Systematic Analysis of Residues in Conserved Region 3 of the Human Papillomavirus 16 E7 Oncoprotein

Although remarkable biological diversity is exhibited by viruses, as obligate intracellular parasites, they rely on host cell functions. As such, viruses typically must overcome a set of host barriers that prevent infection. For human papillomaviruses (HPV) one of these barriers is the state of terminal differentiation of the host cell. For that purpose HPVs encode two major oncoproteins, E6 and E7, which combine their efforts to effectively uncouple cellular differentiation from the cell cycle arrest. The E7 proteins have no intrinsic enzymatic activity or DNA binding ability, but they bind and manipulate numerous host proteins. E7 is a modular oncoprotein and contains three conserved regions (CR), of which CR3 is a highly structured zinc-binding domain, and is the primary focus of this thesis. In order to study this highly structured portion of E7, we created a panel of surface-exposed mutants with the primary aim of preserving the structure of CR3, but disrupting potential cellular interactions. Although it was known that E7 CR3 exists as a dimer, the functional significance of dimerization had not been established. Using our panel of novel E7 mutants, we established that E7 does not need to dimerize in order to transform primary baby rat kidney cells. Furthermore, we utilized the same panel of surface-exposed mutants to characterize the contribution of CR3 in deregulation of one of the most vital interacting partners of E7, the retinoblastoma tumor suppressor (pRb). We establish that CR3 binds pRb independently of the well characterized high-affinity binding site in vivo, and show that this interaction is functionally important in overcoming the cell cycle arrest. We also find that E7 has additional mechanisms for targeted pRb degradation, aside from the known pathway. Additionally, we identify a novel binding partner of E7, the p190RhoGAP, and characterize this interaction using the surface-exposed mutants. We find that the interaction of E7 with p190RhoGAP contributes to deregulation of the RhoA GTPase and the actin cytoskeleton, and therefore likely represents an important aspect of HPV induced tumorigenesis. Considered together, these studies have expanded our knowledge of known processes and illuminated novel pathways utilized by HPV E7 in carcinogenesis

Characterization and Genetic Manipulation of D-cysteine Desulfhydrase from Solanum lycopersicum

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. ACC deaminases break down ACC, the immediate precursor of ethylene in plants, into ammonia and α-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. It was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather that it utilizes D-cysteine as a substrate, and in fact encodes a D-cysteine desulfhydrase. Kinetic characterization of the enzyme has shown that it is similar to other previously characterized D-cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering two amino acid residues within the predicted active site changed the enzyme from D-cysteine desulfhydrase to ACC deaminase. Concomitantly, it was shown that by altering two amino acids residues at the same position within the active site of ACC deaminase from Pseudomonas putida UW4 changed this enzyme into D-cysteine desulfhydrase

Reference evapotranspiration estimate with limited weather data across a range of Mediterranean climates

The standard FAO Penman–Monteith (PM-ETo) method for computing the reference evapotranspiration (ETo), in addition to air temperature, needs data on solar radiation or sunshine duration, relative humidity and wind speed which are often lacking and/or do not respect appropriate quality requirements. Hence, in many cases, ETo has to be estimated with limited weather data using maximum and minimum temperature only. Essentially, two procedures are used when no more than temperature data are available: (i) the well-known Hargreaves–Samani equation (HS), or (ii) the PM-ETo method with weather parameters estimated from the limited available data, called PM temperature (PMT) method. The application of these temperature-based approaches often led to contradictory results for various climates and world regions. The data used in the analysis refer to 577 weather stations available through the CLIMWAT database. The results, confirmed by various statistical indicators, emphasized that: (a) in hyper-arid and arid zones, the performance of HS and PMT methods are similar, with root mean square errors (RMSEs) around 0.60–0.65 mm d 1; (b) in semi-arid to humid climates, the PMT method produced better results than HS, with RMSE smaller than 0.52 mm d 1; (c) the performance of PMT method could be improved when adopting the corrections for aridity/humidity in the estimation of the dew point temperature from minimum temperature data. The spatial elaboration of results indicated high variability of ETo estimates by different methods. Thus, a site-specific analysis using daily datasets of sufficient quality is needed for the validation and calibration of temperature methods for ETo estimate. Maps presenting indicative results on under/over estimation of ETo by both temperature methods may be useful for their more accurate application over different Mediterranean climate

