ŠTO O PROIZVODIMA BILJNOG PODRIJETLA NA HRVATSKOM TRŽIŠTU GOVORE ANALIZE OSTATAKA PESTICIDA?

Sredstva za zaštitu bilja pripravci su koji se sastoje ili koji sadržavaju aktivne tvari i dodatne nepesticidne tvari. Jedna od najuobičajenijih metoda za zaštitu bilja i biljnih proizvoda od utjecaja štetnih organizama svakako je uporaba aktivnih tvari u sredstvima za zaštitu bilja. Međutim, moguća posljedica njihove uporabe može biti prisutnost ostatka na tretiranim proizvodima, u životinjama koje se hrane tim proizvodima i u medu koji su proizvele pčele izložene tim tvarima. Analize ostataka pesticida u proizvodima biljnog i životinjskog podrijetla provode se u analitičkim laboratorijima i koriste se multirezidualne metode (MRM) ili pojedinačne metode (SRM). Rezultati analiza ocjenjuju se sukladno Uredbi 396/2005 za konvencionalne proizvode i na temelju Uredbe (EZ) 889/2008 za proizvode iz ekološke proizvodnje

Influence of Dance on the Movement of Persons With Parkinson’s Disease

Uvod: Neka istraživanja ukazuju na to da ples utječe na poboljšanje hoda i balansa osoba s Parkinsonovom bolešću te kako utjecaj glazbe i ritma facilitira kretanje i brzinu kod ljudi s Parkinsonovom bolešću. Blagotvorni učinci ritma i glazbe proizlaze iz aktiviranja neuralnih struktura uključenih u kretanje i određivanje vremena. Metode: Rad je sustavni pregled literature pretraživanjem relevantnih članaka baza podataka PubMed, PE-Dro, LILACS i Cochrane library od 2007. do 2020. godine. Kriteriji su istraživanja koja se bave utjecajem tanga na hod i balans kod osoba s Parkinsonovom bolešću primjenom standardiziranih testova Berg balans skale (BBS), testa Time up and go (TUG) i Unified Parkinsonʼs Disease Rating Scale III (UPDRS-III). Iz sustavnog pregleda literature od ukupno 65 pronađenih radova uključeno je šest članaka koji zadovoljavaju zadane kriterije. Rezultati: Uspoređujući trajanje intervencija, istraživanja su se provodila uglavnom kratkoročno i intenzivno te dugoročno i umjereno. Prakticiranjem tanga u kratkoročnom ili dugoročnom razdoblju dolazi ne samo do poboljšanja balansa nego i povećanja tjelesnog fitnesa. Rezultati kratkoročnih istraživanja pokazala su statistički značajne razlike kod testova UPDRS-III i BBS u korist skupina koje su kao intervenciju imale tango, dok se kod dugoročnih istraživanja uočavaju poboljšanja u balansu i hodu koja se nisu pokazala kod kratkoročne i intenzivne intervencije tangom. Zaključak: Dokazi o poboljšanju funkcionalne mobilnosti i balansa potiču na proširivanje spektra rehabilitacije kod osoba s Parkinsonovom bolešću jer je tango ples koji svojim ritmom, glazbom i širinom koraka itekako pronalazi mjesto u rehabilitaciji osoba s Parkinsonovom bolešću.Introduction: Some research suggest that dancing improves gait and balance in people with Parkinson’s disease. Also suggest influence of music and rhythm on speed and facilitation of movement in people with Parkinson’s disease. The beneficial effects of rhythm and music result from the activation of neural structures involved in movement and timing. Methods: This paper is a systematic review of the literature searched through relevant database Pubmed, PEDro, LILACS and Cochrane library published in the period from 2007 to 2020. Our criteria for including papers in review were research on tango impact on gait and balance in people with Parkinson’s disease, use of standard tests as Berg balance scale (BBS), Time up and go test (TUG) and Unified Parkinsonʼs Disease Rating Scale III (UPDRS-III). After preformed review only 6 researches were selected in further analysis. Results: Comparing the duration of interventions, the researches were conducted mainly in the short term and intensive and in the long term and moderate. Tango dancing in the short term or long term leads to an improvement not only in balance but also in improvements in body fitness. The results from short term researches showed statistically significant differences in UPDRS III and BBS in tango dancing groups. While in long-term studies we observed improvements in balance and gait which was not shown in short term and intensive tango intervention. Conclusion: Evidence of improved functional mobility and balance encourages the expansion of the spectrum of rehabilitation in people with Parkinson’s disease. Because tango is a dance that, with its rhythm, music, and step width, finds a place in the rehabilitation of people with Parkinson’s disease

