Small molecules targeting endocytic uptake and recycling pathways

Over the past years a growing number of studies highlighted the pivotal role of intracellular trafficking in cell physiology. Among the distinct transport itineraries connecting the endocytic system, both internalization (endocytosis) and recycling (endocytic recycling) pathways were found fundamental to ensure cellular sensing, cell-to-cell communication, cellular division, and collective cell migration in tissue specific-contexts. Consistently, the dysregulation of endocytic trafficking pathways is correlated with several human diseases including both cancers and neurodegeneration. Aimed at suppress specific intracellular trafficking routes involved in disease onset and progression, huge efforts have been made to identify small molecule inhibitors with suitable pharmacological properties for in vivo administration. Here, we review most used drugs and recently discovered small molecules able to block endocytosis and endocytic recycling pathways. We characterize such pharmacological inhibitors by emphasizing their target specificity, molecular affinity, biological activity and efficacy in both in vitro and in vivo experimental models

How PI3K-derived lipids control cell division

To succeed in cell division, intense cytoskeletal and membrane remodeling are required to allow accurate chromosome segregation and cytoplasm partitioning. Spatial restriction of the actin dynamics and vesicle trafficking define the cell symmetry and equivalent membrane scission events, respectively. Protein complexes coordinating mitosis are recruited to membrane microdomains characterized by the presence of the phosphatidylinositol lipid members (PtdIns), like PtdIns(3,4,5)P3, PtdIns(4,5)P2 and PtdIns(3)P. These PtdIns represent a minor component of cell membranes, defining membrane domain identity, ultimately controlling cytoskeleton and membrane dynamics during mitosis. The coordinated presence of PtdIns(3,4,5)P3 at the cell poles and PtdIns(4,5)P2 at the cleavage furrow controls the polarity of the actin cytoskeleton leading to symmetrical cell division. In the endosomal compartment, the trafficking of PtdIns(3)P positive vesicles allows the recruitment of the protein machinery required for the abscission

Small molecules targeting endocytic uptake and recycling pathways

Over the past years a growing number of studies highlighted the pivotal role of intracellular trafficking in cell physiology. Among the distinct transport itineraries connecting the endocytic system, both internalization (endocytosis) and recycling (endocytic recycling) pathways were found fundamental to ensure cellular sensing, cell-to-cell communication, cellular division, and collective cell migration in tissue specific-contexts. Consistently, the dysregulation of endocytic trafficking pathways is correlated with several human diseases including both cancers and neurodegeneration. Aimed at suppress specific intracellular trafficking routes involved in disease onset and progression, huge efforts have been made to identify small molecule inhibitors with suitable pharmacological properties for in vivo administration. Here, we review most used drugs and recently discovered small molecules able to block endocytosis and endocytic recycling pathways. We characterize such pharmacological inhibitors by emphasizing their target specificity, molecular affinity, biological activity and efficacy in both in vitro and in vivo experimental models

MRI-targeted or standard biopsy for prostate-cancer diagnosis

Background Multiparametric magnetic resonance imaging (MRI), with or without targeted biopsy, is an alternative to standard transrectal ultrasonography-guided biopsy for prostate-cancer detection in men with a raised prostate-specific antigen level who have not undergone biopsy. However, comparative evidence is limited. Methods In a multicenter, randomized, noninferiority trial, we assigned men with a clinical suspicion of prostate cancer who had not undergone biopsy previously to undergo MRI, with or without targeted biopsy, or standard transrectal ultrasonography-guided biopsy. Men in the MRI-targeted biopsy group underwent a targeted biopsy (without standard biopsy cores) if the MRI was suggestive of prostate cancer; men whose MRI results were not suggestive of prostate cancer were not offered biopsy. Standard biopsy was a 10-to-12-core, transrectal ultrasonography-guided biopsy. The primary outcome was the proportion of men who received a diagnosis of clinically significant cancer. Secondary outcomes included the proportion of men who received a diagnosis of clinically insignificant cancer. Results A total of 500 men underwent randomization. In the MRI-targeted biopsy group, 71 of 252 men (28%) had MRI results that were not suggestive of prostate cancer, so they did not undergo biopsy. Clinically significant cancer was detected in 95 men (38%) in the MRI-targeted biopsy group, as compared with 64 of 248 (26%) in the standard-biopsy group (adjusted difference, 12 percentage points; 95% confidence interval [CI], 4 to 20; P=0.005). MRI, with or without targeted biopsy, was noninferior to standard biopsy, and the 95% confidence interval indicated the superiority of this strategy over standard biopsy. Fewer men in the MRI-targeted biopsy group than in the standard-biopsy group received a diagnosis of clinically insignificant cancer (adjusted difference, -13 percentage points; 95% CI, -19 to -7; P<0.001). Conclusions The use of risk assessment with MRI before biopsy and MRI-targeted biopsy was superior to standard transrectal ultrasonography-guided biopsy in men at clinical risk for prostate cancer who had not undergone biopsy previously. (Funded by the National Institute for Health Research and the European Association of Urology Research Foundation; PRECISION ClinicalTrials.gov number, NCT02380027 .)

