Auto-sumoylation of the Ubc9 E2 SUMO-conjugating Enzyme Extends Cellular Lifespan.

Calorie restriction (CR) provides anti-aging benefits through diverse processes, such as reduced metabolism and growth and increased mitochondrial activity. Although controversy still exists regarding CR-mediated lifespan effects, many researchers are seeking interventions that mimic the effects of CR. Yeast has proven to be a useful model system for aging studies, including CR effects. We report here that yeast adapted through in vitro evolution to the severe cellular stress caused by loss of the Ulp2 SUMO-specific protease exhibit both enhanced growth rates and replicative lifespan, and they have altered gene expression profiles similar to those observed in CR. Notably, in certain evolved ulp2Δ lines, a dramatic increase in the auto-sumoylation of Ubc9 E2 SUMO-conjugating enzyme results in altered regulation of multiple targets involved in energy metabolism and translation at both transcriptional and post-translational levels. This increase is essential for the survival of aged cells and CR-mediated lifespan extension. Thus, we suggest that high Ubc9 auto-sumoylation exerts potent anti-aging effects by promoting efficient energy metabolism-driven improvements in cell replication abilities. This potential could be therapeutically explored for the development of novel CR-mimetic strategies.

Structural analysis of the YqeY proteins from Campylobacter jejuni and Vibrio parahaemolyticus.

YqeY is a functionally and structurally uncharacterized protein that is ubiquitously expressed in bacteria. To gain structural insights into the function of YqeY, we determined the crystal structures of the Campylobacter jejuni and Vibrio parahaemolyticus YqeY proteins (cjYqeY and vpYqeY, respectively) and analyzed the structural and functional roles of conserved residues via a mutational study. Both cjYqeY and vpYqeY were found to adopt a two-domain structure consisting of an N-terminal four-α-helix domain and a C-terminal three-α-helix domain, with a relatively flexible interdomain orientation. The YqeY structure is unique in its linkage of the two α-helix domains although the C-terminal YqeY domain is structurally homologous to the terminal appendages of glutaminyl-tRNA synthetase and tRNA-dependent amidotransferase. We identified six conserved YqeY residues (Y67, R72, E82, Y89, P91, and G119) and evaluated their roles in protein stability via alanine mutation using a thermal shift assay. Residues Y67, R72, Y89, and P91 were shown to be required to maintain the structural integrity of YqeY. In contrast, residues E82 and G119 were not found to be essential for protein stability and are highly likely to contribute to the biological function of YqeY.

A novel G3BP1-GFP reporter human lung cell system enabling real-time monitoring of stress granule dynamics for in vitro lung toxicity assessment.

Under various cellular stress conditions, including exposure to toxic chemicals, RNA-binding proteins (RBPs), including Ras GTPase-activating protein-binding protein 1 (G3BP1), aggregate and form stress granule complexes, which serve as hallmarks of cellular stress. The existing methods for analyzing stress granule assembly have limitations in the rapid detection of dynamic cellular stress and ignore the effects of constitutively overexpressed RBP on cellular stress and stress-related processes. Therefore, to overcome these limitations, we established a G3BP1-GFP reporter in a human lung epithelial cell line using CRISPR/Cas9-based knock-in as an alternative system for stress granule analysis. We showed that the G3BP1-GFP reporter system responds to stress conditions and forms a stress granule complex similar to that of native G3BP1. Furthermore, we validated the stress granule response of an established cell line under exposure to various household chemicals. Overall, this novel G3BP1-GFP reporter human lung cell system is capable of monitoring stress granule dynamics in real time and can be used for assessing the lung toxicity of various substances in vitro.

Comparison of mortality and short-term outcomes between classic, intubation-surfactant-extubation, and less invasive surfactant administration methods of surfactant replacement therapy.

