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W artykule omówiono kwestie dotyczące raportowania niefinansowego, które stało się obowiązkowe dla dużych przedsiębiorców od 2017 roku w Polsce. Praca omawia podstawy prawne, zmiany, jakie zaszły w raportowaniu ESG oraz nowe standardy, jakie zostały wprowadzone. Autorki poruszają także temat statystyk, z których jasno wynika rosnący trend raportowania niefinansowego również wśród tych podmiotów, które nie zostały do tego zobowiązane. Koniec niniejszego artykułu zwieńczony został informacjami na temat organizowanego od 2007 roku konkursu zrównoważonego rozwoju, w ramach którego sprawdzane są raporty niefinansowe za poszczególne lata.

To (1) describe the prevalence of cardiometabolic disease (CMD) at spinal cord injury (SCI) rehabilitation discharge; (2) compare this with controls without SCI; and (3) identify factors associated with increased CMD.Multicenter, prospective observational study.Five National Institute on Disability, Independent Living, and Rehabilitation Research Model SCI Rehabilitation Centers.SCI (n=95): patients aged 18-70 years, with SCI (neurologic levels of injury C2-L2, American Spinal Injury Association Impairment Scale grades A-D), and enrolled within 2 months of initial rehabilitation discharge. Control group (n=1609): age/sex/body mass index-matched entries in the National Health and Nutrition Examination Education Survey (2016-2019) (N=1704).None MAIN OUTCOME MEASURES: Percentage of participants with SCI with CMD diagnosis, prevalence of CMD determinants within 2 months of rehabilitation discharge, and other significant early risk associations were analyzed using age, sex, body mass index, insulin resistance (IR) by fasting glucose and Homeostasis Model Assessment (v.2), fasting triglycerides, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol, total cholesterol, and resting blood pressure (systolic and diastolic).Participants with SCI had significantly higher diastolic blood pressure and triglycerides than those without SCI, with lower fasting glucose and HDL-C. A total of 74.0% of participants with SCI vs 38.5% of those without SCI were obese when applying population-specific criteria (P.05). Low HDL-C was measured in 54.2% of participants with SCI vs 15.4% of those without (P.05). IR was not significantly different between groups. A total of 31.6% of participants with SCI had ≥3 CMD determinants, which was 40.7% higher than those without SCI (P.05). Interplay of lipids and lipoproteins (ie, total cholesterol:HDL-C ratio and triglyceride:HDL-C ratio) were associated with elevated risk in participants with SCI for myocardial infarction and stroke. The only significant variable associated with CMD was age (P.05).Individuals with SCI have an increased CMD risk compared with the general population; obesity, IR, and low HDL-C are the most common CMD risk determinants; age is significantly associated with early CMD.

Along with different life strategies, organisms have evolved dramatic cellular composition differences. Understanding the molecular basis and fitness effects of these differences is key to elucidating the fundamental characteristics of life. TOR/insulin pathways are key regulators of cell size, but whether their activity determines cell size in a systemic or tissue-specific manner awaits exploration. To that end, we measured cells in four tissues in genetically modified Drosophila melanogaster (rictorΔ2 and Mnt1) and corresponding controls. While rictorΔ2 flies lacked the Rictor protein in TOR complex 2, downregulating the functions of this element in TOR/insulin pathways, Mnt1 flies lacked the transcriptional regulator protein Mnt, weakening the suppression of downstream signalling from TOR/insulin pathways. rictorΔ2 flies had smaller epidermal (leg and wing) and ommatidial cells and Mnt1 flies had larger cells in these tissues than the controls. Females had consistently larger cells than males in the three tissue types. In contrast, dorsal longitudinal flight muscle cells (measured only in males) were not altered by mutations. We suggest that mutations in cell cycle control pathways drive the evolution of systemic changes in cell size throughout the body, but additional mechanisms shape the cellular composition of some tissues independent of these mutations.

Along with differences in life histories, metazoans have also evolved vast differences in cellularity, involving changes in the molecular pathways controlling the cell cycle. The extent to which the signalling network systemically determines cellular composition throughout the body and whether tissue cellularity is organized locally to match tissue-specific functions are unclear. We cultured genetic lines of Drosophila melanogaster on food with and without rapamycin to manipulate the activity of target of rapamycin (TOR)/insulin pathways and evaluate cell-size changes in five types of adult cells: wing and leg epidermal cells, ommatidial cells, indirect flight muscle cells and Malpighian tubule epithelial cells. Rapamycin blocks TOR multiprotein complex 1, reducing cell growth, but this effect has been studied in single cell types. As adults, rapamycin-treated flies had smaller bodies and consistently smaller cells in all tissues. Regardless, females eclosed with larger bodies and larger cells in all tissues than males. Thus, differences in TOR activity and sex were associated with the orchestration of cell size throughout the body, leading to differences in body size. We postulate that the activity of TOR/insulin pathways and their effects on cellularity should be considered when investigating the origin of ecological and evolutionary patterns in life histories.

Technical details of histological techniques and cell size measurements and additional statistical results

The intrinsic sources of mortality relate to the ability to meet the metabolic demands of tissue maintenance and repair, ultimately shaping ageing patterns. Anti-ageing mechanisms compete for resources with other functions, including those involved in maintaining functional plasma membranes. Consequently, organisms with smaller cells and more plasma membranes should devote more resources to membrane maintenance, leading to accelerated intrinsic mortality and ageing. To investigate this unexplored trade-off, we reared Drosophila melanogaster larvae on food with or without rapamycin (a TOR pathway inhibitor) to produce small- and large-celled adult flies, respectively, and measured their mortality rates. Males showed higher mortality than females. As expected, small-celled flies (rapamycin) showed higher mortality than their large-celled counterparts (control), but only in early adulthood. Contrary to predictions, the median lifespan was similar between the groups. Rapamycin administered to adults prolonged life; thus, the known direct physiological effects of rapamycin cannot explain our results. Instead, we invoke indirect effects of rapamycin, manifested as reduced cell size, as a driver of increased early mortality. We conclude that cell size differences between organisms and the associated burdens of plasma membrane maintenance costs may be important but overlooked factors influencing mortality patterns in nature.

