Urinary microbiota dynamics during antibiotic therapy determined by 16S rRNA gene sequencing

Urinarne infekcije predstavljaju jednu od najčešćih infekcija uzrokovanu bakterijama. Većina ih je uzrokovana bakterijom Escherichia coli i češće su prisutne kod žena prvenstveno zbog anatomskih razlika. Napredak tehnologije sekvencioniranja omogućio nam je direktno sekvencioniranje okolišnih uzoraka čime se omogućilo sekvencioniranje bakterija koje se ne mogu kultivirati u laboratoriju. Svrha ovog rada bila je praćenje promjene urinarnog mikrobioma uslijed djelovanja antibiotika Cefalina kod pacijentice s urinarnom infekcijom. Praćenje se provodilo sekvencioniranjem 16S rRNA gena, točnije V3 i V4 varijabilnih regija pomoću posebno dizajniranih početnica. Pokazano je da uzorak prije terapije ima najmanju bioraznolikost pri čemu uzročnik infekcije, nepoznata bakterija iz porodice Enterobacteriaeceae, prevladava sa zastupljenošću većom od 95%. Korištenjem antibiotika dolazi do naglog zamjenjivanja uropatogenih bakterija sa bakterijskim rodovima uobičajenim u urinarnom mikrobiomu, najviše Lactobacillus vrstama. Ta bakterijska vrsta ima zaštitnu ulogu, te pomaže u održavanju normalne mikrobiote.Urinary tract infections (UTI) are among the most common bacterial infections. Most UTIs are caused by uropathogenic Escherichia coli and they are more often present in women primarily due to anatomical reasons. Progress in sequencing technology allowed us to sequence environmental samples which enable sequencing of the non-culturable bacteria. The purpose of this study was to monitor changes in urinary microbiome in patient with UTI who receives antibiotic therapy. Monitoring was carried out by sequencing 16S rRNA gene, specifically V3 and V4 variable region using specially designed primes. It is shown that the pre-treatment sample has the lowest biodiversity. In that sample prevails an unknown Enterobacteiaceae bacteria, which is the cause of infection, with over 95%. The use of antibiotics leads to the rapid replace of urophatogenic bacteria with bacterial genera common in urinary microbiome – Lactobacillus. They have a protective role and help maintain normal microbiota

Comparison of Zn Content in Rapid-acting Insulin and Biphasic Suspension by FAAS

Various insulin analogs (rapid-acting and intermediate-acting insulin) have been investigated for the determination of zinc content by flame atomic absorption spectrometry. This paper presents the validation of a method and comparison of zinc content in the insulin samples studied. The method was linear (r)(^2) = 0.9997), the limit of detection was 0.0098 mg L(^{–1}) and the limit of quantification was 0.0296 mg L(^{–1}), the precision (as relative standard deviation) was up to 7.4 %, and the accuracy was within a range of 95.6 % to 100.1 % for the recovery of fortified insulin samples. The zinc content in the insulin samples ranged from 14.9 mg L(^{–1}) to 16.3 mg L(^{–1}) for rapid-acting insulin and 18.7 mg L(^{–1}) to 19.9 mg L(^{–1}) for intermediate-acting insulin. A higher zinc content was found in the intermediate-acting insulins than in the rapid-acting insulin analogs ((p) < 0.05). The obtained results should be considered in the establishment of new or improvement of currently available procedures used to assure the quality, safety and efficacy of insulin products. This work is licensed under a Creative Commons Attribution 4.0 International License

Ružička days : International conference 16th Ružička Days “Today Science – Tomorrow Industry” : Proceedings

Proceedings contains articles presented at Conference divided into sections: open lecture (1), chemical analysis and synthesis (3), chemical and biochemical engineering (8), food technology and biotechnology (8), medical chemistry and pharmacy (3), environmental protection (11) and meeting of young chemists (2)

