Real-world Outcomes of Dual HER2 Blockade Therapy in Metastatic HER2-Positive Breast Cancer: from Induction to Maintenance.

BACKGROUND AND OBJECTIVE: Dual human epidermal growth factor receptor 2 (HER2) blockade with trastuzumab and pertuzumab combined with taxane-based chemotherapy (Cht) has been the standard first-line treatment for HER2-positive metastatic breast cancer (mBC) for years, due to the impressive results of the CLEOPATRA study. Real-world (RW) studies have become critical for assessing treatment effectiveness and safety in real-life circumstances. The aim of this study was to analyze the treatment outcomes of first-line therapy for HER2-positive mBC in RW clinical practice, specifically focusing on the use of maintenance endocrine therapy (ET) in hormone receptor positive (HR-positive) patients. METHODS: This retrospective analysis included 106 HER2-positive mBC patients treated with trastuzumab and pertuzumab combined with taxane-based Cht from October 2015 to December 2020 at the University Hospital Centre Zagreb. RESULTS: At a median follow-up of 30 months, median progression-free survival (PFS) was 25 months for the total population (95% confidence interval [CI] 16 - not analyzed). Patients with de novo mBC had longer median PFS than patients with recurrent disease (not reached vs. 18 months; hazard ratio 1.99; 95% CI 0.69-3.64, p<0.022). Age, hormone receptor positivity, visceral involvement, number of Cht cycles and previous adjuvant trastuzumab did not impact PFS. Most HR-positive patients (N=55, 88.7%) received maintenance ET after induction Cht. CONCLUSION: This retrospective study provides additional data on patient characteristics, treatment and outcomes of RW HER2-positive mBC patients treated with pertuzumab and trastuzumab as first-line therapy. In our institution, maintenance ET after induction Cht has become standard clinical practice.

Comparison of exoskeleton microbial communities of co-occurring native and invasive crayfish species.

Host-associated microbial communities are an important determinant of individual fitness and have recently been highlighted as one of the factors influencing the success of invasive species. Invasive hosts introduce their microbes into the new environment, and then both the host and its associated microbes enter into a series of interactions with the native macroscopic and microscopic biota. As these processes are largely unexplored, we aimed to compare the exoskeletal microbial communities of co-occurring and phylogenetically related crayfish: the native narrow-clawed crayfish Pontastacus leptodactylus and the invasive signal crayfish Pacifastacus leniusculus from the recently invaded Korana River, Croatia. The results of high-throughput 16S rRNA sequencing showed that the exoskeletal microbiome of both species is very diverse, significantly influenced by the local environment and dominated by low abundance bacterial families from the phylum Proteobacteria. Furthermore, the exoskeletal microbiomes of the crayfish species differed significantly in the composition and abundance of Amplicon Sequence Variants (ASVs), suggesting that they are to some extent shaped by species-specific intrinsic factors, despite sharing a common habitat. However, over 95% of the bacterial genera associated with the exoskeleton were detected in the exoskeleton samples of both native and invasive crayfish. We paid particular attention to two known crayfish pathogens, Aphanomyces astaci and Saprolegnia parasitica, and find that both species carry low amounts of both pathogens. On the side, we find that a non-standard ddPCR protocol outperforms standard qPCR test for A. astaci under low concentration conditions. Taken together, our results indicate the possibility of bidirectional mixing and homogenisation of exoskeleton microbiome. As such, they can serve as a baseline in future detangling of the processes that act together to shape the microbiomes of co-occuring native and invasive congeners during biological invasions.

Sage, Rosemary, and Bay Laurel Hydrodistillation By-Products as a Source of Bioactive Compounds.

Essential oils from Mediterranean wild plants are widely used, but the hydrodistillation residues produced in parallel with these essential oils are significantly understudied and underexploited. Since there are only fragmentary data in the literature, we have, for the first time, systematically analyzed the chemical composition of the by-products obtained after hydrodistillation of sage, bay laurel, and rosemary leaves, i.e., hydrolates, water residues, and solid residues. The chemical composition of the hydrolates changed compared to their respective essential oils towards the dominance of more hydrophilic, oxygenated compounds, such as camphor in sage, 1,8-cineole in bay laurel, and berbenone in rosemary. However, some compounds, mostly sesquiterpenes, which were present in considerable amounts in essential oils, were absent or only present in very small amounts in the hydrolates. Furthermore, both the water and the solid residues were rich in polyphenols, such as procyanidins in bay laurel and rosmarinic acid in rosemary and sage. In conclusion, we demonstrate the valuable chemical composition of sage, rosemary, and bay laurel hydrodistillation by-products and discuss a wide range of their possible applications.