Background: Intubation-Surfactant-Extubation (InSurE) and less invasive surfactant administration (LISA) are alternative surfactant replacement therapy methods for reducing the complications associated with invasive mechanical ventilation. This study aimed to compare the Classic, InSurE, and LISA methods in Very-Low-Birth-Weight infants (VLBWIs) in South Korea. Methods: The Korean Neonatal Network (KNN) enrolled VLBWIs born between January 1, 2019 and December 31, 2020. They were analyzed retrospectively to compare the duration of respiratory support, length of hospitalization, mortality, and short-term outcomes of the three groups. Results: The duration of invasive ventilator support was shorter in the following order: InSurE (3.99 ± 11.93 days), LISA (8.78 ± 29.32 days), and the Classic group (22.36 ± 29.94 days) (p = 0.014, p < 0.01) and InSurE had the shortest hospitalization (64.91 ± 24.07 days, p < 0.05) although the results couldn't adjust for confounding factor because of irregular distribution. InSurE had the lower risk of intraventricular hemorrhage (IVH) grade II-IV [odds ratio (OR) 0.524 [95% confidence interval (CI): 0.287-0.956], p = 0.035] than in the Classic group. Mortality was lower in the InSurE [OR 0.377 (95% CI: 0.146-0.978), p = 0.045] and LISA [OR 0.296 (95% CI: 0.102-0.862), p = 0.026] groups than in the Classic group. There was a reduced risk of moderate to severe bronchopulmonary dysplasia (BPD) [OR 0.691 (95% CI: 0.479-0.998, p = 0.049), OR 0.544 (95% CI: 0.355-0.831, p = 0.005), respectively], pulmonary hypertension [OR 0.350 (95% CI: 0.150-0.817, p = 0.015), OR 0.276 (95% CI: 0.107-0.713, p = 0.008), respectively], periventricular leukomalacia (PVL) [OR 0.382 (95% CI: 0.187-0.780, p = 0.008), OR 0.246 (95% CI: 0.096-0.627, p = 0.003), respectively], and patent ductus arteriosus (PDA) with treatment [OR 0.628 (95% CI: 0.454-0.868, p = 0.005), OR 0.467 (95% CI: 0.313-0.696, p < 0.001) respectively] in the InSurE and LISA groups compared to the Classic group. Conclusion: InSurE showed the lowest duration of invasive ventilator support, length of hospitalization. InSurE and LISA exhibited reduced mortality and decreased risks of moderate to severe BPD, pulmonary hypertension, PVL, and PDA with treatment compared to the Classic group.

Structural and Biochemical Analysis of the Recombination Mediator Protein RecR from Campylobacter jejuni.

The recombination mediator complex RecFOR, consisting of the RecF, RecO, and RecR proteins, is needed to initiate homologous recombination in bacteria by positioning the recombinase protein RecA on damaged DNA. Bacteria from the phylum Campylobacterota, such as the pathogen Campylobacter jejuni, lack the recF gene and trigger homologous recombination using only RecR and RecO. To elucidate the functional properties of C. jejuni RecR (cjRecR) in recombination initiation that differ from or are similar to those in RecF-expressing bacteria, we determined the crystal structure of cjRecR and performed structure-based binding analyses. cjRecR forms a rectangular ring-like tetrameric structure and coordinates a zinc ion using four cysteine residues, as observed for RecR proteins from RecF-expressing bacteria. However, the loop of RecR that has been shown to recognize RecO and RecF in RecF-expressing bacteria is substantially shorter in cjRecR as a canonical feature of Campylobacterota RecR proteins, indicating that cjRecR lost a part of the loop in evolution due to the lack of RecF and has a low RecO-binding affinity. Furthermore, cjRecR features a larger positive patch and exhibits substantially higher ssDNA-binding affinity than RecR from RecF-expressing bacteria. Our study provides a framework for a deeper understanding of the RecOR-mediated recombination pathway.

Development of Two Korean IACUC Guidance Documents to Foster Implementation of the Three Rs.