Razvoj i terapijski potencijal bispecifičnih protutijela

Cilj istraživanja Cilj ovog specijalističkog rada je opisati svojstva bispecifičnih protutijela odobrenih za kliničku uporabu i onih u kliničkim istraživanjima, analizirati metode njihove proizvodnje te dati uvid u terapijske mogućnosti ovih protutijela. Materijali i metode Rad daje pregled razvoja bispecifičnih protutijela te njihove terapijske primjene. Pregledom znanstvene literature detaljnije su opisane metode za ispravno povezivanje lakih i teških lanaca. Opisana su do sad odobrena bispecifična protutijela te njihova terapijska primjena. Navedena su i protutijela koja se nalaze u različitim fazama razvoja. Rezultati Bispecifična protutijela kombiniraju dva ili više elemenata, koji prepoznaju antigene, u jednu cjelinu sa sposobnošću vezanja na dva ili više ciljeva. Djelovanjem na više meta istovremeno povećava se učinkovitost terapije te smanjuje potreba za učestalom primjenom lijeka. Kako bi se povezali različiti teški i laki lanci u jednu molekulu, razvijene su nove metode. Neke od tih metoda su kvadroma tehnologija, metoda "dugme u rupu", zajednički laki lanac, CrossMAb tehnologija, SEED, WuxyBody i dr. Zahvaljujući ovim metodama danas je za kliničku primjenu odobreno sedam bispecifičnih protutijela, a osim njih u pretkliničkim ispitivanjima nalazi se više od 180 bispecifičnih protutijela, dok ih je u različitim fazama kliničkih ispitivanja više od 50. Zaključak Razvojem biotehnologije i genetičkog inženjerstva otkrivene su nove metode ispravnog povezivanja lakih i teških lanaca koje su omogućile razvoj funkcionalnih bispecifičnih protutijela koja se koriste u kliničkoj praksi. Bispecifična protutijela pokazala su znatan napredak u učinkovitosti u odnosu na dosadašnju terapiju. Napredak je vidljiv u ishodima liječenja, vremenu potrebnom za postizanje terapijskog odgovora te u vremenu do pogoršanja bolesti. Osim boljih rezultata liječenja, bispecifična protutijela su se pokazala i kao isplativija nego dosadašnja terapija.Objective The aim of this expert thesis is to describe the properties of bispecific antibodies approved for clinical use and those in clinical trials, to analyse the methods of their production and to provide insight into the therapeutic possibilities of such antibodies. Materials and methods The thesis provides an overview of the development of bispecific antibodies and their therapeutic applications. The review of the scientific literature describes in more detail the methods for the correct linking of light and heavy chains. The thesis describes the approved bispecific antibodies and their therapeutic use, as well as those that are in different stages of development. Results Bispecific antibodies combine two or more antigen-recognizing elements into a single molecule with the ability to link to two or more targets. By acting on multiple targets simultaneously, the effectiveness of the therapy increases and the need for frequent drug administration is reduced. In order to link different heavy and light chains into one molecule, new methods have been developed. Some of these methods are quadroma technology, buttonhole technique, common light chain, CrossMAb technology, SEED, WuxyBody, etc. Thanks to these methods, today there are seven bispecific antibodies approved for clinical use; in addition to these, there are more than 180 bispecific antibodies approved in preclinical trials, as well as more than 50 in different stages of clinical trials. Conclusion The development of biotechnology and genetic engineering has facilitated the discovery of new methods for correct linking of light and heavy chains that have enabled the development of functional bispecific antibodies used in clinical practice. Bispecific antibodies have shown significant improvement in efficacy compared to previous therapy. Progress is evident in treatment outcomes, in the time it takes to achieve a therapeutic response, and in the time to disease worsening. In addition to better treatment results, bispecific antibodies have also been shown to be more cost-effective than the previous therapy

Razvoj i terapijski potencijal bispecifičnih protutijela

Cilj istraživanja Cilj ovog specijalističkog rada je opisati svojstva bispecifičnih protutijela odobrenih za kliničku uporabu i onih u kliničkim istraživanjima, analizirati metode njihove proizvodnje te dati uvid u terapijske mogućnosti ovih protutijela. Materijali i metode Rad daje pregled razvoja bispecifičnih protutijela te njihove terapijske primjene. Pregledom znanstvene literature detaljnije su opisane metode za ispravno povezivanje lakih i teških lanaca. Opisana su do sad odobrena bispecifična protutijela te njihova terapijska primjena. Navedena su i protutijela koja se nalaze u različitim fazama razvoja. Rezultati Bispecifična protutijela kombiniraju dva ili više elemenata, koji prepoznaju antigene, u jednu cjelinu sa sposobnošću vezanja na dva ili više ciljeva. Djelovanjem na više meta istovremeno povećava se učinkovitost terapije te smanjuje potreba za učestalom primjenom lijeka. Kako bi se povezali različiti teški i laki lanci u jednu molekulu, razvijene su nove metode. Neke od tih metoda su kvadroma tehnologija, metoda "dugme u rupu", zajednički laki lanac, CrossMAb tehnologija, SEED, WuxyBody i dr. Zahvaljujući ovim metodama danas je za kliničku primjenu odobreno sedam bispecifičnih protutijela, a osim njih u pretkliničkim ispitivanjima nalazi se više od 180 bispecifičnih protutijela, dok ih je u različitim fazama kliničkih ispitivanja više od 50. Zaključak Razvojem biotehnologije i genetičkog inženjerstva otkrivene su nove metode ispravnog povezivanja lakih i teških lanaca koje su omogućile razvoj funkcionalnih bispecifičnih protutijela koja se koriste u kliničkoj praksi. Bispecifična protutijela pokazala su znatan napredak u učinkovitosti u odnosu na dosadašnju terapiju. Napredak je vidljiv u ishodima liječenja, vremenu potrebnom za postizanje terapijskog odgovora te u vremenu do pogoršanja bolesti. Osim boljih rezultata liječenja, bispecifična protutijela su se pokazala i kao isplativija nego dosadašnja terapija.Objective The aim of this expert thesis is to describe the properties of bispecific antibodies approved for clinical use and those in clinical trials, to analyse the methods of their production and to provide insight into the therapeutic possibilities of such antibodies. Materials and methods The thesis provides an overview of the development of bispecific antibodies and their therapeutic applications. The review of the scientific literature describes in more detail the methods for the correct linking of light and heavy chains. The thesis describes the approved bispecific antibodies and their therapeutic use, as well as those that are in different stages of development. Results Bispecific antibodies combine two or more antigen-recognizing elements into a single molecule with the ability to link to two or more targets. By acting on multiple targets simultaneously, the effectiveness of the therapy increases and the need for frequent drug administration is reduced. In order to link different heavy and light chains into one molecule, new methods have been developed. Some of these methods are quadroma technology, buttonhole technique, common light chain, CrossMAb technology, SEED, WuxyBody, etc. Thanks to these methods, today there are seven bispecific antibodies approved for clinical use; in addition to these, there are more than 180 bispecific antibodies approved in preclinical trials, as well as more than 50 in different stages of clinical trials. Conclusion The development of biotechnology and genetic engineering has facilitated the discovery of new methods for correct linking of light and heavy chains that have enabled the development of functional bispecific antibodies used in clinical practice. Bispecific antibodies have shown significant improvement in efficacy compared to previous therapy. Progress is evident in treatment outcomes, in the time it takes to achieve a therapeutic response, and in the time to disease worsening. In addition to better treatment results, bispecific antibodies have also been shown to be more cost-effective than the previous therapy