Физикален третман на пострауматски контрактури на лакот кај деца – наше искуство

The most common complications of elbow trauma are contractures and neurovascular injuries. The complications can be a result of the initial injury, but they can also be a result of a surgical treatment. In addition to orthopedic treatment of elbow fractures, physical therapy and rehabilitation play a significant role in treatment of posttraumatic contractures. To determine the effects of physical therapy and rehabilitation of posttraumatic elbow contractures in children. This was a retrospective cross-sectional study conducted in the University Clinic for Physical Medicine and Rehabilitation, Skopje in the period 01.01.2021 – 01.07.2022. A total of 52 children were included, at the age between 2 and 13 years who had a posttraumatic elbow contracture, limited range of motion, pain and/or limitations in accomplishing daily activities. Depending on the clinical finding, children underwent a relevant physical therapy (kinesitherapy, functional therapy, electrotherapy, thermotherapy, hydrotherapy and magnetotherapy) in duration of three weeks. For assessing the effects of the rehabilitation therapy, the range of motion of the elbow and forearm was examined along with the Flynn’s scale in all children, prior to and after completion of the physical treatment. Applied physical treatment resulted in a significant improvement in all analyzed movements such as: elbow flexion (p=0.00001), elbow extension (p=0.00001), forearm pronation (p=0.00001), forearm supination (p=0.0000) and Flynn’s scale (p=0.0000). After completion of the rehabilitation treatment, excellent results were registered in 41 (85%) children, moderate in 10 (19.23%) and favorable in 1 (1.92%). Timely and adequate application of physical therapy and rehabilitation can significantly improve the final outcome in treatment of posttraumatic elbow contractures in children. A combination of different physical procedures adequately applied and personalized can significantly improve the range of motion of the elbow.  Најчестите компликации на траумата на лактот се контрактури и невроваскуларни повреди. Компликациите може да бидат резултат на иницијална повреда, но може да бидат и резултат на хируршки третман. Покрај ортопедскиот третман на фрактури на лактот, физикална терапија и рехабилитација играат значајна улога во третманот на посттрауматски контрактури. Целта на оваа студија беше да се утврдат ефектите од физикална терапија и рехабилитација на посттрауматски контрактури на лактот кај децата. Материјали и методи: Ова беше ретроспективна студија на пресек спроведена на Универзитетската клиника за физикална медицина и рехабилитација, Скопје во периодот 01.01.2021 – 01.07.2022 година. Вклучени се вкупно 52 деца, на возраст меѓу 2 и 13 години кои имале посттрауматска контрактура на лактот, ограничен опсег на движења, болка и/или ограничувања во извршувањето на секојдневните активности. Во зависност од клиничкиот наод, на децата им беше извршена соодветна физикална терапија (кинезитерапија, функционална терапија, електротерапија, термотерапија, хидротерапија и магнетотерапија) во времетраење од три недели. За проценка на ефектите од терапијата за рехабилитација, опсегот на движење на лактот и подлактицата беше испитувана и Флиновата скала кај сите деца, пред и по завршувањето на физичкиот третман. Резултати: Применетиот физикален третман резултираше со значително подобрување на сите анализирани движења како што се: флексија на лактот (p=0,00001), екстензија на лактот (p=0,00001), пронација на подлактицата (p=0,00001), супинацција на подлактицата (p=0,0000's) и Флинова скала (p=0,0000). По завршувањето на рехабилитациониот третман, одлични резултати се забележани кај 41 (85%) дете, умерени кај 10 (19,23%) и поволни кај 1 (1,92%). Заклучок: Навремената и адекватна примена на физикална терапија и рехабилитација може значително да го подобри крајниот исход во третманот на посттрауматски контрактури на лактот кај децата. Комбинацијата на различни физички процедури соодветно применети и персонализирани може значително да го подобри опсегот на движење на лактот

