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Pulmonary hypertension contributes to morbidity and mortality in both the term newborn infant, referred to as persistent pulmonary hypertension of the newborn (PPHN), and the premature infant, in the setting of abnormal pulmonary vasculature development and arrested growth. In the term infant, PPHN is characterized by the failure of the physiological postnatal decrease in pulmonary vascular resistance that results in impaired oxygenation, right ventricular failure, and pulmonary-to-systemic shunting. The pulmonary vasculature is either maladapted, maldeveloped, or underdeveloped. In the premature infant, the mechanisms are similar in that the early onset pulmonary hypertension (PH) is due to pulmonary vascular immaturity and its underdevelopment, while late onset PH is due to the maladaptation of the pulmonary circulation that is seen with severe bronchopulmonary dysplasia. This may lead to cor-pulmonale if left undiagnosed and untreated. Neonatologist performed echocardiography (NPE) should be considered in any preterm or term neonate that presents with risk factors suggesting PPHN. In this review, we discuss the risk factors for PPHN in term and preterm infants, the etiologies, and the pathophysiological mechanisms as they relate to growth and development of the pulmonary vasculature. We explore the applications of NPE techniques that aid in the correct diagnostic and pathophysiological assessment of the most common neonatal etiologies of PPHN and provide guidelines for using these techniques to optimize the management of the neonate with PPHN.

BackgroundChanges in systemic and cerebral hemodynamics in preterm infants during early transitional circulation are complex and may differ between infants with or without intraventricular hemorrhage (IVH).MethodIn total, 43 infants born at median (range) 25 + 5 (23 + 3–31) had continuous near-infrared spectroscopy (NIRS) monitoring of tissue oxygenation index (TOI) and cerebrovascular reactivity within the first 48 h of life. Measurements of left and right cardiac outputs (LVO, RVO) and patent ductus arteriosus (PDA) were collected at 6, 12, 24, and 48 h of life.ResultsLVO increased within the first 48 h in the IVH (P = 0.007) and no-IVH (P < 0.001) groups. The pattern of change in LVO and RVO was not different between these two groups. TOI was lower in the IVH (P < 0.001) group. A positive correlation between TOI and LVO (P = 0.003) and a negative correlation between the tissue oxygen reactivity index (TOx) and LVO (P = 0.04) were observed at 24 h of life in the IVH group. PDA diameter was not different between IVH groups at any time interval.ConclusionCerebral oxygenation was lower and cerebrovascular reactivity was passive to systemic blood flow at 24 h in infants who developed an IVH.

Subplate neurons (SPNs) are thought to play a role in nascent sensory processing in neocortex. To better understand how heterogeneity within this population relates to emergent function, we investigated the synaptic connectivity of Lpar1-EGFP SPNs through the first postnatal week in whisker somatosensory cortex (S1BF). These SPNs comprise of two morphological subtypes: fusiform SPNs with local axons and pyramidal SPNs with axons that extend through the marginal zone. The former receive translaminar synaptic input up until the emergence of the whisker barrels, a timepoint coincident with significant cell death. In contrast, pyramidal SPNs receive local input from the subplate at early ages but then – during the later time window – acquire input from overlying cortex. Combined electrical and optogenetic activation of thalamic afferents identified that Lpar1-EGFP SPNs receive sparse thalamic innervation. These data reveal components of the postnatal network that interpret sparse thalamic input to direct the emergent columnar structure of S1BF.

Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetic tools for remote control of targeted cell populations using chemical actuators that bind to modified receptors. Despite the popularity of DREADDs in neuroscience and sleep research, potential effects of the DREADD actuator clozapine-N-oxide (CNO) on sleep have never been systematically tested. Here, we show that intraperitoneal injections of commonly used CNO doses (1, 5, and 10 mg/kg) alter sleep in wild-type male laboratory mice. Using electroencephalography (EEG) and electromyography (EMG) to analyse sleep, we found a dose-dependent suppression of rapid eye movement (REM) sleep, changes in EEG spectral power during non-REM (NREM) sleep, and altered sleep architecture in a pattern previously reported for clozapine. Effects of CNO on sleep could arise from back-metabolism to clozapine or binding to endogenous neurotransmitter receptors. Interestingly, we found that the novel DREADD actuator, compound 21 (C21, 3 mg/kg), similarly modulates sleep despite a lack of back-metabolism to clozapine. Our results demonstrate that both CNO and C21 can modulate sleep of mice not expressing DREADD receptors. This implies that back-metabolism to clozapine is not the sole mechanism underlying side effects of chemogenetic actuators. Therefore, any chemogenetic experiment should include a DREADD-free control group injected with the same CNO, C21, or newly developed actuator. We suggest that electrophysiological sleep assessment could serve as a sensitive tool to test the biological inertness of novel chemogenetic actuators. Scientists have developed ways to remotely turn on and off populations of neurons in the brain to test the role they play in behaviour. One technique that is frequently used is chemogenetics. In this approach, specific neurons are genetically modified to contain a special ‘designer receptor’ which switches cells on or off when its corresponding ‘designer drug’ is present. Recent studies have shown that the drug most commonly used in these experiments, clozapine-N-oxide (CNO), is broken down into small amounts of clozapine, an antipsychotic drug that binds to many natural receptors in the brain and modulates sleep. Nevertheless, CNO is still widely believed to not affect animals’ sleep-wake patterns which in turn could influence a range of other brain activities and behaviours. However, there have been reports of animals lacking designer receptors still displaying unusual behaviours when administered CNO. This suggests that the breakdown of CNO to clozapine may cause off-target effects which could be skewing the results of chemogenetic studies. To investigate this possibility, Traut, Mengual et al. treated laboratory mice that do not have a designer receptor with three doses of CNO, and one dose of a new designer drug called compound-21 (C21) that is not broken down to clozapine. They found that high and medium doses of CNO, but also C21 altered the sleep-wake patterns of the mice and their brain activity during sleep. These findings show that CNO and C21 both have sleep-modulating effects on the brain and suggest that these effects are not only due to the production of clozapine, but the drugs binding to off-target natural receptors. To counteract this, Traut, Mengual et al. recommend optimizing the dose of drugs given to mice, and repeating the experiment on a control group which do not have the designer receptor. This will allow researchers to determine which behavioural changes are the result of turning on or off the neuron population of interest, and which are artefacts caused by the drug itself. They also suggest testing how newly developed designer drugs impact sleep before using them in behavioural experiments. Refining chemogenetic studies in these ways may yield more reliable insights about the role specific groups of cells have in the brain.

Microbial biofertilizers, which include microorganisms that improve soil nutrients and make them easier to cultivate, are eco-friendly alternatives to chemical fertilisers, encouraging plant growth and supporting sustainable agriculture. The purpose of the study was to evaluate the health of crops measured by the normalized difference vegetation index (NDVI) and yield, influenced by the combination of biomass from specific cyanobacteria (MACC-612, Nostoc linckia ) and plant growth promoter bacteria (PGPB). Using a factorial design in a complete randomized block configuration, four replications were performed. The experimental design included the testing of three concentrations of microalgae (untreated, 0.3 g/L N. linckia , and 1 g/L N. linckia ) and two PGPBs (untreated, Azospirillum lipoferum , and Pseudomonas fluorescens ). Experiments in the field were conducted for three consecutive years (2021, 2022, and 2023). The results show that the combined application of N. linckia and PGPB to soil treatment has significantly improved plant health and yield characteristics. The combined use of 0.3 g/L N. linckia and A. lipoferum has improved the health of plants (NDVI), seed count per cob, thousand-seed weight, and total yields, achieving a significant increase of yield by 1.4 fold for 2021, 1.37 fold for 2022, and 1.39 fold for 2023. These results demonstrate that applying low concentrations of N. linckia (0.3 g/L) along with A. lipoferum provide a costeffective solution without compromising the benefits. Consequently, the integration of cyanobacteria and PGPB represents a promising approach to improve crop growth and yield while minimizing environmental impacts.

Intensive agriculture uses continuous chemical fertilizers to increase crop yields, but excessive use of fertilizers leads to environmental pollution, permanent changes in physicochemical conditions in soil ecology, deterioration of soil biological health, leaching of nutrients, surface and groundwater pollution and eutrophication. Plant growth-promoting rhizobacteria (PGPR) are becoming increasingly important for ensuring crop safety, increasing nutrient uptake and output, lowering fertilizer costs, preventing environmental contamination and promoting sustainable agriculture and agricultural resources. Therefore, the purpose of this study was to identify and evaluate the effects of fifteen bacteria strains that were isolated from various acidic rhizospheric soils as biofertilizers on soil biological properties. Growth, yield and quality traits were analyzed, and various PGPR were identified using 16S ribosomal RNA of Turkish oregano. Fifteen bacterial inoculations with 1-aminocyclopropane-1-carboxylate (ACC) deaminase, N2-fixing, P-solubilizing and/or IAA-producing genes were used in the experiment, which was carried out in a randomized block design with five replicates (each with three pots) and a control without inoculation. Increased biological activity in soil inoculated with bacteria with multiple traits was confirmed by high C and N content in microbial biomass, urease, dehydrogenase and acid and alkaline phosphatase activities. Essential oil content, oil yield, thymol and carvacrol contents increased by 0.5–40.1%, 5.9–71.9%, 0.07–16.7% and 0.3–9.2%, respectively, as a result of bacterial inoculation. Oil content ranged from 2.02% to 2.83%; carvacrol (66.1–72.2%) was the main constituent, followed by thymol (14.5–16.9%) and linalool (1.38–3.68%). Two large PGPR groups were formed based on genetic distance analysis. Responses were variable and depended on the inoculant strain and the parameters being evaluated. The results indicate PGPR has clear potential for improving the yield of cultivated aromatic and essential oil plants, such as oregano.