In Korea, Institutional Animal Care and Use Committees (IACUCs) have been legally required to apply the Three Rs principles (i.e. replacement, reduction and refinement) and undertake the ethical review of animal study protocols, since 2008. According to Korean law, each IACUC is required to appoint at least one lay member recommended by a non-governmental animal protection organisation, who participates in the ethical review process as part of this role. Despite the importance of the Three Rs and the ethical review process, limited information and practical resources are available for IACUC members in the Korean language, particularly for lay members who are inexperienced in animal experimentation. In January 2020, the Animal and Plant Quarantine Agency announced the funding for a six-month research project to develop guidance to assist IACUC members in carrying out effective and efficient protocol reviews in line with Korean legislative requirements. This funding was awarded for the production of two IACUC guidance documents - 'Guide for Animal Study Protocols' and 'Guide for the IACUC Lay Member' - which were published in December 2020. These guidance documents aim to foster the implementation of the Three Rs and provide practical resources for IACUC members, researchers and other relevant personnel. This paper describes the framework for animal use in Korea and the overall production of these two IACUC Guidance Documents.

A Bivalent Inactivated Vaccine Prevents Enterovirus 71 and Coxsackievirus A16 Infections in the Mongolian Gerbil.

Hand-foot-and-mouth disease (HFMD) is a viral infectious disease that occurs in children under 5 years of age. Its main causes are coxsackievirus (CV) and enterovirus (EV). Since there are no efficient therapeutics for HFMD, vaccines are effective in preventing the disease. To develop broad coverage against CV and EV, the development of a bivalent vaccine form is needed. The Mongolian gerbil is an efficient and suitable animal model of EV71 C4a and CVA16 infection used to investigate vaccine efficacy following direct immunization. In this study, Mongolian gerbils were immunized with a bivalent inactivated EV71 C4a and inactivated CVA16 vaccine to test their effectiveness against viral infection. Bivalent vaccine immunization resulted in increased Ag-specific IgG antibody production; specifically, EV71 C4a-specific IgG was increased with medium and high doses and CVA16-specific IgG was increased with all doses of immunization. When gene expression of T cell-biased cytokines was analysed, Th1, Th2, and Th17 responses were found to be highly activated in the high-dose immunization group. Moreover, bivalent vaccine immunization mitigated paralytic signs and increased the survival rate following lethal viral challenges. When the viral RNA content was determined from various organs, all three doses of bivalent vaccine immunization were found to significantly decrease viral amplification. Upon histologic examination, EV71 C4a and CVA16 induced tissue damage to the heart and muscle. However, bivalent vaccine immunization alleviated this in a dose-dependent manner. These results suggest that the bivalent inactivated EV71 C4a/CVA16 vaccine could be a safe and effective candidate HFMD vaccine.

Unique dimeric structure of the DUF2891 family protein CJ0554 from Campylobacter jejuni.

Campylobacter jejuni is a pathogenic bacterium that causes enteritis and Guillain-Barre syndrome in humans. To identify a protein target for the development of a new therapeutic against C. jejuni infection, each gene product of C. jejuni must be functionally characterized. The cj0554 gene of C. jejuni encodes a DUF2891 family protein with unknown functions. To provide functional insights into CJ0554, we determined and analyzed the crystal structure of the CJ0554 protein. CJ0554 adopts an (α/α)6-barrel structure, which consists of an inner α6 ring and an outer α6 ring. CJ0554 assembles into a dimer in a unique top-to-top orientation that is not observed in its structural homologs, N-acetylglucosamine 2-epimerase superfamily members. Dimer formation was verified by analyzing CJ0554 and its ortholog protein through gel-filtration chromatography. The top of the CJ0554 monomer barrel harbors a cavity, which is connected to that of the second subunit in the dimer structure, generating a larger intersubunit cavity. This elongated cavity accommodates extra nonproteinaceous electron density, presumably as a pseudosubstrate, and is lined with generally catalytically active histidine residues that are invariant in CJ0554 orthologs. Therefore, we propose that the cavity functions as the active site of CJ0554.

Organocatalytic Asymmetric [3 + 2]-Annulations of γ-Sulfonamido/γ-Hydroxy-α,ß-Unsaturated Ketones with Cyclic N-Sulfimines: Synthesis of Chiral Polyheterotricyclic Imidazolidines and Oxazolidines.

