Somatic embryogenesis as a tool for virus elimination in Croatian indigenous grapevine cultivars

Most Croatian indigenous grapevine cultivars and vineyards are infected with a few dominant viruses. The goal of this study was to establish somatic embryogenesis as an efficient method for virus elimination from valuable Croatian cultivars and creating a reliable source of healthy plants. Somatic embryogenesis was induced from immature anthers and somatic embryogenesis-derived plantlets for seven indigenous cultivars were successfully regenerated. This procedure led to the elimination of viruses GFLV, GLRaV-1, GLRaV-3 and GFkV that were initially detected in the field-grown cultivars ‘Plavac mali’ and ‘Babica’ with an elimination success of at least 30%. The described method has the potential for production of virus-free rooted plantlets for all economically important cultivars or superior cultivar clones and for the establishment of a steady source of certified virus-free planting material

Usporedba intraplazmidne rekombinacije u bakterijama Agrobacterium tumefaciens i Escherichia coli

In this work we have constructed a plasmid to compare intraplasmid recombination efficiency in Agrobacterium tumefaciens and Escherichia coli. The plasmid contains two directly repeated copies of spectinomycin resistance gene, one lacking 5’ and the other lacking 3’ end. These two copies share a 570-bp region of homology and are separated by the ampicillin resistance gene. Homologous recombination between repeated copies of incomplete spectinomycin resistance genes results in the restoration of spectinomycin resistance. During this process, ampicillin resistance gene is either deleted or incomplete spectinomycin genes are amplified along with the ampicillin resistance gene. This experimental system enabled us to follow for the first time the generation of deletions and amplifications during intraplasmid recombination in A. tumefaciens. We show here that predominantly RecA-independent mechanism contributes to the formation of deletion and amplification products in both, A. tumefaciens and E. coli. Additionally, deletion and amplification products were detected at similar frequencies, suggesting that amplifications and deletions probably occur by a similar mechanism.U ovom smo radu konstruirali plazmid koji nam je omogućio da usporedimo intraplazmidnu rekombinaciju u bakterijama Agrobacterium tumefaciens i Escherichia coli. Plazmid sadržava dvije istosmjerno ponovljene kopije gena odgovornog za rezistenciju na spektinomicin, pri čemu jednoj kopiji nedostaje 5\u27, a drugoj 3\u27 kraj gena, a međusobno su homologne u duljini od 570 pb. Osim toga, DNA koja se nalazi između ove dvije istosmjerno ponovljene sekvencije sadržava gen koji daje rezistenciju na antibiotik ampicilin. Homolognom rekombinacijom između nepotpunih gena za rezistenciju na spektinomicin nastaje funkcionalni gen, odgovoran za pojavu rezistencije. Pritom može doći do delecije gena za rezistenciju na ampicilin ili njegovog umnožavanja, zajedno s nepotpunim genima za otpornost na spektinomicin. Ovaj eksperimentalni sustav omogućio nam je da po prvi put pratimo pojavu delecija i amplifikacija tijekom intraplazmidne rekombinacije u bakteriji A. tumefaciens. Pokazali smo da delecije i amplifikacije u bakterijama Agrobacterium tumefaciens i Escherichia coli nastaju prvenstveno RecA-neovisnim mehanizmom. Osim toga, ustanovili smo da se delecije i amplifikacije pojavljuju s podjednakom učestalošću, što upućuje na to da je mehanizam oba rekombinacijska događaja sličan

The Repetitive DNA Composition in the Natural Pesticide Producer Tanacetum cinerariifolium: Interindividual Variation of Subtelomeric Tandem Repeats

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.), a plant species endemic to the east Adriatic coast, is used worldwide for production of the organic insecticide, pyrethrin. Most studies concerning Dalmatian pyrethrum have focused on its morphological and biochemical traits relevant for breeding. However, little is known about the chromosomal evolution and genome organization of this species. Our study aims are to identify, classify, and characterize repetitive DNA in the T. cinerariifolium genome using clustering analysis of a low coverage genomic dataset. Repetitive DNA represents about 71.63% of the genome. T. cinerariifolium exhibits linked 5S and 35S rDNA configuration (L-type). FISH reveals amplification of interstitial telomeric repeats (ITRs) in T. cinerariifolium. Of the three newly identified satellite DNA families, TcSAT1 and TcSAT2 are located subterminally on most of T. cinerariifolium chromosomes, while TcSAT3 family is located intercalary within the longer arm of two chromosome pairs. FISH reveals high levels of polymorphism of the TcSAT1 and TcSAT2 sites by comparative screening of 28 individuals. TcSAT2 is more variable than TcSAT1 regarding the number and position of FISH signals. Altogether, our data highlights the dynamic nature of DNA sequences associated with subtelomeres in T. cinerariifolium and suggests that subtelomeres represent one of the most dynamic and rapidly evolving regions in eukaryotic genomes

