Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis.

Improved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.

Germinal centre-driven maturation of B cell response to mRNA vaccination.

Germinal centres (GC) are lymphoid structures in which B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells1-5 (BMPCs). SARS-CoV-2 mRNA vaccination induces a persistent GC response that lasts for at least six months in humans6-8. The fate of responding GC B cells as well as the functional consequences of such persistence remain unknown. Here, we detected SARS-CoV-2 spike protein-specific MBCs in 42 individuals who had received two doses of the SARS-CoV-2 mRNA vaccine BNT162b2 six month earlier. Spike-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of sequencing the B cell receptors of responding blood plasmablasts and MBCs, lymph node GC B cells and plasma cells and BMPCs from eight individuals and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1,540 spike-specific B cell clones. On average, early blood spike-specific plasmablasts exhibited the lowest SHM frequencies. By contrast, SHM frequencies of spike-specific GC B cells increased by 3.5-fold within six months after vaccination. Spike-specific MBCs and BMPCs accumulated high levels of SHM, which corresponded with enhanced anti-spike antibody avidity in blood and enhanced affinity as well as neutralization capacity of BMPC-derived monoclonal antibodies. We report how the notable persistence of the GC reaction induced by SARS-CoV-2 mRNA vaccination in humans culminates in affinity-matured long-term antibody responses that potently neutralize the virus.

ABBV-319: A CD19-targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies.

Glucocorticoids are key components of the current standard-of-care regimens (e.g., R-CHOP, EPOCH-R, Hyper-CVAD) for treatment of B-cell malignancy. However, systemic glucocorticoid treatment is associated with several adverse events. CD19 displays restricted expression in normal B-cells and is up-regulated in B-cell malignancies. ABBV-319 is a CD19-targeting antibody-drug conjugate (ADC) engineered to reduce glucocorticoid-associated toxicities while possessing three distinct mechanisms of action (MOA) to increase therapeutic efficacy: (1) antibody-mediated delivery of glucocorticoid receptor modulator (GRM) payload to activate apoptosis, (2) inhibition of CD19 signaling, and (3) enhanced Fc-mediated effector function via afucosylation of the antibody backbone. ABBV-319 elicited potent GRM-driven anti-tumor activity against multiple malignant B-cell lines in vitro as well as in cell line-derived xenografts (CDXs) and patient-derived xenografts (PDXs) in vivo. Remarkably, a single-dose of ABBV-319 induced sustained tumor regression and enhanced anti-tumor activity compared to repeat dosing of systemic prednisolone at the maximum tolerated dose (MTD) in mice. The unconjugated CD19 monoclonal antibody (mAb) also displayed anti-proliferative activity on a subset of B-cell lymphoma cell lines through the inhibition of PI3K signaling. Moreover, afucosylation of the CD19 mAb enhanced Fc-mediated antibody-dependent cellular cytotoxicity (ADCC), and this activity was maintained after conjugation with GRM payloads. Notably, ABBV-319 displayed superior efficacy compared to afucosylated CD19 mAb in human CD34+ PBMC-engrafted NSG-tg(Hu-IL15) transgenic mice, demonstrating enhanced anti-tumor activity when multiple MOAs are enabled. ABBV-319 also showed durable anti-tumor activity across multiple B-cell lymphoma PDX models, including non-germinal center B-cell (GCB) DLBCL and relapsed lymphoma post R-CHOP treatment. Collectively, these data support the ongoing evaluation of ABBV-319 in Phase I clinical trial (NCT05512390).

Persons with HIV Develop Spike-Specific Lymph Node Germinal Center Responses following SARS-CoV-2 Vaccination.

COVID-19 disproportionately affects persons with HIV (PWH) in worldwide locations with limited access to SARS-CoV-2 vaccines. PWH exhibit impaired immune responses to some, but not all, vaccines. Lymph node (LN) biopsies from PWH demonstrate abnormal LN structure, including dysregulated germinal center (GC) architecture. It is not clear whether LN dysregulation prevents PWH from mounting Ag-specific GC responses in the draining LN following vaccination. To address this issue, we longitudinally collected blood and draining LN fine needle aspiration samples before and after SARS-CoV-2 vaccination from a prospective, observational cohort of 11 PWH on antiretroviral therapy: 2 who received a two-dose mRNA vaccine series and 9 who received a single dose of the Ad26.COV2.S vaccine. Following vaccination, we observed spike-specific Abs, spike-specific B and T cells in the blood, and spike-specific GC B cell and T follicular helper cell responses in the LN of both mRNA vaccine recipients. We detected spike-specific Abs in the blood of all Ad26.COV2.S recipients, and one of six sampled Ad26.COV2.S recipients developed a detectable spike-specific GC B and T follicular helper cell response in the draining LN. Our data show that PWH can mount Ag-specific GC immune responses in the draining LN following SARS-CoV-2 vaccination. Due to the small and diverse nature of this cohort and the limited number of available controls, we are unable to elucidate all potential factors contributing to the infrequent vaccine-induced GC response observed in the Ad26.COV2.S recipients. Our preliminary findings suggest this is a necessary area of future research.

