Microfluidic Transfection System and Temperature Strongly Influence the Efficiency of Transient Transfection.

For the process of transient transfection (TTF), DNA is often transported into the cells using polyplexes. The polyplex uptake and the subsequent transient expression of the gene of interest are of great importance for a successful transfection. In this study, we investigated a 3D-printed microfluidic system designed to facilitate direct TTF for suspension of CHO-K1 cells. The results demonstrate that this system achieves significantly better results than the manual approach. Furthermore, the effect of both post-transfection incubation time (t) and temperature (T) on polyplex uptake was explored in light of the membrane phase transitions. Attention was paid to obtaining the highest possible transfection efficiency (TFE), viability (V), and viable cell concentration (VCC). Our results show that transfection output measured as product of VCC and TFE is optimal for t = 1 h at T = 22 °C. Moreover, post-transfection incubation at T = 22 °C with short periods of increased T at T = 40 °C were observed to further increase the output. Finally, we found that around T = 19 °C, the TFE increases strongly. This is the membrane phase transition T of CHO-K1 cells, and those results therefore suggest a correlation between membrane order and permeability (and in turn, TFE).

Heterochronic shift in gene expression leads to ontogenetic morphological divergence between two closely related polyploid species.

Heterochrony-alteration to the rate or timing of development-is an important mechanism of trait differentiation associated with speciation. Heterochrony may explain the morphological divergence between two polyploid species, June sucker (Chasmistes liorus) and Utah sucker (Catostomus ardens). The larvae of both species have terminal mouths; however, as adults, June sucker and Utah sucker develop subterminal and ventral mouths, respectively. We document a difference in the timing of shape development and a corresponding change in the timing of gene expression, suggesting the distinctive mouth morphology in June suckers may result from paedomorphosis. Specifically, adult June suckers exhibit an intermediate mouth morphology between the larval (terminal) and ancestral (ventral) states. Endemic and sympatric Chasmistes/Catostomus pairs in two other lakes also are morphologically divergent, but genetically similar. These species pairs could have resulted from the differential expression of genes and corresponding divergence in trait development. Paedomorphosis may lead to adaptive diversification in Catostomids.

Revolutionizing patient care: the harmonious blend of artificial intelligence and surgical tradition.

Surgery has undergone remarkable evolution over the past decades, propelled by unprecedented technological advancement. Despite these changes, the role of surgeons and their irreplaceable qualities remains pivotal. This article delves into the intersection of surgery and artificial intelligence (AI), underscoring the enduring significance of human expertise and values. The potential of AI to learn and improve over time holds great promise for enhancing various facets of surgery, including diagnostics, personalized treatment, preoperative planning, real-time support in the operating room, and comprehensive postoperative analytics of the outcome. However, it is essential to emphasize the continued importance of the surgeon's role to uphold universal surgical principles. This includes a commitment to minimalism and the use of evidence-based practice, ensuring optimal outcomes and standardized procedures. By recognizing the synergies between AI and traditional surgical approaches, we can navigate the evolving landscape of surgery to achieve the highest standards of patient care.

Preclinical models of maternal asthma and progeny outcomes: a scoping review.

There is an increased risk of adverse perinatal outcomes in the ∼17% of women with asthma during pregnancy. The mechanisms linking maternal asthma and adverse outcomes are largely unknown, but reflect joint effects of genetics and prenatal exposure to maternal asthma. Animal models are essential to understand the underlying mechanisms independent of genetics and comorbidities, and enable safe testing of interventions. This scoping review aimed to explore the methodology, phenotype, characteristics, outcomes and quality of published studies using preclinical maternal asthma models. MEDLINE (PubMed), Embase (Elsevier) and Web of Science were systematically searched using previously validated search strings for maternal asthma and for animal models. Two reviewers independently screened titles and abstracts, full texts, and then extracted and assessed the quality of each study using the Animal Research: Reporting of In Vivo Experiments (ARRIVE) 2.0 guidelines. Out of 3618 studies identified, 39 were eligible for extraction. Most studies were in rodents (86%) and all were models of allergic asthma. Maternal and progeny outcomes included airway hyperresponsiveness, airway resistance, inflammation, lung immune cells, lung structure and serum immunoglobulins and cytokines. Experimental design (100%), procedural details (97%) and rationale (100%) were most often reported. Conversely, data exclusion (21%), blinding (18%) and adverse events (8%) were reported in a minority of studies. Species differences in physiology and timing of development, the use of allergens not relevant to humans and a lack of comparable outcome measures may impede clinical translation. Future studies exploring models of maternal asthma should adhere to the minimum core outcomes set presented in this review.

