Hybrid Mucin‐Vaterite Microspheres for Delivery of Proteolytic Enzyme Chymotrypsin

Abstract While the enteral delivery of proteolytic enzymes is widely established for combating many diseases as an alternative to antibiotic treatment, their local delivery only emerges as administration route enabling sustained release in a controlled manner on site. The latest requires the development of drug delivery systems suitable for encapsulation and preservation of enzymatic proteolytic activity. This study proposes hybrid microspheres made of mucin and biodegradable porous crystals of calcium carbonate (CC) as the carriers for chymotrypsin (CTR) delivery. CTR is impregnated into CC and hybrid CC/mucin (CCM) microspheres by means of sorption without any chemical modification. The loading of the CC with mucin enhances CTR retention on hybrid microspheres (adsorption capacity of ≈8.7 mg g−1 vs 4.7 mg g−1), recharging crystal surface due to the presence of mucin and diminishing the average pore diameter of the crystals from 25 to 8 nm. Mucin also retards recrystallization of vaterite into nonporous calcite improving stability of CCM microspheres upon storage. Proteolytic activity of CTR is preserved in both CC and CCM microspheres, being pH dependent. Temperature‐induced inactivation of CTR significantly diminishes by CTR encapsulation into CC and CCM microspheres. Altogether, these findings indicate promises of hybrid mucin‐vaterite microspheres for mucosal application of proteases

Immobilization of Antioxidant Enzyme Catalase on Porous Hybrid Microparticles of Vaterite with Mucin

Catalase is one of the crucial antioxidant enzymes with diverse applications in textile, food industries, wastewater treatment, cosmetics, and pharmaceutics, which, however, is highly sensitive to environmental challenges. Resisting the loss of activity and prolongation of formulation storage can be achieved via the catalase entrapment into insoluble carriers. Affordable and degradable vaterite is proposed as amicable material for catalase immobilization. To improve the carrier properties of the vaterite, it was co‐precipitated with mucin from the pig's stomach producing ca 5 μm hybrid mucin/vaterite microparticles. Catalase is impregnated into the crystals by means of adsorption without chemical modifications. The presence of mucin matrix partially hinders catalase penetration into the crystals and reduces the adsorption capacity (for 0.1 mg mL−1 catalase, ca 2.3 vs ca 1.5 mg g−1 for pristine and hybrid microparticles, respectively) but significantly promotes the protection of antioxidant activity upon storage and under the action of temperature, organic solvent (acetonitrile), and proteolytic enzyme (trypsin). Hybrid microcrystals are pH‐sensitive and better retain the enzyme at pH 3–5 due to catalase‐mucin complexation. Immobilized catalase can be used for 5–6 consecutive cycles until it loses catalytic activity. Altogether, these findings indicate promises of hybrid mucin/vaterite microparticles for immobilization of antioxidant enzymes

A phase III, randomized, double-blind, multicenter study to compare the efficacy, safety, pharmacokinetics, and immunogenicity between SB8 (proposed bevacizumab biosimilar) and reference bevacizumab in patients with metastatic or recurrent nonsquamous non-small cell lung cancer

Objectives: Efficacy, safety, pharmacokinetics (PK), and immunogenicity of the biosimilar candidate SB8 was compared to its reference product bevacizumab (BEV) in patients with metastatic or recurrent nonsquamous non―small cell lung cancer. Methods: Patients were randomized (1:1) in a phase III, double-blind study to receive intravenous SB8 or BEV 15 mg/kg with paclitaxel/carboplatin every 3 weeks for 24 weeks, followed by SB8 or BEV maintenance monotherapy. The primary endpoint was best overall response rate (ORR) by 24 weeks. Secondary endpoints included survival outcomes, safety, PK, and immunogenicity. Results: 763 patients (SB8, n = 379; BEV, n = 384) were randomized; baseline characteristics were well balanced. Best ORR in the FAS was 47.6% and 42.8%, and best ORR in the PPS was 50.1% and 44.8% for SB8 and BEV, respectively. The risk ratio of best ORR was 1.11 (90% CI, 0.975−1.269), and the risk difference in best ORR was 5.3% (95% CI, −2.2%–12.9%). Median survival outcomes were comparable between SB8 and BEV: progression-free survival was 8.50 vs 7.90 months, respectively (HR [95% CI], 0.99 [0.83–1.18]; p = 0.9338); overall survival was 14.90 vs 15.80 months, respectively (HR [95% CI], 1.03 [0.83–1.28]; p = 0.7713); and duration of response was 7.70 vs 7.00 months, respectively (HR [95% CI], 1.05 [0.81–1.37]; p = 0.6928). Severity and incidence of treatment-emergent adverse events, PK, and immunogenicity were comparable between SB8 and BEV. Conclusion: This study demonstrated equivalence between SB8 and BEV in terms of best ORR risk ratio, with comparable safety, PK, and immunogenicity

Large family with both parents affected by distinct BRCA1 mutations: implications for genetic testing

Although the probability of both parents being affected by BRCA1 mutations is not negligible, such families have not been systematically described in the literature. Here we present a large breast-ovarian cancer family, where 3 sisters and 1 half-sister inherited maternal BRCA1 5382insC mutation while the remaining 2 sisters carried paternal BRCA1 1629delC allele. No BRCA1 homozygous mutations has been detected, that is consistent with the data on lethality of BRCA1 knockout mice. This report exemplifies that the identification of a single cancer-predisposing mutation within the index patient may not be sufficient in some circumstances. Ideally, all family members affected by breast or ovarian tumor disease have to be subjected to the DNA testing, and failure to detect the mutation in any of them calls for the search of the second cancer-associated allele

The negative elongation factor NELF promotes induced transcriptional response of Drosophila ecdysone-dependent genes