Provjera autentičnosti ekstrakta lista biljke Ginkgo biloba L. i proizvoda koji ga sadrže na hrvatskom tržištu putem analize količine i omjera ginkoflavonoglikozida (kvercetin, kempferol i izoramnetin) i terpenskih trilaktona u svrhu otkrivanja krivotvorenih pripravaka opasnih za zdravlje korisnika

Ginkgo biloba L. is the eldest plant growing on the Earth; preparations made of its leaves and seeds represent an integral part of the Chinese medicine for over a millennium. The plant species was first discovered by Linnaeus in 1771, its name thereby originating from the Latin words bis (two) and lobus (lobe), which duly illustrate the specific shape of its leaf. Contemporary Ginkgo biloba L. plant based pharmaceuticals mostly comprise extracts recovered from leaves harvested during fall, when the concentration of active components reaches its peak. Recent investigations have managed to establish the chemical composition of the plant leaf, together with the mechanisms underlying its beneficial effects on rheological profile of the blood and acceleration of its flow. High price of these preparations and their vast popularity have soon become an incentive for counterfeiting Ginkgo biloba L. extracts and the release of bogus drugs comprising cheaper extracts coming from other plants. Namely, modern Ginkgo biloba L.-based medicinal products and food supplements comprise extracts recovered from the plant leaf that get to be standardized according to its key pharmacological active components, most often flavone glycosides (represented in the share of 22%-27%) and terpene trilactones (represented in the share of 6%-7%). The flavonoids that predominate such preparations and are most relevant from the pharmacological standpoint are quercetin, kaempferol and isorhamnetin, their total amount and mutual ratios, thereby being an unquestionable indicator of the extract authenticity. Therefore, most of the analyses aiming at verifying the authenticity of a given Ginkgo biloba L.-based product boil down to the analysis of these parameters. Counterfeiting involves partial or full replacement of the Ginkgo biloba L. extract (GBE) with a cheaper plant extract of a similar composition, the latter occasionally being enriched with an additional amount of flavonoids, most often quercetin, not originating from the Ginkgo biloba L. plant. The aim of this study was to verify the authenticity and quality of Ginkgo biloba L.-based products circulating on the Croatian market. To that effect, 10 samples of products produced by various manufacturers were analyzed in a certified laboratory. The parameters based on which the authenticity of the preparations was assessed were the shares of aglycones of typical ginkgo flavone glycosides, that is to say, quercetin, kaempferol and isorhamnetin, and mutual ratios of the established quantities of quercetin to kaempferol as the key clues to unmasking Ginkgo extracts counterfeiting. The amount of ginkgo flavone glycosides was established using high performance liquid chromatography. The analysis proved 80% of the samples analyzed to be conformant to the label statements as regards the total amount of flavone glycosides and their mutual ratios. In 20% of the samples, the ratio of quercetin to kaempferol deviated from normal values; on top of that, the presence of the phytoestrogen genistein, one of the components typically comprised by the Sophora japonica L. plant, was also proven, documenting counterfeiting of the GBE and its replacement by the Sophora japonica L. extracts in the samples under consideration. Due to the untrue label statements descriptive of these products, the information on the presence of pharmacologically active genistein was neglected to be mentioned despite its unfavorable health impact that can be expected in some consumer groups. The results of this study indicated the frequency of counterfeiting the Ginkgo biloba L.