Impact of nutrients and trace elements on freshwater microbial communities in Croatia: identifying bacterial bioindicator taxa.

Since aquatic microbial communities promptly respond to environmental changes, it is now evident that they can complement traditional taxa such as fish, macroinvertebrates and algae as bioindicators of water quality. The aim of this study was to correlate the physico-chemical parameters of water with the microbial community structure and the occurrence of putative bioindicator taxa. Thirty-five water samples were collected throughout Croatia and their physico-chemical parameters, including the concentration of trace elements using the high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS), and the composition of the microbial communities by high-throughput sequencing of the 16S rRNA marker gene, were analysed in parallel. Partial least squares regression (PLS-R) modelling revealed that a number of microbial taxa were positively correlated with some of the water parameters. For example, some taxa from the phylum Proteobacteria were positively correlated with the ion content of the water (e.g. Erythrobacter, Rhodobacteraceae, Alteromonadaceae), while some Firmicutes taxa, such as the well-known faecal indicators Enterococcus and Clostridium, were correlated with nutrient content (ammonium and total phosphorus). Among the trace elements, uranium was positively correlated with a highest number of microbial taxa. The results obtained will aid in development of protocols for eDNA-based biological assessment of water quality.

Marine Pollutant Tributyltin Affects DNA Methylation and Fitness of Banded Murex (Hexaplex trunculus) Populations.

Banded murex, Hexaplex trunculus, is a marine gastropod whose reproductive fitness can be severely affected by very low concentrations of antifouling compound tributyltin (TBT). TBT has strong xenoandrogen impacts on snails, causing the development of imposex (e.g., the superimposition of male sexual characteristic in females), thereby affecting the fitness of entire populations. TBT is also known as a DNA-demethylating agent and an obesogenic factor. The aim of this study was to unravel the interactions between TBT bioaccumulation, phenotypic responses, and epigenetic and genetic endpoints in native populations of H. trunculus. Seven populations inhabiting environments along the pollution gradient were sampled in the coastal eastern Adriatic. These included sites of intense marine traffic and boat maintenance activity and sites with low anthropogenic impact. Populations inhabiting intermediately and highly polluted sites exhibited higher TBT burdens, higher incidences of imposex, and higher wet masses of snails than populations in lowly polluted sites. Other morphometric traits and cellular biomarker responses did not show clear differentiation among populations in relation to marine traffic/pollution intensity. An analysis of methylation sensitive amplification polymorphism (MSAP) revealed environmentally driven population differentiation and higher epigenetics than genetic within-population diversity. Moreover, decreases in genome-wide DNA methylation coincided with the imposex level and snail mass, suggesting an epigenetic background of the animal phenotypic response.

Tracing the oomycete pathogen Saprolegnia parasitica in aquaculture and the environment.

Saprolegnia parasitica causes saprolegniosis, a disease responsible for significant economic losses in aquaculture and declines of fish populations in the wild, but the knowledge of its distribution and prevalence in the environment is limited. We developed a fast, sensitive and specific S. parasitica droplet digital PCR (ddPCR) assay and demonstrated its applicability for the detection and quantification of the pathogen in environmental samples: swab DNA collected from the host (trout skin, surface of eggs) and environmental DNA extracted from water. The developed assay was used to assess how abiotic (i.e. physico-chemical parameters of the water) and biotic (health status of the host) factors influence the S. parasitica load in the environment. The pathogen load in water samples was positively correlated with some site-specific abiotic parameters such as electrical conductivity (EC) and calcium, while fluorides were negatively correlated, suggesting that physico-chemical parameters are important for determining S. parasitica load in natural waters. Furthermore, skin swabs of injured trout had significantly higher pathogen load than swabs collected from healthy fish, confirming that S. parasitica is a widespread opportunistic pathogen. Our results provide new insights into various environmental factors that influence the distribution and abundance of S. parasitica.

First report of potato virus S and potato virus Y in tomatoes from Croatia.