For many decades, Duhem has been considered a paradigmatic instrumentalist, and while some commentators have argued against classifying him in this way, it still seems prevalent as an interpretation of his philosophy of science. Yet such a construal bears scant resemblance to the views presented in his own works—so little, indeed, that it might be said to constitute no more than a mere phantom with respect to his actual thought. In this article, we aim to deconstruct this phantom, tracing the sources of the misconceptions surrounding his ideas and pinpointing the sources and/or causes of its proliferation. We subsequently point out and discuss those elements of his philosophy that, taken together, support the view of him as a scientific realist of a sophisticated kind. Finally, we defend our own interpretation of his thought against suggestions to the effect that it is oriented towards neither instrumentalism nor scientific realism.

Genetic analysis of an adult patient with an unusual course of Ketosis-Prone Diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the 5’-UTR of PDX1, a beta-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm stage. Metabolomic analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the definitive endoderm to the pancreatic progenitor stage, and RNA-sequencing showed defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR-Cas9 manipulation of the PDX1 variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the PDX1 5’-UTR, leading to decreased PDX1 expression at the early pancreatic progenitor stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical beta-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset beta-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multi-omics analysis of patient-specific stem cells for clinical discovery.

With a wide range of available cytotoxic therapeutics, the main focus of current cancer research is to deliver them specifically to the cancer cells, minimizing toxicity against healthy tissues. Targeted therapy utilizes different carriers for cytotoxic drugs, combining a targeting molecule, typically an antibody, and a highly toxic payload. For the effective delivery of such cytotoxic conjugates, a molecular target on the cancer cell is required. Various proteins are exclusively or abundantly expressed in cancer cells, making them a possible target for drug carriers. Fibroblast growth factor receptor 1 (FGFR1) overexpression has been reported in different types of cancer, but no FGFR1-targeting cytotoxic conjugate has been approved for therapy so far. In this study, the FGFR1-targeting peptide previously described in the literature was reformatted into a peptibody-peptide fusion with the fragment crystallizable (Fc) domain of IgG1. PeptibodyC19 can be effectively internalized into FGFR1-overexpressing cells and does not induce cells' proliferation. The main challenge for its use as a cytotoxic conjugate is a cysteine residue located within the targeting peptide. A standard drug-conjugation strategy based on the maleimide-thiol reaction involves modification of cysteines within the Fc domain hinge region. Applied here, however, may easily result in the modification of the targeting peptide with the drug, limiting its affinity to the target and therefore the potential for specific drug delivery. To investigate if this is the case, we have performed conjugation reactions with different auristatin derivatives (PEGylated and unmodified) under various conditions. By controlling the reduction conditions and the type of cytotoxic payload, different numbers of cysteines were substituted, allowing us to avoid conjugating the drug to the targeting peptide, which could affect its binding to FGFR1. The optimized protocol with PEGylated auristatin yielded doubly substituted peptibodyC19, showing specific cytotoxicity toward the FGFR1-expressing lung cancer cells, with no effect on cells with low FGFR1 levels. Indeed, additional cysteine poses a risk of unwanted modification, but changes in the type of cytotoxic payload and reaction conditions allow the use of standard thiol-maleimide-based conjugation to achieve standard Fc hinge region cysteine modification, analogously to antibody-drug conjugates.

New 6-CF3-1H-pyrazolo[3,4-b]quinolines with a methyl and/or phenyl group attached to the pyrazole core (Molx (x = 1, 2, 3, 4)) were synthesized and characterized in terms of their optoelectronic applications: photovoltaic and electroluminescence. The fluorescence emissions of the investigated phenyl-decorated pyrazoloquinolines is caused by the photoinduced charge transfer p process occurring between the phenyl substituent and the pyrazoloquinoline core, while 1,3-dimethyl-6-CF3-1H-pyrazolo[3,4-b]quinoline exhibits an π,π*-type emission. The number of phenyls and their substitution positions modulate both emission properties and HOMO energy levels. Next, the bulk heterojunction BHJ solar cells based on 1H-pyrazolo[3,4-b] quinoline derivatives with architecture ITO/PEDOT:PSS/PDT + Molx/Al were fabricated. The organic active layer was a blend of Molx and poly(3-decylthiophene-2,5-diyl). The complex refractive index and the layer thickness of the organic solar cells were determined using a spectroscopic ellipsometer Woollam M2000 (J.A. Woollam Co., Inc., Lincoln, NE, USA) and CompleteEASE software. For solar devices with the best value of power efficiency of approximately 0.38%, the thickness of the active layer (Mol3 + PDT) was 111 nm, with a short-circuit current density of JSC = 32.81 μA/cm2 and an open–circuit voltage of VOC = 0.78 V. Finally, we demonstrated double-layer light-emitting diodes with an organic active layer (Molx + PVK) and an electron transporting material layer, ETM (2-[3,5-bis(4-phenyl-2-quinolyl)phenyl]-4-phenylquinoline (Tris-Q). Bright bluish-green light originating from the active layer was observed in the double-layer device, ITO/PEDOT:PSS/active layer/ETM/Ca/A. The active layer was a mixture of PV-doped 1H-pyrazolo[3, 4-b]quinoline dyes. An OLED device was constructed by employing Molx as an emitter, which gave a deep bluish-green emission with the spectra range of 481–506 nm. The best value of the maximum brightness at approximately 1436.0 cd/m2 was achieved for a diode based on Mol3 (1-phenyl-3-phenyl-6-CF3-1H-pyrazolo[3,4-b]quinoline) and [R1 = Ph, R3 = Ph and R6 = CF3]. The current efficiency was up to 1.26 cd/A at 506 nm with a CIE of 0.007, 0.692.

