Effect of cryopreservation on Odc1 and RhoA genes expression in diapausing mouse blastocysts.

The possibility of embryo cryopreservation is important for applying the genome resource banking (GRB) concept to those mammalian species that exhibit embryonal diapause in their early development. Odc1 encodes ODC1, which is a key enzyme in polyamine synthesis. RhoA is an essential part of Rho/ROCK system. Both Odc1 and RhoA play an important role in preimplantation embryo development. Studying these systems in mammalian species with obligate or experimentally designed embryonic diapause may provide insight into the molecular machinery underlying embryo dormancy and re-activation. The effect of cryopreservation procedures on the expression of the Odc1 and RhoA in diapausing embryos has not been properly studied yet. The purpose of this work is to address the possibility of cryopreservation diapausing embryos and to estimate the expression of the Odc1 and RhoA genes in diapausing and non-diapausing embryos before and after freeze-thaw procedures using ovariectomized progesterone treated mice as a model. Both diapausing and non-diapausing in vivo-derived embryos continued their development in vitro after freezing-thawing as evidenced by blastocoel re-expansion. Although cryopreservation dramatically decreased the expression of the Odc1 and RhoA genes in non-diapausing embryos, no such effects have been observed in diapausing embryos where these genes were already at the low level before freeze-thaw procedures. Future studies may attempt to facilitate the re-activation of diapausing embryos, for example frozen-thawed ones, specifically targeting Odc1 or Rho/ROCK system.

Program freezing of diapausing embryos in the mouse.

Embryonal diapause is a characteristic feature of about 130 mammalian species. However, very few studies have addressed cryopreservation of diapausing embryos. This work is aimed to apply program freezing to blastocysts obtained from CD1 mice, which were at diapause state after ovariectomy and the subsequent hormonal therapy. Blastocysts collected from non-operated mice of the same strain served as controls. Some diapausing as well as non-diapausing frozen-thawed blastocysts demonstrated blastocoel re-expansion after 24 h of in vitro culture (IVC) indicating their viability after cryopreservation. Raman spectroscopy assessment of phenylalanine accumulation revealed that the fraction of new synthesized proteins was lower for non-frozen as well as for frozen-thawed diapausing blastocysts compared to non-diapausing ones. Although protein metabolism was reduced in diapausing embryos, most of the protein synthesis remained active. Cell number increased after 24 h of IVC in non-frozen as well as in the frozen-thawed blastocysts of the control but not of the diapause group. However, cell numbers were increased in frozen-thawed diapausing blastocysts after 47 h of IVC in a medium supplemented with putrescine. This indicates viability of frozen-thawed diapausing embryos after cryopreservation. Besides, protein metabolism was not affected by cryopreservation in diapausing and non-diapausing murine embryos indicating their viability. Our results demonstrated the possibility of successful cryopreservation of diapausing murine embryos.

Quality of life with cemiplimab plus chemotherapy for first-line treatment of advanced non-small cell lung cancer: Patient-reported outcomes from phase 3 EMPOWER-Lung 3.

BACKGROUND: EMPOWER-Lung 3, a randomized 2:1 phase 3 trial, showed clinically meaningful and statistically significant overall survival improvement with cemiplimab plus platinum-doublet chemotherapy versus placebo plus chemotherapy for first-line treatment of advanced non-small cell lung cancer. This study evaluated patient-reported outcomes (PROs). METHODS: PROs were assessed at day 1 (baseline), the start of each treatment cycle (every 3 weeks) for the first six doses, and then at start of every three cycles, using the European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life-Core 30 (QLQ-C30) and Quality of Life-Lung Cancer Module (QLQ-LC13) questionnaires. Prespecified analyses included a longitudinal mixed-effect model comparing treatment arms and a time to definitive clinically meaningful deterioration (TTD) analysis performed for global health status/quality of life (GHS/QoL) and all scales from the questionnaires. Between-arm TTD comparisons were made using a stratified log-rank test and proportional hazards model. RESULTS: A total of 312 patients were assigned to receive cemiplimab plus platinum-doublet chemotherapy and 154 to receive placebo plus chemotherapy; 391 (83.9%) were male and the median age was 63.0 years (range, 25-84). For pain symptoms (EORTC QLQ-C30), a statistically significant overall improvement from baseline (-4.98, 95% confidence interval [CI] -8.36 to -1.60, p = .004) and a statistically significant delay in TTD (hazard ratio, 0.39; 95% CI, 0.26-0.60, p < .0001) favoring cemiplimab plus chemotherapy were observed. Statistically significant delays in TTD, all favoring cemiplimab plus chemotherapy, were also observed in functioning and symptom scales. A significant overall improvement from baseline in GHS/QoL was seen for cemiplimab plus chemotherapy compared with nonsignificant overall change from baseline for placebo plus chemotherapy (1.69, 95% CI, 0.20-3.19 vs. 1.08, 95% CI, -1.34 to 3.51; between arms, p = .673). No analyses yielded statistically significant PRO results favoring placebo plus chemotherapy for any QLQ-C30 or QLQ-LC13 scale. CONCLUSION: Cemiplimab plus chemotherapy resulted in significant overall improvement in pain symptoms and delayed TTD in cancer-related and lung cancer-specific symptoms and functions.