The first organocatalytic asymmetric [3 + 2]-annulation of γ-sulfonamido-α,ß-unsaturated ketones with cyclic N-sulfimines has been developed, and enantioenriched functionalized polyheterotricyclic imidazolidines were obtained in good yields and with excellent enantioselectivities. This approach was also extended to the asymmetric [3 + 2]-annulation of γ-hydroxy-α,ß-unsaturated ketones, affording enantioenriched polyheterotricyclic oxazolidines. In addition, base-catalyzed [3 + 2]-annulations of γ-sulfonamido/γ-hydroxy-α,ß-unsaturated ketones with cyclic N-sulfimines were re-investigated under mild reaction conditions for the synthesis of racemic polyheterotricyclic imidazolidines and oxazolidines with excellent diastereoselectivities.

Absolute response of a proton detector composed of a microchannel plate assembly and a charge-coupled device to laser-accelerated multi-MeV protons.

The absolute response of a real-time proton detector, composed of a microchannel plate (MCP) assembly, an imaging lens, and a charge-coupled device (CCD) camera, is calibrated for the spectral characterization of laser-accelerated protons, using a Thomson parabola spectrometer (TPS). A slotted CR-39 plate was used as an absolute particle-counting detector in the TPS, simultaneously with the MCP-CCD detector to obtain a calibration factor (count/proton). In order to obtain the calibration factor as a function of proton energy for a wide range of proton numbers, the absolute response was investigated for different operation parameters of the MCP-CCD detector, such as MCP voltage, phosphor voltage, and CCD gain. A theoretical calculation for the net response of the MCP was in good agreement with the calibrated response of the MCP-CCD detector, and allows us to extend the response to higher proton energies. The response varies in two orders of magnitude, showing an exponential increase with the MCP voltage and almost linear increase with the phosphor voltage and the CCD gain. The calibrated detector enabled characterization of a proton energy spectrum in a wide dynamic range of proton numbers. Moreover, two MCP assemblies having different structures of MCP, phosphor screen, and optical output window have been calibrated, and the difference in the absolute response was highlighted. The highly-sensitive detector operated with maximum values of the parameters enables measuring a single proton particle and evaluating an absolute spectrum at high proton energies in a single laser shot. The absolute calibrations can be applied for the spectral measurement of protons using different operating voltages and gains for optimized response in a large range of proton energy and number.

Characterization of Rotavirus Infection in Hospitalized Children under 5 with Acute Gastroenteritis 5 Years after Introducing the Rotavirus Vaccines in South Korea.

We herein characterized rotavirus infection in hospitalized children under 5 years of age with gastroenteritis after introducing rotavirus vaccines in South Korea from 20 February 2012, to 31 March 2013. Enzyme-linked fluorescent immunoassay was performed to detect rotavirus antigens. G and P genotyping was performed using nested multiplex PCR. For the failed PCR samples, sequencing was conducted. We performed a test-negative case-control study to estimate vaccine effectiveness. Vaccine effectiveness was measured using a multivariate logistic regression model. Rotavirus was detected in 16 (13.2%) of the 121 patients, with a seasonal peak in April 2012. The dominant genotypes detected were G3P[8] (33.3%) and G4P[6] (26.7%), and vaccine effectiveness against rotavirus hospitalization was 84.9% [95% CI: 23.2−97.0] in the complete vaccinated group. A higher prevalence of rotavirus infection was observed among children with siblings than those without siblings (p < 0.001). Also, the presence of siblings was significantly associated with a history of nonvaccination (p < 0.001). In conclusion, the prevalence of rotavirus followed a decreasing trend, and there was no evidence of emergences of nonvaccine-type strains. Vaccine effectiveness against rotavirus hospitalization was 84.9%. Although children with siblings were more susceptible to rotavirus infection, they were less likely to receive vaccination against rotavirus.

Particulate matter exposure exacerbates cellular damage by increasing stress granule formation in respiratory syncytial virus-infected human lung organoids.