Disruption of proteostasis causes IRE1 mediated reprogramming of alveolar epithelial cells.

Disruption of alveolar type 2 cell (AEC2) protein quality control has been implicated in chronic lung diseases, including pulmonary fibrosis (PF). We previously reported the in vivo modeling of a clinical surfactant protein C (SP-C) mutation that led to AEC2 endoplasmic reticulum (ER) stress and spontaneous lung fibrosis, providing proof of concept for disruption to proteostasis as a proximal driver of PF. Using two clinical SP-C mutation models, we have now discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop a reprogrammed cell state that heretofore has been seen only as a response to lung injury. Using single-cell RNA sequencing in vivo and organoid-based modeling, we show that this state arises de novo from intrinsic AEC2 dysfunction. The cell-autonomous AEC2 reprogramming can be attenuated through inhibition of inositol-requiring enzyme 1 (IRE1α) signaling as the use of an IRE1α inhibitor reduced the development of the reprogrammed cell state and also diminished AEC2-driven recruitment of granulocytes, alveolitis, and lung injury. These findings identify AEC2 proteostasis, and specifically IRE1α signaling through its major product XBP-1, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.

Trastuzumab deruxtecan in patients with metastatic non-small-cell lung cancer (DESTINY-Lung01): primary results of the HER2-overexpressing cohorts from a single-arm, phase 2 trial.

BACKGROUND: DESTINY-Lung01 is a multicentre, open-label, phase 2 study evaluating the antitumour activity and safety of trastuzumab deruxtecan, a HER2-directed antibody-drug conjugate, in patients with HER2-overexpressing or HER2 (ERBB2)-mutant unresectable or metastatic non-small-cell lung cancer (NSCLC). The results of the HER2-mutant cohort (cohort 2) have been reported elsewhere. Herein, we report the primary analysis of cohorts 1 and 1A, which aimed to evaluate the activity and safety of trastuzumab deruxtecan 5·4 mg/kg and 6·4 mg/kg in patients with HER2-overexpressing NSCLC. METHODS: Patients aged 18 years or older with unresectable or metastatic (or both unresectable and metastatic) non-squamous NSCLC who had relapsed following or were refractory to standard treatment or for whom no standard treatment was available, with an HER2 immunohistochemistry score of 3+ or 2+ (without known HER2 mutations) and an Eastern Cooperative Oncology Group performance status score of 0 or 1, were enrolled at 20 specialist hospitals in France, Japan, the Netherlands, Spain, and the USA. Patients were assigned to cohorts sequentially, first to cohort 1, to receive trastuzumab deruxtecan 6·4 mg/kg (cohort 1), then to cohort 1A, to receive trastuzumab deruxtecan 5·4 mg/kg, both administered intravenously once every 3 weeks. The primary endpoint was confirmed objective response rate by independent central review and was assessed in the full analysis set, which included all patients who signed an informed consent form and were enrolled in the study. Safety was assessed in all enrolled patients who received at least one dose of trastuzumab deruxtecan. This trial is registered with ClinicalTrials.gov, NCT03505710, and is ongoing (closed to recruitment). FINDINGS: Between Aug 27, 2018, and Jan 28, 2020, 49 patients were enrolled in cohort 1 (median age 63·0 years [IQR 58·0-68·0], 30 [61%] male, 19 [39%] female, and 31 [63%] White), and from June 16 to Dec 9, 2020, 41 patients were enrolled in cohort 1A (median age 62·0 years [IQR 56·0-66·0], 22 [54%] male, 19 [46%] female, and 31 [76%] White). As of data cutoff (Dec 3, 2021), the median treatment duration was 4·1 months (IQR 1·4-7·1) in cohort 1 and 5·5 months (1·4-8·7) in cohort 1A, and median follow-up was 12·0 months (5·4-22·4) in cohort 1 and 10·6 months (4·5-13·5) in cohort 1A. Confirmed objective response rate by independent central review was 26·5% (95% CI 15·0-41·1; 13 of 49, all partial responses) in cohort 1 and 34·1% (20·1-50·6; 14 of 41; two complete responses and 12 partial responses) in cohort 1A. The most common treatment-emergent adverse events of grade 3 or worse were neutropenia (12 [24%] of 49 in cohort 1, none in cohort 1A), pneumonia (six [12%] and two [5%], respectively), fatigue (six [12%] and three [7%], respectively), and disease progression (six [12%] and four [10%], respectively). Drug-related treatment-emergent adverse events of grade 3 or worse occurred in 26 (53%) of 41 patients in cohort 1 and nine (22%) of 49 patients in cohort 1A. Drug-related serious adverse events were reported in ten (20%) patients and three (7%) patients, respectively. Deaths due to treatment-emergent adverse events occurred in ten (20%) patients in cohort 1 (disease progression in six (12%) patients and bronchospasm, hydrocephalus, respiratory failure, and pneumonitis in one [2%] patient each), and in seven (17%) patients in cohort 1A (due to disease progression in four (10%) patients and dyspnoea, malignant neoplasm, and sepsis in one (2%) patient each). One death due to a treatment-emergent adverse event was determined to be due to study treatment by the investigator, which was in cohort 1 (pneumonitis). Independent adjudication of interstitial lung disease or pneumonitis found that drug-related interstitial lung disease or pneumonitis occurred in ten (20%) patients in cohort 1 (two [4%] grade 1, five [10%] grade 2, and three [6%] grade 5) and two (5%) patients in cohort 1A (one [2%] grade 2 and one [2%] grade 5). An additional patient in cohort 1A had grade 4 pneumonitis after the data cutoff, which was subsequently adjudicated as drug-related grade 5 interstitial lung disease or pneumonitis. INTERPRETATION: Given the low antitumour activity of existing treatment options in this patient population, trastuzumab deruxtecan might have the potential to fill a large unmet need in HER2-overexpressing NSCLC. Our findings support further investigation of trastuzumab deruxtecan in patients with HER2-overexpressing NSCLC. FUNDING: Daiichi Sankyo and AstraZeneca.