Cemiplimab Plus Chemotherapy Versus Chemotherapy Alone in Advanced NSCLC: 2-Year Follow-Up From the Phase 3 EMPOWER-Lung 3 Part 2 Trial.

INTRODUCTION: EMPOWER-Lung 3 part 2 (NCT03409614), a double-blind, placebo-controlled phase 3 study, investigated cemiplimab (antiprogrammed cell death protein 1) plus chemotherapy versus placebo plus chemotherapy in patients with advanced NSCLC without EGFR, ALK, or ROS1 aberrations, with either squamous or nonsquamous histology, irrespective of programmed death-ligand 1 levels. At primary analysis, after 16.4 months of follow-up, cemiplimab plus chemotherapy improved median overall survival (OS) versus chemotherapy alone (21.9 versus 13.0 mo, hazard ratio [HR] = 0.71, 95% confidence interval [CI]: 0.53-0.93, p = 0.014). Here, we report protocol-specified final OS and 2-year follow-up results. METHODS: Patients (N = 466) were randomized 2:1 to receive histology-specific platinum-doublet chemotherapy, with 350 mg cemiplimab (n = 312) or placebo (n = 154) every 3 weeks for up to 108 weeks. Primary end point was OS; secondary end points included progression-free survival and objective response rates. RESULTS: After 28.4 months of median follow-up, median OS was 21.1 months (95% CI: 15.9-23.5) for cemiplimab plus chemotherapy versus 12.9 months (95% CI: 10.6-15.7) for chemotherapy alone (HR = 0.65, 95% CI: 0.51-0.82, p = 0.0003); median progression-free survival was 8.2 months (95% CI: 6.4-9.0) versus 5.5 months (95% CI: 4.3-6.2) (HR = 0.55, 95% CI: 0.44-0.68, p < 0.0001), and objective response rates were 43.6% versus 22.1%, respectively. Safety was generally consistent with previously reported data. Incidence of treatment-emergent adverse events of grade 3 or higher was 48.7% with cemiplimab plus chemotherapy and 32.7% with chemotherapy alone. CONCLUSIONS: At protocol-specified final OS analysis with 28.4 months of follow-up, the EMPOWER-Lung 3 study continued to reveal benefit of cemiplimab plus chemotherapy versus chemotherapy alone in patients with advanced squamous or nonsquamous NSCLC, across programmed death-ligand 1 levels.

Biochemical and Genomic Characterization of Two New Strains of Lacticaseibacillus paracasei Isolated from the Traditional Corn-Based Beverage of South Africa, Mahewu, and Their Comparison with Strains Isolated from Kefir Grains.