Abstract For many years it was believed that promoter-proximal RNA-polymerase II (Pol II) pausing manages the transcription of genes in Drosophila development by controlling spatiotemporal properties of their activation and repression. But the exact proteins that cooperate to stall Pol II in promoter-proximal regions of developmental genes are still largely unknown. The current work describes the molecular mechanism employed by the Negative ELongation Factor (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). According to our data, the NELF complex is recruited to the promoters and enhancers of 20E-dependent genes. Its presence at the regulatory sites of 20E-dependent genes correlates with observed interaction between the NELF-A subunit and the ecdysone receptor (EcR). The complete NELF complex is formed at the 20E-dependent promoters and participates in both their induced transcriptional response and maintenance of the uninduced state to keep them ready for the forthcoming transcription. NELF depletion causes a significant decrease in transcription induced by 20E, which is associated with the disruption of Pol II elongation complexes. A considerable reduction in the promoter-bound level of the Spt5 subunit of transcription elongation factor DSIF was observed at the 20E-dependent genes upon NELF depletion. We presume that an important function of NELF is to participate in stabilizing the Pol II-DSIF complex, resulting in a significant impact on transcription of its target genes. In order to directly link NELF to regulation of 20E-dependent genes in development, we show the presence of NELF at the promoters of 20E-dependent genes during their active transcription in both embryogenesis and metamorphosis. We also demonstrate that 20E-dependent promoters, while temporarily inactive at the larval stage, preserve a Pol II paused state and bind NELF complex

Maternal Hyperhomocysteinemia Disturbs the Mechanisms of Embryonic Brain Development and Its Maturation in Early Postnatal Ontogenesis

Maternal hyperhomocysteinemia causes the disruption of placental blood flow and can lead to serious disturbances in the formation of the offspring’s brain. In the present study, the effects of prenatal hyperhomocysteinemia (PHHC) on the neuronal migration, neural tissue maturation, and the expression of signaling molecules in the rat fetal brain were described. Maternal hyperhomocysteinemia was induced in female rats by per os administration of 0.15% aqueous methionine solution in the period of days 4–21 of pregnancy. Behavioral tests revealed a delay in PHHC male pups maturing. Ultrastructure of both cortical and hippocampus tissue demonstrated the features of the developmental delay. PHHC was shown to disturb both generation and radial migration of neuroblasts into the cortical plate. Elevated Bdnf expression, together with changes in proBDNF/mBDNF balance, might affect neuronal cell viability, positioning, and maturation in PHHC pups. Reduced Kdr gene expression and the content of SEMA3E might lead to impaired brain development. In the brain tissue of E20 PHHC fetuses, the content of the procaspase-8 was decreased, and the activity level of the caspase-3 was increased; this may indicate the development of apoptosis. PHHC disturbs the mechanisms of early brain development leading to a delay in brain tissue maturation and formation of the motor reaction of pups

Maternal Hyperhomocysteinemia Produces Memory Deficits Associated with Impairment of Long-Term Synaptic Plasticity in Young Rats

Maternal hyperhomocysteinemia (HCY) is a common pregnancy complication caused by high levels of the homocysteine in maternal and fetal blood, which leads to the alterations of the cognitive functions, including learning and memory. In the present study, we investigated the mechanisms of these alterations in a rat model of maternal HCY. The behavioral tests confirmed the memory impairments in young and adult rats following the prenatal HCY exposure. Field potential recordings in hippocampal slices demonstrated that the long-term potentiation (LTP) was significantly reduced in HCY rats. The whole-cell patch-clamp recordings in hippocampal slices demonstrated that the magnitude of NMDA receptor-mediated currents did not change while their desensitization decreased in HCY rats. No significant alterations of glutamate receptor subunit expression except GluN1 were detected in the hippocampus of HCY rats using the quantitative real-time PCR and Western blot methods. The immunofluorescence microscopy revealed that the number of synaptopodin-positive spines is reduced, while the analysis of the ultrastructure of hippocampus using the electron microscopy revealed the indications of delayed hippocampal maturation in young HCY rats. Thus, the obtained results suggest that maternal HCY disturbs the maturation of hippocampus during the first month of life, which disrupts LTP formation and causes memory impairments

Maternal Hyperhomocysteinemia Produces Memory Deficits Associated with Impairment of Long-Term Synaptic Plasticity in Young Rats

Maternal hyperhomocysteinemia (HCY) is a common pregnancy complication caused by high levels of the homocysteine in maternal and fetal blood, which leads to the alterations of the cognitive functions, including learning and memory. In the present study, we investigated the mechanisms of these alterations in a rat model of maternal HCY. The behavioral tests confirmed the memory impairments in young and adult rats following the prenatal HCY exposure. Field potential recordings in hippocampal slices demonstrated that the long-term potentiation (LTP) was significantly reduced in HCY rats. The whole-cell patch-clamp recordings in hippocampal slices demonstrated that the magnitude of NMDA receptor-mediated currents did not change while their desensitization decreased in HCY rats. No significant alterations of glutamate receptor subunit expression except GluN1 were detected in the hippocampus of HCY rats using the quantitative real-time PCR and Western blot methods. The immunofluorescence microscopy revealed that the number of synaptopodin-positive spines is reduced, while the analysis of the ultrastructure of hippocampus using the electron microscopy revealed the indications of delayed hippocampal maturation in young HCY rats. Thus, the obtained results suggest that maternal HCY disturbs the maturation of hippocampus during the first month of life, which disrupts LTP formation and causes memory impairments

Increased Susceptibility of the CD57⁻ NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential, Activation Degree, and Death Induction Response

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57−NKG2C+ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57− NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3+NKG2C+ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3+ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3+NKG2C+ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded