Characterization of the zebrafish gabra1sa43718/sa43718 germline loss of function allele confirms a function for Gabra1 in motility and nervous system development.

Mutation of the GABRA1 gene is associated with neurodevelopmental defects and epilepsy. GABRA1 encodes for the α1 subunit of the γ-aminobutyric acid type A receptor (GABAAR), which regulates the fast inhibitory impulses of the nervous system. Multiple model systems have been developed to understand the function of GABRA1, but these models have produced complex and, at times, incongruent data. Thus, additional model systems are required to validate and substantiate previous results. We sought to provide initial phenotypic analysis of a novel germline mutant allele. Our analysis provides a solid foundation for the future use of this allele to characterize gabra1 functionally and pharmacologically using zebrafish. We investigated the behavioral swim patterns associated with a nonsense mutation of the zebrafish gabra1 (sa43718 allele) gene. The sa43718 allele causes a decrease in gabra1 mRNA expression, which is associated with light induced hypermotility, one phenotype previously associated with seizure like behavior in zebrafish. Mutation of gabra1 was accompanied by decreased mRNA expression of gabra2, gabra3, and gabra5, indicating a reduction in the expression of additional α sub-units of the GABAAR. Although multiple sub-units were decreased, larvae continued to respond to pentylenetetrazole (PTZ), indicating that a residual GABAAR exists in the sa43718 allele. Proteomics analysis demonstrated that mutation of gabra1 is associated with abnormal expression of proteins that regulate synaptic vesicle fusion, vesicle transport, synapse development, and mitochondrial protein complexes. These data support previous studies performed in a zebrafish nonsense allele created by CRISPR/Cas9 and validate that loss of function mutations in the gabra1 gene result in seizure-like phenotypes with abnormal development of the GABA synapse. Our results add to the existing body of knowledge as to the function of GABRA1 during development and validate that zebrafish can be used to provide complete functional characterization of the gene.

Hepatitis C virus-induced differential transcriptional traits in host cells after persistent infection elimination by direct-acting antivirals in cell culture.

Chronic hepatitis C virus infection (HCV) causes liver inflammation and fibrosis, leading to the development of severe liver disease, such as cirrhosis or hepatocellular carcinoma (HCC). Approval of direct-acting antiviral drug combinations has revolutionized chronic HCV therapy, with virus eradication in >98% of the treated patients. The efficacy of these treatments is such that it is formally possible for cured patients to carry formerly infected cells that display irreversible transcriptional alterations directly caused by chronic HCV Infection. Combining differential transcriptomes from two different persistent infection models, we observed a major reversion of infection-related transcripts after complete infection elimination. However, a small number of transcripts were abnormally expressed in formerly infected cells. Comparison of the results obtained in proliferating and growth-arrested cell culture models suggest that permanent transcriptional alterations may be established by several mechanisms. Interestingly, some of these alterations were also observed in the liver biopsies of virologically cured patients. Overall, our data suggest a direct and permanent impact of persistent HCV infection on the host cell transcriptome even after virus elimination, possibly contributing to the development of HCC.

Abnormal chondrocyte development in a zebrafish model of cblC syndrome restored by an MMACHC cobalamin binding mutant.

Variants in the MMACHC gene cause combined methylmalonic acidemia and homocystinuria cblC type, the most common inborn error of intracellular cobalamin (vitamin B12) metabolism. cblC is associated with neurodevelopmental, hematological, ocular, and biochemical abnormalities. In a subset of patients, mild craniofacial dysmorphia has also been described. Mouse models of Mmachc deletion are embryonic lethal but cause severe craniofacial phenotypes such as facial clefts. MMACHC encodes an enzyme required for cobalamin processing and variants in this gene result in the accumulation of two metabolites: methylmalonic acid (MMA) and homocysteine (HC). Interestingly, other inborn errors of cobalamin metabolism, such as cblX syndrome, are associated with mild facial phenotypes. However, the presence and severity of MMA and HC accumulation in cblX syndrome is not consistent with the presence or absence of facial phenotypes. Thus, the mechanisms by which mutations in MMACHC cause craniofacial defects are yet to be completely elucidated. Here we have characterized the craniofacial phenotypes in a zebrafish model of cblC (hg13) and performed restoration experiments with either a wildtype or a cobalamin binding deficient MMACHC protein. Homozygous mutants did not display gross morphological defects in facial development but did have abnormal chondrocyte nuclear organization and an increase in the average number of neighboring cell contacts, both phenotypes were fully penetrant. Abnormal chondrocyte nuclear organization was not associated with defects in the localization of neural crest specific markers, sox10 (RFP transgene) or barx1. Both nuclear angles and the number of neighboring cell contacts were fully restored by wildtype MMACHC and a cobalamin binding deficient variant of the MMACHC protein. Collectively, these data suggest that mutation of MMACHC causes mild to moderate craniofacial phenotypes that are independent of cobalamin binding.

