Type I Interferon Signaling Disrupts the Hepatic Urea Cycle and Alters Systemic Metabolism to Suppress T Cell Function.

Infections induce complex host responses linked to antiviral defense, inflammation, and tissue damage and repair. We hypothesized that the liver, as a central metabolic hub, may orchestrate systemic metabolic changes during infection. We infected mice with chronic lymphocytic choriomeningitis virus (LCMV), performed RNA sequencing and proteomics of liver tissue, and integrated these data with serum metabolomics at different infection phases. Widespread reprogramming of liver metabolism occurred early after infection, correlating with type I interferon (IFN-I) responses. Viral infection induced metabolic alterations of the liver that depended on the interferon alpha/beta receptor (IFNAR1). Hepatocyte-intrinsic IFNAR1 repressed the transcription of metabolic genes, including Otc and Ass1, which encode urea cycle enzymes. This led to decreased arginine and increased ornithine concentrations in the circulation, resulting in suppressed virus-specific CD8+ T cell responses and ameliorated liver pathology. These findings establish IFN-I-induced modulation of hepatic metabolism and the urea cycle as an endogenous mechanism of immunoregulation. VIDEO ABSTRACT.

Newborn Screening for Severe T and B Cell Lymphopenia Using TREC/KREC Detection: A Large-Scale Pilot Study of 202,908 Newborns.

Newborn screening (NBS) for severe inborn errors of immunity (IEI), affecting T lymphocytes, and implementing measurements of T cell receptor excision circles (TREC) has been shown to be effective in early diagnosis and improved prognosis of patients with these genetic disorders. Few studies conducted on smaller groups of newborns report results of NBS that also include measurement of kappa-deleting recombination excision circles (KREC) for IEI affecting B lymphocytes. A pilot NBS study utilizing TREC/KREC detection was conducted on 202,908 infants born in 8 regions of Russia over a 14-month period. One hundred thirty-four newborns (0.66‰) were NBS positive after the first test and subsequent retest, 41% of whom were born preterm. After lymphocyte subsets were assessed via flow cytometry, samples of 18 infants (0.09‰) were sent for whole exome sequencing. Confirmed genetic defects were consistent with autosomal recessive agammaglobulinemia in 1/18, severe combined immunodeficiency - in 7/18, 22q11.2DS syndrome - in 4/18, combined immunodeficiency - in 1/18 and trisomy 21 syndrome - in 1/18. Two patients in whom no genetic defect was found met criteria of (severe) combined immunodeficiency with syndromic features. Three patients appeared to have transient lymphopenia. Our findings demonstrate the value of implementing combined TREC/KREC NBS screening and inform the development of policies and guidelines for its integration into routine newborn screening programs.

Evaluation of a novel 177Lu-labelled therapeutic Affibody molecule with a deimmunized ABD domain and improved biodistribution profile.

PURPOSE: Fusion of Affibody molecules with an albumin-binding domain (ABD) provides targeting agents, which are suitable for radionuclide therapy. To facilitate clinical translation, the low immunogenic potential of such constructs with targeting properties conserved is required. METHODS: The HER2-targeting Affibody molecule ZHER2:2891 was fused with a deimmunized ABD variant and DOTA was conjugated to a unique C-terminal cysteine. The novel construct, PEP49989, was labelled with 177Lu. Affinity, specificity, and in vivo targeting properties of [177Lu]Lu-PEP49989 were characterised. Experimental therapy in mice with human HER2-expressing xenografts was evaluated. RESULTS: The maximum molar activity of 52 GBq/µmol [177Lu]Lu-PEP49989 was obtained. [177Lu]Lu-PEP49989 bound specifically to HER2-expressing cells in vitro and in vivo. The HER2 binding affinity of [177Lu]Lu-PEP49989 was similar to the affinity of [177Lu]Lu-ABY-027 containing the parental ABD035 variant. The renal uptake of [177Lu]Lu-PEP49989 was 1.4-fold higher, but hepatic and splenic uptake was 1.7-2-fold lower than the uptake of [177Lu]Lu-ABY-027. The median survival of xenograft-bearing mice treated with 21 MBq [177Lu]Lu-PEP49989 (> 90 days) was significantly longer than the survival of mice treated with vehicle (38 days) or trastuzumab (45 days). Treatment using a combination of [177Lu]Lu-PEP49989 and trastuzumab increased the number of complete tumour remissions. The renal and hepatic toxicity was minimal to mild. CONCLUSION: In preclinical studies, [177Lu]Lu-PEP49989 demonstrated favourable biodistribution and a strong antitumour effect, which was further enhanced by co-treatment with trastuzumab.