Virus and Virus-like Pathogens in the Grapevine Virus Collection of Croatian Autochthonous Grapevine Cultivars

Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses (arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leafroll-associated viruses 1, 2, and 3, and grapevine viruses A and B), a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of “flavescence dorée” phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of “flavescence dorée”, makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years

Reversible "brain atrophy" in patients with Cushing's disease

During the past 25 years, we came across 60 patients with corticotroph pituitary adenomas and Cushing's disease. Neuroradiological examination showed prominent volume loss of the brain parenchyma, unexpected for the patient's age. This "brain atrophy" appeared to regress after surgical removal of pituitary adenoma and normalization of cortisol level. Observed difference between degree of "brain atrophy" in the Cushing's disease group and in the control group was statistically significant (p < 0.001). The degree of "brain atrophy" correlated well with the duration of Cushing's disease. Partial reversibility of "brain atrophy" was noticed during the 2nd, 3rd and 4th year after surgery and normalization of cortisol level. Increased cortisol level is one of the causative factors in pathogenesis of "brain atrophy". Loss of brain volume is at least partially reversible after normalization of cortisol levels

Surgical treatment of prolactinomas - our experience [Prolaktinomi - kirurško ili medikamentozno liječenje?]

The dilemma of whether to apply surgical or drug treatment to prolactinomas has been ongoing for the past 30 years. The aim of this study is to compare the early postoperative values of prolactin (PRL) in two groups of patients with prolactinomas: those who underwent primary surgical-treatment, and those who underwent surgery after a dopamine agonist (DA) therapy. We present the results of surgical treatment on a series of 161 patients with prolactinomas. Surgery was the primary treatment in 65 patients, while 96 patients had surgery following a long-term treatment with a DA. All surgically treated prolactinomas were operated in the standard transsphenoidal, microsurgical approach. The criteria for hyperprolactinemia remission was a PRL level under 25 ng/ml. Early normalization of PRL was achieved in 92% of those patients who underwent primary surgical-treatment, yet it was achieved in only 42% of patients who were operated on after receiving a long-term drug treatment with a DA. The highest prevalence of postoperative normalization of PRL was achieved in a group of patients with microadenomas who were primarily operated on (98%). The worst results in postoperative normalization of PRL were found in the group of patients with macroadenomas who received a long-term drug treatment with a DA first. These results show our surgical experience in treating prolactinomas. Using surgical treatment, the best clinical outcome was achieved with microprolactinomas and intrasellar, well-confined macroprolactinomas. Nevertheless, we stress the need of an individualized approach and recommend treatment in multidisciplinary centres for pituitary diseases

Smjerovi razvoja kirurškog liječenja tumora hipofize

Transsphenoidal approach to pituitary tumors was for a long time reserved for sellar region tumors, whereas tumors expanding into extrasellar region were treated by transcranial approach. With the development of surgical equipment and gaining surgical experience, indications for the transsphenoidal approach and its modifications have become wider and ever more frequently used for pituitary tumors with extrasellar expansion. During the twenty-five years of performing pituitary surgery, we have operated on more than 1400 tumors of the sellar region using the transsphenoidal approach and its modifications. The novel transsphenoidal endoscopic approach is strongly taking more place in pituitary surgery, advancing the possibilities of the standard transsphenoidal approach. A review is presented of surgical approaches to pituitary tumors.Transsfenoidni pristup tumorima hipofize dugo je bio rezerviran za intraselarne neoplazme, dok se ekstraselarno proširene tumore rješavalo transkranijskim pristupom. Zahvaljujući razvoju kirurške opreme i instrumentarija te većem iskustvu operatera indikacije za ovaj pristup i njegove modalitete postale su šire pa se češće rabe i za ekstraselarno proširene tumore. Tijekom 25 godina bavljenja kirurgijom hipofize operirali smo preko 1400 tumora selarne regije rabeći transsfenoidni pristup i njegove modalitete. Novi endoskopski transsfenoidni pristup zauzima sve više mjesta u kirurškom liječenju hipofize te tako povećava lepezu mogućnosti proširenog transsfenoidnog pristupa. Prikazani su kirurški pristupi liječenju tumora hipofize

Prolaktinomi - kirurško ili medikamentozno liječenje?