Background The COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost 1 million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. Results We present the ‘Mini-XT’ miniaturized tagmentation-based library preparation protocol available on protocols.io ( https://doi.org/10.17504/protocols.io.bvntn5en ). SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. The final version of Mini-XT has been used to sequence 4384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. Conclusions The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes eightfold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.

Doc number: 14 Abstract Background: During cerebral cortex development, multipotent neural progenitor cells generate a variety of neuronal subtypes in a fixed temporal order. How a single neural progenitor cell generates the diversity of cortical projection neurons in a temporal sequence is not well understood. Based on their function in developmental timing in other systems, Dicer and microRNAs are potential candidate regulators of cellular pathways that control lineage progression in neural systems. Results: Cortex-specific deletion of Dicer results in a marked reduction in the cellular complexity of the cortex, due to a pronounced narrowing in the range of neuronal types generated by Dicer-null cortical stem and progenitor cells. Instead of generating different classes of lamina-specific neurons in order over the 6-day period of neurogenesis, Dicer null cortical stem and progenitor cells continually produce one class of deep layer projection neuron. However, gliogenesis in the Dicer-null cerebral cortex was not delayed, despite the loss of multipotency and the failure of neuronal lineage progression. Conclusions: We conclude that Dicer is required for regulating cortical stem cell multipotency with respect to neuronal diversity, without affecting the larger scale switch from neurogenesis to gliogenesis. The differences in phenotypes reported from different timings of Dicer deletion indicate that the molecular pathways regulating developmental transitions are notably dosage sensitive.

Tubulin alpha 8 (Tuba8) is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG) syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself.

Key Points The subplate zone is a highly dynamic structure that contains diverse cell populations that are derived from cortical (ventricular and subventricular zones) and extracortical (rostro-medial telencephalic wall and ganglionic eminence) sources. Interneurons may be underrepresented in the postnatal subplate. Subplate cells in rodents and primates share similarities, such as an early birth date and their location below the cortical plate, but they exhibit marked differences in relative cell survival times, molecular expression profiles and cell morphologies. Subplate cells pioneer axonal projections from the cortex to subcortical targets, but there are species differences in the targets that they innervate. Ablation of the subplate by excitotoxicity or immunotoxicity impairs circuit-level maturation of the primary sensory cortex, and an absence of subplate neurons prevents thalamic afferents from crossing the pallial–subpallial boundary and invading the cortex. Transcriptomic evidence highlights the relative maturity of embryonic and fetal subplate cells and suggests novel roles for subplate neurons in the secretion of various extracellular molecules involved in axon pathfinding, cell survival or differentiation, and synaptic plasticity. Histological, MRI and transcriptomic evidence points towards a role for the subplate in schizophrenia and autism. Whether this is causal or a consequence of earlier malformations remains unclear. The subplate is a transient cortical zone that forms during mammalian brain development and has a crucial role in the formation of intracortical and extracortical circuits. Here, Hoerder-Suabedissen and Molnár review the changing architecture and cellular diversity of this zone in developing mouse and primate brains. Subplate neurons have an essential role in cortical circuit formation. They are among the earliest formed neurons of the cerebral cortex, are located at the junction of white and grey matter, and are necessary for correct thalamocortical axon ingrowth. Recent transcriptomic studies have provided opportunities for monitoring and modulating selected subpopulations of these cells. Analyses of mouse lines expressing reporter genes have demonstrated novel, extracortical subplate neurogenesis and have shown how subplate cells are integrated under the influence of sensory activity into cortical and extracortical circuits. Recent studies have revealed that the subplate is involved in neurosecretion and modification of the extracellular milieu.