Starch is a natural polysaccharide for which the technological quality depends on the genetic basis of the plant and the environmental conditions of the cultivation. Growing plants under cover without soil has many advantages for controlling the above-mentioned conditions. The present research focuses on determining the effect of under cover hydroponic potato cultivation on the physicochemical properties of accumulated potato starch (PS). The plants were grown in the hydroponic system, with (greenhouse, GH) and without recirculation nutrient solution (foil tunnel, FT). The reference sample was PS isolated from plants grown in a tunnel in containers filled with mineral soil (SO). The influence of the cultivation method on the elemental composition of the starch molecules was noted. The cultivation method also influenced the protein and amylose content of the PS. Considering the chromatic parameters, PS-GH and PS-FT were brighter and whiter, with a tinge of blue, than PS-SO. PS-SO was also characterized by the largest average diameters of granules, while PS-GH had the lowest crystallinity. PS-SO showed a better resistance to the combined action of elevated temperature and shear force. There was a slight variation in the gelatinization temperature values. Additionally, significant differences for enthalpy and the retrogradation ratio were observed. The cultivation method did not influence the glass transition and melting.

Targeted therapies are a promising alternative to conventional chemotherapy, with an increasing number of therapeutics targeting specific molecular aberrancies in cancer cells. One of the emerging targets for directed cancer treatments is fibroblast growth factor receptors (FGFRs), which are known to be involved in the pathogenesis and progression of multiple cancer types, specially in lung, bladder, and breast cancers. Here, we are demonstrating the development of the FGFR1-targeting agent based on the interactome screening approach, based on the isolation of binding regions from ligands interacting with the receptor. The parallel analysis by FGFR1 pull-down of chymotryptic peptides coupled with MS analysis, and PepSpot analysis yielded equivalent peptide sequences from FGF4, one of the FGFR1 ligands. Three sequences served as a basis for peptibody (Fc-fusion) generation, to overcome clinical limitations of peptidic agents, and two of them showed favorable FGFR1-binding in vitro and FGFR1-dependent internalization into cells. To validate if developed FGFR1-targeting peptibodies can be used for drug delivery, similar to the well-established concept of antibody–drug conjugates (ADCs), peptibodyF4_1 was successfully conjugated with monomethylauristatin E (MMAE), and has shown significant and specific toxicity toward FGFR1-expressing lung cancer cell lines, with nanomolar EC50 values. Essentially, the development of new effective FGFR1 binders that comprise the naturally occurring FGFR-recognition peptides and Fc region ensuring high plasma stability, and long bloodstream circulation is an interesting strategy expanding targeted anticancer agents’ portfolio. Furthermore, identifying peptides effectively binding the receptor from sequences of its ligands is not limited to FGFRs and is an approach versatile enough to be a basis for a new peptide/peptibodies development strategy.

Structure-selective endonucleases cleave branched DNA substrates. Slx1 is unique among structure-selective nucleases because it can cleave all branched DNA structures at multiple sites near the branch point. The mechanism behind this broad range of activity is unknown. The present study structurally and biochemically investigated fungal Slx1 to define a new protein interface that binds the non-cleaved arm of branched DNAs. The DNA arm bound at this new site was positioned at a sharp angle relative to the arm that was modeled to interact with the active site, implying that Slx1 uses DNA bending to localize the branch point as a flexible discontinuity in DNA. DNA binding at the new interface promoted a disorder-order transition in a region of the protein that was located in the vicinity of the active site, potentially participating in its formation. This appears to be a safety mechanism that ensures that DNA cleavage occurs only when the new interface is occupied by the non-cleaved DNA arm. Models of Slx1 that interacted with various branched DNA substrates were prepared. These models explain the way in which Slx1 cuts DNA toward the 3′ end away from the branch point and elucidate the unique ability of Slx1 to cleave various DNA structures.

Holliday junctions (HJs) are four-way DNA structures that occur in DNA repair by homologous recombination. Specialized nucleases, termed resolvases, remove (i.e., resolve) HJs. The bacterial protein RuvC is a canonical resolvase that introduces two symmetric cuts into the HJ. For complete resolution of the HJ, the two cuts need to be tightly coordinated. They are also specific for cognate DNA sequences. Using a combination of structural biology, biochemistry, and a computational approach, here we show that correct positioning of the substrate for cleavage requires conformational changes within the bound DNA. These changes involve rare high-energy states with protein-assisted base flipping that are readily accessible for the cognate DNA sequence but not for non-cognate sequences. These conformational changes and the relief of protein-induced structural tension of the DNA facilitate coordination between the two cuts. The unique DNA cleavage mechanism of RuvC demonstrates the importance of high-energy conformational states in nucleic acid readouts., Holliday junctions (HJs) are four-way DNA structures that occur in DNA repair by homologous recombination which are removed by nucleases such as RuvC. Here the authors provide structural and biochemical details on the steps required for the completion of the process.

Overexpression of fibroblast growth factor receptor 1 (FGFR1) is a common aberration in lung and breast cancers and has necessitated the design of drugs targeting FGFR1-dependent downstream signaling and FGFR1 ligand binding. To date, the major group of drugs being developed for treatment of FGFR1-dependent cancers are small-molecule tyrosine kinase inhibitors; however, the limited specificity of these drugs has led to increasing attempts to design molecules targeting the extracellular domain of FGFR1. Here, we used the phage display technique to select cyclic peptides F8 (ACSLNHTVNC) and G10 (ACSAKTTSAC) as binders of the fibroblast growth factor 1 (FGF1)-FGFR1 interface. ELISA and in vitro cell assays were performed to reveal that cyclic peptide F8 is more effective in preventing the FGF1-FGFR1 interaction, and also decreases FGF1-induced proliferation of BA/F3 FGFR1c cells by over 40%. Such an effect was not observed for BA/F3 cells lacking FGFR1. Therefore, cyclic peptide F8 can act as a FGF1-FGFR1 interaction antagonist, and may be suitable for further development for potential use in therapies against FGFR1-expressing cancer cells.