Child development education in the Neonatal Unit: Understanding parent developmental literacy needs, priorities and preferences.

OBJECTIVE: To describe child development knowledge needs, priorities, and preferences for education to enhance developmental literacy among parents with children admitted to the neonatal unit (NNU). METHODS: Two separate cohorts completed a survey; 1) Parents with children graduated from Australian NNUs (n = 316); 2) Parents with infants' inpatient at two South Australian NNUs (n = 209). RESULTS: Parents considered it extremely important to understand child development (Graduates: 80%; Inpatients: 71%). Inpatient parents reported lower child development knowledge. Almost half (42%) of graduate parents described the child development education provided by neonatal staff as poor or inadequate. There was consistency in preferences for developmental literacy education provision. Parents desired education to commence during NNU and continue post discharge. Priorities included content specific to preterm birth and how to support child development over the first two years of life. Individualised education by a Neonatal Nurse/Midwife was most preferred. CONCLUSION: Mothers and fathers value guidance to support their child's development during NNU admission and early childhood. Our study highlights the importance of improved early developmental literacy education for parents with children admitted to the neonatal unit. PRACTICE IMPLICATIONS: Our findings can be used to inform the creation of future educational resources targeting improved parent developmental literacy.

Evidence-Based Medicine and Observational Studies.

Evidence-based medicine drives medical decision-making in the modern era, which has historically favored randomized control trials. Despite their notoriety, randomized control trials have multiple disadvantages when applied to spinal surgery. Observational studies are popular in spinal surgery literature and are seen in various forms, such as retrospective studies and prospective cohort studies. For researchers, learners, and practicing spine surgeons, this paper describes options for study design when applied to spinal surgery.

Longitudinal trends in domperidone dispensing to mothers of very preterm infants and its association with breast milk feeding at infant discharge: a retrospective study.

OBJECTIVE: This study aims: (a) to evaluate patterns of domperidone dispensing to mothers of very preterm (<32 weeks gestation) infants born before and after 2014 when international recommendations were made to limit its use and (b) to examine characteristics associated with domperidone dispensing and impacts on breast milk feeding rates at infant hospital discharge. DESIGN: Retrospective audit using linked electronic medical records and hospital pharmacy records. SETTING: Tertiary-referral neonatal intensive care unit at the Women's and Children's Hospital in South Australia. PATIENTS: Mothers of preterm infants admitted to neonatal intensive care from January 2004 to December 2018. MAIN OUTCOME MEASURES: Rate of domperidone dispensing compared pre-2014 and post-2014 recommendations using interrupted time series analyses, and breast milk feeding rates at infant discharge based on domperidone treatment status, adjusted for other factors known to influence breast milk production. RESULTS: Overall, domperidone was dispensed to 691 (41%) of 1688 mothers. Prior to 2014 recommendations, the proportion of women dispensed domperidone was stable. Following the recommendations, there was a significant reduction in trend (-2.55% per half year, 95% CI -4.57% to -0.53%;), reflecting less domperidone dispensing.Breast milk feeding rates at discharge remained consistently lower in infants of women dispensed domperidone than those who were not (adjusted OR 0.58, 95% CI 0.45 to 0.75). CONCLUSION: Domperidone dispensing in mothers of hospitalised very preterm infants has declined over time following international regulatory warnings. Breast milk feeding rates remain lower in mothers prescribed domperidone, suggesting further research is needed to optimise lactation support for mothers of very preterm infants.

Thresholds for Red Blood Cell Transfusion in Preterm Infants: Evidence to Practice.

Rapid blood loss with circulatory shock is dangerous for the preterm infant as cardiac output and oxygen-carrying capacity are simultaneously imperilled. This requires prompt restoration of circulating blood volume with emergency transfusion. It is recommended that clinicians use both clinical and laboratory responses to guide transfusion requirements in this situation. For preterm infants with anemia of prematurity, it is recommended that clinicians use a restrictive algorithm from one of two recently published clinical trials. Transfusion outside these algorithms in very preterm infants is not evidence-based and is actively discouraged.