Lacticaseibacillus paracasei (formerly Lactobacillus paracasei) is a nomadic lactic acid bacterium (LAB) that inhabits a wide variety of ecological niches, from fermented foodstuffs to host-associated microenvironments. Many of the isolated L. paracasei strains have been used as single-strain probiotics or as part of a symbiotic consortium within formulations. The present study contributes to the exploration of different strains of L. paracasei derived from non-conventional isolation sources-the South African traditional fermented drink mahewu (strains MA2 and MA3) and kefir grains (strains KF1 and ABK). The performed microbiological, biochemical and genomic comparative analyses of the studied strains demonstrated correlation between properties of the strains and their isolation source, which suggests the presence of at least partial strain adaptation to the isolation environments. Additionally, for the studied strains, antagonistic activities against common pathogens and against each other were observed, and the ability to release bioactive peptides with antioxidant and angiotensin I-converting enzyme inhibitory (ACE-I) properties during milk fermentation was investigated. The obtained results may be useful for a deeper understanding of the nomadic lifestyle of L. paracasei and for the development of new starter cultures and probiotic preparations based on this LAB in the future.

Cemiplimab plus chemotherapy versus chemotherapy alone in non-small cell lung cancer: a randomized, controlled, double-blind phase 3 trial.

First-line cemiplimab (anti-programmed cell death-1 (PD-1)) monotherapy has previously shown significant improvement in overall survival (OS) and progression-free survival (PFS) versus chemotherapy in patients with advanced non-small cell lung cancer (aNSCLC) and PD-ligand 1 (PD-L1) expression ≥50%. EMPOWER-Lung 3 ( NCT03409614 ), a double-blind, placebo-controlled, phase 3 study, examined cemiplimab plus platinum-doublet chemotherapy as first-line treatment for aNSCLC, irrespective of PD-L1 expression or histology. In this study, 466 patients with stage III/IV aNSCLC without EGFR, ALK or ROS1 genomic tumor aberrations were randomized (2:1) to receive cemiplimab 350 mg (n = 312) or placebo (n = 154) every 3 weeks for up to 108 weeks in combination with four cycles of platinum-doublet chemotherapy (followed by pemetrexed maintenance as indicated). In total, 57.1% (266/466 patients) had non-squamous NSCLC, and 85.2% (397/466 patients) had stage IV disease. The primary endpoint was OS. The trial was stopped early per recommendation of the independent data monitoring committee, based on meeting preset OS efficacy criteria: median OS was 21.9 months (95% confidence interval (CI), 15.5-not evaluable) with cemiplimab plus chemotherapy versus 13.0 months (95% CI, 11.9-16.1) with placebo plus chemotherapy (hazard ratio (HR) = 0.71; 95% CI, 0.53-0.93; P = 0.014). Grade ≥3 adverse events occurred with cemiplimab plus chemotherapy (43.6%, 136/312 patients) and placebo plus chemotherapy (31.4%, 48/153 patients). Cemiplimab is only the second anti-PD-1/PD-L1 agent to show efficacy in aNSCLC as both monotherapy and in combination with chemotherapy for both squamous and non-squamous histologies.

Exoproteome Analysis of Antagonistic Interactions between the Probiotic Bacteria Limosilactobacillus reuteri LR1 and Lacticaseibacillus rhamnosus F and Multidrug Resistant Strain of Klebsiella pneumonia.

The expansion of multiple drug resistant (MDR) strains of Klebsiella pneumoniae presents an immense threat for public health. Annually, this microorganism causes thousands of lethal nosocomial infections worldwide. Currently, it has been shown that certain strains of lactic acid bacteria (LAB) can efficiently inhibit growth of K. pneumoniae and the formation of its biofilms; however, the active principle of such action remains unknown. In the current article, the growth inhibition of MDR K. pneumoniae by two LAB-Limosilactobacillus reuteri LR1 and Lacticaseibacillus rhamnosus F-is demonstrated, and the nature of this inhibition studied at the level of exoproteome. This article shows that the exoproteomes of studied LAB contains both classically and non-classically secreted proteins. While for L. reuteri LR1 the substantial portion of classically secreted proteins was presented by cell-wall-degrading enzymes, for L. rhamnosus F only one out of four classically secreted proteins was presented by cell-wall hydrolase. Non-classically secreted proteins of both LAB were primarily metabolic enzymes, for some of which a possible moonlighting functioning was proposed. These results contribute to knowledge regarding antagonistic interaction between LAB and pathogenic and opportunistic microorganisms and set new perspectives for the use of LAB to control the spread of these microorganisms.

Development of Antioxidant and Antihypertensive Properties during Growth of Lactobacillus helveticus, Lactobacillus rhamnosus and Lactobacillus reuteri on Cow's Milk: Fermentation and Peptidomics Study.