Potato virus Y (PVY, genus Potyvirus) is an economically important aphid-transmissible virus with a very wide host range reported in many tomato-growing areas (Rivarez et al. 2021). Potato virus S (PVS, genus Carlavirus) has a limited host range (Lin et al. 2014) and occurs in tomato (Predajna et al. 2017), mostly in mixed infections with other viruses. In 2021, greenhouse tomatoes from Vidovec (46° 17' 3.4'' N, 16° 15' 37.0'' E) in the northwestern and Sedlarica (45° 54' 23.0'' N, 17° 12' 0.5'' E) in the eastern regions of Croatia were surveyed for virus-like diseases. In total, 30 plants were sampled (12 from Vidovec and 18 from Sedlarica) showing symptoms of mild mottling, leaf rugosity and mild bronzing followed by leaf necroses later in the season. Nucleic acids were extracted from leaves by adapted CTAB procedure (Murray and Thompson 1980) and DNase treated. Four representative samples from Vidovec and four from Sedlarica were pooled for high throughput sequencing (HTS). After rRNA depletion (RiboMinus™ Plant Kit for RNA-Seq, Invitrogen) and polyA tailing, two location specific libraries (PCR-cDNA sequencing kit, Oxford Nanopore Technologies) were prepared for nanopore HTS on MinION Mk1C device. From Vidovec samples, 459,285 raw reads (mean length 354 nt) were obtained and 206,718 (mean length 446 nt) from Sedlarica and mapped (Minimap2, v.2.17) against Kraken2 viral genome sequences database (https://benlangmead.github.io/aws-indexes/k2). The number of reads mapped to PVS genome was 1004 from Vidovec (coverage depth 1.56) and those mapped to PVY genome were 781 (coverage depth 0.99) and 57 (coverage depth 1), from Vidovec and Sedlarica, respectively. The PVS complete consensus genome from Vidovec (ON468562, 8485 nt) had 99.09% nucleotide identity (BLASTn) to a potato isolate from the Netherlands (MF418030). The PVY consensus genome sequences from Vidovec (ON505007, 9698 nt) and Sedlarica (ON505008, 9698 nt) had respectively 98.37% and 98.48% identities to a tomato isolate from Slovakia (MW685827). Reverse transcription polymerase chain reaction (RT-PCR) was performed for all 30 samples and amplicons were Sanger sequenced, with primers PVS-7773F/PVS-3'endR for a 720 nt PVS genome portion spanning the 3'-part of the CP and a complete 11K gene (Lin et al. 2014) and PVY-2F/2R primers for a 510 nt portion of PVY CP gene (Aramburu et al. 2006). Only one tomato out of 12 ('Borana') from Vidovec harbored PVS in the mixed infection with PVY. Two additional tomatoes from Vidovec and two from Sedlarica were infected solely by PVY. Amplicon sequences of PVS (ON651427) and PVY (ON707000-4, ON734067-8) had 100% identity with the HTS assembled sequences. The PVS isolate from Croatia grouped with PVSO (ordinary) strain in phylogenetic analysis and the PVY isolates from both sites grouped with the PVY-NTN strain (Cox and Jones 2010). Although PVY is considered to be widespread in tomato (Nikolic et al. 2018; Rivarez et al. 2021), this is its first report from Croatia. PVS, newly reported from Croatia here, is probably not associated with the symptoms recorded because the same symptomatology was observed in the singly and mixed infected 'Borana' tomato plants. The occurrence of PVY in the geographically distant (100 km apart) Vidovec and Sedlarica, suggests that it is widespread in the continental Croatia where tomatoes are commercially grown in plastic greenhouses. Further analyses are needed to elucidate PVY and PVS epidemiology and impact on the local tomato production.

Variations in the Sporulation Efficiency of Pathogenic Freshwater Oomycetes in Relation to the Physico-Chemical Properties of Natural Waters.

Oomycete pathogens in freshwaters, such as Saprolegnia parasitica and Aphanomyces astaci, are responsible for fish/crayfish population declines in the wild and disease outbreaks in aquaculture. Although the formation of infectious zoospores in the laboratory can be triggered by washing their mycelium with natural water samples, the physico-chemical properties of the water that might promote sporulation are still unexplored. We washed the mycelia of A. astaci and S. parasitica with a range of natural water samples and observed differences in sporulation efficiency. The results of Partial Least Squares Regression (PLS-R) multivariate analysis showed that SAC (spectral absorption coefficient measured at 254 nm), DOC (dissolved organic carbon), ammonium-N and fluoride had the strongest positive effect on sporulation of S. parasitica, while sporulation of A. astaci was not significantly correlated with any of the analyzed parameters. In agreement with this, the addition of environmentally relevant concentrations of humic acid, an important contributor to SAC and DOC, to the water induced sporulation of S. parasitica but not of A. astaci. Overall, our results point to the differences in ecological requirements of these pathogens, but also present a starting point for optimizing laboratory protocols for the induction of sporulation.

Immune Response in Crayfish Is Species-Specific and Exhibits Changes along Invasion Range of a Successful Invader.