Predicting mortality in severe Covid-19 Pneumonia: the role of right ventricular dysfunction.

PURPOSE: There is evidence that COVID-19 can have a clinically significant effect on the right ventricle (RV). Our objective was to enhance the efficiency of assessing RV dilation for diagnosing ACP by utilizing both linear measurements and qualitative assessment and its usefulness as an independent predictor of mortality. METHODS: This is an observational, retrospective and single-center study of the Intensive Care Unit of the Sanatorio de Los Arcos in Buenos Aires, Argentina from March 2020 to January 2022. All patients admitted with acute respiratory distress syndrome due to COVID-19 pneumonia (C-ARDS) on mechanical ventilation who were assessed by transthoracic echocardiography (TTE) were included. RESULTS: A total of 114 patients with C-ARDS requiring invasive mechanical ventilation were evaluated by echocardiography. 12.3% had RV dilation defined as a RV basal diameter greater than 41 mm, and 87.7% did not. Acute cor pulmonale (ACP) defined as RV dilation associated with paradoxical septal motion was found in 6.1% of patients. 7% had right ventricular systolic dysfunction according to qualitative evaluation. The different RV echocardiographic variables were studied with a logistic regression model as independent predictors of mortality. In the multivariate analysis, both the RV basal diameter and the presence of ACP showed to be independent predictors of in-hospital mortality with OR of 3.16 (95% CI 1.36-7.32) and 3.64 (95% CI 1.05-12.65) respectively. CONCLUSION: An increase in the RV basal diameter and the presence of ACP measured by TTE are independent predictors of in-hospital mortality in patients with C-ARDS.

Short Axis In-Plane Ultrasound-Guided Technique for Internal Jugular Vein Cannulation.

BACKGROUND: Real-time ultrasound (US)-guided venipuncture has become the standard of care due to its reduced complications and higher success rate. There are various techniques for US-guided cannulation of the internal jugular vein (IJV); the transversal and longitudinal views are the most widely used. There is a less commonly used technique that combines the benefits of both methods. DISCUSSION: Two main techniques for central line US-guided cannulation of the IJV are based on the location of the probe relative to the vessel and are known as the transversal view and the longitudinal view. The transversal view may make it difficult to identify the tip of the needle as it enters the vein, and the longitudinal view may not allow for visualization of surrounding structures. A third and less commonly used technique, the short axis in-plan view, aims to combine the benefits of both previous methods. In this technique, the entire needle can be tracked in real time as it enters the IJV, while also providing visualization of surrounding structures. CONCLUSIONS: The short axis in-plane view technique for central venous line placements allows for real-time US-guided needle venipuncture with simultaneous visualization of surrounding structures.

Ultrasound-guided cerebral resuscitation in patients with severe traumatic brain Injury.

Traumatic brain injury (TBI) is a worldwide public health concern given its significant morbidity and mortality, years of potential life lost, reduced quality of life and elevated healthcare costs. The primary injury occurs at the moment of impact, but secondary injuries might develop as a result of brain hemodynamic abnormalities, hypoxia, and hypotension. The cerebral edema and hemorrhage of the injured tissues causes a decrease in cerebral perfusion pressure (CPP), which leads to higher risk of cerebral ischemia, herniation and death. In this setting, our role as physicians is to minimize damage by the optimization of the CPP and therefore to reduce mortality and improve neurological outcomes. Performing a transcranial doppler ultrasound (TCD) allows to estimate cerebral blood flow velocities and identify states of low flow and high resistance. We propose to include TCD as an initial assessment and further monitoring tool for resuscitation guidance in patients with severe TBI. We present an Ultrasound-Guided Cardio-cerebral Resuscitation (UGCeR) protocol in Patients with Severe TBI.