Razvoj i terapijski potencijal bispecifičnih protutijela

Cilj istraživanja Cilj ovog specijalističkog rada je opisati svojstva bispecifičnih protutijela odobrenih za kliničku uporabu i onih u kliničkim istraživanjima, analizirati metode njihove proizvodnje te dati uvid u terapijske mogućnosti ovih protutijela. Materijali i metode Rad daje pregled razvoja bispecifičnih protutijela te njihove terapijske primjene. Pregledom znanstvene literature detaljnije su opisane metode za ispravno povezivanje lakih i teških lanaca. Opisana su do sad odobrena bispecifična protutijela te njihova terapijska primjena. Navedena su i protutijela koja se nalaze u različitim fazama razvoja. Rezultati Bispecifična protutijela kombiniraju dva ili više elemenata, koji prepoznaju antigene, u jednu cjelinu sa sposobnošću vezanja na dva ili više ciljeva. Djelovanjem na više meta istovremeno povećava se učinkovitost terapije te smanjuje potreba za učestalom primjenom lijeka. Kako bi se povezali različiti teški i laki lanci u jednu molekulu, razvijene su nove metode. Neke od tih metoda su kvadroma tehnologija, metoda "dugme u rupu", zajednički laki lanac, CrossMAb tehnologija, SEED, WuxyBody i dr. Zahvaljujući ovim metodama danas je za kliničku primjenu odobreno sedam bispecifičnih protutijela, a osim njih u pretkliničkim ispitivanjima nalazi se više od 180 bispecifičnih protutijela, dok ih je u različitim fazama kliničkih ispitivanja više od 50. Zaključak Razvojem biotehnologije i genetičkog inženjerstva otkrivene su nove metode ispravnog povezivanja lakih i teških lanaca koje su omogućile razvoj funkcionalnih bispecifičnih protutijela koja se koriste u kliničkoj praksi. Bispecifična protutijela pokazala su znatan napredak u učinkovitosti u odnosu na dosadašnju terapiju. Napredak je vidljiv u ishodima liječenja, vremenu potrebnom za postizanje terapijskog odgovora te u vremenu do pogoršanja bolesti. Osim boljih rezultata liječenja, bispecifična protutijela su se pokazala i kao isplativija nego dosadašnja terapija.Objective The aim of this expert thesis is to describe the properties of bispecific antibodies approved for clinical use and those in clinical trials, to analyse the methods of their production and to provide insight into the therapeutic possibilities of such antibodies. Materials and methods The thesis provides an overview of the development of bispecific antibodies and their therapeutic applications. The review of the scientific literature describes in more detail the methods for the correct linking of light and heavy chains. The thesis describes the approved bispecific antibodies and their therapeutic use, as well as those that are in different stages of development. Results Bispecific antibodies combine two or more antigen-recognizing elements into a single molecule with the ability to link to two or more targets. By acting on multiple targets simultaneously, the effectiveness of the therapy increases and the need for frequent drug administration is reduced. In order to link different heavy and light chains into one molecule, new methods have been developed. Some of these methods are quadroma technology, buttonhole technique, common light chain, CrossMAb technology, SEED, WuxyBody, etc. Thanks to these methods, today there are seven bispecific antibodies approved for clinical use; in addition to these, there are more than 180 bispecific antibodies approved in preclinical trials, as well as more than 50 in different stages of clinical trials. Conclusion The development of biotechnology and genetic engineering has facilitated the discovery of new methods for correct linking of light and heavy chains that have enabled the development of functional bispecific antibodies used in clinical practice. Bispecific antibodies have shown significant improvement in efficacy compared to previous therapy. Progress is evident in treatment outcomes, in the time it takes to achieve a therapeutic response, and in the time to disease worsening. In addition to better treatment results, bispecific antibodies have also been shown to be more cost-effective than the previous therapy

Peptidomimetics

Podložnost proteolitičkim enzimima, polarnost koja otežava transport kroz lipidni dvosloj te prevelika konformacijska sloboda ograničavaju terapeutsku primjenu peptida. Primjenom njihovih mimetika moguće je prevladati spomenute nedostatke, tako da dizajn i sinteza peptidomimetika predstavljaju moćno oruđe za pripravu lijekova koji djeluju kao inhibitori enzima ili ligandi za različite receptore. Dizajn peptidomimetika bazira se na: (i) zamjeni dijela peptidne okosnice nepeptidnim fragmentom, (ii) modifikaciji bočnih ogranaka i(li) (iii) ograničavanju konformacijske slobode, s ciljem oponašanja onog fragmenta peptidne sekundarne strukture koji je odgovoran za molekulsko prepoznavanje. Na taj se način oponaša biološka aktivnost prirodnih peptida, a moguće je ostvariti i poboljšanu selektivnost, efikasnost, biodostupnost i produljenu biološku aktivnost.The low metabolic stability towards proteolytic enzymes, polar character that disables their transport through lipide bilayer as well as the conformational freedom limit their therapeutic application. Since those adversities might be prevailed by using of their mimetics, peptidomimetic design and synthesis present a powerfull tools for the preparation of drugs acting as enzyme inhibitors or receptor ligands. The design of peptidomimetics is based on: (i) the replacement of the peptide backbone with non-peptide fragment, (ii) the modification of the side-chains and/or (iii) the locking the conformational freedom in order to mimic secondary structure element responsible for molecular recognition. This approach not only enables the mimicking of the biological activity, but also improve selectivity, efficiency, bioavailability and activity

