Functional expression of a blood tolerant laccase in Pichia pastoris

Background: Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory. Results: A blood-active laccase (ChU-B mutant) fused to the native/evolved -factor prepro-leader was cloned under the control of two different promoters (PAOX1 and PGAP) and expressed in Pichia pastoris. The most active construct, which contained the PAOX1 and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the -factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers. Conclusions: The tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications.(VLID)90678

The Importance of Centres for Gerontology of County Institutes of Public Health and Gerontology Centres in Croatia

Praćenje utvrđenih i evaluiranih zdravstvenih potreba starijih osoba prioritetni je gerontološki javnozdravstveni program, čija primjena je indikator napretka ili propusta u zaštiti zdravlja cjelokupnog pučanstva. Stoga vođenje Registra utvrđenih i evaluiranih zdravstvenih potreba starijih osoba za svoje područje djelovanja osiguravaju centri za gerontologiju Zavoda za javno zdravstvo. Svrha je izrada primjerenih normi i Programa zdravstvenih postupaka i mjera te primarne, sekundarne i tercijarne prevencije u zaštiti zdravlja starijih osoba. Za individualnu stariju osobu, zdravstvene se potrebe zadovoljavaju s udjelom od 68% u gerontološkim centrima izvaninstitucijske skrbi za starije u lokalnoj zajednici gdje žive jer, svaka peta starija osoba u Hrvatskoj ovisna je o tuđoj pomoći, odnosno socijalno-medicinskoj skrbi. Centri za gerontologiju ZZJZ-a i gerontološki centri po županijama Hrvatske omogućuju reduciranje razlike između utvrđenih i zadovoljenih zdravstvenih potreba starijih osoba. Pritom je nužna trajna edukacija iz gerontologije i gerijatrije u dodiplomskoj i poslijediplomskoj izobrazbi stručnjaka različitog profi la koji se skrbe za zaštitu zdravlja starijih osoba, a poglavito kao subspecijalizacija gerontologija iz javnog zdravstva i gerijatrija iz interne medicine.Monitoring of identifi ed and evaluated health care needs of the elderly is becoming a priority public health measure, as an indicator of progress or failure in health care of the population at large. Therefore, the implementation of the Register of Identifi ed and Evaluated Health Care Needs of the Elderly is ensured by the respective Centre for Gerontology of the Institute of Public Health. The objective is to design appropriate standards, the Programme of Medical Procedures and Measures, and primary, secondary and tertiary prevention in elderly health care. As every fi fth elderly person in Croatia requires social-medical care, sixty-eight percent of health care needs of the elderly are managed at outpatient gerontology centres in local communities. Centres for Gerontology of the Institute of Public Health and regional gerontology centres will reduce a difference between identifi ed and covered health care needs of the elderly. This, however, requires continuing undergraduate and postgraduate education in gerontology and geriatrics for the variety of professionals engaged in elderly health care, especially as a public health subspecialt

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference

The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better "evolvability"of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (Tm) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in Tm compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a Tm 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.Spanish Ministry of Economy and Competitiveness through grant BIO-2013-44963R. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledge

Gerontology – A Public Health Subspecialty

Gerontologija kao uža specijalizacija iz specijalizacije javnoga zdravstva sukladna je Programu specijalističkoga i subspecijalističkog usavršavanja po UEMS-u (European Union of Medical Specialists) i EuMaG-u (European Master in Gerontology). Hrvatsko društvo za javno zdravstvo HLZ-a u svibnju 2005. godine i Hrvatsko društvo za gerontologiju i gerijatriju HLZ-a prihvatili su predloženi Program subspecijalističkog usavršavanja iz gerontologije. Razrađene su potrebne vještine i umijeća iz područja gerontologije koje specijalist javnoga zdravstva mora sadržajno svladati u 12 mjeseci. U izvedbi programa nužna je primjena interdisciplinarnoga gerontološkog pristupa, uz nužno razumijevanje i poznavanje hrvatske i europske gerontološke doktrine, vještine praktične implementacije gerontološke datoteke, poznavanje gerontoloških normi, legislative te mreže i uloge gerontološke institucijske i izvaninstitucijske skrbi u zaštiti zdravlja starijih. Nužno je upoznavanje s praktičnim aspektima vođenja Registra praćenja i proučavanja zdravstvenih potreba i funkcionalne sposobnosti starijih osoba po županijama/regijama Hrvatske i Grada Zagreba, Registra za Alzheimerovu bolest, Registra hrvatskih stogodišnjaka, planiranjem Programa primarne, sekundarne i tercijarne prevencije za starije, ulogom Centara za gerontologiju županijskih zavoda za javno zdravstvo i djelatnosti gerontoloških centara po županijama Hrvatske i Grada Zagreba, Programom zdravstvenih mjera i postupaka u zaštiti zdravlja starijih ljudi, menadžmentom za starije; gerontogogijom i gerontehnološkim metodama, gerontološkim javnozdravstvenim problemima morbiditeta i mortaliteta starije dobi, četiri “N” u gerijatriji (nepokretnost, nestabilnost, nesamostalnost, nekotrolirano mokrenje) i prevencijom gerijatrijskoga “domino” efekta. Tijekom subspecijalističkog usavršavanja stjecat će se i specifi čna znanja o utvrđivanju rizičnih čimbenika nastanka bolesnog starenja te negativnoga zdravstvenog ponašanja i evaluacije provedbe Programa zadovoljenja zdravstvenih potreba starijih osoba sa suradnim i srodnim resursima skrbi za starije. Provjera znanja temeljit će se radom gerontološkoga treninga kroz gerontološke kolokvije, praktičnom provedbom od gerontoloških centara izvaninstitucijske skrbi za starije te u domovima za starije, do drugih interdisciplinarnih gerontoloških radilišta. Po završetku subspecijalističkog usavršavanja obvezatna je izrada diplomskoga rada iz gerontologije.Gerontology is a public health subspecialty in accordance with UEMS and EUMAG. In May 2005, the Croatian Society for Public Health and the Croatian Society for Gerontology and Geriatrics of the Croatian Medical Association accepted the draft Programme of Subspecialty in Gerontology. The requisite skills and knowledge from the fi eld of gerontology, which a public health specialist should acquire over a 12- month period, were elaborated. An interdisciplinary approach to gerontology, along with the understanding and knowledge of Croatian and European gerontologic doctrine, implementation of gerontologic database, knowledge of gerontologic standards and legislation, as well as of the network and role of gerontologic institutions and non-institutionalised care, are indispensable in the implementation of this programme. Public health specialists should get familiar with some practical aspects of keeping of the Register of Monitoring and Studying of Health Needs and Functional Capabilities of the Elderly by county/region of Croatia and the City of Zagreb, Register of Alzheimer’s Disease, Register of Croatian Centenaires, planning of the Programme of Primary, Secondary and Tertiary Prevention for the Elderly, role of Centres for Gerontology of County Institutes of Public Health, Programme of Medical Measures and Procedures in Elderly Health Care, management for the elderly, gerontology and its methods, gerontologic-public health problems related to morbidity and mortality in this age group, four „I’s” in geriatrics (immobility, instability, incontinence and intellectual impairment), and prevention of the geriatric “domino” effect. During this subspecialisation, specifi c knowledge related to the defi nition of risk factors for debilitating aging and negative health behaviour will be acquired and the programme for meeting of health needs of the elderly will be evaluated in cooperation with related institutions. The examinations, i.e. colloquia, will be based on training in gerontology and conducted by gerontology centres, old people’s homes and other interdisciplinary gerontology centres. Upon subspecialisation, a bachelor’s thesis in gerontology is required

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L−1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.This work was supported by the European Union Project grant H2020-BBI-PPP-2015-2-720297-ENZOX2; the Spanish projects PID2019-106166RB-100-OXYWAVE, PID2020-118968RB-100-LILI, PID2021-123332OB-C21 and PID2019-107098RJ-I00, funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (AEI)/doi: 10.13039/501100011033/; the “Comunidad de Madrid” Synergy CAM project Y2018/BIO-4738-EVOCHIMERA-CM; the Generalitat Valenciana projects CIPROM/2021/079-PROMETEO and SEJI/2020/007; and the PIE-CSIC projects PIE-202040E185 and PIE-201580E042. P.G.d.S. thanks the Ministry of Science, Innovation and Universities (Spain) for her FPI scholarship (BES-2017-080040) and the Ministry of Science and Innovation for her contract as part of the PTQ2020-011037 project funded by MCIN/AEI/10.13039/501100011033 within the NextGenerationEU/PRTR. D.G.-P. thanks Juan de la Cierva Incorporación contract Ref. No.: IJC2020-043725-I, funded by MCIN/AEI/10.13039/501100011033, and the EU NextGenerationEU/PRTR program. K.Ś. thanks to Ministerio de Ciencia e Innovación and Fondo Social Europeo for a Ramón y Cajal contract (Ref. RYC2020-030596-I). We thank the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance with the BL13-XALOC beamline

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission

peer-reviewedHere for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 mu A and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.PUBLISHEDpeer-reviewe

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Combined searches for the production of supersymmetric top quark partners in proton-proton collisions at root s=13 TeV

A combination of searches for top squark pair production using proton-proton collision data at a center-of-mass energy of 13 TeV at the CERN LHC, corresponding to an integrated luminosity of 137 fb(-1) collected by the CMS experiment, is presented. Signatures with at least 2 jets and large missing transverse momentum are categorized into events with 0, 1, or 2 leptons. New results for regions of parameter space where the kinematical properties of top squark pair production and top quark pair production are very similar are presented. Depending on themodel, the combined result excludes a top squarkmass up to 1325 GeV for amassless neutralino, and a neutralinomass up to 700 GeV for a top squarkmass of 1150 GeV. Top squarks with masses from 145 to 295 GeV, for neutralino masses from 0 to 100 GeV, with a mass difference between the top squark and the neutralino in a window of 30 GeV around the mass of the top quark, are excluded for the first time with CMS data. The results of theses searches are also interpreted in an alternative signal model of dark matter production via a spin-0 mediator in association with a top quark pair. Upper limits are set on the cross section for mediator particle masses of up to 420 GeV

Probing effective field theory operators in the associated production of top quarks with a Z boson in multilepton final states at root s=13 TeV

Peer reviewe

Measurement of B-c(2S)(+) and B-c*(2S)(+) cross section ratios in proton-proton collisions at root s=13 TeV

Peer reviewe