Carotid flow as a surrogate of the left ventricular stroke volume.

Transthoracic echocardiography (TTE) is a fundamental tool for hemodynamic monitoring in critical patients. It allows evaluating the left ventricle's stroke volume based on the measurement of the velocity-time integral (VTI) of the left ventricle outflow tract (LVOT). However, in the intensive care unit obtaining adequate echocardiographic views may present a challenge. We propose to measure, as a surrogate of the stroke volume, the carotid flow with a novel technique. This is an observational, prospective, and simple blind study, conducted in the intensive care unit of Sanatorio de los Arcos and Hospital Aleman, in Buenos Aires, Argentina. We measured the carotid systodiastolic flow (CSD) VTI and the carotid systolic flow (CS) VTI at the level of the left supraclavicular fossa and we compared it with the LVOT VTI obtained by TTE. We evaluated 43 subjects. Spearman's correlation coefficient between LVOT VTI and CS VTI was 0.81 (95% CI 0.67-0.89) and between LVOT VTI and CSD VTI was 0.89 (95% CI 0.81-0.94). The Bland-Altman method analysis of the 5-chamber apical window LVOT VTI compared to the CSD VTI showed a bias of - 0.2 (95% CI - 0.82 to 0.43), with a concordance interval between - 4.2 (95% CI - 5.2 to - 3.1) and 3.8 cm (95% CI 2.7 to 4.9). The percentage error was 37.9%. Almost 100% of the values fell within the concordance limits, and no trend was observed in bias across the spectrum of mean variables. Although the CSD VTI could not be interchangeable with the LVOT VTI, it could be considered as its surrogate.

Delayed hemorrhage after pediatric stereo-electroencephalography: delayed occurrence or delayed diagnosis?

BACKGROUND: Stereo-electroencephalography (SEEG) is a well-known invasive diagnostic method for drug-resistant epilepsy (DRE). Its rate of complications is relatively low, being the intracranial hemorrhage (ICH) the most relevant. Most centers perform immediate imaging studies after SEEG to rule out complications. However, delayed intracranial hemorrhages (DIH) can occur despite normal imaging studies in the immediate postoperative period. METHODS: We performed a retrospective review of DRE pediatric patients operated on SEEG between April 2016 and December 2020 in our institution. After implantation, an immediate postoperative CT was performed to check electrode placement and rule out acute complications. An additional MRI was performed 24 h after surgery. We collected all postoperative hemorrhages and considered them as major or minor according to Wellmer´s classification. RESULTS: Overall, 25 DRE patients were operated on SEEG with 316 electrodes implanted. Three ICHs were diagnosed on postoperative imaging. Two of them were asymptomatic requiring no treatment, while the other needed surgical evacuation after clinical worsening. The total risk of hemorrhage per procedure was 12%, but just one third of them were clinically relevant. Two hemorrhages were not visible on immediate postoperative CT, being incidentally diagnosed in the 24 h MRI. We recorded them as DIH and are reported in detail. CONCLUSION: Few reports of DIH after SEEG exist in the literature. It remains unclear whether these cases are late occurring hemorrhages or immediate postoperative hemorrhages undiagnosed on initial imaging. According to our findings, we recommend to perform additional late postoperative imaging to diagnose these cases and manage them accurately.

Activation of WNT signaling restores the facial deficits in a zebrafish with defects in cholesterol metabolism.