Persistent Megalocystic Ovary Following in Vitro Fertilization in a Postpartum Patient with Polycystic Ovarian Syndrome

SummaryObjectiveOvarian hyperstimulation syndrome (OHSS) is more severe when pregnancy occurs, as the developing pregnancy produces human chorionic gonadotropin, which stimulates the ovary's persistent growth. If no pregnancy occurs, the syndrome will typically resolve within 1 week. In a maintained pregnancy, slow resolution of symptoms usually occurs over 1-2 months.Case ReportA 31-year-old woman, gravida 2, para 1, aborta 1, with polycystic ovary syndrome underwent in vitro fertilization (IVF) with clomiphene citrate and follicle-stimulating hormone/gonadotropin releasing hormone-antagonist stimulation. During transvaginal oocyte retrieval, enlarged bilateral ovaries were noted. She had an episode of OHSS after IVF/embryo transfer, for which paracentesis was performed three times. Pregnancy was achieved. Throughout antenatal examinations, bilateral ovaries were enlarged. She delivered a healthy baby by cesarean section at term. However, 1 month after delivery, the bilateral ovary had not shrunk, and levels of tumor markers CA125 and CA199 were 50.84 and 41.34 U/mL, respectively. At laparotomy for suspected malignancy, both adnexae formed “kissing ovaries”, which were multinodulated with yellow serous fluid. Specimens from wedge resection submitted for frozen section showed a benign ovarian cyst. The final pathology report showed bilateral follicle cysts.ConclusionWith the increasing use of gonadotropins in the management of infertility, ovarian enlargement secondary to hyperstimulation is common. Generally, symptoms appear between the 6th and 13th weeks of pregnancy and disappear thereafter. The hyperstimulated ovary often subsides after the first trimester. This case is unusual as the megalocystic ovary persisted after delivery. To the best of our knowledge, we report the first case of enlarged bilateral ovaries persisting 2 months after delivery

Pediatric Moyamoya Disease and Syndrome in Italy: A Multicenter Cohort

Background: Moyamoya is a rare progressive cerebral arteriopathy, occurring as an isolated phenomenon (moyamoya disease, MMD) or associated with other conditions (moyamoya syndrome, MMS), responsible for 6–10% of all childhood strokes and transient ischemic attacks (TIAs). Methods: We conducted a retrospective multicenter study on pediatric-onset MMD/MMS in Italy in order to characterize disease presentation, course, management, neuroradiology, and outcome in a European country. Results: A total of 65 patients (34/65 women) with MMD (27/65) or MMS (38/65) were included. About 18% (12/65) of patients were asymptomatic and diagnosed incidentally during investigations performed for an underlying condition (incMMS), whereas 82% (53/65) of patients with MMD or MMS were diagnosed due to the presence of neurological symptoms (symptMMD/MMS). Of these latter, before diagnosis, 66% (43/65) of patients suffered from cerebrovascular events with or without other manifestations (ischemic stroke 42%, 27/65; TIA 32%, 21/65; and no hemorrhagic strokes), 18% (12/65) of them reported headache (in 4/12 headache was not associated with any other manifestation), and 26% (17/65) of them experienced multiple phenotypes (≥2 among: stroke/TIA/seizures/headache/others). Neuroradiology disclosed ≥1 ischemic lesion in 67% (39/58) of patients and posterior circulation involvement in 51% (30/58) of them. About 73% (47/64) of patients underwent surgery, and 69% (45/65) of them received aspirin, but after diagnosis, further stroke events occurred in 20% (12/61) of them, including operated patients (11%, 5/47). Between symptom onset and last follow-up, the overall patient/year incidence of stroke was 10.26% (IC 95% 7.58–13.88%). At last follow-up (median 4 years after diagnosis, range 0.5–15), 43% (26/61) of patients had motor deficits, 31% (19/61) of them had intellectual disability, 13% (8/61) of them had epilepsy, 11% (7/61) of them had behavioral problems, and 25% (13/52) of them had mRS > 2. The proportion of final mRS > 2 was significantly higher in patients with symptMMD/MMS than in patients with incMMS (p = 0.021). Onset age <4 years and stroke before diagnosis were significantly associated with increased risk of intellectual disability (p = 0.0010 and p = 0.0071, respectively) and mRS > 2 at follow-up (p = 0.0106 and p = 0.0009, respectively). Conclusions: Moyamoya is a severe condition that may affect young children and frequently cause cerebrovascular events throughout the disease course, but may also manifest with multiple and non-cerebrovascular clinical phenotypes including headache (isolated or associated with other manifestations), seizures, and movement disorder. Younger onset age and stroke before diagnosis may associate with increased risk of worse outcome (final mRS > 2)