Urinary microbiota dynamics during antibiotic therapy determined by 16S rRNA gene sequencing

Urinarne infekcije predstavljaju jednu od najčešćih infekcija uzrokovanu bakterijama. Većina ih je uzrokovana bakterijom Escherichia coli i češće su prisutne kod žena prvenstveno zbog anatomskih razlika. Napredak tehnologije sekvencioniranja omogućio nam je direktno sekvencioniranje okolišnih uzoraka čime se omogućilo sekvencioniranje bakterija koje se ne mogu kultivirati u laboratoriju. Svrha ovog rada bila je praćenje promjene urinarnog mikrobioma uslijed djelovanja antibiotika Cefalina kod pacijentice s urinarnom infekcijom. Praćenje se provodilo sekvencioniranjem 16S rRNA gena, točnije V3 i V4 varijabilnih regija pomoću posebno dizajniranih početnica. Pokazano je da uzorak prije terapije ima najmanju bioraznolikost pri čemu uzročnik infekcije, nepoznata bakterija iz porodice Enterobacteriaeceae, prevladava sa zastupljenošću većom od 95%. Korištenjem antibiotika dolazi do naglog zamjenjivanja uropatogenih bakterija sa bakterijskim rodovima uobičajenim u urinarnom mikrobiomu, najviše Lactobacillus vrstama. Ta bakterijska vrsta ima zaštitnu ulogu, te pomaže u održavanju normalne mikrobiote.Urinary tract infections (UTI) are among the most common bacterial infections. Most UTIs are caused by uropathogenic Escherichia coli and they are more often present in women primarily due to anatomical reasons. Progress in sequencing technology allowed us to sequence environmental samples which enable sequencing of the non-culturable bacteria. The purpose of this study was to monitor changes in urinary microbiome in patient with UTI who receives antibiotic therapy. Monitoring was carried out by sequencing 16S rRNA gene, specifically V3 and V4 variable region using specially designed primes. It is shown that the pre-treatment sample has the lowest biodiversity. In that sample prevails an unknown Enterobacteiaceae bacteria, which is the cause of infection, with over 95%. The use of antibiotics leads to the rapid replace of urophatogenic bacteria with bacterial genera common in urinary microbiome – Lactobacillus. They have a protective role and help maintain normal microbiota

Comparison of zinc content in various samples of insulin by flame atomic absorption spectroscopy