Activation of Alveolar Epithelial ER Stress by ß-Coronavirus Infection Disrupt Surfactant Homeostasis in Mice: Implications for Covid-19 Respiratory Failure.

COVID-19 syndrome is characterized by acute lung injury, hypoxemic respiratory failure, and high mortality. Alveolar Type 2 (AT2) cells are essential for gas exchange, repair, and regeneration of distal lung epithelium. We have shown that the causative agent, SARS-CoV-2 and other ß-coronavirus genus members induce an ER stress response in vitro, however the consequences for host AT2 function in vivo are less understood. To study this, two murine models of coronavirus infection were employed- mouse hepatitis virus-1 (MHV-1) in A/J mice and a mouse adapted SARS-CoV-2 strain. MHV-1 infected mice exhibited dose-dependent weight loss with histological evidence of distal lung injury accompanied by elevated bronchoalveolar lavage fluid (BALF) cell counts and total protein. AT2 cells showed evidence of both viral infection and increased BIP/GRP78 expression, consistent with activation of the unfolded protein response (UPR). The AT2 UPR included increased IRE1α signaling and a biphasic response in PERK signaling accompanied marked reductions in AT2 and BALF surfactant protein (SP-B, SP-C) content, increases in surfactant surface tension, and emergence of a re-programmed epithelial cell population (Krt8+, Cldn4+). The loss of a homeostatic AT2 endophenotype was attenuated by treatment with the IRE1α inhibitor OPK711. As proof-of-concept, C57BL6 mice infected with mouse-adapted SARS-CoV-2 demonstrated similar lung injury and evidence of disrupted surfactant homeostasis. We conclude that lung injury from ß-coronavirus infection results from an aberrant host response activating multiple AT2 UPR pathways, altering surfactant metabolism/function, and changing AT2 endophenotypes offering a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and acute respiratory failure.

The hormonal profile in women using oral contraceptives is not as stable as previously thought: a temporal analysis of serum endogenous and exogenous hormones.

Oral contraceptive pills are used by approximately 250 million women worldwide, however a clear understanding of the concentrations of endogenous and exogenous hormones across a 28-day oral contraceptive pill pack is not well described. In our study of 16 female participants taking various monophasic oral contraceptive pills, we found significant fluctuations in endogenous and exogenous hormone levels throughout the pill cycle, challenging the previous assumption of hormonal stability in oral contraceptive users. The results from this study have wide ranging implications for research and treatment in women's health including: considerations in research design and interpretation for studies including women taking oral contraceptives, the potential for more precise and personalized methods of dosing to reduce unwanted side effects and adverse events, and the potential treatment of a variety of disorders ranging from musculoskeletal to neurological with exogenous hormones.