Recent studies showed that SARS-CoV-2 can infect adult human pancreas and trigger pancreatic damage. Here, using human fetal pancreas samples and 3D differentiation of human pluripotent cells into pancreatic endocrine cells, we determined that SARS-CoV-2 receptors ACE2, TMPRSS2, and NRP1 are expressed in precursors of insulin-producing pancreatic β-cells, rendering them permissive to SARS-CoV-2 infection. We also show that SARS-CoV-2 enters and undergoes efficient replication in human multipotent pancreatic and endocrine progenitors

Ectotherms can become physiologically challenged when performing oxygen-demanding activities (e.g., flight) across differing environmental conditions, specifically temperature and oxygen levels. Achieving a balance between oxygen supply and demand can also depend on the cellular composition of organs, which either evolves or changes plastically in nature, however, this hypothesis has rarely been examined, especially in tracheated flying insects. The relatively large cell membrane area of small cells should increase the rates of oxygen and nutrient fluxes in cells, however, it does also increase the costs of cell membrane maintenance. To address the effects of cell size on flying insects, we measured the wing-beat frequency in two cell-size phenotypes of Drosophila melanogaster when flies were exposed to two temperatures (warm/hot) combined with two oxygen conditions (normoxia/hypoxia). The cell-size phenotypes were induced by rearing 15 isolines on either standard food (large cells) or rapamycin-enriched food (small cells). Rapamycin supplementation (downregulation of TOR activity) produced smaller flies with smaller wing epidermal cells. Flies generally flapped their wings at a slower rate in cooler (warm treatment) and less-oxygenated (hypoxia) conditions, but the small-cell-phenotype flies were less prone to oxygen limitation than the large-cell-phenotype flies and did not respond to the different oxygen conditions under the warm treatment. We suggest that ectotherms with small-cell life strategies can maintain physiologically demanding activities (e.g., flight) when challenged by oxygen-poor conditions, but this advantage may depend on the correspondence among body temperatures, acclimation temperatures and physiological thermal limits.

Despite the fact that there is a growing awareness to the callosal connections between hemispheres the two hemispheres of the brain are commonly treated as independent structures when peripheral or cortical manipulations are applied to one of them. The contralateral hemisphere is often used as a within-animal control of plastic changes induced onto the other side of the brain. This ensures uniform conditions for producing experimental and control data, but it may overlook possible interhemispheric interactions. In this paper we provide, for the first time, direct proof that cortical, experience-dependent plasticity is not a unilateral, independent process. We mapped metabolic brain activity in rats with 2-[14C] deoxyglucose (2DG) following experience-dependent plasticity induction after a month of unilateral (left), partial whiskers deprivation (only row B was left). This resulted in ∼45% widening of the cortical sensory representation of the spared whiskers in the right, contralateral barrel field (BF). We show that the width of 2DG visualized representation is less than 20% when only contralateral stimulation of the spared row of whiskers is applied in immobilized animals. This means that cortical map remodeling, which is induced by experience-dependent plasticity mechanisms, depends partially on the contralateral hemisphere. The response, which is observed by 2DG brain mapping in the partially deprived BF after standard synchronous bilateral whiskers stimulation, is therefore the outcome of at least two separately activated plasticity mechanisms. A focus on the integrated nature of cortical plasticity, which is the outcome of the emergent interactions between deprived and non-deprived areas in both hemispheres may have important implications for learning and rehabilitation. There is also a clear implication that there is nothing like “control hemisphere” since any plastic changes in one hemisphere have to have influence on functioning of the opposite one.

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials. One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

A chronic inability to maintain blood glucose homeostasis leads to diabetes, which can damage multiple organs. The pancreatic islets regulate blood glucose levels through the coordinated action of islet cell-secreted hormones, with the insulin released by β-cells playing a crucial role in this process. Diabetes is caused by insufficient insulin secretion due to β-cell loss, or a pancreatic dysfunction. The restoration of a functional β-cell mass might, therefore, offer a cure. To this end, major efforts are underway to generate human β-cells de novo, in vitro, or in vivo. The efficient generation of functional β-cells requires a comprehensive knowledge of pancreas development, including the mechanisms driving cell fate decisions or endocrine cell maturation. Rapid progress in single-cell RNA sequencing (scRNA-Seq) technologies has brought a new dimension to pancreas development research. These methods can capture the transcriptomes of thousands of individual cells, including rare cell types, subtypes, and transient states. With such massive datasets, it is possible to infer the developmental trajectories of cell transitions and gene regulatory pathways. Here, we summarize recent advances in our understanding of endocrine pancreas development and function from scRNA-Seq studies on developing and adult pancreas and human endocrine differentiation models. We also discuss recent scRNA-Seq findings for the pathological pancreas in diabetes, and their implications for better treatment.

Fibroblast growth factor receptors (FGFRs) are emerging targets for directed cancer therapy. Presented here is a new FGFR1-targeting conjugate, the peptibodyF2, which employs peptibody, a fusion of peptide and the Fc fragment of human IgG as a selective targeting agent and drug carrier. Short peptide based on FGF2 sequence was used to construct a FGFR1-targeting peptibody. We have shown that this peptide ensures specific delivery of peptibodyF2 into FGFR1-expressing cells. In order to use peptibodyF2 as a delivery vehicle for cytotoxic drugs, we have conjugated it with MMAE, a drug widely used in antibody&ndash, drug conjugates for targeted therapy. Resulting conjugate shows high and specific cytotoxicity towards FGFR1-positive cells, i.e., squamous cell lung carcinoma NCI-H520, while remaining non-toxic for FGFR1-negative cells. Such peptibody&ndash, drug conjugate can serve as a basis for development of therapy for tumors with overexpressed or malfunctioning FGFRs.

This paper summarises a little over 100 years of research on the synthesis and the photophysical and biological properties of 1H-pyrazolo[3,4-b]quinolines that was published in the years 1911–2021. The main methods of synthesis are described, which include Friedländer condensation, synthesis from anthranilic acid derivatives, multicomponent synthesis and others. The use of this class of compounds as potential fluorescent sensors and biologically active compounds is shown. This review intends to summarize the abovementioned aspects of 1H-pyrazolo[3,4-b]quinoline chemistry. Some of the results that are presented in this publication come from the laboratories of the authors of this review.