-based products found on the Croatian market to be deemed substantial. Therefore, a more rigorous and more thorough control of these products and sanctioning of irresponsible manufacturers and distributers is proposed, so as to contribute to a higher market representation of high-quality products, as well as to avoid health risks and downsize the rate of their counterfeiting.Ginkgo biloba L. najstarija je biljka na zemlji, a pripravci od njegovih listova i sjemenaka dio su kineske medicine već više tisuća godina. Vrstu je 1771. godine otkrio Linnaeus, a ime je dobila od latinskih riječi bis, tj. dva i lobus zbog specifičnog oblika lista. Današnji farmaceutski oblici biljke Ginkgo biloba L. sadrže uglavnom ekstrakte iz listova ubranih ujesen kada je najveća koncentracija aktivnih komponenata. Suvremenim istraživanjima određen je kemijski sastav njegovog lista, a utvrđeni su i mehanizmi njegovog pozitivnog djelovanja na reološka svojstva krvi i ubrzanje njezinog protoka. Visoka cijena i velika popularnost ubrzo su postali poticaj za krivotvorenje ekstrakta biljke Ginkgo biloba L. jeftinijim ekstraktima drugih biljaka. Naime, suvremeni medicinski proizvodi i dodaci prehrani na osnovi biljke Ginkgo biloba L. sadrže ekstrakte njegovog lista koji se standardiziraju prema njegovim ključnim farmakološki aktivnim sastavnicama; najčešće je to 22%-27% flavonoid glikozida i 6%-7% terpenskih trilaktona. Kvercetin, kempferol i izoramnetin su dominantni i farmakološki najvažniji flavonoidi, a njihova ukupna količina i međusobni omjeri nedvojben su pokazatelj autentičnosti ekstrakta. Stoga se i većina analiza za utvrđivanje ispravnosti proizvoda koji sadrže biljku Ginkgo biloba L. temelji na analizi ovih parametara. Krivotvorenja podrazumijevaju djelomičnu ili potpunu zamjenu ektrakta Ginkgo biloba L. nekim jeftinijim biljnim ekstraktom sličnog sastava uz eventualno obogaćivanje dodatnom količinom flavonoida, najčešće kvercetina, koji ne potječu od biljke Ginkgo biloba L. Svrha ovoga rada bila je provjeriti autentičnost i kakvoću pripravaka Ginkgo biloba L. na hrvatskom tržištu. Analizirano je ukupno 10 uzoraka različitih proizvođača. Kao pokazatelji njihove ispravnosti određivani su udjeli aglikona karakterističnih ginkoflavonoglikozida: kvercetin, kempferol i izoramnetin te omjeri nađenih količina kvercetina i kempferola kao ključni pokazatelji krivotvorenja ginkgo-ekstrakta. Količina ginkoflavonoglikozida određena je metodom tekućinske kromatografije visoke djelotvornosti. Analizom je utvrđeno da 80% uzoraka odgovara navodima deklaracije u pogledu ukupne količine flavonoglikozida kao i u pogledu njihovog međusobnog omjera. U 20% uzoraka omjer kvercetina i kempferola odstupao je od uobičajenih vrijednosti, a dokazano je i prisustvo fitoestrogena genisteina, jedne od karakterističnih sastavnica biljke Sophora japonica L., čijim ekstraktom je krivotvoren ginkgo-ekstrakt u analiziranim uzorcima. Zbog neistinite deklaracije na proizvodima koji su sadržavali ove ekstrakte izostalo je i obvezno upozorenje o prisutnosti farmakološki aktivnog genisteina koji može izazvati neželjene zdravstvene posljedice kod pojedinih skupina potrošača. Rezultati ovoga rada ukazuju na značajnu pojavnost krivotvorenja proizvoda biljke Ginkgo biloba L. na hrvatskom tržištu te se zbog toga predlaže njihova temeljitija kontrola kao i sankcioniranje neodgovornih proizvođača i trgovaca, čime bi se doprinijelo većem udjelu kvalitetnijih proizvoda i izbjegli zdravstveni rizici te smanjila pojavnost prijevare

Flower honey as a potential bio-indicator of environmental pollution by pesticides in the Varaždin County