Inborn errors of cholesterol metabolism occur as a result of mutations in the cholesterol synthesis pathway (CSP). Although mutations in the CSP cause a multiple congenital anomaly syndrome, craniofacial abnormalities are a hallmark phenotype associated with these disorders. Previous studies have established that mutation of the zebrafish hmgcs1 gene (Vu57 allele), which encodes the first enzyme in the CSP, causes defects in craniofacial development and abnormal neural crest cell (NCC) differentiation. However, the molecular mechanisms by which the products of the CSP disrupt NCC differentiation are not completely known. Cholesterol is known to regulate the activity of WNT signaling, an established regulator of NCC differentiation. We hypothesized that defects in cholesterol synthesis are associated with reduced WNT signaling, consequently resulting in abnormal craniofacial development. To test our hypothesis we performed a combination of pharmaceutical inhibition, gene expression assays, and targeted rescue experiments to understand the function of the CSP and WNT signaling during craniofacial development. We demonstrate reduced expression of four canonical WNT downstream target genes in homozygous carriers of the Vu57 allele and reduced axin2 expression, a known WNT target gene, in larvae treated with Ro-48-8071, an inhibitor of cholesterol synthesis. Moreover, activation of WNT signaling via treatment with WNT agonist I completely restored the craniofacial defects present in a subset of animals carrying the Vu57 allele. Collectively, these data suggest interplay between the CSP and WNT signaling during craniofacial development.

Host phosphatidic acid phosphatase lipin1 is rate limiting for functional hepatitis C virus replicase complex formation.

Hepatitis C virus (HCV) infection constitutes a significant health burden worldwide, because it is a major etiologic agent of chronic liver disease, cirrhosis and hepatocellular carcinoma. HCV replication cycle is closely tied to lipid metabolism and infection by this virus causes profound changes in host lipid homeostasis. We focused our attention on a phosphatidate phosphate (PAP) enzyme family (the lipin family), which mediate the conversion of phosphatidate to diacylglycerol in the cytoplasm, playing a key role in triglyceride biosynthesis and in phospholipid homeostasis. Lipins may also translocate to the nucleus to act as transcriptional regulators of genes involved in lipid metabolism. The best-characterized member of this family is lipin1, which cooperates with lipin2 to maintain glycerophospholipid homeostasis in the liver. Lipin1-deficient cell lines were generated by RNAi to study the role of this protein in different steps of HCV replication cycle. Using surrogate models that recapitulate different aspects of HCV infection, we concluded that lipin1 is rate limiting for the generation of functional replicase complexes, in a step downstream primary translation that leads to early HCV RNA replication. Infection studies in lipin1-deficient cells overexpressing wild type or phosphatase-defective lipin1 proteins suggest that lipin1 phosphatase activity is required to support HCV infection. Finally, ultrastructural and biochemical analyses in replication-independent models suggest that lipin1 may facilitate the generation of the membranous compartment that contains functional HCV replicase complexes.

Hcfc1a regulates neural precursor proliferation and asxl1 expression in the developing brain.

BACKGROUND: Precise regulation of neural precursor cell (NPC) proliferation and differentiation is essential to ensure proper brain development and function. The HCFC1 gene encodes a transcriptional co-factor that regulates cell proliferation, and previous studies suggest that HCFC1 regulates NPC number and differentiation. However, the molecular mechanism underlying these cellular deficits has not been completely characterized. METHODS: Here we created a zebrafish harboring mutations in the hcfc1a gene (the hcfc1aco60/+ allele), one ortholog of HCFC1, and utilized immunohistochemistry and RNA-sequencing technology to understand the function of hcfc1a during neural development. RESULTS: The hcfc1aco60/+ allele results in an increased number of NPCs and increased expression of neuronal and glial markers. These neural developmental deficits are associated with larval hypomotility and the abnormal expression of asxl1, a polycomb transcription factor, which we identified as a downstream effector of hcfc1a. Inhibition of asxl1 activity and/or expression in larvae harboring the hcfc1aco60/+ allele completely restored the number of NPCs to normal levels. CONCLUSION: Collectively, our data demonstrate that hcfc1a regulates NPC number, NPC proliferation, motor behavior, and brain development.

Mutations in THAP11 cause an inborn error of cobalamin metabolism and developmental abnormalities.