Comparative Preclinical Evaluation of HYNIC-Modified Designed Ankyrin Repeat Proteins G3 for the 99mTc-Based Imaging of HER2-Expressing Malignant Tumors.

HER2 status determination is a necessary step for the proper choice of therapy and selection of patients for the targeted treatment of cancer. Targeted radiotracers such as radiolabeled DARPins provide a noninvasive and effective way for the molecular imaging of HER2 expression. This study aimed to evaluate tumor-targeting properties of three 99mTc-labeled DARPin G3 variants containing Gly-Gly-Gly-Cys (G3C), (Gly-Gly-Gly-Ser)3-Cys ((G3S)3C), or Glu-Glu-Glu-Cys (E3C) amino acid linkers at the C-terminus and conjugated to the HYNIC chelating agent, as well as to compare them with the clinically evaluated DARPin G3 labeled with 99mTc(CO)3 using the (HE)3-tag at the N-terminus. The labeling of DARPin G3-HYNIC variants provided radiochemical yields in the range of 50-80%. Labeled variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 0.5-3 nM. There was no substantial influence of the linker and HYNIC chelator on the binding of 99mTc-labeled DARPin G3 variants to HER2 in vitro; however, [99mTc]Tc-G3-(G3S)3C-HYNIC had the highest affinity. Comparative biodistribution of [99mTc]Tc-G3-G3C-HYNIC, [99mTc]Tc-G3-(G3S)3C-HYNIC, [99mTc]Tc-G3-E3C-HYNIC, and [99mTc]Tc-(HE)3-G3 in healthy CD1 mice showed that there was a strong influence of the linkers on uptake in normal tissues. [99mTc]Tc-G3-E3C-HYNIC had an increased retention of activity in the liver and the majority of other organs compared to the other conjugates. The tumor uptake of [99mTc]Tc-G3-(G3S)3C-HYNIC and [99mTc]Tc-(HE)3-G3 in Nu/j mice bearing SKOV-3 xenografts was similar. The specificity of tumor targeting in vivo was demonstrated for both tracers. [99mTc]Tc-G3-(G3S)3C-HYNIC provided comparable, although slightly lower tumor-to-lung, tumor-to spleen and tumor-to-liver ratios than [99mTc]Tc-(HE)3-G3. Radiolabeling of DARPin G3-HYNIC conjugates with 99mTc provided the advantage of a single-step radiolabeling procedure; however, the studied HYNIC conjugates did not improve imaging contrast compared to the 99mTc-tricarbonyl-labeled DARPin G3. At this stage, [99mTc]Tc-(HE)3-G3 remains the most promising candidate for the clinical imaging of HER2-overexpressing cancers.

Influence of Molecular Design on the Tumor Targeting and Biodistribution of PSMA-Binding Tracers Labeled with Technetium-99m.

Previously, we designed the EuK-based PSMA ligand BQ0413 with an maE3 chelator for labeling with technetium-99m. It showed efficient tumor targeting, but our preclinical data and preliminary clinical results indicated that the renal excretion levels need to be decreased. We hypothesized that this could be achieved by a decrease in the ligand's total negative charge, achieved by substituting negatively charged glutamate residues in the chelator with glycine. The purpose of this study was to evaluate the tumor targeting and biodistribution of two new PSMA inhibitors, BQ0411 and BQ0412, compared to BQ0413. Conjugates were radiolabeled with Tc-99m and characterized in vitro, using PC3-pip cells, and in vivo, using NMRI and PC3-pip tumor-bearing mice. [99mTc]Tc-BQ0411 and [99mTc]Tc-BQ0412 demonstrated PSMA-specific binding to PC3-pip cells with picomolar affinity. The biodistribution pattern for the new conjugates was characterized by rapid excretion. The tumor uptake for [99mTc]Tc-BQ0411 was 1.6-fold higher compared to [99mTc]Tc-BQ0412 and [99mTc]Tc-BQ0413. [99mTc]Tc-BQ0413 has demonstrated predominantly renal excretion, while the new conjugates underwent both renal and hepatobiliary excretion. In this study, we have demonstrated that in such small targeting ligands as PSMA-binding EuK-based pseudopeptides, the structural blocks that do not participate in binding could have a crucial role in tumor targeting and biodistribution. The presence of a glycine-based coupling linker in BQ0411 and BQ0413 seems to optimize biodistribution. In conclusion, the substitution of amino acids in the chelating sequence is a promising method to alter the biodistribution of [99mTc]Tc-labeled small-molecule PSMA inhibitors. Further improvement of the biodistribution properties of BQ0413 is needed.