Exposure to atmospheric particulate matter (PM) increases morbidity and mortality in respiratory diseases by causing various adverse health effects; however, the effects of PM exposure on cellular stress under virus-infected conditions remain unclear. The effects of PM under 10 µm (PM10) and diesel PM (DPM) on respiratory syncytial virus (RSV) infection were investigated in human two-dimensional lung epithelial cells and human three-dimensional lung organoids mimicking the lung tissue. We evaluated the formation of stress granules, which are important in cellular adaptation to various stress conditions. Furthermore, we investigated the effects of repeated exposure to PM10 and DPM on DNA damage and cell death during viral infection. PM10 and DPM did not cause stress granule formation in the absence of RSV infection but drastically increased stress granule formation and signal transduction during RSV infection in human lung epithelial cells and human lung organoids. Further, repeated exposure to PM10 and DPM caused cell death by severely damaging DNA under RSV infection conditions. Thus, PM10 and DPM induce severe lung toxicity under stress conditions, such as viral infection, suggesting that the effects of PMs under various stressful conditions should be examined to accurately predict the lung toxicity of PM.

Wearable Photomedicine for Neonatal Jaundice Treatment Using Blue Organic Light-Emitting Diodes (OLEDs): Toward Textile-Based Wearable Phototherapeutics.

Neonatal jaundice is a very common disease in newborns and can lead to brain damage or death in severe cases. Phototherapy with light-emitting diode (LED) arrays is widely used as the easiest and fastest way to relieve jaundice in newborns, but it has distinct disadvantages such as loss of water in the patient, damage to the retina, and separation from parents. In this paper, a novel light source-based phototherapy for neonatal jaundice is proposed using a textile-based wearable organic light-emitting diode (OLED) platform that can move flexibly and conform to the curvature of the human body. The soft and flexible textile-based blue OLED platform is designed to have a peak wavelength of 470 nm, suitable for jaundice treatment, and shows performance (>20 µW cm-2 nm- 1 ) suitable for intensive jaundice treatment even at low voltage (<4.0 V). The textile-based OLEDs fabricated in this study exhibit an operating reliability of over 100 h and low-temperature operation (<35 °C). The results of an in vitro jaundice treatment test using a large-area blue OLED confirm that the bilirubin level decreases to 12 mg dL-1 with 3 h of OLED irradiation.

Clinical and growth outcomes after meconium-related ileus improved with Gastrografin enema in very low birth weight infants.

BACKGROUND: Meconium-related ileus in very low birth weight infants can lead to increased morbidity or mortality and prolonged hospitalization without prompt diagnosis and treatment. This study primarily aimed to identify the incidence of and factors associated with meconium-related ileus and secondarily sought to investigate clinical and growth outcomes after water-soluble contrast media (Gastrografin) enema. METHODS: We retrospectively reviewed medical records of very low birth weight infants born between February 2009 and March 2019 in the neonatal intensive care unit of a single medical center. Perinatal factors, clinical outcomes, and growth outcomes were compared between the group with meconium-related ileus that received Gastrografin enema and the control group. RESULTS: Twenty-four (6.9%) patients were diagnosed with meconium-related ileus among 347 very low birth weight infants. All achieved successful evacuation of meconium with an average of 2.8 (range: 1-8) Gastrografin enema attempts without procedure-related complications. Initiation of Gastrografin enema was performed at mean 7.0 days (range: 2-16) after birth. Incidences of moderate to severe bronchopulmonary dysplasia were higher and the duration of mechanical ventilation and need for oxygen were longer in the meconium-related ileus group (P = 0.039, 0.046, 0.048, respectively). Meconium-related ileus infants took more time to start enteral feeding and the nothing per oral time was longer (P = 0.001 and 0.018, respectively). However, time to achieve full enteral feeding and Z-scores for weight and height at 37 weeks and at 6 months corrected age did not differ between the two groups. CONCLUSIONS: Gastrografin enema in very low birth weight infants with meconium-related ileus was an effective and safe medical management. Following Gastrografin enema, very low birth weight infants with meconium-related ileus achieved similar subsequent feeding progress and similar growth levels as the control groups without meconium-related ileus.