Andrzej Kisielewicz’s praise of common senseI present here the evaluation and comments on the position of Andrzej Kisi­elewicz presented in his latest book, Logic and Arguments: A Practical Course in Critical Thinking. I recognize that this book can be of great benefit to a very diverse group of potential recipients. It is original in design, inspiring and even provoking the reader to discuss some of its main theses. I am debating some of the author’s views, in particular I draw attention to the relationship between logic and formal logic presented by Kisielewicz. I point to the similarity of some of the radical assessments presented in Logic and Arguments to one of the criticisms by Kazimierz Twardowski published a hundred years earlier.

The manuscript comprises the analysis of several photophysical (absorption and emission spectra, quantum efficiency, fluorescence life times) and electrochemical (oxidation potentials) properties of four fluorine-substituted 1,3-diphenyl-1 H -pyrazolo[3,4- b ]quinoline ( PQ ) fluorescent dyes. The experimental results are compared with those obtained by DFT calculations. Introduction of fluorine atoms in 1 H -pyrazolo[3,4- b ]quinoline molecule modifies some of its properties, like fluorescence quantum efficiency, HOMO and LUMO levels, absorption band position. The basicity of PQ molecule has been tuned by distance dependent electron-withdrawing effect of fluorine. This leads to the increased resistance of PQ fluorine derivatives to proton donors. The high fluorescence yield of 8-fluoro-1,3-diphenyl-1 H -pyrazolo[3,4- b ]quinoline is preserved for 0.1 M TFA concentration, which is enough to quench the emission of unsubstituted 1,3-diphenyl-1 H -pyrazolo[3,4- b ]quinoline.

A series of 6-substituted-1,3-diphenyl-1H-pyrazolo[3,4-b]quinoxalines were prepared using a new synthetic pathway: reductive cyclization of appropriate 5-(o-nitrophenyl)-pyrazoles with ferrous oxalate or triphenylphosphine. The main advantage of this procedure is that, contrary to the older protocols of pyrazolo[3,4-b]quinoxaline synthesis, this method allows for a substituent to be introduced to the carbocyclic ring without the formation of isomers. The pyrazole ring can also be modified to some extent. Furthermore, we propose a new mechanism for the oldest reported pyrazolo[3,4-b]quinoxaline synthesis, based on the condensation between o-phenylenediamine and 3,4-pyrazolin-5-diones.

In this work, we report on second order nonlinear optical (NLO) susceptibilities of some substituted 4-(5-nitro-1,3-benzoxazol-2-yl)aniline chromophores that were embedded into photopolymer matrices. Depending on the photo-induced nitrogen laser (wavelength 371 nm) power density of photo-solidification we found an optimal photo-solidification parameter to achieve maximum second harmonic generation (SHG). The influence of the π-conjugated bonds on the output second order susceptibility and HOMO-LUMO level positions is analysed. The performed experiments are compared with the theoretically calculated microscopic hyper-polarizabilities obtained within the B3LYP DFT approach. The possible operation by the output SHG efficiency versus the photoinduced laser power density is presented. The role of the intra-molecular and surrounding polymers in the observed nonlinear optical behaviour is explored.

Mixed acidic-alkaline refluxate is a major pathogenic factor in chronic esophagitis progressing to Barrett's esophagus (BE). We hypothesized that epidermal growth factor (EGF) can interact with COX-2 and peroxisome proliferator-activated receptor-γ (PPARγ) in rats surgically prepared with esophagogastroduodenal anastomosis (EGDA) with healthy or removed salivary glands to deplete salivary EGF. EGDA rats were treated with 1) vehicle, 2) EGF or PPARγ agonist pioglitazone with or without EGFR kinase inhibitor tyrphostin A46, EGF or PPARγ antagonist GW9662 respectively, 3) ranitidine or pantoprazole, and 4) the selective COX-2 inhibitor celecoxib combined with pioglitazone. At 3 mo, the esophageal damage and the esophageal blood flow (EBF) were determined, the mucosal expression of EGF, EGFR, COX-2, TNFα, and PPARγ mRNA and phospho-EGFR/EGFR protein was analyzed. All EGDA rats developed chronic esophagitis, esophageal ulcerations, and intestinal metaplasia followed by a fall in the EBF, an increase in the plasma of IL-1β, TNFα, and mucosal PGE2content, the overexpression of COX-2-, and EGF-EGFR mRNAs, and proteins, and these effects were aggravated by EGF and attenuated by pioglitazone. The rise in EGF and COX-2 mRNA was inhibited by pioglitazone but reversed by pioglitazone cotreated with GW9662. We conclude that 1) EGF can interact with PG/COX-2 and the PPARγ system in the mechanism of chronic esophagitis; 2) the deleterious effect of EGF involves an impairment of EBF and the overexpression of COX-2 and EGFR, and 3) agonists of PPARγ and inhibitors of EGFR may be useful in the treatment of chronic esophagitis progressing to BE.NEW & NOTEWORTHY Rats with EGDA exhibited chronic esophagitis accompanied by a fall in EBF and an increase in mucosal expression of mRNAs for EGF, COX-2, and TNFα, and these effects were exacerbated by exogenous EGF and reduced by removal of a major source of endogenous EGF with salivectomy or concurrent treatment with tyrphostin A46 or pioglitazone combined with EGF. Beneficial effects of salivectomy in an experimental model of BE were counteracted by PPARγ antagonist, whereas selective COX-2 inhibitor celecoxib synergistically with pioglitazone reduced severity of esophageal damage and protected esophageal mucosa from reflux. We propose the cross talk among EGF/EGFR, PG/COX-2, and proinflammatory cytokines with PPARγ pathway in the mechanism of pathogenesis of chronic esophagitis progressing to BE and EAC.