Deliver on Time or Pay the Fine: Scheduling in Membrane Trafficking

Membrane trafficking is all about time. Automation in such a biological process is crucial to ensure management and delivery of cellular cargoes with spatiotemporal precision. Shared molecular regulators and differential engagement of trafficking components improve robustness of molecular sorting. Sequential recruitment of low affinity protein complexes ensures directionality of the process and, concomitantly, serves as a kinetic proofreading mechanism to discriminate cargoes from the whole endocytosed material. This strategy helps cells to minimize losses and operating errors in membrane trafficking, thereby matching the appealed deadline. Here, we summarize the molecular pathways of molecular sorting, focusing on their timing and efficacy. We also highlight experimental procedures and genetic approaches to robustly probe these pathways, in order to guide mechanistic studies at the interface between biochemistry and quantitative biology

Possible ocular involvement in pulmonary alveolar proteinosis

To the Editors: Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterised by lipoproteinaceous material accumulation in the alveoli. In the most frequent form of the disease (acquired PAP), a neutralising auto-antibody against granulocytemacrophage colony-stimulating factor (GM-CSF) causes defects in the functioning of alveolar macrophages, including impairment of the catabolism of surfactant lipids and proteins [1]. The clinical manifestations of PAP are progressive dyspnoea, cough or fatigue. Available reports have never disclosed any ocular involvement. Herein, we describe, in a patient with acquired PAP, a retinal lesion resembling adult-onset foveomacular vitelliform dystrophy (AOFVD), a macular degeneration characterised by a generally autosomal dominant inheritance pattern and yellow- grey macular deposits in the deeper retinal layers. A 47-year-old male was referred to the Dept of Ophthalmology (University of Ferrara, Ferrara, Italy) due to a progressive loss of vision in his right eye. His medical history was positive for the recent onset of acquired PAP, which was treated with a therapeutic whole-lung lavage. At the ophthalmological examination, his best-corrected visual acuity was 20/32 in the right eye and 20/20 in the left eye. Ophthalmoscopic evaluation was unremarkable in the left eye, whereas the right eye revealed a round-shaped, yellowish subfoveal lesion. On fluorescein angiography, this lesion appeared centrally hypofluorescent with a marginal irregular hyperfluorescence in the early arteriovenous phase and totally hyperfluorescent in the late venous phase. Interestingly, late-phase indocyanine green angiography disclosed a diffuse hypofluorescence in the macular region of both eyes. Optical coherence tomography (OCT) was abnormal only in the right eye, showing an anterior displacement of the photoreceptor layer by a hyperreflective structure located over a hyporeflective space above the retinal pigment epithelium layer. No evidence of a familial inheritance pattern, an unusual OCT presentation and, above all, AOFVD onset shortly after the disclosure of acquired PAP prompted us to consider a not purely coincidental association between the two diseases. Although no data about ocular tissues are available, animal models of PAP manifest a number of more subtle but important extrapulmonary abnormalities [2]. Furthermore, another study [3] has emphasised the role of GM-CSF, which is released by retinal pigment epithelial cells, as a regulator of macrophage and granulocyte function in the eye, and lipofuscin (the likely aetiology of the vitelliform appearance of the fovea in AOFVD) is thought to be the indigestible residues of rod outer segments that have been phagocytosed by macrophages [4]. Lastly, transgenic mice with dysregulated GM-CSF production exhibit a wide range of ocular lesions [5]. Ophthalmological investigations in other patients with pulmonary alveolar proteinosis, but not necessarily with ocular symptoms, could elucidate the possible relationship between this disease and macular disorders. P. Perri, C. Campa, S. D’Angelo, C. Costagliola, C. Incorvaia and A. Sebastiani Dept of Ophthalmology, University of Ferrara, Ferrara, Italy. REFERENCES 1 Trapnell BC, Whitsett JA, Nakata K. Pulmonary alveolar proteinosis. N Engl J Med 2003; 349: 2527-2539. 2 Seymour JF, Presneill JJ. Pulmonary alveolar proteinosis: progress in the first 44 years. Am J Respir Crit Care Med 2002; 166: 215-235. 3 Crane IJ, Kuppner MC, McKillop-Smith S, Wallace CA, Forrester JV. Cytokine regulation of granulocytemacrophage colony-stimulating factor (GM-CSF) production by human retinal pigment epithelial cells. Clin Exp Immunol 1999; 115: 288-293. 4 Kennedy CJ, Rakoczy PE, Constable IJ. Lipofuscin of the retinal pigment epithelium: a review. Eye 1995; 9: 763-771. 5 Lang RA, Metcalf D, Cuthbertson RA, et al. Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF) develop accumulations of macrophages, blindness, and a fatal syndrome of tissue damage. Cell 1987; 51: 675-686