PCSK6 and Survival in Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by limited treatment options and high mortality. A better understanding of the molecular drivers of IPF progression is needed. Objectives: To identify and validate molecular determinants of IPF survival. Methods: A staged genome-wide association study was performed using paired genomic and survival data. Stage I cases were drawn from centers across the United States and Europe and stage II cases from Vanderbilt University. Cox proportional hazards regression was used to identify gene variants associated with differential transplantation-free survival (TFS). Stage I variants with nominal significance (P < 5 × 10-5) were advanced for stage II testing and meta-analyzed to identify those reaching genome-wide significance (P < 5 × 10-8). Downstream analyses were performed for genes and proteins associated with variants reaching genome-wide significance. Measurements and Main Results: After quality controls, 1,481 stage I cases and 397 stage II cases were included in the analysis. After filtering, 9,075,629 variants were tested in stage I, with 158 meeting advancement criteria. Four variants associated with TFS with consistent effect direction were identified in stage II, including one in an intron of PCSK6 (proprotein convertase subtilisin/kexin type 6) reaching genome-wide significance (hazard ratio, 4.11 [95% confidence interval, 2.54-6.67]; P = 9.45 × 10-9). PCSK6 protein was highly expressed in IPF lung parenchyma. PCSK6 lung staining intensity, peripheral blood gene expression, and plasma concentration were associated with reduced TFS. Conclusions: We identified four novel variants associated with IPF survival, including one in PCSK6 that reached genome-wide significance. Downstream analyses suggested that PCSK6 protein plays a potentially important role in IPF progression.

Two type I topoisomerases maintain DNA topology in human mitochondria.

Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.

Microplastics and plastisphere at surface waters in the Southwestern Caribbean sea.

Pollution generated by plastic waste has brought an environmental problem characterized by the omnipresence of smaller pieces of this material known as microplastics (MP). This issue was addresses by collecting samples with 250 µm pore size nets in two marine-coastal sectors of Southwestern Caribbean Sea during two contrasting seasons. Higher concentrations were found in rainy season than in dry season, reaching respectively 1.72 MP/m3 and 0.22 MP/m3. Within each sector, there were differences caused firstly by localities of higher concentrations of semi-closed water bodies localities during rainy season (Ciénaga Grande de Santa Marta and La Caimanera marsh), and secondly by lower concentrations of localities with less influenced of flow rates during dry season (Salamanca and Isla Fuerte). Moreover, the lowest concentration in dry season corresponding to La Caimanera marsh reflects how the community environmental management might decrease MP pollution. In both sectors and seasons, the particles of 0.3 mm (0.3-1.4 mm) size class dominated over those of 1.4 mm (1.4-5.0 mm) (reaching each respectively 1.33 MP/m3 and 0.39 MP/m3), with a dominance of fibers, except in the rainy season in Magdalena, where they were films. Using the FTIR technique, polypropylene was identified as the most abundant polymer in both sectors. The composition of the assemblage of microorganisms attached to microplastics presented higher richness and differed from that of free-living planktonic microbes. The most abundant members of the plastisphere were proteobacteria whose major representation was the pathogenic genus Vibrio, while the cyanobacteria dominated in seawater samples.

Adaptation and validation of an abbreviated version of the SIPAT integrated psychosocial risk scale in patients with liver cirrhosis candidates for liver transplantation (SIPAT-11). / Adaptación y validación de una versión abreviada de la escala SIPAT de riesgo psicosocial integrado en pacientes con cirrosis hepática candidatos a trasplante hepático (SIPAT-11).

INTRODUCTION: Patients with liver cirrhosis who are candidates for liver transplantation must be evaluated both clinically and socially in order to obtain the optimal outcomes and avoid futile therapeutic measures. For the evaluation of the social aspects in these patients, no validated scale in Spanish is available. The SIPAT (Stanford Integrated Psychosocial Assessment for Transplantation) scale is an instrument that measures the social, family and psychological aspects in candidates for solid organ transplantation. The objective of this study is to adapt and validate an abbreviated version of the SIPAT scale in Spanish for patients with liver cirrhosis. MATERIAL AND METHODS: Prospective observational study carried out in the Hepatology Unit of the La Fe Unversity Hospital in Valencia, by questionnaire validation methodology. To analyze the reliability of the questionnaire, the internal consistency of all variables was calculated, for variability an exploratory factor analysis, and for stability the test-retest test was carried out. RESULTS: 96 patients who were admitted for decompensated cirrhosis to the Hepatology Unit of the La Fe Hospital in Valencia between November 1, 2017 and January 31, 2017 were selected. 84% were men, the mean age was 60.01 (SD 10.12) years. In 73.2% of those admitted, the etiology of cirrhosis was alcoholic. 14.4% had a Child's stage A, 57.7% B and 27.8% C. The internal consistency of all variables reached a Cronbach's Alpha of 0.766. In the exploratory factor analysis, 6 dimensions of the questionnaire were identified that explain 84.27% of the total variability. To see the stability of the instrument, the measurement was repeated at 2 and 6 months of follow-up, obtaining in the test-retest a kappa agreement of 0.612 and 0.565 respectively. CONCLUSION: The SIPAT-11 questionnaire has good psychometric characteristics in cirrhotic patients who are candidates for liver transplantation. It is easy to complete and can be administered by professionals who are not specialists in the area of ​​Mental Health.