Preclinical Evaluation of HER2-Targeting DARPin G3: Impact of Albumin-Binding Domain (ABD) Fusion.

Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in several cancers. The use of the DARPin G3 for radionuclide therapy is complicated by its high renal reabsorption after clearance via the glomeruli. We tested the hypothesis that a fusion of the DARPin G3 with an albumin-binding domain (ABD) would prevent rapid renal excretion and high renal reabsorption resulting in better tumour targeting. Two fusion proteins were produced, one with the ABD at the C-terminus (G3-ABD) and another at the N-terminus (ABD-G3). Both variants were labelled with 177Lu. The binding properties of the novel constructs were evaluated in vitro and their biodistribution was compared in mice with implanted human HER2-expressing tumours. Fusion with the ABD increased the retention time of both constructs in blood compared with the non-ABD-fused control. The effect of fusion with the ABD depended strongly on the order of the domains in the constructs, resulting in appreciably better targeting properties of [177Lu]Lu-G3-ABD. Our data suggest that the order of domains is critical for the design of targeting constructs based on scaffold proteins.

Comparison of glycosyl donors: a supramer approach.

The development of new methods for chemical glycosylation commonly includes comparison of various glycosyl donors. An attempted comparison of chemical properties of two sialic acid-based thioglycoside glycosyl donors, differing only in the substituent at O-9 (trifluoroacetyl vs chloroacetyl), at different concentrations (0.05 and 0.15 mol·L-1) led to mutually excluding conclusions concerning their relative reactivity and selectivity, which prevented us from revealing a possible influence of remote protective groups at O-9 on glycosylation outcome. According to the results of the supramer analysis of the reaction solutions, this issue might be related to the formation of supramers of glycosyl donors differing in structure hence chemical properties. These results seem to imply that comparison of chemical properties of different glycosyl donors may not be as simple and straightforward as it is usually considered.

Changes in the tumor oxygenation but not in the tumor volume and tumor vascularization reflect early response of breast cancer to neoadjuvant chemotherapy.

BACKGROUND: Breast cancer neoadjuvant chemotherapy (NACT) allows for assessing tumor sensitivity to systemic treatment, planning adjuvant treatment and follow-up. However, a sufficiently large number of patients fail to achieve the desired level of pathological tumor response while optimal early response assessment methods have not been established now. In our study, we simultaneously assessed the early chemotherapy-induced changes in the tumor volume by ultrasound (US), the tumor oxygenation by diffuse optical spectroscopy imaging (DOSI), and the state of the tumor vascular bed by Doppler US to elaborate the predictive criteria of breast tumor response to treatment. METHODS: A total of 133 patients with a confirmed diagnosis of invasive breast cancer stage II to III admitted to NACT following definitive breast surgery were enrolled, of those 103 were included in the final analysis. Tumor oxygenation by DOSI, tumor volume by US, and tumor vascularization by Doppler US were determined before the first and second cycle of NACT. After NACT completion, patients underwent surgery followed by pathological examination and assessment of the pathological tumor response. On the basis of these, data regression predictive models were created. RESULTS: We observed changes in all three parameters 3 weeks after the start of the treatment. However, a high predictive potential for early assessment of tumor sensitivity to NACT demonstrated only the level of oxygenation, ΔStO2, (ρ = 0.802, p ≤ 0.01). The regression model predicts the tumor response with a high probability of a correct conclusion (89.3%). The "Tumor volume" model and the "Vascularization index" model did not accurately predict the absence of a pathological tumor response to treatment (60.9% and 58.7%, respectively), while predicting a positive response to treatment was relatively better (78.9% and 75.4%, respectively). CONCLUSIONS: Diffuse optical spectroscopy imaging appeared to be a robust tool for early predicting breast cancer response to chemotherapy. It may help identify patients who need additional molecular genetic study of the tumor in order to find the source of resistance to treatment, as well as to correct the treatment regimen.