The dilemma of whether to apply surgical or drug treatment to prolactinomas has been ongoing for the past 30 years. The aim of this study is to compare the early postoperative values of prolactin (PRL) in two groups of patients with prolactinomas: those who underwent primary surgical-treatment, and those who underwent surgery after a dopamine agonist (DA) therapy. We present the results of surgical treatment on a series of 161 patients with prolactinomas. Surgery was the primary treatment in 65 patients, while 96 patients had surgery following a long-term treatment with a DA. All surgically treated prolactinomas were operated in the standard transsphenoidal, microsurgical approach. The criteria for hyperprolactinemia remission was a PRL level under 25 ng/ml. Early normalization of PRL was achieved in 92% of those patients who underwent primary surgical-treatment, yet it was achieved in only 42% of patients who were operated on after receiving a long-term drug treatment with a DA. The highest prevalence of postoperative normalization of PRL was achieved in a group of patients with microadenomas who were primarily operated on (98%). The worst results in postoperative normalization of PRL were found in the group of patients with macroadenomas who received a long-term drug treatment with a DA first. These results show our surgical experience in treating prolactinomas. Using surgical treatment, the best clinical outcome was achieved with microprolactinomas and intrasellar, well-confined macroprolactinomas. Nevertheless, we stress the need of an individualized approach and recommend treatment in multidisciplinary centres for pituitary diseases.Dilema, operirati ili medikamentozno liječiti prolaktinome prisutna je već 30 godina. Cilj ovog istraživanja je usporediti normalizaciju ranih postoperativnih vrijednosti prolaktina (PRL) u dvije skupine bolesnika sa prolaktinomima: primarno kirurški liječenih i onih koji su kirurški liječeni nakon prethodne terapije agonistima dopamina (DA). U ovom članku prezentirani su rezultati operacijskog liječenja 161 bolesnika s prolaktinomom. Operacija je bila primarni način liječenja u 65 bolesnika, dok je 96 bolesnika operirano nakon dugotrajne medikamentozne terapije s DA. Svi kirurški tretirani prolaktinomi operirani su standardnim, transsfenoidalnim, mikrokirurškim pristupom od strane jednog operatera. Kao definiciju remisije hiperprolaktinemije koristili smo normalizaciju koncentracije bazalnih vrijednosti prolaktina (PRL) ispod 25 ng/mL. U primarno operiranih bolesnika postignuta je rana postoperativna normalizacija PRL u 92% slučajeva, a u operiranih nakon dugotrajne medikamentozne terapije sa DA u samo 42% slučajeva. Najbolje rezultate u normalizaciji koncentracije prolaktina postigli smo u skupini bolesnika sa mikroadenomima koji su primarno operirani (98%), a najlošije rezultate u skupini bolesnika s makroadenomima koji su prethodno bili na dugotrajnoj medikamentoznoj terapiji s DA (17,3%). Ovi rezultati prikazuju naše iskustvo u kiruškom liječenju prolaktinoma. Najbolji klinički rezultat imali smo sa mikroprolaktinomima i intraselarnim, dobro ograničenim makroprolaktinomima. Ističemo potrebu multidisciplinarnog i individualnog pristupa u izboru metode liječenja

Surgical Treatment of Prolactinomas – Our Experience

The dilemma of whether to apply surgical or drug treatment to prolactinomas has been ongoing for the past 30 years. The aim of this study is to compare the early postoperative values​​ of prolactin (PRL) in two groups of patients with prolactinomas: those who underwent primary surgical-treatment, and those who underwent surgery after a dopamine agonist (DA) therapy. We present the results of surgical treatment on a series of 161 patients with prolactinomas. Surgery was the primary treatment in 65 patients, while 96 patients had surgery following a long-term treatment with a DA. All surgically treated prolactinomas were operated in the standard transsphenoidal, microsurgical approach. The criteria for hyperprolactinemia remission was a PRL level under 25 ng/ml. Early normalization of PRL was achieved in 92% of those patients who underwent primary surgical-treatment, yet it was achieved in only 42% of patients who were operated on after receiving a long-term drug treatment with a DA. The highest prevalence of postoperative normalization of PRL was achieved in a group of patients with microadenomas who were primarily operated on (98%). The worst results in postoperative normalization of PRL were found in the group of patients with macroadenomas who received a long-term drug treatment with a DA first. These results show our surgical experience in treating prolactinomas. Using surgical treatment, the best clinical outcome was acieved with microprolactinomas and intrasellar, well-confined macroprolactinomas. Nevertheless, we stress the need of an individualized approach and recommend treatment in multidisciplinary centres for pituitary diseases