Bioactive peptides derived from milk proteins are an active research area. Exhibiting numerous positive physiological effects on digestive, cardiovascular, immune and nervous systems, these peptides thought to be one of the most promising ingredients for functional food. Generally, these peptides are inactive within the parent proteins and can be liberated during milk fermentation by the specific proteolytic systems of various Lactobacillus spp. Here we present the study of milk fermentation by Lactobacillus helveticus NK1, Lactobacillus rhamnosus F and Lactobacillus reuteri LR1 strains. It was demonstrated that the antioxidant activity of the milk fermented by these strains concomitantly increased with the strains' proteolytic activity. For the angiotensin I-converting enzyme (ACE) inhibitory activity, the same tendency was not observed. Although the proteolytic activity of L. helveticus NK1 was two times higher than that of L. rhamnosus F, the milk fermented by these strains showed comparable ACE inhibition. The analysis of the peptide profiles of the fermented milk samples allowed us to hypothesize that some previously unreported peptides can be produced by L. rhamnosus F. In addition, it was demonstrated that these potential ACE-inhibiting peptides originated from the C-terminus of αS2-casein.

Sperm cryopreservation in the Far-Eastern wildcat (Prionailurus bengalensis euptilurus).

The Far-Eastern wildcat (Prionailurus bengalensis euptilurus) is a rare and poorly investigated nondomestic felid species. An attempt of freezing and cryopreserving Far-Eastern wildcat spermatozoa in CaniPlus Freeze (CPF) medium is reported. Sperm was collected by electroejaculation from five adult Far-Eastern wildcat captive-born males. Epididymal spermatozoa from five adult randomly bred domestic cat males were used as a reference. The viability of frozen-thawed spermatozoa evaluated by double staining with SYBR Green I and PI followed by the subsequent confocal laser scanning microscopy (CLSM) was 38.2% ± 3.0% for the domestic cat and 38.0% ± 10.2% for the Far-Eastern wildcat. The motility of frozen-thawed spermatozoa was 30.8% ± 9.8% for the domestic cat and 33.7% ± 15.1% for the Far-Eastern wildcat. Sperm morphology was assessed by light microscopy. The total percentage of normal spermatozoa after freezing and thawing was 51.9 ± 5.9 for the domestic cat and 55.0% ± 6.4% for the Far-Eastern wildcat. Defects of flagella were the most frequently observed abnormalities in both species (32.2% ± 4.8% and 30.8% ± 4.4% of all reported anomalies for the domestic cat and Far-Eastern wildcat, respectively). Domestic cat epididymal and Far-Eastern ejaculatory spermatozoa fertilized in vitro-matured oocytes of the domestic cat (30.0% ± 5.5% and 35.5% ± 15.0%, respectively). Taken together, these results suggest that the freezing of Far-Eastern wildcat spermatozoa with CPF medium is a suitable method for Felidae cryopreservation.

Characteristics and fertility of domestic cat epididymal spermatozoa cryopreserved with two different freezing media.