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway.

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.

Oncogenic TYK2 P760L kinase is effectively targeted by combinatorial TYK2, mTOR and CDK4/6 kinase blockade.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase/signal transducer and activator of transcription pathway, which is central in cytokine signaling. Previously, germline TYK2 mutations have been described in two patients developing de novo T-cell acute lymphoblastic leukemias (T-ALL) or precursor B-ALL. The mutations (P760L and G761V) are located within the regulatory pseudokinase domain and lead to constitutive activation of TYK2. We demonstrate the transformation capacity of TYK2 P760L in hematopoietic cell systems including primary bone marrow cells. In vivo engraftment of TYK2 P760L-expressing cell lines led to development of leukemia. A kinase inhibitor screen uncovered that oncogenic TYK2 acts synergistically with the PI3K/AKT/mTOR and CDK4/6 pathways. Accordingly, the TYK2-specific inhibitor deucravacitinib (BMS986165) reduces cell viability of TYK2 P760L-transformed cell models and ex vivo cultured TYK2 P760L-mutated patient- derived xenograft cells most efficiently when combined with mTOR or CDK4/6 inhibitors. Our study thereby pioneers novel treatment options for patients suffering from TYK2-driven acute leukemia.

Feasibility of Co-Targeting HER3 and EpCAM Using Seribantumab and DARPin-Toxin Fusion in a Pancreatic Cancer Xenograft Model.

Pancreatic cancer (PC) is one of the most aggressive malignancies. A combination of targeted therapies could increase the therapeutic efficacy in tumors with heterogeneous target expression. Overexpression of the human epidermal growth factor receptor type 3 (HER3) and the epithelial cell adhesion molecule (EpCAM) in up to 40% and 30% of PCs, respectively, is associated with poor prognosis and highlights the relevance of these targets. Designed ankyrin repeat protein (DARPin) Ec1 fused with the low immunogenic bacterial toxin LoPE provides specific and potent cytotoxicity against EpCAM-expressing cancer cells. Here, we investigated whether the co-targeting of HER3 using the monoclonal antibody seribantumab (MM-121) and of EpCAM using Ec1-LoPE would improve the therapeutic efficacy in comparison to the individual agents. Radiolabeled 99mTc(CO)3-Ec1-LoPE showed specific binding with rapid internalization in EpCAM-expressing PC cells. MM-121 did not interfere with the binding of Ec1-LoPE to EpCAM. Evaluation of cytotoxicity indicated synergism between Ec1-LoPE and MM-121 in vitro. An experimental therapy study using Ec1-LoPE and MM-121 in mice bearing EpCAM- and HER3-expressing BxPC3 xenografts demonstrated the feasibility of the therapy. Further development of the co-targeting approach using HER3 and EpCAM could therefore be justified.

Preclinical Evaluation of a Novel High-Affinity Radioligand [99mTc]Tc-BQ0413 Targeting Prostate-Specific Membrane Antigen (PSMA).

