS allowed specifically assessing SA at a declarative level and on all possible functional domains. More interestingly, it seems also able to assess both emergent and anticipatory SA, thus overcoming some important limits of other current assessment methods. Our findings are consistent with a holistic perspective of the patient with sABI because thanks to the combined use of assessing tools, the SAMAS can provide an accurate diagnosis of ISA, thus better addressing the neurorehabilitation treatment and, accordingly, reducing the possible occurrence of its primary and secondary implications.

Coma Recovery Scale-Revised With and Without the Emotional Stimulation of Caregivers.

The Coma Recovery Scale-Revised (CRS-R) is the gold standard of responsiveness assessment in patients with disorder of consciousness. The purpose of this study is to search for the efficacy of the caregivers' involvement in the evaluation of responsiveness in these patients. Responsiveness assessment was performed in 15 patients with CRS-R. The CRS-R was administered with and without the emotional stimulation of the primary caregiver at different times. Our preliminary findings seem to suggest that, including also the caregivers during CRS-R assessment, may obtain better responsiveness scoring than that obtained by professionals and might reduce the misdiagnosis rate.

Résultats à l'échelle d'évaluation d'éveil lors d'un coma avec ou sans la stimulation affective de personnes soignantes. L'échelle d'évaluation d'éveil lors d'un coma (Coma Recovery Scale-Revised) demeure la norme de référence en matière d'évaluation de la réactivité de patients aux prises avec des troubles de la conscience. L'objectif de cette étude est d'analyser l'impact de l'implication de personnes soignantes dans l'évaluation de la réactivité de ces patients. Une telle analyse a été effectuée chez quinze patients soumis à l'échelle d'évaluation d'éveil lors d'un coma, et ce, avec ou sans la stimulation affective d'une personne soignante et à différents moments. À cet égard, nos constatations préliminaires semblent indiquer que les scores de réactivité à cette échelle pourraient, en présence de personnes soignantes, dépasser ceux obtenus en compagnie de professionnels et ainsi réduire les taux de diagnostics erronés.

The Suv420h histone methyltransferases regulate PPAR-γ and energy expenditure in response to environmental stimuli.

Obesity and its associated metabolic abnormalities have become a global emergency with considerable morbidity and mortality. Epidemiologic and animal model data suggest an epigenetic contribution to obesity. Nevertheless, the cellular and molecular mechanisms through which epigenetics contributes to the development of obesity remain to be elucidated. Suv420h1 and Suv420h2 are histone methyltransferases responsible for chromatin compaction and gene repression. Through in vivo, ex vivo, and in vitro studies, we found that Suv420h1 and Suv420h2 respond to environmental stimuli and regulate metabolism by down-regulating peroxisome proliferator-activated receptor gamma (PPAR-γ), a master transcriptional regulator of lipid storage and glucose metabolism. Accordingly, mice lacking Suv420h proteins activate PPAR-γ target genes in brown adipose tissue to increase mitochondria respiration, improve glucose tolerance, and reduce adipose tissue to fight obesity. We conclude that Suv420h proteins are key epigenetic regulators of PPAR-γ and the pathways controlling metabolism and weight balance in response to environmental stimuli.