CblX (MIM309541) is an X-linked recessive disorder characterized by defects in cobalamin (vitamin B12) metabolism and other developmental defects. Mutations in HCFC1, a transcriptional co-regulator which interacts with multiple transcription factors, have been associated with cblX. HCFC1 regulates cobalamin metabolism via the regulation of MMACHC expression through its interaction with THAP11, a THAP domain-containing transcription factor. The HCFC1/THAP11 complex potentially regulates genes involved in diverse cellular functions including cell cycle, proliferation, and transcription. Thus, it is likely that mutation of THAP11 also results in biochemical and other phenotypes similar to those observed in patients with cblX. We report a patient who presented with clinical and biochemical phenotypic features that overlap cblX, but who does not have any mutations in either MMACHC or HCFC1. We sequenced THAP11 by Sanger sequencing and discovered a potentially pathogenic, homozygous variant, c.240C > G (p.Phe80Leu). Functional analysis in the developing zebrafish embryo demonstrated that both THAP11 and HCFC1 regulate the proliferation and differentiation of neural precursors, suggesting important roles in normal brain development. The loss of THAP11 in zebrafish embryos results in craniofacial abnormalities including the complete loss of Meckel's cartilage, the ceratohyal, and all of the ceratobranchial cartilages. These data are consistent with our previous work that demonstrated a role for HCFC1 in vertebrate craniofacial development. High throughput RNA-sequencing analysis reveals several overlapping gene targets of HCFC1 and THAP11. Thus, both HCFC1 and THAP11 play important roles in the regulation of cobalamin metabolism as well as other pathways involved in early vertebrate development.

Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment.

Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment.IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments.

Soluble levels and endogenous vascular gene expression of KLOTHO are related to inflammation in human atherosclerotic disease.

Atherosclerosis is a chronic inflammatory disorder affecting the artery wall. Klotho, an anti-aging factor expressed in the vessel walls that participates in the maintenance of vascular homeostasis, can be down-regulated by inflammation. In this proof-of-concept work we seek to characterize the arterial KLOTHO expression in the vascular wall, as well as the serum concentration of this protein, in a group of patients with clinical atherosclerotic disease. In addition, we aim to analyze the relationship between Klotho and inflammation. Vascular samples were obtained from 27 patients with atherosclerotic disease under an elective vascular surgery procedure, and from 11 control subjects (cadaveric organ donation programme). qRT-PCR was performed to analyze the gene expression of KLOTHO, TNF-α, IL-6, and IL-10 Serum levels of soluble KLOTHO were measured by ELISA. As compared with control subjects, serum concentrations and vascular expression of Klotho were lower in patients with atherosclerotic vascular disease, whereas inflammatory status was significantly higher. There was a negative and significant correlation between inflammatory parameters and Klotho. After controlling for the effect of other variables, partial correlation showed a direct relationship between vascular KLOTHO gene expression and IL-10 mRNA levels, whereas there was a negative association with serum LDL concentrations and vascular TNF-α expression. Our study indicates an inverse interrelationship between inflammation and Klotho in atherosclerosis. Further studies are necessary to elucidate whether the inflammatory state causes Klotho deficiency or, on the contrary, reduction of Klotho could be responsible for greater inflammation, and finally, to investigate the potential clinical relevance of this association.

Passive leg raising test using the carotid flow velocity-time integral to predict fluid responsiveness.

PURPOSE: The passive leg raising test (PLR) is a noninvasive method widely adopted to assess fluid responsiveness. We propose to explore if changes in the carotid flow assessed by echo-Doppler can predict fluid responsiveness after a PLR. METHODS: We conducted a performance diagnostic study in two intensive care units from Argentina between February and April 2022. We included patients with signs of tissular hypoperfusion that required fluid resuscitation. We labeled the patients as fluid responders when we measured, after a fluid bolus, an increase greater than 15% in the left ventricle outflow tract (LVOT) VTI in an apical 5-chamber view and we compared those results with the carotid flow (CF) velocity-time integral (VTI) from the left supraclavicular region in a semi-recumbent position and during the PLR. RESULTS: Of the 62 eligible patients, 50 patients (80.6%) were included. The area under the ROC curve for a change in CF VTI during the PLR test was 0.869 (95% CI 0.743-0.947). An increase of at least of 11% in the CF VTI with the PLR predicted fluid-responsiveness with a sensitivity of 77.3% (95% CI 54.6-92.2%) and specificity of 78.6% (95% CI 59-91.7%). The positive predictive value was 73.9% (95% CI 57.4-85.6%) and the negative predictive value was 81.5% (95% CI 66.5-90.7%). The positive likelihood ratio was 3.61 and the negative likelihood ratio was 0.29. CONCLUSION: An increase greater than 11% in CF VTI after a PLR may be useful to predict fluid responsiveness among critically ill patients.

Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography.

Plus-strand RNA viruses are proficient at remodeling host cell membranes for optimal viral genome replication and the production of infectious progeny. These ultrastructural alterations result in the formation of viral membranous organelles and may be observed by different imaging techniques, providing nanometric resolution. Guided by confocal and electron microscopy, this study describes the generation of wide-field volumes using cryogenic soft-X-ray tomography (cryo-SXT) on SARS-CoV-2-infected human lung adenocarcinoma cells. Confocal microscopy showed accumulation of double-stranded RNA (dsRNA) and nucleocapsid (N) protein in compact perinuclear structures, preferentially found around centrosomes at late stages of the infection. Transmission electron microscopy (TEM) showed accumulation of membranous structures in the vicinity of the infected cell nucleus, forming a viral replication organelle containing characteristic double-membrane vesicles and virus-like particles within larger vesicular structures. Cryo-SXT revealed viral replication organelles very similar to those observed by TEM but indicated that the vesicular organelle observed in TEM sections is indeed a vesiculo-tubular network that is enlarged and elongated at late stages of the infection. Overall, our data provide additional insight into the molecular architecture of the SARS-CoV-2 replication organelle.

A modified subcostal view: a novel method for measuring the LVOT VTI.

PURPOSE: The velocity time integral (VTI) of the left ventricular outflow tract (LVOT) obtained in the apical view by echocardiography can be regarded as a surrogate for the stroke volume. In critically ill patients it is often difficult to obtain an appropriate apical view to assess the VTI. The subcostal view is more accessible, but while it allows a qualitative assessment of the heart, is not adequate for estimating a reliable LVOT VTI, given the inappropriate angle between the Doppler signal and the flow through the LVOT. We present a new modified subcostal view that allows a proper LVOT VTI measurement. METHODS: This is a single-centre experimental, retrospective, and observational study using data from patients in a tertiary-care centre. We included adult patients admitted to the intensive care unit in the period from June 2020 to January 2022, who were evaluated by echocardiography and whose LVOT VTI was measured aligned with the Doppler signal in both the apical five-chamber view and the modified subcostal view. RESULTS: A total of 30 patients were evaluated in the study period by ultrasonography. The Bland-Altman method analysis of the LVOT VTI measured in the apical view compared with that obtained in the subcostal view showed a bias of 0.8 (95% CI 0.39-1.21) with a 95% limit of agreement between - 1.35 (95% CI - 2.06 to - 0.64) and 2.96 (95% CI 2.25-3.67). The percentage error was calculated to be 23%. The Pearson correlation coefficient for the two forms of measurements showed an R value of 0.98 (95% CI 0.96-0.99). CONCLUSION: The LVOT VTI measured in a modified subcostal view is useful for estimating the value of the LVOT VTI obtained in an apical view.

Biocultural Calendars Across Four Ethnolinguistic Communities in Southwestern South America.