Cilj: U ovom istraživanju uzorci cvjetnoga meda korišteni su kao bio-pokazatelji onečišćenja okoliša pesticidima. Materijal i metode: Ukupno je prikupljeno 40 (4x10) uzoraka sa 4 različite prostorne lokacije (subregije), u ukupno 16 općina, od proizvođača meda u Varaždinskoj županiji, kao i 20 (2x10) uzoraka s dvije različite lokacije proizvođača u susjednoj županiji, registriranih kao "ekoloških proizvođači", a udaljenih od poljoprivrednih površina pod konvencionalnom proizvodnjom. Analize su provedene korištenjem vezanog sustava plinske kromatografije i spektrometrije masa (GCMS) i tekućinskom kromatografijom visoke djelotvornosti (HPLC). Rezultati: Identificirano je i kvantificirano 78 aktivnih sastojaka pesticide, koji su dio od ukupno cca 400 pesticida koji se analiziraju u voću i povrću, a za koje je laboratorij raspolagao analitičkom opremom koja i dijelom određuje i vrstu i broj pesticida koje se detektiraju. Rezultati analiza ukazali su na mogući uzročno posljedični odnos između konvencionalnih poljoprivrednih aktivnosti u Varaždinskoj županiji s identificiranim i kvantificiranim aktivnim sastojcima pesticida. Iz rezultata dobivenih na udjelima identificiranih i kvantificiranih ostataka pesticida, s obzirom na dopustivost, vidljivo je da je udio dopuštenih pesticida u uzorcima cvjetnoga meda iznosio 55,2%, dok je udio ilegalnih pesticida iznosio 44,8%. Analizirajući broj otkrivenih insekticida i fungicida, ukupno na sve 4 lokacije utvrđeno je da cvjetni med sadrži 13 različitih vrsta insekticida i 3 vrste fungicida. Zaključak: Rezultati i statistička analiza podataka pokazuju da je cvjetni med dobar bio-indikator onečišćenja okoliša pesticidima, te stoga ova studija može poslužiti kao osnova za znanstvenike i stručnjake koji se bave istraživanjem štetnih učinaka pesticida na okoliš i ljudsko zdravlje.Aim: In this research samples of floral honey were used as bio-indicators of environmental pollution by pesticides. Material and methods: A total of 40 (4x10) samples from bee farms were collected at 4 different locations (sub-regions) including 16 municipalities in the Varaždin County, as well as 20 (2x10) samples from two locations – bee-farms registered as "ecological producers" in the neighborhood County, both located far away from conventional agricultural production. Analyses were carried out using the gas chromatography joint system with mass spectrometry (GCMS) and high-performance liquid chromatography (HPLC). Results: Identification and quantification of 78 pesticide active ingredients were performed which are part of a total of approximately 400 pesticides that are usually analyzed in fruits and vegetables whereby the laboratory had available analytical equipment that also determined the number of detected pesticides. Due to identified and quantified active ingredients of pesticides, the results of analyses might indicate a possible cause-effect relationship with conventional agricultural activities in the Varaždin County. It was evident from the obtained results on the shares of identified and quantified pesticide residues, and with respect to admissibility, that the share of permitted pesticides in the samples of floral honey accounted for 55.2%, while the share of illegal pesticides accounted for 44.8%. Analyzing the number of detected insecticides and fungicides, on a total of all 4 locations (sub-regions), it was found that floral honey contained 13 different types of insecticides and 3 types of fungicides. Conclusion: The results and statistical data analysis showed that floral honey is a good bio-indicator of environmental pollution by pesticides, therefore this study can serve as a basis for scientists and experts engaged in the research of harmful effects of pesticides on the environment and human health