Prognostic role of the ratio of natural killer cells to regulatory T cells in patients with multiple myeloma treated with lenalidomide and dexamethasone.

Despite advances in treating newly diagnosed multiple myeloma (NDMM), a biomarker-driven personalized approach remains an unmet need. A combination of lenalidomide and dexamethasone (RD) is a widely available chemotherapeutic option for NDMM. We aimed to find a circulating immune cell-based biomarker to predict prognosis following RD in patients with NDMM. Clinical data and peripheral blood samples of 71 consecutive NDMM patients treated with RD were retrospectively analyzed. Peripheral blood samples were taken at the time of diagnosis. Immune cell populations, including natural killer (NK) cells, T cells, and their subpopulations, were identified by flow cytometry. In univariable analysis, four variables, including low expression (third or lower quartile) of NK cells, high expression (first or greater quartile) of regulatory T (Treg) cells, female sex, and lambda light chain type, could be plausible factors in predicting poor progression-free survival (PFS). With use of the ratio of NK cells to Treg cells (NK/Treg) as a biomarker, the median PFS of patients with low NK/Treg (less than first quartile, n = 18) was significantly inferior to that of patients with high NK/Treg (first or greater quartile, n = 53): 19.8 months versus 57.3 months, p = 0.047. In multivariable analysis, low NK/Treg was significantly associated with poor PFS (hazard ratio: 2.877, 95% confidence interval: 0.001-1.009, p = 0.048), even after adjusting for other confounding factors. NK/Treg at the time of diagnosis might be a useful immune cell biomarker for clinical decision-making for the use of RD in NDMM. Further investigations are needed to improve outcomes of NDMM patients based on the understanding of the role of NK/Treg.

Incidence, Clinical Characteristics, and Genotype Distribution of Rotavirus in a Neonatal Intensive Care Unit 5 Years After Introducing Rotavirus Vaccine.

BACKGROUND: Rotavirus (RV) is a common cause of viral gastroenteritis in children worldwide. We aimed to investigate the incidence, symptoms, and genotype of RV infection in a neonatal intensive care unit (NICU) in South Korea 5 years after the introduction of RV vaccination to evaluate its effect on newborn infants. METHODS: A total of 431 fecal specimens were collected from patients admitted to NICU between April 20, 2012 and September 10, 2013. Enzyme-linked immunoassays were used to detect RV antigen. Nested multiplex polymerase chain reaction was used for genotyping. RESULTS: The overall incidence of RV infection was 43.9% and was significantly higher in preterm infants, infants born in the study hospital, low birth weight infants, and cesarean births (P < 0.05). Symptoms of diarrhea, poor feeding, abdominal distension, and apnea were significantly higher in infants with RV infection than those without infection. RV infection gradually increased depending on infant care at home, postpartum clinic, or hospital (26.0, 45.1, and 60.2%, respectively; P = 0.000). The dominant RV genotype in the NICU was G4P[6] at 95.4%. CONCLUSION: Current RV vaccines did not affect the incidence of RV infection in newborn and preterm infants in the NICU. Most RV-positive patients in the NICU had symptoms, and the incidence of RV infection was relatively higher in hospitals and postpartum clinics with group life than home. The dominant RV genotype was G4P[6] across study groups.

Pulmonary fibrosis model using micro-CT analyzable human PSC-derived alveolar organoids containing alveolar macrophage-like cells.

Human lung organoids (hLOs) are useful for disease modelling and drug screening. However, a lack of immune cells in hLOs limits the recapitulation of in vivo cellular physiology. Here, we generated hLOs containing alveolar macrophage (AMφ)-like cells derived from pluripotent stem cells (PSC). To bridge hLOs with advanced human lung high-resolution X-ray computed tomography (CT), we acquired quantitative micro-CT images. Three hLO types were observed during differentiation. Among them, alveolar hLOs highly expressed not only lung epithelial cell markers but also AMφ-specific markers. Furthermore, CD68+ AMφ-like cells were spatially organized on the luminal epithelial surface of alveolar hLOs. Bleomycin-treated alveolar hLOs showed upregulated expression of fibrosis-related markers and extracellular matrix deposits in the alveolar sacs. Alveolar hLOs also showed structural alterations such as excessive tissue fraction under bleomycin treatment. Therefore, we suggest that micro-CT analyzable PSC-derived alveolar hLOs are a promising in vitro model to predict lung toxicity manifestations, including fibrosis.