Immunity is an important component of invasion success since it enables invaders' adaptation to conditions of the novel environment as they expand their range. Immune response of invaders may vary along the invasion range due to encountered parasites/microbial communities, conditions of the local environment, and ecological processes that arise during the range expansion. Here, we analyzed changes in the immune response along the invasion range of one of the most successful aquatic invaders, the signal crayfish, in the recently invaded Korana River, Croatia. We used several standard immune parameters (encapsulation response, hemocyte count, phenoloxidaze activity, and total prophenoloxidaze) to: i) compare immune response of the signal crayfish along its invasion range, and between species (comparison with co-occurring native narrow-clawed crayfish), and ii) analyze effects of specific predictors (water temperature, crayfish abundance, and body condition) on crayfish immune response changes. Immune response displayed species-specificity, differed significantly along the signal crayfish invasion range, and was mostly affected by water temperature and population abundance. Specific immune parameters showed density-dependent variation corresponding to increased investment in them during range expansion. Obtained results offer baseline insights for elucidating the role of immunocompetence in the invasion success of an invertebrate freshwater invader.

Essential Oils of Sage, Rosemary, and Bay Laurel Inhibit the Life Stages of Oomycete Pathogens Important in Aquaculture.

Saprolegnia parasitica, the causative agent of saprolegniosis in fish, and Aphanomyces astaci, the causative agent of crayfish plague, are oomycete pathogens that cause economic losses in aquaculture. Since toxic chemicals are currently used to control them, we aimed to investigate their inhibition by essential oils of sage, rosemary, and bay laurel as environmentally acceptable alternatives. Gas Chromatography-Mass Spectrometry (GC-MS) analysis showed that the essential oils tested were rich in bioactive volatiles, mainly monoterpenes. Mycelium and zoospores of A. astaci were more sensitive compared to those of S. parasitica, where only sage essential oil completely inhibited mycelial growth. EC50 values (i.e., concentrations of samples at which the growth was inhibited by 50%) for mycelial growth determined by the radial growth inhibition assay were 0.031-0.098 µL/mL for A. astaci and 0.040 µL/mL for S. parasitica. EC50 values determined by the zoospore germination inhibition assay were 0.007-0.049 µL/mL for A. astaci and 0.012-0.063 µL/mL for S. parasitica. The observed inhibition, most pronounced for sage essential oil, could be partly due to dominant constituents of the essential oils, such as camphor, but more likely resulted from a synergistic effect of multiple compounds. Our results may serve as a basis for in vivo experiments and the development of environmentally friendly methods to control oomycete pathogens in aquaculture.

Effect of Enzymatic, Ultrasound, and Reflux Extraction Pretreatments on the Chemical Composition of Essential Oils.

The effect of different hydrodistillation pretreatments, namely, reflux extraction, reflux extraction with the addition of cell wall-degrading enzymes, and ultrasound, on the yield and chemical composition of essential oils of sage, bay laurel, and rosemary was examined. All pretreatments improved essential oil yield compared to no-pretreatment control (40-64% yield increase), while the oil quality remained mostly unchanged (as shown by statistical analysis of GC-MS results). However, enzyme-assisted reflux extraction pretreatment did not significantly outperform reflux extraction (no-enzyme control), suggesting that the observed yield increase was mostly a consequence of reflux extraction and enzymatic activity had only a minute effect. Thus, we show that ultrasound and reflux extraction pretreatments are beneficial in the production of essential oils of selected Mediterranean plants, but the application of enzymes has to be carefully re-evaluated.

Biomarker response of Mediterranean mussels Mytilus galloprovincialis regarding environmental conditions, pollution impact and seasonal effects.

The complexity of seasonally and spatially variable environments, coupled with complex biological interactions, makes it difficult to pinpoint biological responses to specific environmental stressors, including chemical pollution. To disentangle causative factors and reveal biomarker responses, we applied biomarker-based multivariate approaches to 15 native populations of Mediterranean mussel Mytilus galloprovincialis in spring and autumn. In addition, we used a subset of these populations in transplant experiments between clean and polluted environments in nature and in lab mesocosms. The extent of biomarker responses in native populations is affected by season, and significantly lower variability across seasons was observed among mussels from clean than from polluted sites. Results of paired block designed transplant experiment demonstrated both regional and pollution effect, with mussels uniformly exhibiting higher responses on more impacted sites in each of the Adriatic regions. Biomarker status of mussels varied among Adriatic regions in dependence on the set of environmental variables, and between clean and polluted sites in dependence on measured concentrations of metals in mussels' tissue. Results of the mesocosm experiment revealed distinctive biomarker responses of two populations of different origin when exposed to common conditions. Multivariate description of biomarker activity and application of specific experiments allowed us to link environmental condition, exposure to pollution and seasonality to mussels' biomarker responses.