Mogućnost zaštite pasulja od Xanthomonas campestris pv. phaseoli primenom bakarnih preparata i aktivatora otpornosti

The efficacy of several new formulations of copper compounds, namely Cuprozin 35 WP (copper-oxychloride), Cuproxat (copper-sulphate), Funguran OH (copper-hydroxide) and the plant activator Bion (acibenzolar-S-methyl), and their combinations with dithiocarbamates (Dithane M-70) was estimated in controlling Xanthomonas campestris pv. phaseoli (artificial inoculation) in field conditions in two localities during 2006. In the locality Zemun, the efficacy of copper compounds ranged from 92.7% to 98.5%. The plant activator Bion 50 WG exhibited similar efficacy (94.4-97.1%). Combinations of Funguran OH and Dithane M-70, applied at different concentrations, also showed high efficacy (98.3-99.3%), as well as the combinations of Bion 50 WG and the other bactericides (95.5-96.8%). There was no significant difference between the efficacies achieved by the compounds applied individually and their combinations, except Cuproxat, which exhibited decreased efficacy at lower concentration. In the locality Smederevska Palanka, the efficacy of copper compounds was 95.0-98.2%, while Bion achieved 96.8-97.7% efficacy. Combinations of copper-hydroxide (Funguran OH) and dithiocarbamates (Dithane M-70) also showed high efficacy (98.1- 99.4%) but without a significant difference. The efficacy of combinations of Bion and copper- hydroxide, and Bion and mancozeb was 97.9-98.9%. There was no significant difference in the efficacies of the bactericides tested or the efficacies of their combinations in that locality. Our investigation confirmed high efficacy of acibenzolar-S-methyl, which was equal to the efficacy of standard bactericide treatment. This compound therefore offers a very good alternative to conventional chemicals used for controlling bacterial diseases in beans.U radu je ispitivana efikasnost novijih formulacija bakarnih jedinjenja; Cuprozin 35 WP (bakar-oksihlorid), Cuproxat (bakar-sulfat), Funguran OH (bakar-hidroksid) i aktivatora otpornosti Bion-a (acibenzolar-S-metil), i njihovih kombinacija sa ditiokarbamatima (Dithane M-70), u suzbijanju Xanthomonas campestris pv. phaseoli (veštačka inokulacija). Ispitivanja su obavljena tokom 2006. godine u poljskim uslovima na dva lokaliteta. Na lokalitetu Zemun, efikasnost bakarnih preparata kretala se 92.7-98.5%. Sličnu efikasnost ispoljio je i activator otpornosti Bion 50 WG (94.4-97.1%). Zadovoljavajuću efikasnost (98.3-99.3%) imale su I kombinacije preparata Funguran OH i Dithane M-70 pri različitim koncentracijama primene, kao i kombinacije Bion-a sa drugim preparatima (95.5-96.8%). Statistički značajne razlike u efikasnosti primenjenih preparata i njihovih mešavina nisu zabeležene, izuzev kod preparata Cuproxat, koji je ispoljio slabiju efikasnost pri nižoj koncentraciji primene. Na lokalitetu Smederevska Palanka efikasnost bakarnih jedinjenja iznosila je 95.0-98.2%, a za Bion 96.8-97.7%. Kombinacije bakar-hidroksida (Funguran OH) i ditiokarbamata (Dithane M-70) ispoljile su nešto veću efikasnost (98.1-99.4%), ali razlike nisu bile statistički značajne. Efikasnost Bion-a u mešavini sa bakar-hidroksidom i mankozebom iznosila je 97.9-98.9%. Na ovom lokalitetu nisu konstatovane statistički značajne razlike između samostalno primenjenih preparata i njihovih kombinacija. U ovim ispitivanjima potvrđena je i visoka efikasnost acibenzolar-S-metil-a (preparat Bion), koja je bila na nivou efikasnosti standardnih baktericida. Zbog toga ovo jedinjenje predstavlja dobru alternativu konvencionalnom hemijskom suzbijanju bakterioza pasulja