The manuscript comprises the analysis of pyrazole derivatives: (5-methyl-4-nitro-2-phenyl-2H-pyrazol-3-yl)-phenyl-amines and (2H-pyrazol-3-yl)-(2-nitrophenyl)-amines molecular structure as obtained for from single-crystal X-ray diffraction measurements and DFT calculations. High temperature requirements for the reductive cyclization of the derivatives, leading to substituted 1H-pyrazolo[3,4-b]quinoxalines were discussed and explained. Our results show that one of the important factors responsible for low reactivity of the studied compounds is the existence of intramolecular hydrogen bonds. Alternative conditions for the synthesis involving microwave irradiation were proposed and tested within the study.

Fibroblast growth factor 1 (FGF1) has been shown to interact with integrin &alpha, v&beta, 3 through a specific binding site, involving Arg35 residue. The FGF1 mutant (R35E) with impaired integrin binding was found to be defective in its proliferative response, although it was still able to interact with FGF receptors (FGFR) and heparin and induce the activation of downstream signaling pathways. Here, we demonstrate that the lack of mitogenic potential of R35E mutant is directly caused by its decreased thermodynamic stability and susceptibility to proteolytic degradation. Introduction of three stabilizing mutations into R35E variant compensated the effect of destabilizing R35E mutation and restored the proliferation potential of FGF1. Moreover, the stabilized R35E variant regained both anti-apoptotic and wound healing activities, while remaining defective in binding to integrin &alpha, 3. Our results suggest that the thermodynamic stability and resistance to degradation, rather than the interaction with integrin are required for mitogenic response of FGF1.

Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are members of the Hsp100 family of ring-forming hexameric AAA+ chaperones that promote the solubilization of aggregated proteins and the propagation of prions. ClpB and Hsp104 cooperate with cognate Hsp70 chaperones for substrate targeting and activation of ATPase and substrate threading, achieved by transient Hsp70 binding to the repressing ClpB/Hsp104 M-domain. Fundamental differences in ATPase regulation and disaggregation mechanisms have been reported; however, these differences are raising doubts regarding the working principle of this AAA+ chaperone. In particular, unique functional plasticity was suggested to specifically enable Hsp104 to circumvent Hsp70 requirement for derepression in protein disaggregation and prion propagation. We show here that both ClpB and Hsp104 cooperation with Hsp70 is crucial for efficient protein disaggregation and, in contrast to earlier claims, cannot be circumvented by activating M-domain mutations. Activation of ClpB and Hsp104 requires two signals, relief of M-domain repression and substrate binding, leading to increased ATPase subunit coupling. These data demonstrate that ClpB and Hsp104 operate by the same basic mechanism, underscore a dominant function of Hsp70 in regulating ClpB/Hsp104 activity, and explain a plethora of in vivo studies showing a crucial function of Hsp70 in proteostasis and prion propagation.

Anna Szlachcic, Malgorzata Zakrzewska, Michal Lobocki, Piotr Jakimowicz, Jacek Otlewski Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Abstract: Fibroblast growth factor receptors (FGFRs) are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V), was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE), and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE) upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. Keywords: fibroblast growth factor 1, FGF receptor, targeted cancer therapy, cytotoxic conjugates, FGFR-dependent cancer, MMAE, auristatin

Anna Szlachcic, Malgorzata Zakrzewska, Michal Lobocki, Piotr Jakimowicz, Jacek Otlewski Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Abstract: Fibroblast growth factor receptors (FGFRs) are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V), was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE), and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE) upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. Keywords: fibroblast growth factor 1, FGF receptor, targeted cancer therapy, cytotoxic conjugates, FGFR-dependent cancer, MMAE, auristatin

Purpose– Narrative therapy uses psychological thinking to help people reconnect with important aspects of their lives that have become subjugated by a dominant problem story. Collective narrative practices (CNPs) such as the tree of life (ToL) metaphor apply these principles to communities. Limited research suggests that ToL groups benefit service users and staff in inpatient settings. The purpose of this paper is to explore the utility of a pilot ToL group, run in an inpatient treatment ward.Design/methodology/approach– Nine participants attended a seven-week ToL group. Eight service users and two facilitators contributed to the evaluation. Qualitative data were derived from interviews and questionnaires and analysed from a social constructionist perspective using inductive thematic analysis.Findings– Three themes were generated: first, a sense of community – service user and staff relationships developed both inside and outside of the group, through sharing personal experiences. Second, rediscovering identity – participants reconnected with important aspects of their lives, which helped them to plan their recovery. Third, usefulness of the metaphor – service users and staff reflected on useful aspects of the ToL metaphor and how groups could be improved in the future. Data collection methods were key limitations of the study. Further groups should be conducted, whilst considering means of developing CNPs and their evaluation in this setting.Originality/value– Few evaluations of ToL groups have been conducted in inpatient settings. This research outlines considerations for setting up and evaluating ToL groups and highlights their value for service users and staff.

The objective of this study was to identify the relationship between metabolic syndrome (MetS) and pressure ulcers (PrU), after controlling for demographic and injury characteristics, socioeconomic factors, health behaviors and fatigue among participants with spinal cord injury (SCI). This cross-sectional study recruited 350 participants with SCI from a hospital in the western region of the USA. Blood tests and physical examination were performed. Waist circumference, high-density cholesterol, triglycerides, blood pressure and fasting glucose were used to diagnose MetS according to Criteria for Clinical Diagnosis of Metabolic Syndrome defined by the American Heart Association. All other variables were self-reported. Three-stage multivariate logistic regression models were conducted to evaluate the effects of three sequential sets of predictors, including demographic/injury, socio-environmental/behavioral and health factors, basing the order of analysis on the Theoretical Risk and Prevention Model. The prevalence of PrU and MetS was 11.0% (n=36) and 35.3% (n=115), respectively. Ethnicity, smoking, alcohol consumption and MetS were statistically associated with PrU in the full model. Participants who were non-Hispanic (odds ratio (OR)=10.30, 95% confidence interval (CI): 3.46–30.65), smokers (OR=2.69, 95% CI: 1.00–7.27) and drank over 30 drinks per month (OR=5.26, 95% CI: 1.24–22.26) had greater odds of having a PrU compared with those who were Hispanic, non-smokers and non-drinkers, respectively. We also observed a positive association between MetS and PrU (ORMetS=3.71, 95% CI: 1.45–9.52), even after controlling for all other factors. Participants who had MetS had higher odds of PrU than those without MetS after adjusting for multiple covariates. Unhealthy behaviors such as smoking and excess drinking were positively associated with PrU.