Radionuclide imaging using radiolabeled inhibitors of prostate-specific membrane antigen (PSMA) can be used for the staging of prostate cancer. Previously, we optimized the Glu-urea-Lys binding moiety using a linker structure containing 2-napththyl-L-alanine and L-tyrosine. We have now designed a molecule that contains mercaptoacetyl-triglutamate chelator for labeling with Tc-99m (designated as BQ0413). The purpose of this study was to evaluate the imaging properties of [99mTc]Tc-BQ0413. PSMA-transfected PC3-pip cells were used to evaluate the specificity and affinity of [99mTc]Tc-BQ0413 binding in vitro. PC3-pip tumor-bearing BALB/C nu/nu mice were used as an in vivo model. [99mTc]Tc-BQ0413 bound specifically to PC3-pip cells with an affinity of 33 ± 15 pM. In tumor-bearing mice, the tumor uptake of [99mTc]Tc-BQ0413 (38 ± 6 %IA/g in PC3-pip 3 h after the injection of 40 pmol) was dependent on PSMA expression (3 ± 2 %IA/g and 0.9 ± 0.3 %IA/g in PSMA-negative PC-3 and SKOV-3 tumors, respectively). We show that both unlabeled BQ0413 and the commonly used binder PSMA-11 enable the blocking of [99mTc]Tc-BQ0413 uptake in normal PSMA-expressing tissues without blocking the uptake in tumors. This resulted in an appreciable increase in tumor-to-organ ratios. At the same injected mass (5 nmol), the use of BQ0413 was more efficient in suppressing renal uptake than the use of PSMA-11. In conclusion, [99mTc]Tc-BQ0413 is a promising probe for the visualization of PSMA-positive lesions using single-photon emission computed tomography (SPECT).

Effects of Treadmill Running at Different Light Cycles in Mice with Metabolic Disorders.

Type 2 diabetes mellitus accounts for about 90% of cases of diabetes and is considered one of the most important problems of our time. Despite a significant number of studies on glucose metabolism, the molecular mechanisms of its regulation in health and disease remain insufficiently studied. That is why non-drug treatment of metabolic disorders is of great relevance, including physical activity. Metabolic changes under the influence of physical activity are very complex and are still difficult to understand. This study aims to deepen the understanding of the effect of physical exercise on metabolic changes in mice with diabetes mellitus. We studied the effect of forced treadmill running on body weight and metabolic parameters in mice with metabolic disorders. We developed a high-fat-diet-induced diabetic model of metabolic disorders. We exposed mice to forced treadmill running for 4 weeks. We determined glucose and insulin levels in the blood plasma biochemically and analyzed Glut-4 and citrate synthase in M. gastrocnemius muscle tissue using Western blotting. The research results show that daily treadmill running has different effects on different age groups of mice with metabolic disorders. In young-age animals, forced running has a more pronounced effect on body weight. At week 12, young obese mice had a 17% decrease in body weight. Body weight did not change in old mice. Moreover, at weeks 14 and 16, the decrease in body weight was more significant in the young mice (by 17%) compared to the old mice (by 6%) (p < 0.05). In older animals, it influences the rate of glucose uptake. At 60 min, the blood glucose in the exercised older mice decreased to 14.46 mmol/L, while the glucose concentration in the non-exercised group remained at 17 mmol/L. By 120 min, in mice subjected to exercise, the blood glucose approached the initial value (6.92 mmol/L) and amounted to 8.35 mmol/L. In the non-exercised group, this difference was 45%. The effects of physical activity depend on the time of day. The greater effect is observed when performing shift training or exercise during the time when animals are passive (light phase). In young mice, light phase training had a significant effect on increasing the content of Glut-4 in muscle tissue (84.3 ± 11.3%, p < 0.05 with control group-59.3 ± 7.8%). In aged mice, shift training caused an increase in the level of Glut-4 in muscle tissue (71.3 ± 4.1%, p < 0.05 with control group-56.4 ± 10,9%). In the group of aged mice, a lower CS level was noticed in all groups in comparison with young mice. It should also be noted that we observed that CS increased during exercise in the group of young mice, especially during light phase training. The CS content in the light phase subgroup (135.8 ± 7.0%) was higher than in the dark phase subgroup (113.3 ± 7.7%) (p = 0.0006). The CS decreased in aged chow-fed mice and increased in the high-fat-fed group. The CS content in the chow diet group (58.2 ± 5.0%) was 38% lower than in the HFD group (94.9 ± 8.8%).

Synthesis, 123I-Radiolabeling Optimization, and Initial Preclinical Evaluation of Novel Urea-Based PSMA Inhibitors with a Tributylstannyl Prosthetic Group in Their Structures.

Prostate-specific membrane antigen (PSMA) has been identified as a target for the development of theranostic agents. In our current work, we describe the design and synthesis of novel N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-L-lysine (DCL) urea-based PSMA inhibitors with a chlorine-substituted aromatic fragment at the lysine ε-nitrogen atom, a dipeptide including two phenylalanine residues in the L-configuration as the peptide fragment of the linker, and 3- or 4-(tributylstannyl)benzoic acid as a prosthetic group in their structures for radiolabeling. The standard compounds [127I]PSMA-m-IB and [127I]PSMA-p-IB for comparative and characterization studies were first synthesized using two alternative synthetic approaches. An important advantage of the alternative synthetic approach, in which the prosthetic group (NHS-activated esters of compounds) is first conjugated with the polypeptide sequence followed by replacement of the Sn(Bu)3 group with radioiodine, is that the radionuclide is introduced in the final step of synthesis, thereby minimizing operating time with iodine-123 during the radiolabeling process. The obtained DCL urea-based PSMA inhibitors were radiolabeled with iodine-123. The radiolabeling optimization results showed that the radiochemical yield of [123I]PSMA-p-IB was higher than that of [123I]PSMA-m-IB, which were 74.9 ± 1.0% and 49.4 ± 1.2%, respectively. The radiochemical purity of [123I]PSMA-p-IB after purification was greater than 99.50%. The initial preclinical evaluation of [123I]PSMA-p-IB demonstrated a considerable affinity and specific binding to PC-3 PIP (PSMA-expressing cells) in vitro. The in vivo biodistribution of this new radioligand [123I]PSMA-p-IB showed less accumulation than [177Lu]Lu-PSMA-617 in several normal organs (liver, kidney, and bone). These results warrant further preclinical development, including toxicology evaluation and experiments in tumor-bearing mice.

The emerging role of radionuclide molecular imaging of HER2 expression in breast cancer.

Targeting of human epidermal growth factor type 2 (HER2) using monoclonal antibodies, antibody-drug conjugates and tyrosine kinase inhibitors extends survival of patients with HER2-expressing metastatic breast cancer. High expression of HER2 is a predictive biomarker for such specific treatment. Accurate determination of HER2 expression level is necessary for stratification of patients to targeted therapy. Non-invasive in vivo radionuclide molecular imaging of HER2 has a potential of repetitive measurements, addressing issues of heterogeneous expression and conversion of HER2 status during disease progression or in response to therapy. Imaging probes based of several classes of targeting proteins are currently in preclinical and early clinical development. Both preclinical and clinical data suggest that the most promising are imaging agents based on small proteins, such as single domain antibodies or engineered scaffold proteins. These agents permit a very specific high-contrast imaging at the day of injection.

JAK-STAT core cancer pathway: An integrative cancer interactome analysis.

Through a comprehensive review and in silico analysis of reported data on STAT-linked diseases, we analysed the communication pathways and interactome of the seven STATs in major cancer categories and proposed rational targeting approaches for therapeutic intervention to disrupt critical pathways and addictions to hyperactive JAK/STAT in neoplastic states. Although all STATs follow a similar molecular activation pathway, STAT1, STAT2, STAT4 and STAT6 exert specific biological profiles associated with a more restricted pattern of activation by cytokines. STAT3 and STAT5A as well as STAT5B have pleiotropic roles in the body and can act as critical oncogenes that promote many processes involved in cancer development. STAT1, STAT3 and STAT5 also possess tumour suppressive action in certain mutational and cancer type context. Here, we demonstrated member-specific STAT activity in major cancer types. Through systems biology approaches, we found surprising roles for EGFR family members, sex steroid hormone receptor ESR1 interplay with oncogenic STAT function and proposed new drug targeting approaches of oncogenic STAT pathway addiction.

Exhaustive Conformational Search for Sialyl Cation Reveals Possibility of Remote Participation of Acyl Groups.