Efikasnost bakarnih preparata u suzbijanju Xanthomonas campestris pv. vesicatoria, prouzrokovača bakteriozne pegavosti lišća paprika

The efficacy of several new formulations of copper compounds in controlling Xanthomonas campestris pv. vesicatoria in pepper was estimated in field conditions using artificial inoculation. The efficacies of Cuprozin 35 WP (copper-oxychloride) and Blauvit (copper-hydroxide) were tested in the localities Dobanovci and Kupinovo in 2005. Another two formulations, Cuproxat (copper-sulfate) and Fungohem SC (copper-hydroxide), were tested along with the previous two in the localities Zemun and Smederevska Palanka in 2006. Both bactericides tested in 2005 exhibited high efficacy in controlling X. c. pv. vesicatoria in both localities. However, there were no significant differences in the efficacy of Cuprozin 35 WP at higher concentration (74.3%-78.7%) and Blauvit (74.6%-78.9%) in the two trials. Cuprozin 35 WP decreased efficacy, but a satisfactory effectiveness was still achieved at lower concentration (66.5%-66.5%). In the experiments conducted in 2006, higher concentration of Fungohem SC showed the highest efficacy (86.1%-89.1%) in controlling bacterial spot of pepper. Blauvit, Cuprozin 35 WP and Cuproxat also exhibited high efficacy. However, there were no significant differences in the efficacy of these bactericides between the two localities. Fungohem SC applied at lower concentration was less effective (77.2%-80.0%) but its efficacy was still good enough. Our experiments showed that copper bactericides based on copper-sulfate, copper-oxychloride and copper-hydroxide exhibited satisfactory efficacy in controlling the causal agent of bacterial spot of pepper in our climatic conditions.Ispitivana je efikasnost novijih formulacija bakarnih preparata u suzbijanju Xanthomonas campestris pv. vesicatoria na paprici u uslovima veštačke inokulacije u polju. U 2005-oj godini, na lokalitetima Dobanovci i Kupinovo, ispitivana je efikasnost preparata Cuprozin 35 WP (bakar- oksihlorid) i Blauvit (bakar- hidroksid). Tokom 2006. godine osim ovih preparata primenjeni su i Cuproxat (bakar-sulfat trobazni) i Fungohem SC (bakar-hidroksid), na lokalitetima Zemun i Smederevska Palanka. Preparati ispitivani u 2005-oj godini ispoljili su zadovoljavajuć u efikasnost u suzbijanju X. c. pv. vesicatoria na oba lokaliteta. Između efikasnosti preparata Cuprozin 35 WP pri višoj koncentraciji primene (74,3%-78,7%) i preparata Blauvit (74,6%-78,9%) nije zabeležena statistički značajna razlika u oba ogleda. Nešto nižu, ali zadovoljavajuć u efikasnost ispoljio je Cuprozin 35 WP pri nižoj koncentraciji primene (66,5%-66,5%). U ogledima izvedenim tokom 2006. godine, najveću efikasnost u suzbijanju prouzrokovača bakteriozne pegavosti paprike ispoljio je preparat Fungohem SC (86,1%-89,1%) pri višoj koncentraciji primene. Preparati Blauvit, Cuprozin 35 WP i Cuproxat pokazali su takođe visoku efikasnost tako da statistički značajne razlike nisu zabeležene između tretmana ovim preparatima na oba lokaliteta. Nešto nižu, mada zadovoljavajuću efikasnost imao je preparat Fungohem SC primenjen u nižoj koncentraciji (77,2%-80,0%). Naši ogledi pokazali su da preparati na bazi bakarsulfata trobaznog, bakar- oksihlorida i bakar- hidroksida u našim klimatskim uslovima ispoljavaju zadovoljavajuć­u efikasnost u suzbijanju prouzrokovača bakteriozne pegavosti paprike