Neighbor-Aware Non-Orthogonal Multiple Access Scheme for Energy Harvesting Internet of Things.

In a non-orthogonal multiple access (NOMA) environment, an Internet of Things (IoT) device achieves a high data rate by increasing its transmission power. However, excessively high transmission power can cause an energy outage of an IoT device and have a detrimental effect on the signal-to-interference-plus-noise ratio of neighbor IoT devices. In this paper, we propose a neighbor-aware NOMA scheme (NA-NOMA) where each IoT device determines whether to transmit data to the base station and the transmission power at each time epoch in a distributed manner with the consideration of its energy level and other devices' transmission powers. To maximize the aggregated data rate of IoT devices while keeping an acceptable average energy outage probability, a constrained stochastic game model is formulated, and the solution of the model is obtained using a best response dynamics-based algorithm. Evaluation results show that NA-NOMA can increase the average data rate up to 22% compared with a probability-based scheme while providing a sufficiently low energy outage probability (e.g., 0.05).

Assessment of optimal chest compression depth during neonatal cardiopulmonary resuscitation: a randomised controlled animal trial.

AIM: The study aimed to examine the optimal anterior-posterior depth which will reduce the time to return of spontaneous circulation and improve survival during chest compressions. Asphyxiated neonatal piglets receiving chest compression resuscitated with a 40% anterior-posterior chest depth compared with 33%, 25% or 12.5% will have reduced time to return of spontaneous circulation and improved survival. METHODS: Newborn piglets (n=8 per group) were anaesthetised, intubated, instrumented and exposed to 45 min normocapnic hypoxia followed by asphyxia and cardiac arrest. Piglets were randomly allocated to four intervention groups ('anterior-posterior 12.5% depth', 'anterior-posterior 25% depth', 'anterior-posterior 33% depth' or 'anterior-posterior 40% depth'). Chest compressions were performed using an automated chest compression machine with a rate of 90 per minute. Haemodynamic and respiratory parameters, applied compression force, and chest compression depth were continuously measured. RESULTS: The median (IQR) time to return of spontaneous circulation was 600 (600-600) s, 135 (90-589) s, 85 (71-158)* s and 116 (63-173)* s for the 12.5%, 25%, 33% and 40% depth groups, respectively (*p<0.001 vs 12.5%). The number of piglets that achieved return of spontaneous circulation was 0 (0%), 6 (75%), 7 (88%) and 7 (88%) in the 12.5%, 25%, 33% and 40% anterior-posterior depth groups, respectively. Arterial blood pressure, central venous pressure, carotid blood flow, applied compression force, tidal volume and minute ventilation increased with greater anterior-posterior chest depth during chest compression. CONCLUSIONS: Time to return of spontaneous circulation and survival were similar between 25%, 33% and 40% anterior-posterior depths, while 12.5% anterior-posterior depth did not result in return of spontaneous circulation or survival. Haemodynamic and respiratory parameters improved with increasing anterior-posterior depth, suggesting improved organ perfusion and oxygen delivery with 33%-40% anterior-posterior depth. TRIAL REGISTRATION NUMBER: PTCE0000193.

Relationship between DNA mismatch repair and CRISPR/Cas9-mediated knock-in in the bovine ß-casein gene locus.

OBJECTIVE: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine ß-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). METHODS: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine ß-casein gene locus, followed by treatment of the cells with CdCl2 for 24 hours. After 3 days of CdCl2 treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). RESULTS: Treatment with CdCl2 decreased the mRNA expression of RAD51 and MMR-related genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. CONCLUSION: Treatment with CdCl2 inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.