Study protocol of the WashT Trial: transfusion with washed versus unwashed red blood cells to reduce morbidity and mortality in infants born less than 28 weeks' gestation - a multicentre, blinded, parallel group, randomised controlled trial.

INTRODUCTION: Many extremely preterm newborns develop anaemia requiring a transfusion, with most receiving three to five transfusions during their admission. While transfusions save lives, the potential for transfusion-related adverse outcomes is an area of growing concern. Transfusion is an independent predictor of death and is associated with increased morbidity, length of hospital stay, risk of infection and immune modulation. The underlying mechanisms include adverse pro-inflammatory and immunosuppressive responses. Evidence supports an association between transfusion of washed red cells and fewer post-transfusion complications potentially through removal of chemokines, lipids, microaggregates and other biological response modifiers. However, the clinical and cost-effectiveness of washed cells have not been determined. METHODS AND ANALYSIS: This is a multicentre, randomised, double-blinded trial of washed versus unwashed red cells. Infants <28 weeks' gestation requiring a transfusion will be enrolled. Transfusion approaches will be standardised within each study centre and will occur as soon as possible with a recommended fixed transfusion volume of 15 mL/kg whenever the haemoglobin is equal to or falls below a predefined restrictive threshold, or when clinically indicated. The primary outcome is a composite of mortality and/or major morbidity to first discharge home, defined as one or more of the following: physiologically defined bronchopulmonary dysplasia; unilateral or bilateral retinopathy of prematurity grade >2, and; necrotising enterocolitis stage ≥2. To detect a 10% absolute reduction in the composite outcome from 69% with unwashed red blood cell (RBCs) to 59% with washed RBCs with 90% power, requires a sample size of 1124 infants (562 per group). Analyses will be performed on an intention-to-treat basis with a prespecified statistical analysis plan. A cost-effectiveness analysis will also be undertaken. ETHICS AND DISSEMINATION: Ethics approval has been obtained from the Women's and Children's Health Network Human Research Ethics Committee (HREC/12/WCHN/55). The study findings will be disseminated through peer-reviewed articles and conferences. TRIAL REGISTRATION NUMBER: ACTRN12613000237785 Australian New Zealand Clinical Trials Registry.

Intrahepatic cholestasis of pregnancy - Diagnosis and management: A consensus statement of the Society of Obstetric Medicine of Australia and New Zealand (SOMANZ): Executive summary.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy liver disease, characterised by pruritus and increased total serum bile acids (TSBA), Australian incidence 0.6-0.7%. ICP is diagnosed by non-fasting TSBA ≥19 µmol/L in a pregnant woman with pruritus without rash without a known pre-existing liver disorder. Peak TSBA ≥40 and ≥100 µmol/L identify severe and very severe disease respectively, associated with spontaneous preterm birth when severe, and with stillbirth, when very severe. Benefit-vs-risk for iatrogenic preterm birth in ICP remains uncertain. Ursodeoxycholic acid remains the best pharmacotherapy preterm, improving perinatal outcome and reducing pruritus, although it has not been shown to reduce stillbirth.

Inviting Newton to Visit the Delivery Room. The Role of Gravity During Childbirth.

Throughout history, the upright position was the norm for most deliveries. However, due to cultural reasons, this practice was changed in the 17th century, and since then, the majority of deliveries have been conducted with the parturient lying on her back. The aim of this review article is to challenge the nowadays recumbent birth position and to emphasize the added value of gravity to the childbirth. Physiologically there is a strong argument for delivering in an upright position due to the significant role of gravity. The baby's presenting part does not move back between contractions as happens in today's recumbent position when due to the anatomy of the pelvis, the presenting part moves uphill. In this position, the second stage is shorter, and the signal for active pushing occurs spontaneously. It is recommended that any parturient should be informed about the risks and benefits of each birthing option to decide the birthing position that suits her.

PKCζ activation promotes maturation of cord blood T cells towards a Th1 IFN-γ propensity.