Sphaerospora molnari (myxozoa) kod šaranske mlađi

Sferosporidioza škrga je obolenje riba izazvano parazitom Sphaerospora molnari koji napada škrge i kožu. Prvo pojavljivanje sferosporidioze škrga kod šaranskih mladunaca utvrđeno je u Mađarskoj još 1972, zatim u Češkoj i Poljskoj, dok je kod nas obolenje prisutno od sredine osamdesetih godina prošlog veka. Molnar, koji je prvi је izučavao patogeni efekat ovog uzročnika, najpre ga je identifikovao kao Sphaerospora carassi. Češki istraživači Lom et Dycova detektovali su uzročnika sferosporidioze škrga iz škržnog materijala obolelih mladunaca šarana pomoću histološke sekcije tkiva i predložili da se parazitu da ime Sphaerospora molnari. Ovo obolenje je dosta često kod mladunaca ribnjačkog šarana i amura, pri čemu intenzitet infestacije može dostići čak i do 100%. Cilj ovog rada jeste da se utvrdi prisustvo obolenja izazvanog parazitom Sphaerospora molnari i da se isprate kliničke i patohistološke promene kod infestiranih mladunaca šarana. Istraživanja su sprovedena na 18 šaranskih ribnjaka u Srbiji, od 2008. do 2012. godine, a u sklopu sistemskog monitoringa najznačajnijih protozooza šarana. Mladunci šarana pregledani su tokom čitavog vegetacionog perioda. Praćene su kliničke promene, i uzimani su uzorci za nativnu mikroskopiju koja je rađena pomoću svetlosnog mikroskopa. Od inficiranih jedinki uzimano je tkivo škrga za patohistološku analizu koja je sprovedena klasičnom metodologijom, fiksiranjem u 10% formalinu, sečenjem 5 μm velikih isečaka koji su kalupljeni u parafin i bojenjem isečaka pomoću H&E. Prisustvo S. molnari ustanovljeno je kod mladunaca šarana od 20 dana do 3 meseca starosti. Na škrgama su bili prisutni razvojni stadijumi i zrele spore što se moglo uočiti na stratifikovanom epitelu škržnih filamenata. Spore su invadirale epitel i formirale velike klastere. Akumulacija razvojnih stadijuma i zrelih spora bila je prisutna je i kod dvostrukog sloja epitelnih ćelija koje pokrivaju sekundarne lamele, i to najčešće između unutrašnjeg i spoljašnjeg omotača izazivajući tako distenziju tkiva. Zaražene lamele podležu nekrozi, što dovodi do kretanja spora prema spolja. Veličina spora iznosila je 10 x 10 μm. Klinički, obolenje se manifestovalo pojavom beličastih depozita na škrgama kao posledica agregacije parazita na njima, pri čemu paraziti mogu da zauzmu i do 80% površine slojevitog epitela, prekrivajući pločice i lukove škrga. Pritisak parazita koji se razmnožavaju je takav da vrši ćelijsku deformaciju tkiva i na kraju se uočava istančanost citoplazme ćelija šktžnog epitela u obliku mreže. Pošto spore prekrivaju najveći deo respiratornog epitela, smanjuju otpornost organizma i stvaraju uslove za razvoj drugih uzročnika obolenja (prvenstveno trematoda), što Sphaerosporu molnari svrstava u patogene parazite. Lokalizacija, veličina spora odnosno razvojnih stadijuma S. molnari, kao i kliničke i patohistološke promene zabeležene tokom ovog istraživanja odgovaraju rezultatima koje su opisali ostali istraživači koji su se bavili ovom problematikom. Pošto ne postoji ni jedno adekvatno terapeutsko sredstvo, kontrola sferopsoridioze i dalje se bazira na pridržavanju osnovnih sanitarno–profilaktičkih mera, kao što su isušivanje objekata, izmrzavanje, mehanička obrada tla i dezinfekcija krečom