Finding convenient ways for the stereoselective α-sialylation is important due to the high practical significance of α-sialic acid-containing glycans and neoglycoconjugates. It was proposed that sialylation stereoselectivity is determined by the structure of the sialyl cation (also known in biochemistry as "sialosyl cation"), a supposed intermediate in this reaction. Here we design a new approach for studying the conformational space of highly flexible sialyl cation and find 1625 unique conformers including those stabilized by covalent remote participation (also known as long-range participation) of 4-O-acetyl (4-OAc), 5-N-trifluoroacetyl (5-NTFA), as well as 7,8,9-OAc from both α and ß sides. The most energetically stable sialyl cation conformers are featured by 4-OAc participation, closely followed by 5-NTFA- and 7-OAc-stabilized conformers; unstabilized sialyl cation conformers are ∼10 kcal mol-1 less stable than the 4-OAc-stabilized ones. Analysis of all the obtained conformers by means of substituents positions, side chain conformations and ring puckering led us to a new "eight-conformer hypothesis" which describes interconversions among the most important sialyl cation conformers and predicts that stronger remote participation of acyl groups favors ß-anomers. Thus, selective synthesis of the desired α-sialosides requires minimization of acyl groups participation.

Identification of a Novel de Novo Variant in the CASZ1 Causing a Rare Type of Dilated Cardiomyopathy.

A new de novo frameshift variant has been identified in the CASZ1 gene leading to severe dilated cardiomyopathy. METHODS: The proband was analyzed with WES NGS, post-mortem, using dried blood spots on filters. The variant was verified with Sanger sequencing for the proband and her parents. RESULTS: We reported a proband with a new de novo frameshift mutation, c.3781del (p.(Trp1261GlyfsTer29)), in the CASZ1 gene. The clinical presentation was similar to the severe phenotype described in previous studies. CONCLUSIONS: In this study, we described a new case with a frameshift mutation in CASZ1 causing a severe phenotype of dilated cardiomyopathy.

Time- and Spectrally-Resolved Photoluminescence Study of Alloyed CdxZn1-xSeyS1-y/ZnS Quantum Dots and Their Nanocomposites with SPIONs in Living Cells.

Magnetic-luminescent composites based on semiconductor quantum dots (QDs) and superparamagnetic iron oxide nanoparticles (SPIONs) can serve as a platform combining visualization and therapy. Here, we report the construction of QD-SPION nanocomposites based on synthesized SPIONs and alloyed QDs (CdxZn1-xSeyS1-y)/ZnS solubilized with L-cysteine molecules. The study of the spectral-luminescence characteristics, the kinetics of luminescence decay show the composite's stability in a solution. After incubation with HeLa cells, QDs, SPIONs, and their composites form clusters on the cell surface and associate with endosomes inside the cells. Component-wise analysis of the photoluminescence decay of cell-associated QDs/SPIONs provides information about their localization and aggregate status.

Direct In Vivo Comparison of 99mTc-Labeled Scaffold Proteins, DARPin G3 and ADAPT6, for Visualization of HER2 Expression and Monitoring of Early Response for Trastuzumab Therapy.

Non-invasive radionuclide molecular visualization of human epidermal growth factor receptor type 2 (HER2) can provide stratification of patients for HER2-targeting therapy. This method can also enable monitoring of the response to such therapies, thereby making treatment personalized and more efficient. Clinical evaluation in a phase I study demonstrated that injections of two scaffold protein-based imaging probes, [99mTc]Tc-(HE)3-G3 and [99mTc]Tc-ADAPT6, are safe, well-tolerated and cause a low level of radioactivity in healthy tissue. The goal of this preclinical study was to select the best probe for stratification of patients and response monitoring. Biodistribution of both tracers was compared in mice bearing SKOV-3 xenografts with high HER2 expression or MDA-MB-468 xenografts with very low expression. Changes in accumulation of the probes in SKOV-3 tumors 24 h after injection of trastuzumab were evaluated. Both [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3 permitted high contrast imaging of HER2-expressing tumors and a clear discrimination between tumors with high and low HER2 expression. However, [99mTc]Tc-ADAPT6 has better preconditions for higher sensitivity and specificity of stratification. On the other hand, [99mTc]Tc-(HE)3-G3 is capable of detecting the decrease of HER2 expression on response to trastuzumab therapy only 24 h after injection of the loading dose. This indicates that the [99mTc]Tc-(HE)3-G3 tracer would be better for monitoring early response to such treatment. The results of this study should be considered in planning of further clinical development of HER2 imaging probes.