Određivanje ohratoksina A u bijelom i crnom vinu

Mikotoksini su sekundarni produkti plijesni i vrlo česti kontaminanti hrane. Najčešće su njima kontaminirane žitarice, ali istraživanja ukazuju na to da i druge vrste namirnica mogu biti kontaminirane mikotoksinima. Vino je namirnica koja je najčešće kontaminirana ohratoksinom A (OTA), koji nastaje kao produkt plijesni nastale na grožđu ili kasnije tijekom njegove prerade, a kao i većina drugih mikotoksina, štetno utječe na zdravlje ljudi i životinja, te se njihova prisutnost u hrani redovito provjerava. U ovome radu analizirali smo bijela i crna vina iz podregije koprivničko-đurđevačkog vinogorja na prisutnost ohratoksina A kojeg sintetiziraju plijesni iz skupine Aspergillus ochraceus i Penicillium viridicatum. Kao članica Europske Unije, Hrvatska je prihvatila Uredbu 1881/2006 o utvrđivanju najvećih dopuštenih količina određenih kontaminanata u hrani koja jednoznačno utvrđuje najviše dopuštene koncentracije ohratoksina A u određenim vrstama hrane, uključujući i vino. Ukupno je analizirano 34 uzorka vina, 19 bijelih vina i 15 uzoraka crnih vina. Vrijednosti OTA kretale su se u rasponu od 0,269 do 1,696 g/L za bijelo vino i od 0,254 do 0,565 g/L za crno vino. Nakon izolacije, kvantifikacija ohratoksina A iz vina provedena je imunoenzimskom tehnikom ELISA. Sve dobivene vrijednosti ohratoksina A u analiziranim uzorcima vina niže su od najveće dozvoljene količine koja iznosi 2 g/L, te su ocijenjena sukladnim i prihvatljivim za konzumaciju. Obzirom na dobivene rezultate može se zaključiti da je evidentna prisutnost ohratoksina A u vinima, te je neophodno kontinuirao pratiti njegove vrijednosti u vinima na tržištu, a sve u svrhu zaštite zdravlja potrošača

Determiniranje teških metala u različitim vrstama riba uzorkovanih na hrvatskim tržnicama i mogući utjecaj na zdravlje

The purpose of this study was to determine and to compare the amount of heavy metals in the meat of white (N = 39) and blue (N = 35) sea fish, considering the spring (N = 38) and fall (N = 36) catch period to study the possible type and seasonal differences especially in Hg concentrations in fish, and to estimate if the amounts registered can have a harmful effect on consumer health. The fish were chosen as an indicator of eco-system contamination with mercury and other heavy metals. The amount of heavy metals, lead (Pb) and cadmium (Cd), in both groups was lower than the Highest Permissible Amounts regulated by law in Croatia and the European Union. Mercury (Hg) exceeded the maximum allowed value in just a number of samples 3/74 (0.04%), while it is relevant for arsenic (As) to be present in almost any sample, although it has simultaneously not been regulated by law. The statistically significant value of arsenic exists in blue fish during the spring period, and the amount of mercury is higher in white fish compared to blue fish. Also a statistically significant difference in mercury levels was recorded during fall compared to spring,. Given the dietary habits and frequent fish consumption in Croatia, there is no risk of harmful health effects.Cilj ovoga rada bio je odrediti i usporediti količinu teških metala u 74 uzorka bijele (N = 39) i plave ribe (N = 35), uzorkovanih u proljetnom (N = 38) i jesenskom razdoblju (N = 36), kako bi se istražila moguća razlika u koncentracijama teških metala, posebice žive, ovisno o sezoni ulova ili tipu ribe i procijenilo mogu li determinirane količine imati štetnih utjecaja na zdravlje potrošača. Riba je izabrana, budući da je ona indikator zagađenja teškim metalima u eko sustavu. Količina teških metala, olova (Pb) i kadmija (Cd), u obje grupe uzoraka, bila je niža od maksimalnih dopuštenih koncentracija prema hrvatskih i europskim zakonima. Količina žive (Hg) bila je više od maksimalnih dopuštenih koncentracija u vrlo malom broju uzoraka 3/74 (0,04%). Međutim, u uzrocima su zabilježene relevantne količine arsena (As), iako količine arsena nisu regulirane hrvatskim propisima. Multivarijatna statistička obrada (neparametrijski Mann-Whitney U test) pokazala je statistički značajne razlike u količini arsena u proljetnom razdoblju u odnosu na jesensko razdoblje, kao što je i izmjerena količina žive statistički značajno bila viša u bijeloj u odnosu na plavu ribu, te ona izmjerena u jesenskom razdoblju u odnosu na proljetno. Uzevši, međutim, u obzir potrošačke navike i učestalost konzumacije ribe u Hrvatskoj, izmjerene koncentracije nisu povezane s rizikom štetnih posljedica na zdravlje potrošača