Since the mid-20th century, the so-called Great Acceleration (sensu Steffen et al., 2007, https://doi.org/10.1579/0044-7447(2007)36[614:TAAHNO]2.0.CO;2) has amplified processes of ecosystem degradation, extinction of biological species, displacement of local peoples, losses of languages, and cultural diversity. These losses are still underperceived by the academic community, and by a global society that is disconnected from biocultural diversity. To reconnect society with biocultural diversity, we integrate temporal and spatial dimensions of seasonal cycles, by combining two conceptual frameworks: ecological calendars and the "3Hs" model of the biocultural ethic (sensu Rozzi, 2012, https://doi.org/10.5840/enviroethics20123414). The latter values the vital links between human and other-than-human co-inhabitants, their life habits (e.g., cultural practices of humans or life cycles of other-than-human species), and the structure and processes of their shared habitats. This integration enhances an understanding of links between cultural practices and the life cycles of biocultural keystone species. As a synthesis, we use the term biocultural calendars to emphasize their co-constitutive nature that result from interactions between dynamic biophysical and cultural processes embedded in specific ecosystems and cultures. These calendars link astronomical, biological, and cultural seasonal cycles that sustain life and enhance the integration of Indigenous and scientific knowledge to confront challenges of climate change faced from local to global scales. To illustrate this integration, we examine cultural practices and socio-environmental changes across four contrasting ethnolinguistic communities in southwestern South America, from southern to northern Chile along a marked climatic gradient to show the broad application of the concept of biocultural calendars.

Missense and nonsense mutations of the zebrafish hcfc1a gene result in contrasting mTor and radial glial phenotypes.

Mutations in the HCFC1 transcriptional co-factor protein are the cause of cblX syndrome and X-linked intellectual disability (XLID). cblX is the more severe disorder associated with intractable epilepsy, abnormal cobalamin metabolism, facial dysmorphia, cortical gyral malformations, and intellectual disability. In vitro, murine Hcfc1 regulates neural precursor (NPCs) proliferation and number, which has been validated in zebrafish. However, conditional deletion of mouse Hcfc1 in Nkx2.1 + cells increased cell death, reduced Gfap expression, and reduced numbers of GABAergic neurons. Thus, the role of this gene in brain development is not completely understood. Recently, knock-in of both a cblX (HCFC1) and cblX-like (THAP11) allele were created in mice. Knock-in of the cblX-like allele was associated with increased expression of proteins required for ribosome biogenesis. However, the brain phenotypes were not comprehensively studied due to sub-viability. Therefore, a mechanism underlying increased ribosome biogenesis was not described. We used a missense, a nonsense, and two conditional zebrafish alleles to further elucidate this mechanism during brain development. We observed contrasting phenotypes at the level of Akt/mTor activation, the number of radial glial cells, and the expression of two downstream target genes of HCFC1, asxl1 and ywhab. Despite these divergent phenotypes, each allele studied demonstrates with a high degree of face validity when compared to the phenotypes reported in the literature. Collectively, these data suggest that individual mutations in the HCFC1 protein result in differential mTOR activity which may be associated with contrasting cellular phenotypes.

Characterization of the zebrafish gabra1sa43718/sa43718 germline loss of function allele confirms a function for Gabra1 in motility and nervous system development.

Mutation of the GABRA1 gene is associated with neurodevelopmental defects and epilepsy. GABRA1 encodes for the α1 subunit of the gamma-aminobutyric acid type A receptor (GABAAR), which regulates the fast inhibitory impulses of the nervous system. Multiple model systems have previously been developed to understand the function of GABRA1 during development, but these models have produced complex and at times incongruent data. Thus, additional model systems are required to validate and substantiate previously published results. We investigated the behavioral swim patterns associated with a nonsense mutation of the zebrafish gabra1 (sa43718 allele) gene. The sa43718 allele causes a decrease in gabra1 mRNA expression, which is associated with light induced hypermotility, one phenotype associated with seizure like behavior in zebrafish. Mutation of gabra1 was accompanied by decreased mRNA expression of gabra2, gabra3, and gabra5, indicating a reduction in the expression of additional alpha sub-units of the GABAAR. Although multiple sub-units were decreased in total expression, larvae continued to respond to pentylenetetrazole (PTZ) indicating that a residual GABAAR exists in the sa43718 allele. Proteomics analysis demonstrated that nonsense mutation of gabra1 is associated with abnormal expression of proteins that regulate proton transport, ion homeostasis, vesicle transport, and mitochondrial protein complexes. These data support previous studies performed in a zebrafish nonsense allele created by CRISPR/Cas9 and validate that loss of function mutations in the gabra1 gene result in seizure like phenotypes with abnormal function of inhibitory synapses.