Two pyrazoline-based fluorescent and colorimetric probes have been synthesized and their photophysical properties have been investigated by means of electronic absorption and emission spectroscopy. The compounds differ from each other by the presence of a phenyl or thiophene end group attached to the α,β-unsaturated ketone. The probes detect sulphite anions in the Michael addition reaction to the α,β-unsaturated ketone with a high selectivity and good sensitivity (7.56 μM for phenyl and 4.87 μM for the thiophene counterpart). Here, the optical response is based on the recovery of triphenylpyrazoline fluorescence, which is largely blue-shifted as compared to weak charge transfer emission of the sensors. This large hypsochromic shift of the emission maximum along with a strong fluorescence enhancement (up to Isulphite/I0 = 43) can be an advantage in terms of the accurate evaluation of the fluorescence intensity ratio. The pyrazoline-based sensor decorated with thiophene is able to detect sulphite species in river water solutions with a good selectivity and sensitivity.

The work demonstrates the photophysical properties of 1-(2-pyridyl)-3-phenyl-pyrazolo[3,4-b]quinoline with the focus on the effect of pyridyl substituent on excited-state deactivation of the pyrazoloquinoline core, and on the molecular logic gate implementation. The deactivation mechanism of the singlet excited state was investigated by means of UV-vis absorption, steady-state and time-resolved fluorescence spectroscopy as well as nanosecond laser flash photolysis. The maximum of absorption (emission) in nonpolar cyclohexane is located at 385 nm (421 nm) and only slightly depends on solvent polarity. The compound exhibits a moderate fluorescence (up to 42%) and a low-temperature phosphorescence (77 K). The studies reveal that the pyridyl group modifies the deactivation mechanism by triggering efficient intersystem crossing. Moreover, the effect of various chemical species: proton, electron-donating amine, and dihydrogenphosphate anion on the absorption and emission of the investigated dye was evaluated. The interpretation of the fluorescence behaviour in the presence or absence of the chemical inputs leads to the realization of unimolecular logic circuits that are able to perform advanced arithmetic operations, such as subtraction (XOR/INH) or IMP/INH logic circuit. The experimental results were supported by quantum-chemical calculations.

The analysis of literature revealed a certain lack of knowledge concerning Cornelian pectins, as well as a significant gap in the description of properties of apple pectin, and therefore the purpose of this study was to examine and compare the physicochemical properties of these compounds. Pectins from both sources were obtained using water extraction. Mass distribution was determined for each solution, physicochemical properties were measured using osmometry, DLS, and intrinsic viscosity; Atomic Force Microscopy (AFM) photos of the particles were recorded, together with their FT-IR spectra, and the ζ-potential was measured. Additionally both compounds were compared by X-ray diffraction revealing the amorphousness of the samples. Both polysaccharides exhibit polyion properties, but the results reveal considerable differences between them: industry-grade apple pectins have higher weight average molecular mass with strong dispersion, the Cornelian cherry pectins, unlike industry-grade apple ones, are poorly soluble in water. The Cornelian pectins’ inability to aggregate is quite an unexpected behaviour, as is the presence of protein complexes, which are absent in industry-grade apple pectin.

HD is mainly a late-onset disorder, however, subtle symptoms in patients may occur years or even decades prior to diagnosis. Such changes at a molecular level may begin much earlier, even in stem cells. Here, we present a study defining the transcriptional profiles and early HD alterations in human HD-iPS cell lines. For the RNA sequencing analysis, we used three clonal HD lines with 71 CAG repeats, three juvenile HD clonal lines with 109 CAG repeats and control lines. HD-iPSC lines (71Q and 109Q) were compared with control lines where 82 significantly deregulated mRNAs were identified (30 downregulated and 52 upregulated). In addition 71Q lines were compared with control lines yielding 113 significantly deregulated mRNAs (33 downregulated and 80 upregulated). In the last group, in which 109Q lines were compared with control lines, 169 significantly deregulated mRNAs were identified (90 downregulated and 79 upregulated). The analysis revealed mRNAs which occurred in both HD lines (ex. OTOGL, TRIM69) but also many unique mRNAs, deregulated in 71Q (ex. PIWIL2, HIST1H3C) or HD109Q-iPSC lines (ex. TP53, CDKN1A). RNA sequencing was also focused on circular RNA (circRNA) profiling. Nearly 100 significantly deregulated circRNAs were identified, also showing many transcriptomic differences between 71Q and 109Q lines. The high-throughput RNA screening was followed by bioinformatics analyses, such as differential expression analysis and also over-representation and enrichment analyses which demonstrated several affected biological processes in iPSC. These processes were related with central nervous system development, disruption of the apoptosis pathway, enhanced DNA methylation and negative regulation of Wnt signaling pathway. RNA-seq and in silico analyses were then followed by experimental validation of the most deregulated and the most interesting mRNAs and digital droplet PCR for chosen circRNAs.