Flower honey as a potential bio-indicator of environmental pollution by pesticides in the Varaždin County

Cilj: U ovom istraživanju uzorci cvjetnoga meda korišteni su kao bio-pokazatelji onečišćenja okoliša pesticidima. Materijal i metode: Ukupno je prikupljeno 40 (4x10) uzoraka sa 4 različite prostorne lokacije (subregije), u ukupno 16 općina, od proizvođača meda u Varaždinskoj županiji, kao i 20 (2x10) uzoraka s dvije različite lokacije proizvođača u susjednoj županiji, registriranih kao "ekoloških proizvođači", a udaljenih od poljoprivrednih površina pod konvencionalnom proizvodnjom. Analize su provedene korištenjem vezanog sustava plinske kromatografije i spektrometrije masa (GCMS) i tekućinskom kromatografijom visoke djelotvornosti (HPLC). Rezultati: Identificirano je i kvantificirano 78 aktivnih sastojaka pesticide, koji su dio od ukupno cca 400 pesticida koji se analiziraju u voću i povrću, a za koje je laboratorij raspolagao analitičkom opremom koja i dijelom određuje i vrstu i broj pesticida koje se detektiraju. Rezultati analiza ukazali su na mogući uzročno posljedični odnos između konvencionalnih poljoprivrednih aktivnosti u Varaždinskoj županiji s identificiranim i kvantificiranim aktivnim sastojcima pesticida. Iz rezultata dobivenih na udjelima identificiranih i kvantificiranih ostataka pesticida, s obzirom na dopustivost, vidljivo je da je udio dopuštenih pesticida u uzorcima cvjetnoga meda iznosio 55,2%, dok je udio ilegalnih pesticida iznosio 44,8%. Analizirajući broj otkrivenih insekticida i fungicida, ukupno na sve 4 lokacije utvrđeno je da cvjetni med sadrži 13 različitih vrsta insekticida i 3 vrste fungicida. Zaključak: Rezultati i statistička analiza podataka pokazuju da je cvjetni med dobar bio-indikator onečišćenja okoliša pesticidima, te stoga ova studija može poslužiti kao osnova za znanstvenike i stručnjake koji se bave istraživanjem štetnih učinaka pesticida na okoliš i ljudsko zdravlje.Aim: In this research samples of floral honey were used as bio-indicators of environmental pollution by pesticides. Material and methods: A total of 40 (4x10) samples from bee farms were collected at 4 different locations (sub-regions) including 16 municipalities in the Varaždin County, as well as 20 (2x10) samples from two locations – bee-farms registered as "ecological producers" in the neighborhood County, both located far away from conventional agricultural production. Analyses were carried out using the gas chromatography joint system with mass spectrometry (GCMS) and high-performance liquid chromatography (HPLC). Results: Identification and quantification of 78 pesticide active ingredients were performed which are part of a total of approximately 400 pesticides that are usually analyzed in fruits and vegetables whereby the laboratory had available analytical equipment that also determined the number of detected pesticides. Due to identified and quantified active ingredients of pesticides, the results of analyses might indicate a possible cause-effect relationship with conventional agricultural activities in the Varaždin County. It was evident from the obtained results on the shares of identified and quantified pesticide residues, and with respect to admissibility, that the share of permitted pesticides in the samples of floral honey accounted for 55.2%, while the share of illegal pesticides accounted for 44.8%. Analyzing the number of detected insecticides and fungicides, on a total of all 4 locations (sub-regions), it was found that floral honey contained 13 different types of insecticides and 3 types of fungicides. Conclusion: The results and statistical data analysis showed that floral honey is a good bio-indicator of environmental pollution by pesticides, therefore this study can serve as a basis for scientists and experts engaged in the research of harmful effects of pesticides on the environment and human health