A significant number of babies present transiently with low protein kinase C zeta (PKCζ) levels in cord blood T cells (CBTC), associated with reduced ability to transition from a neonatal Th2 to a mature Th1 cytokine bias, leading to a higher risk of developing allergic sensitisation, compared to neonates whose T cells have 'normal' PKCζ levels. However, the importance of PKCζ signalling in regulating their differentiation from a Th2 to a Th1 cytokine phenotype propensity remains undefined. To define the role of PKCζ signalling in the regulation of CBTC differentiation from a Th2 to a Th1cytokine phenotype we have developed a neonatal T cell maturation model which enables the cells to develop to CD45RA- /CD45RO+ T cells while maintaining the Th2 immature cytokine bias, despite having normal levels of PKCζ. The immature cells were treated with phytohaemagglutinin, but in addition with phorbol 12-myristate 13-acetate (PMA), an agonist which does not activate PKCζ. This was compared to development in CBTC in which the cells were transfected to express constitutively active PKCζ. The lack of PKCζ activation by PMA was monitored by western blot for phospho-PKCζ and translocation from cell cytosol to the membrane by confocal microscopy. The findings demonstrate that PMA fails to activate PKCζ in CBTC. The data show that CBTC matured under the influence of the PKC stimulator, PMA, maintain a Th2 cytokine bias, characterised by robust IL-4 and minimal interferon gamma production (IFN-γ), and lack of expression of transcriptional factor, T-bet. This was also reflected in the production of a range of other Th2/Th1 cytokines. Interestingly, introduction of a constitutively active PKCζ mutant into CBTC promoted development towards a Th1 profile with high IFN-γ production. The findings demonstrate that PKCζ signalling is essential for the immature neonatal T cells to transition from a Th2 to a Th1 cytokine production bias.

Molecular Farming of Pembrolizumab and Nivolumab.

Immune checkpoint inhibitors (ICIs) are a class of immunotherapy agents capable of alleviating the immunosuppressive effects exerted by tumorigenic cells. The programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint is one of the most ubiquitous checkpoints utilized by tumorigenic cells for immune evasion by inducing apoptosis and inhibiting the proliferation and cytokine production of T lymphocytes. Currently, the most frequently used ICIs targeting the PD-1/PD-L1 checkpoint include monoclonal antibodies (mAbs) pembrolizumab and nivolumab that bind to PD-1 on T lymphocytes and inhibit interaction with PD-L1 on tumorigenic cells. However, pembrolizumab and nivolumab are costly, and thus their accessibility is limited in low- and middle-income countries (LMICs). Therefore, it is essential to develop novel biomanufacturing platforms capable of reducing the cost of these two therapies. Molecular farming is one such platform utilizing plants for mAb production, and it has been demonstrated to be a rapid, low-cost, and scalable platform that can be potentially implemented in LMICs to diminish the exorbitant prices, ultimately leading to a significant reduction in cancer-related mortalities within these countries.

Preclinical models of maternal asthma and progeny outcomes: a scoping review protocol.

OBJECTIVE: This scoping review will describe the methodology, phenotype, and characteristics of maternal asthma models used in preclinical studies and the outcomes that have been measured in the mother and progeny. The review This will identify gaps in knowledge of maternal and progeny outcomes following maternal asthma in pregnancy. INTRODUCTION: Maternal asthma affects up to 17% of pregnancies worldwide and is associated with adverse perinatal outcomes in mothers and babies, including pre-eclampsia, gestational diabetes, cesarean section, preterm birth, small for gestational age, nursery admission, and neonatal death. While the associations are well established, the mechanisms linking maternal asthma and adverse perinatal outcomes are largely unknown due to the difficulties of human mechanistic studies. The appropriate selection of animal models is vital to understanding the mechanisms underlying associations between human maternal asthma and adverse perinatal outcomes. INCLUSION CRITERIA: This review will include primary studies published in English where outcomes have been studied in vivo in non-human mammalian species. METHODS: This review will follow the JBI methodology for scoping reviews. We will search MEDLINE (PubMed), Embase, and Web of Science to identify papers published before the end of 2022. Initial keywords will include pregnancy, gestation, asthma , and wheeze , as well as validated search strings to identify papers that describe animal models. Extracted data will include information on methods used to induce maternal asthma; asthmatic phenotypes and characteristics; and maternal, pregnancy, placental, and progeny outcomes. The characteristics of each study will be presented in summary tables and a core outcome list to assist researchers in developing, reporting, and comparing future animal studies of maternal asthma. REVIEW REGISTRATION: Open Science Framework osf.io/trwk5.