Cink ima važnu ulogu u svim fazama metabolizma inzulina, od proizvodnje preko lučenja i iskorištavanja do skladištenja. Interakcija cinka i inzulina može dovesti do konformacijskih promjena i pojačati afinitet vezanja za receptore. Cink ima inzulinomimetičku aktivnost koja je najvjerojatnije posljedica inhibicije proteinskih tirozin fosfataza. On je i kofaktor enzima ključnih za metabolizam glukoze. Cilj ovog rada bio je validirati metodu određivanja cinka u inzulinu AAS-om i usporediti koncentraciju cinka u dva komercijalno dostupna pripravka inzulina u Republici Hrvatskoj. Sadržaj cinka u uzorcima određen je pomoću plamene apsorpcijske spektroskopije. Parametri validacije pokazali su zadovaljajuću linearnost u ispitivanom području (R2 =0,999). Ponovljivost metode, izražena kao relativna standardna devijacija (RSD) bila je između 0,5 % – 1,64 %, reproducibilnost metode između 3,1 % - 5,2 %, a dobivena točnost u rasponu između 95,6 % i 100,1 %. Granica dokazivanja bila je 0,025 mg/L, a granica određivanja 0,076 mg/L što pokazuje da je metoda pouzdana i osjetljiva za određivanje koncentracije cinka u uzorcima inzulina. Koncentracija cinka u komercijalno pribavljenim uzorcima inzulina bila je u rasponu od 0,152mg/L do 0191mg/L. Veći sadržaj cinka dobiven je u brzodjelujućemanalogu inzulina za razliku od sadržaja cinka kod bifazične suspenzije (30% brzodjelujući inzulin i 70% srednjedugo djelujućeg humanog inzulina) gdje je bio nešto niži. Dobiveni rezultati potvrđuju činjenicu da je cink jedan od parametara koji pospješuje brže vezanje inzulina na receptor te određuje duljinu djelovanja inzulina.Zinc plays a significant role in all phases of insulin metabolism, from production through digestion and exploitation to storage. The interaction of zinc and insulin can lead to conformational changes and enhance binding affinity for receptors. Zinc possesses an insulin-mimetic activity that is most likely due to inhibition of protein tyrosine phosphatase. It is a cofactor of crucial enzymes for glucose metabolism. The aim of this research work was to validate the AAS method for determining zinc in insulin samples and to compare a concentration of zinc in two commercially available insulins in Croatia. The concentration of zinc in insulin samples was determined by flame atomic absorption spectroscopy. Validation parameters showed satisfactory linearity in the examined area (R2 = 0.999). The repeatability of the method expressed as the relative standard deviation (RSD) was between 0.5% - 1.64%, the reproducibility of the method was between 3.1% - 5.2%, the accuracy ranging between 95.6% and 100.1 %. The detection limit was 0.025 mg / L, and the determination limit was 0.076 mg / L. That shows the method is reliable and sensitive to determining the concentration of zinc in insulin samples. The concentration of zinc in commercially available insulin samples ranged from 0.152mg / L to 0191mg / L. The higher zinc content was obtained in the fast-acting insulin analog as opposed to the zinc content of the biphasic suspension (30% fast-acting insulin and 70% of the intermediate-acting human insulin) where it was little lower

Peptidomimetics

Podložnost proteolitičkim enzimima, polarnost koja otežava transport kroz lipidni dvosloj te prevelika konformacijska sloboda ograničavaju terapeutsku primjenu peptida. Primjenom njihovih mimetika moguće je prevladati spomenute nedostatke, tako da dizajn i sinteza peptidomimetika predstavljaju moćno oruđe za pripravu lijekova koji djeluju kao inhibitori enzima ili ligandi za različite receptore. Dizajn peptidomimetika bazira se na: (i) zamjeni dijela peptidne okosnice nepeptidnim fragmentom, (ii) modifikaciji bočnih ogranaka i(li) (iii) ograničavanju konformacijske slobode, s ciljem oponašanja onog fragmenta peptidne sekundarne strukture koji je odgovoran za molekulsko prepoznavanje. Na taj se način oponaša biološka aktivnost prirodnih peptida, a moguće je ostvariti i poboljšanu selektivnost, efikasnost, biodostupnost i produljenu biološku aktivnost.The low metabolic stability towards proteolytic enzymes, polar character that disables their transport through lipide bilayer as well as the conformational freedom limit their therapeutic application. Since those adversities might be prevailed by using of their mimetics, peptidomimetic design and synthesis present a powerfull tools for the preparation of drugs acting as enzyme inhibitors or receptor ligands. The design of peptidomimetics is based on: (i) the replacement of the peptide backbone with non-peptide fragment, (ii) the modification of the side-chains and/or (iii) the locking the conformational freedom in order to mimic secondary structure element responsible for molecular recognition. This approach not only enables the mimicking of the biological activity, but also improve selectivity, efficiency, bioavailability and activity