In Huntington disease (HD) subtle symptoms in patients may occur years or even decades prior to diagnosis. HD changes at a molecular level may begin as early as in cells that are non-lineage committed such as stem cells or HD patients induced pluripotent stem cells (iPSCs) offering opportunity to enhance the understanding of the HD pathogenesis. In addition, juvenile HD non-linage committed cells were previously not directly investigated in detail by RNA-seq. In the present manuscript, we define the early HD and juvenile HD transcriptional alterations using 6 human HD iPS cell lines from two patients, one with 71 CAGs and one with 109 CAG repeats. We identified 107 (6 HD lines), 198 (3 HD71Q lines) and 217 (3 HD109Q lines) significantly dysregulated mRNAs in each comparison group. The analyses showed that many of dysregulated transcripts in HD109Q iPSC lines are involved in DNA damage response and apoptosis, such as CCND1, CDKN1A, TP53, BAX, TNFRSF10B, TNFRSF10C, TNFRSF10D, DDB2, PLCB1, PRKCQ, HSH2D, ZMAT3, PLK2, and RPS27L. Most of them were identified as downregulated and their proteins are direct interactors with TP53. HTT probably alters the level of several TP53 interactors influencing apoptosis. This may lead to accumulation of an excessive number of progenitor cells and potential disruption of cell differentiation and production of mature neurons. In addition, HTT effects on cell polarization also demonstrated in the analysis may result in a generation of incorrect progenitors. Bioinformatics analysis of transcripts dysregulated in HD71Q iPSC lines showed that several of them act as transcription regulators during the early multicellular stages of development, such as ZFP57, PIWIL2, HIST1H3C, and HIST1H2BB. Significant upregulation of most of these transcripts may lead to a global increase in expression level of genes involved in pathways critical for embryogenesis and early neural development. In addition, MS analysis revealed altered levels of TP53 and ZFP30 proteins reflecting the functional significance of dysregulated mRNA levels of these proteins which were associated with apoptosis and DNA binding. Our finding very well corresponds to the fact that mutation in the HTT gene may cause precocious neurogenesis and identifies pathways likely disrupted during development.

Melatonin is a tryptophan-derived molecule with pleiotropic activities which is produced in all living organisms. This “sleep” hormone is a free radical scavenger, which activates several anti-oxidative enzymes and mechanisms. Melatonin, a highly lipophilic hormone, can reach body target cells rapidly, acting as the circadian signal to alter numerous physiological functions in the body. This indoleamine can protect the organs against a variety of damaging agents via multiple signaling. This review focused on the role played by melatonin in the mechanism of esophagoprotection, starting with its short-term protection against acute reflux esophagitis and then investigating the long-term prevention of chronic inflammation that leads to gastroesophageal reflux disease (GERD) and Barrett’s esophagus. Since both of these condition are also identified as major risk factors for esophageal carcinoma, we provide some experimental and clinical evidence that supplementation therapy with melatonin could be useful in esophageal injury by protecting various animal models and patients with GERD from erosions, Barrett’s esophagus and neoplasia. The physiological aspects of the synthesis and release of this indoleamine in the gut, including its release into portal circulation and liver uptake is examined. The beneficial influence of melatonin in preventing esophageal injury from acid-pepsin and acid-pepsin-bile exposure in animals as well as the usefulness of melatonin and its precursor, L-tryptophan in prophylactic and supplementary therapy against esophageal disorders in humans, are also discussed.

In the rapidly developing field of targeted cancer therapy there is growing interest towards therapeutics combining two or more compounds to achieve synergistic action and minimize the chance of cancer resistance to treatment. We developed a fibroblast growth factor 2 (FGF2)-conjugate bearing two cytotoxic drugs with independent mode of action: &alpha, amanitin and monomethyl auristatin E. Drugs are covalently attached to the targeting protein in a site-specific manner via maleimide-thiol conjugation and Cu(I)-catalyzed alkyne-azide cycloaddition. The dual warhead conjugate binds to FGF receptor 1 (FGFR1) and utilizes receptor-mediated endocytosis for selective internalization into cancer cells with FGFR1. The developed conjugate displays high cytotoxicity towards all tested FGFR1-positive cell lines. Most importantly, the improved cytotoxic effect of both drugs is observed for lung cancer cell line NCI-H446. The single drug-FGF2 conjugates have no impact on the viability of NCI-H446 cells, whereas the dual warhead-FGF2 conjugate selectively and efficiently kills these FGFR1 positive cancer cells. Due to the diversified mode of action the dual warhead-FGF2 conjugate may overcome the potential acquired resistance of FGFR1-overproducing cancer cells towards single cytotoxic drugs.

In vitro derivation of pancreatic β-cells from human pluripotent stem cells holds promise as diabetes treatment. Despite recent progress, efforts to generate physiologically competent β-cells are still hindered by incomplete understanding of the microenvironment’s role in β-cell development and maturation. Here, we analyze the human mesenchymal and endothelial primary cells from weeks 9-20 fetal pancreas and identify a time point-specific microenvironment that permits β-cell differentiation. Further, we uncover unique factors that guide in vitro development of endocrine progenitors, with WNT5A markedly improving human β-cell differentiation. WNT5A initially acts through the non-canonical (JNK/c-JUN) WNT signaling and cooperates with Gremlin1 to inhibit the BMP pathway during β-cell maturation. Interestingly, we also identify the endothelial-derived Endocan as a SST+ cell promoting factor. Overall, our study shows that the pancreatic microenvironment-derived factors can mimic in vivo conditions in an in vitro system to generate bona fide β-cells for translational applications.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are human neuromuscular disorders associated with mutations of simple repetitive sequences in affected genes. The abnormal expansion of CTG repeats in the 3′-UTR of the DMPK gene elicits DM1, whereas elongated CCTG repeats in intron 1 of ZNF9/CNBP triggers DM2. Pathogenesis of both disorders is manifested by nuclear retention of expanded repeat-containing RNAs and aberrant alternative splicing. The precise determination of absolute numbers of mutant RNA molecules is important for a better understanding of disease complexity and for accurate evaluation of the efficacy of therapeutic drugs. We present two quantitative methods, Multiplex Ligation-Dependent Probe Amplification and droplet digital PCR, for studying the mutant DMPK transcript (DMPKexpRNA) and the aberrant alternative splicing in DM1 and DM2 human tissues and cells. We demonstrate that in DM1, the DMPKexpRNA is detected in higher copy number than its normal counterpart. Moreover, the absolute number of the mutant transcript indicates its low abundance with only a few copies per cell in DM1 fibroblasts. Most importantly, in conjunction with fluorescence in-situ hybridization experiments, our results suggest that in DM1 fibroblasts, the vast majority of nuclear RNA foci consist of a few molecules of DMPKexpRNA.