Effectiveness of Intraoperative Neuromonitoring in a Patient Undergoing a One-Level Transforaminal Lumbar Interbody Fusion: A Case Report.

Posterior lumbar interbody fusion (PLIF) and transforaminal lumbar interbody fusion (TLIF) are common modes of operative treatment of lumbar radiculopathy and spondylolisthesis. An integral part of these procedures is the appropriate placement of pedicle screws to ensure proper fusion. Breach of the medial cortex during pedicle screw fixation can potentially cause permanent impairment for a patient; significant technology and resources have been universally devoted to preventing this complication. Intraoperative neuromonitoring (IONM) is a frequently used tool by spine surgeons, which, along with fluoroscopy, is traditionally thought to reduce the incidence of neurologic injury. Unfortunately, IONM is not infallible and, in certain studies, has not been shown to decrease the risk of neurologic compromise. This case presentation details the clinical course of a 55-year-old who underwent an L4-5 TLIF. Despite benign electromyography recordings intraoperatively, the patient presented postoperatively with a new-onset left foot drop and a CT scan that confirmed bilateral L4 screw malposition with a breach of the medial cortex. We hope to further advance the discussion regarding the dangerous inconsistency of IONM in hopes of identifying a multimodal approach to avoid dreaded complications like this one in the future.

Neonatal Group B Streptococcal Infection in Australia: A Case-control Study.

BACKGROUND: To determine maternal and neonatal risk factors for, and incidence of, neonatal early-onset group B streptococcus (EOGBS) and late-onset (LOGBS) infection in South Australia (SA) and the Northern Territory (NT). METHODS: A case-control study with 2:1 matched controls to cases. The study included tertiary hospitals in South Australia and the Northern Territory, Australia. Retrospective data were collected from a 16-year epoch (2000-2015). RESULTS: Of a total of 188 clinically suspected or confirmed cases, 139 were confirmed, of which 56.1% (n = 78) were EOGBS and 43.9% (n = 61) were LOGBS. The incidence of clinically suspected and confirmed cases of EOGBS was 0.26/1000 live births in SA and 0.73/1000 live births in the NT, and the incidence of confirmed cases was 0.19/1000 for SA and 0.36/1000 for the NT. The incidence of clinically suspected or confirmed LOGBS was 0.18/1000 live births in SA and 0.16/1000 for the NT. The majority of infants with GBS presented with sepsis, pneumonia, or meningitis. Developmental delay was the most commonly recorded long-term complication at 1 year old. Risk factors for EOGBS included maternal GBS carriage, previous fetal death, identifying as Aboriginal and/or Torres Strait Islander, and maternal fever in labor/chorioamnionitis. CONCLUSIONS: GBS remains a leading cause of neonatal morbidity and mortality. Adding previous fetal death to GBS screening guidelines would improve GBS prevention. The introduction of maternal GBS vaccination programs should be guided by country-specific disease epidemiology.

Does donor sex influence the potential for transfusion with washed packed red blood cells to limit transfusion-related immune responses in preterm newborns?