The study represents a comparison of cryopreservation of domestic cat epididymal spermatozoa with two commercially available freezing media: CaniPlus Freeze (CPF) and SpermFreeze (SF). The viability of nonfrozen spermatozoa evaluated by the VitalScreen test was 68.7 ±â€¯3.0%. These figures were lower for the frozen-thawed spermatozoa: 51.2 ±â€¯6.3% for CPF group and 54.4 ±â€¯3.1% for SF group. The motility of nonfrozen spermatozoa was 57.2 ±â€¯4.5%. These figures were reduced in both frozen-thawed groups; however, there was no significant difference in these parameters between CPF (30.8 ±â€¯7.1%) and SF (27.4 ±â€¯8.1%) groups. The percentage of nonprogressively moving motile spermatozoa after freezing-thawing was decreased in both frozen-thawed groups (23.5 ±â€¯5.9 and 12.0 ±â€¯2.4 for CPF and SF frozen correspondingly) as compared with nonfrozen controls (42.1 ±â€¯4.1%). Morphology of spermatozoa was assessed by light microscopy. The mean percentages of normal spermatozoa were 28.5 ±â€¯4.1% for nonfrozen group, 26.0 ±â€¯2.3% for CPF frozen group, and 23.9 ±â€¯1.9% for SF frozen group. The most frequent anomalies in all the three groups were flagella and combined defects. In vitro fertilization (IVF) of domestic cat oocytes with nonfrozen and frozen-thawed spermatozoa produced developing embryos. The percentage of in-vitro-derived embryos was 43.6% after using nonfrozen spermatozoa. Frozen-thawed spermatozoa developed at a similar rate (44.0%) after using SF. However, the rate of embryo development was lower (20.1%) when CPF was used. The in-vitro-derived embryos in the nonfrozen group consisted of 46.9 ±â€¯2.5 cells after 5-day culturing. After cryopreservation with SF and CPF the cell numbers per embryo were 39.9 ±â€¯2.7 and 31.8 ±â€¯3.4 correspondingly. In CPF group these numbers were lower than in nonfrozen controls. Cryopreservation of spermatozoa with either of two freezing media led to a decrease in post-thaw viability and motility of spermatozoa but did not affect the rate or spectrum of their morphological anomalies. The use of CPF, but not SF led to a decrease of sperm fertilizing abilities.

Neurodevelopment and behavior in neonatal OXYS rats with genetically determined accelerated senescence.

Study of the developmental characteristics and mechanisms of senescence is an important field in brain aging research. The OXYS strain was selected from Wistar rats in Novosibirsk, and it serves as a rat model of accelerated aging. Previously, neurodegenerative processes and aberrant behavior were reported in experiments with adult OXYS rats. In our study, neurodevelopmental reflexes, neuronal density in the prefrontal cortex and hippocampus, and global DNA methylation in the hippocampus are compared between OXYS and WAG (Wistar Albino Glaxo) neonatal pups. The development of the righting, forelimb grasp, and cliff avoidance reflexes is delayed, and body weight gain was deferred in neonatal OXYS pups. Neuronal density in the hippocampus does not differ between one-day-old OXYS and WAG pups. On the sixth day, the neuronal density in OXYS pups is reduced in the CA2 hippocampal zone, augmented in CA3 and DG, and unchanged in CA1. Six-day-old OXYS pups have fewer and smaller pyramidal neurons in the prefrontal cortex as compared to WAG controls. Global DNA methylation levels in the hippocampus of OXYS newborns are significantly lower than in the WAG newborn pups. At the age of six days, the global DNA methylation level decreases in WAG pups, but does not change in OXYS pups. Thus, neonatal OXYS rats show delayed neurodevelopment accompanied by changes in the global DNA methylation pattern in the hippocampus and in neuronal density in the hippocampus and the prefrontal cortex. These changes may be related to accelerated senescence in adult OXYS rats.

Embryo cryopreservation and in vitro culture of preimplantation embryos in Campbell's hamster (Phodopus campbelli).

The aims of this study were to compare different protocols of Campbell's hamster (Phodopus campbelli) embryos freezing-thawing and to explore the possibilities of their in vitro culture. First, the embryos were flushed from the reproductive ducts 2 days post coitum at the two-cell stage and cultured in rat one-cell embryo culture medium (R1ECM) for 48 hours. Most (86.7%) of the two-cell embryos developed to blastocysts in R1ECM. Second, the embryos at the two- to eight-cell stages were flushed on the third day post coitum. The eight-cell embryos were frozen in 0.25 mL straws according to standard procedures of slow cooling. Ethylene glycol (EG) was used either as a single cryoprotectant or in a mixture with sucrose. The survival of frozen-thawed embryos was assessed by double staining with fluorescein diacetate and propidium iodide. The use of EG as a single cryoprotectant resulted in fewer alive embryos when compared with control (fresh embryos), but combined use of EG and sucrose improved the survival rate after thawing. Furthermore, granulocyte-macrophage colony-stimulating factor rat (2 ng/mL) improved the rate of the hamster frozen-thawed embryo development in vitro by increasing the final cell number and alleviating nuclear fragmentation. Our data show the first attempt in freezing and thawing Campbell's hamster embryos and report the possibility of successful in vitro culture for this species in R1ECM supplemented with granulocyte-macrophage colony-stimulating factor.