Ibuprofen oral administration increases the intraocular pressure-lowering effect of latanoprost in patients with primary open-angle glaucoma

In patients with primary open-angle glaucoma ibuprofen single-dose significantly enhances the latanoprost-induced intraocular-pressure-lowering effect during the short plasma half-life of ibuprofen. This therapeutic action seems to be obtained via high-affinity activation of FP-subtype receptors located in the ciliary muscle. Experimental evidence indicates that the expression of EP1, EP3 and FP prostanoid receptors is modulated by the PGs levels; high levels of endogenous PGs down-regulate receptor densities, whereas the NSAID-inhibition of PGs synthesis up-regulates the concentration of these receptors in both brain synaptosomes and retinovascular tissues of newborn and adult pigs. In healthy volunteers, it has been demonstrated that PGs receptors are scarcely expressed in ciliary epithelial and ciliary muscle cells, whereas in glaucomatous tissues an over-expression of these receptors occurs. NSAID administration is able to inhibit the PGs synthesis at the level of the iris-ciliary body complex, as demonstrated in experiments conducted on human cultured cells of both trabecular meshwork and ciliary muscle, without affecting IOP levels. It is not so hazardous to hypothesize that at this fall in endogenous PG synthesis there could correspond an increase in the prostanoid receptor expression. Because of latanoprost interacts mainly with FP receptors, when their expression is increased, as probably occurred in our NSAID-treated patients with primary open-angle glaucoma, the hypotensive action of latanoprost should become more relevant. Although the synergism between NSAID and latanoprost could appear as an advantage in glaucoma treatment, conversely it is well-known that NSAIDs long-term administration is responsible for the occurrence of local side effects, ranging from conjunctival hyperaemia to indolent corneal ulceration and melts. In conclusion, because in clinical practice NSAIDs represent one of the most widely utilized medical therapies, both ophthalmologists and general practitioners should schedule supplementary intraocular-pressure checks during the co-administration of NSAIDs and PGs analogues

Physics of compartmentalization: How phase separation and signaling shape membrane and organelle identity

Compartmentalization of cellular functions is at the core of the physiology of eukaryotic cells. Recent evidences indicate that a universal organizing process – phase separation – supports the partitioning of biomolecules in distinct phases from a single homogeneous mixture, a landmark event in both the biogenesis and the maintenance of membrane and non-membrane-bound organelles. In the cell, ‘passive’ (non energy-consuming) mechanisms are flanked by ‘active’ mechanisms of separation into phases of distinct density and stoichiometry, that allow for increased partitioning flexibility and programmability. A convergence of physical and biological approaches is leading to new insights into the inner functioning of this driver of intracellular order, holding promises for future advances in both biological research and biotechnological applications