OBJECTIVE: To evaluate the association of donor sex with transfusion-associated recipient immune responses in preterm newborns receiving unwashed and washed blood. DESIGN: A cohort study using data collected during the Effect of Washed versus Unwashed Packed Red Blood Cell Transfusion on Immune Responses in the Extremely Preterm Newborn randomised trial. SETTING: Participants were recruited from two South Australian hospitals between September 2015 and December 2020. PATIENTS: Preterm newborns (<29 weeks). INTERVENTIONS: Transfusion with unwashed and washed packed red blood cells (PRBCs) from either exclusively male or any female donor for the first three transfusions. MAIN OUTCOMES MEASURES: The primary outcome was the change from baseline in post-transfusion plasma cytokine concentrations, specifically interferon gamma, interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12, IL-17A and tumour necrosis factor (TNF). RESULTS: In total, 153 newborns were evaluated. By the third transfusion, the magnitude of pretransfusion to post-transfusion change in cytokines between the groups differed for IL-6 (p=0.003), IL-12 (p=0.008), IL-17A (p=0.003) and TNF (p=0.007). On post hoc comparison, compared with the unwashed-any female donor group, IL-6 (p<0.05), IL-12 (p<0.05) and IL-17A (p<0.01) were lower in the washed-exclusively male donor group, and IL-6 (p<0.01), IL-12 (p<0.05) and TNF (p<0.01) were lower in the washed-any female donor group. CONCLUSION: These findings suggest that transfusion with unwashed PRBCs from female donors is associated with an increased recipient immune response, an effect that can be ameliorated with pretransfusion washing. Larger randomised controlled studies confirming this mechanistic link between donor sex and transfusion-associated morbidity are warranted. TRIAL REGISTRATION NUMBER: ACTRN12613000237785.

Red cell infusion but not saline is effective for volume expansion in preterm piglets.

BACKGROUND: A common first-line treatment for supporting cardiovascular function in preterm infants is volume expansion using saline, but this does not improve outcomes. This study aimed to determine if volume expansion with saline increases blood volume, blood pressure and cerebral oxygenation; and if volume expansion with packed red blood cells (RBC) is more effective. We hypothesized that RBC infusion is more effective than saline for increasing blood volume and maintaining cardiovascular function and cerebral oxygenation. METHODS: Five groups of preterm piglets (98/115d gestation) were infused with saline (10 or 20 mL/kg) or RBC (10 or 20 mL/kg) or no treatment. Blood volume, blood pressure, central venous pressure, heart rate, carotid flow, cerebral oxygenation, arterial pH, base excess, and lactate levels were assessed for 6 h after treatment started. RESULTS: Both RBC groups had significant increases in blood volume, and improved measures of cardiovascular function, cerebral oxygenation and metabolic acidosis. Saline infusion did not increase blood volume or measures of cardiovascular function, cerebral oxygenation or metabolic acidosis. CONCLUSIONS: The results suggest that the deteriorating cardiovascular function in the hours after birth in preterm piglets, and possibly in premature babies, may be reversed or halted by more effective support of blood volume. IMPACT: Blood volume decreases after birth in preterm piglets and this decrease is associated with deteriorating cardiovascular function and cerebral oxygenation. Infusion of saline does not increase blood volume nor prevent deterioration in cardiovascular function. Infusion of packed red blood cells results in an increase in blood volume and improvements in cardiovascular function and cerebral oxygenation. Deteriorating cardiovascular function in the hours after birth in preterm piglets, and possibly in human preterm neonates, may be reversed or halted by more effective support of blood volume.

Testing the acceptability of stillbirth awareness messages in an SMS program for fathers.

ISSUE ADDRESSED: To raise expectant fathers' awareness of risk factors for stillbirth. METHODS: A set of brief text messages was developed addressing recognised risk factors for stillbirth: avoidance of maternal cigarette smoking, maternal going to sleep on side messaging, awareness of the importance of noticing and reporting changes in foetal movement and fathers' involvement in shared decision making for timing of birth. Eight messages were inserted into the SMS4dads pilot program being conducted by NSW Health. Feedback on the messages was requested. Participants rated the quality of the messages on a three-point Likert scale and provided comments. RESULTS: Overall, 2528 messages were sent to 626 fathers' mobile phones, 45% of fathers replied with 666 ratings and 115 comments evaluating the texts. The quantitative ratings indicated substantial overall approval of the messages. Within the coding category "Evaluation of Message Content," three themes described fathers' reactions and feelings about the smoking, movement, side sleeping and birth timing messages: "important-good information," "not appropriate/anxiety provoking" and "not relevant-obvious." Three themes reflecting the attributes of the messages within the "Service Quality" category were "need more information," "complements public health" and "child voice fit." CONCLUSIONS: Results indicate that the messages are an acceptable way to provide information and suggested actions addressing stillbirth risk factors to fathers-to-be. SO WHAT?: Fathers' awareness of the risk factors for stillbirth can assist mothers to take appropriate actions for a healthy birth. Information on risk factors can be provided to fathers via a father-focused text messaging service.