Erwinia amylovora – prouzrokovač nekroze korenovog vrata stabla jabuke

A large-scale outbreak of fire blight symptoms caused by Erwinia amylovora was recorded in pome fruit trees during 2007. In addition to fruit necrosis and shoot blight as the typical disease symptoms, dark purple necrosis was observed in the root collar area girdling the trunk just above the ground and thus withering the whole apple tree. Since similar symptoms on apple trees could be caused by E. amylovora or one of several phytopathogenic fungi of the genera Phomopsis and Phytophthora, an investigation was conducted to identify the causal agent of this disease. Levan-producing, nonfluorescent bacteria were isolated from diseased samples. The isolated strains produced HR in tobacco leaves and necrosis of artificially inoculated, immature pear fruits, followed by oozing of bacterial exudate, a characterisitic of E. amylovora. Based on the results of pathogenicity tests, biochemical characteristics, ELISA test and PCR analysis, it was confirmed that the investigated strains belonged to E. amylovora, causing the root collar necrosis of apple trees as an atypical symptom of this bacterium in Serbia. The explanation of this symptom may be that the vegetative rootstocks were infected with E. amylovora. Therefore, the development of diagnostic protocols for detection of E. amylovora in apple rootstock is very important for health inspections of planting materials.Tokom 2007. godine zabeležena je masovna pojava bakteriozne plamenjače jabučastih voćaka prouzrokovana bakterijom Erwinia amylovora. Pored nekroze plodova i plamenjače mladara, tipičnih simptoma bolesti, našu pažnju je privukla pojava nekroze mrkoljubičaste boje u zoni korenovog vrata, koja prstenasto zahvata prizemni deo i prouzrokuje izumiranje stabala jabuke. S obzirom na to da uzrok ovakvih patoloških promena može da bude E. amylovora, ali i fitopatogene gljive iz rodova Phomopsis i Phytophthora, preduzeta su istraživanja sa ciljem da se utvrdi etiologija ovog oboljenja jabuke. Iz obolelih uzoraka izolovane su bakterije koje stvaraju levan, ali ne i fluorescentni pigment. Proučavani sojevi prouzrokovali su HR na listovima duvana i nekrozu inokulisanih, nesazrelih plodova kruške, uz obilno stvaranje bakterijskog eksudata, što je karakterističan znak E. amylovora. Na osnovu rezultata dobijenih pri testovima provere patogenosti, ali i rezultata biohemijskih odlika, ELISA testa i molekularnih metoda (PCR), potvrđeno je da proučavani izolati pripadaju bakteriji E. amylovora, koja je uzročnik simptoma nekroze korenovog vrata stabala jabuke, prvi put zabeleženih u Srbiji. Moguće objašnjenje ove pojave je da su vegetativne podloge za kalemljenje bile inficirane ovom bakterijom. Zbog toga je veoma bitno razvijati dijagnostičke protokole za utvrđivanje prisustva E. amylovora u podlogama za kalemljenje jabuke, tokom procesa kontrole zdravstvene ispravnosti sadnog materijala

Conserved region 3 of human papillomavirus 16 E7 contributes to deregulation of the retinoblastoma tumor suppressor

The human papillomavirus (HPV) E7 oncoprotein binds cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. A key target of E7 is the product of the retinoblastoma susceptibility locus (pRb). This interaction results in the release of E2F transcription factors and drives the host cell into the S phase of the cell cycle. E7 binds pRb via a high-affinity binding site in conserved region 2 (CR2) and also targets a portion of cellular pRb for degradation via the proteasome. Evidence suggests that a secondary binding site exists in CR3, and that this interaction influences pRb deregulation. Additionally, evidence suggests that CR3 also participates in the degradation of pRb. We have systematically analyzed the molecular mechanisms by which CR3 contributes to deregulation of the pRb pathway by utilizing a comprehensive series of mutations in residues predicted to be exposed on the surface of HPV16 E7 CR3. Despite differences in the ability to interact with cullin 2, all CR3 mutants degrade pRb comparably to wild-type E7. We identified two specific patches of residues on the surface of CR3 that contribute to pRb binding independently of the high-affinity CR2 binding site. Mutants within CR3 that affect pRb binding are less effective than the wild-type E7 in overcoming pRb-induced cell cycle arrest. This demonstrates that the interaction between HPV16 E7 CR3 and pRb is functionally important for alteration of the cell cycle. © 2012, American Society for Microbiology