Early Endosomal Vps34-Derived Phosphatidylinositol-3-Phosphate Is Indispensable for the Biogenesis of the Endosomal Recycling Compartment

Phosphatidylinositol-3-phosphate (PI3P), a major identity tag of early endosomes (EEs), provides a platform for the recruitment of numerous cellular proteins containing an FYVE or PX domain that is required for PI3P-dependent maturation of EEs. Most of the PI3P in EEs is generated by the activity of Vps34, a catalytic component of class III phosphatidylinositol-3-phosphate kinase (PI3Ks) complex. In this study, we analyzed the role of Vps34-derived PI3P in the EE recycling circuit of unperturbed cells using VPS34-IN1 (IN1), a highly specific inhibitor of Vps34. IN1-mediated PI3P depletion resulted in the rapid dissociation of recombinant FYVE- and PX-containing PI3P-binding modules and endogenous PI3P-binding proteins, including EEA1 and EE sorting nexins. IN1 treatment triggered the rapid restructuring of EEs into a PI3P-independent functional configuration, and after IN1 washout, EEs were rapidly restored to a PI3P-dependent functional configuration. Analysis of the PI3P-independent configuration showed that the Vps34-derived PI3P is not essential for the pre-EE-associated functions and the fast recycling loop of the EE recycling circuit but contributes to EE maturation toward the degradation circuit, as previously shown in Vps34 knockout and knockdown studies. However, our study shows that Vps34-derived PI3P is also essential for the establishment of the Rab11a-dependent pathway, including recycling cargo sorting in this pathway and membrane flux from EEs to the pericentriolar endosomal recycling compartment (ERC). Rab11a endosomes of PI3P-depleted cells expanded and vacuolized outside the pericentriolar area without the acquisition of internalized transferrin (Tf). These endosomes had high levels of FIP5 and low levels of FIP3, suggesting that their maturation was arrested before the acquisition of FIP3. Consequently, Tf-loaded-, Rab11a/FIP5-, and Rab8a-positive endosomes disappeared from the pericentriolar area, implying that PI3P-associated functions are essential for ERC biogenesis. ERC loss was rapidly reversed after IN1 washout, which coincided with the restoration of FIP3 recruitment to Rab11a-positive endosomes and their dynein-dependent migration to the cell center. Thus, our study shows that Vps34-derived PI3P is indispensable in the recycling circuit to maintain the slow recycling pathway and biogenesis of the ERC

Endosomal Phosphatidylinositol-3-phosphate is Essential for the Final Assembly of CMV Virions

Cytomegaloviruses (CMVs) initiate the stepwise establishment of their cytoplasmic virion assembly compartment (cVAC) at the earliest stages of infection by expanding the interface between early endosomes (EEs) and endosomal recycling compartment (ERC) and relocating the Golgi complex. This membranous organelle reorganization involves phosphatidylinositol-3-phosphate (PI(3)P)-containing membrane domains, as PI(3)P is the master regulator of the EE pathway. Our goal was to investigate the contribution of PI(3)P-dependent EE domains to cVAC biogenesis and their role in the consequent progression of the CMV life cycle. Our functional analysis of PI(3)P was based on rapid depletion of PI(3)P production at EE membranes using VPS34-IN1, a specific and reversible inhibitor of Vps34 component of class III PI(3)P kinase, and on saturation of PI(3)P by overexpression of recombinant PI(3)P-binding domains (2xFYVE, p40PX). PI(3)P depletion led to rapid reorganization of the Rab11a-dependent pathway of the ERC, and abolished recycling cargo sorting in this pathway as well as membrane flux from EE to the ERC. Despite these alterations and although PI(3)P+ and Vps34+ endosomes build a substantial part of cVAC, the PI(3)P inhibition did not prevent the establishment of infection, rogression through the early phase of infection, and membrane reorganization associated with cVAC development, as analyzed by confocal imaging and Western blot analysis. However, the drastically reduced viral growth under PI(3)P depletion and the significant inhibition of late infection events (DNA replication, late protein expression) together with the reduced release of infectious virions suggest that PI(3)P is essential for entry into the late phase of infection and the final formation of progeny virions