Chronic Expression of a Clinical SFTPC Mutation Causes Murine Lung Fibrosis with Idiopathic Pulmonary Fibrosis Features.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic interstitial lung disease. A barrier to developing more effective therapies for IPF is the dearth of preclinical models that recapitulate the early pathobiology of this disease. Intratracheal bleomycin, the conventional preclinical murine model of IPF, fails to reproduce the intrinsic dysfunction to the alveolar epithelial type 2 cell (AEC2) that is believed to be a proximal event in the pathogenesis of IPF. Murine fibrosis models based on SFTPC (Surfactant Protein C gene) mutations identified in patients with interstitial lung disease cause activation of the AEC2 unfolded protein response and endoplasmic reticulum stress-an AEC2 dysfunction phenotype observed in IPF. Although these models achieve spontaneous fibrosis, they do so with precedent lung injury and thus are challenged to phenocopy the general clinical course of patients with IPF-gradual progressive fibrosis and loss of lung function. Here, we report a refinement of a murine Sftpc mutation model to recapitulate the clinical course, physiological impairment, parenchymal cellular composition, and biomarkers associated with IPF. This platform provides the field with an innovative model to understand IPF pathogenesis and index preclinical therapeutic candidates.

Rearranging and completing the puzzle: Phylogenomic analysis of bark beetles Dendroctonus reveals new hypotheses about genus diversification.

Studies carried out on bark beetles within Dendroctonus have been extensive and revealed diverse information in different areas of their natural history, taxonomy, evolution, and interactions, among others. Despite these efforts, phylogenetic hypotheses have remained obscured mainly due to limited information analyzed (taxonomic, gene sampling, or both) in studies focused on obtaining evolutionary hypotheses for this genus. With the aim of filling these gaps in the evolutionary history for Dendroctonus, we analyzed ∼1800 loci mapped to a reference genome obtained for 20 of the 21 species recognized to date, minimizing the impact of missing information and improving the assumption of orthology in a phylogenomic framework. We obtained congruent phylogenetic topologies from two phylogenomic inference strategies: loci concatenation (ML framework) and a multispecies coalescent model (MSC) through the analysis of site pattern frequencies (SNPs). Dendroctonus is composed of two major clades (A and B), each containing five and four subclades, respectively. According to our divergence dating analysis, the MRCA for Dendroctonus dates back to the early Eocene, while the MRCA for each major clade diverged in the mid-Eocene. Interestingly, most of the speciation events of extant species occurred during the Miocene, which could be correlated with the diversification of pine trees (Pinus). The MRCA for Dendroctonus inhabited large regions of North America, with all ancestors and descendants of clade A having diversified within this region. The Mexican Transition Zone is important in the diversification processes for the majority of clade A species. For clade B, we identified two important colonization events to the Old World from America: the first in the early Oligocene from the Arctic to Asia (via Beringia), and the second during the Miocene from the Arctic-Western-Alleghany region to Europe and Siberia (also via Beringia). Our genomic analyses also supported the existence of hidden structured lineages within the frontalis complex, and also that D. beckeri represent a lineage independent from D. valens, as previously suggested. The information presented here updates the knowledge concerning the diversification of a genus with remarkable ecological and economic importance.

Supporting responsive parenting in real-world implementation: minimal effective dose of the Video Interaction Project.

BACKGROUND: The Video Interaction Project (VIP) is a healthcare-based intervention that provides real-time video-feedback of parent-child play and reading interactions to families with children aged 0 to 36 months. Although evidence from randomized controlled trials demonstrates improved early relational health, including responsive parenting, after three to five VIP visits, the minimal effective dose in real-world implementations is unknown. This study aimed to determine the minimal effective dose of VIP during a real-world implementation for changing responsive parenting behaviors. METHODS: We performed a longitudinal prospective study of 183 dyads at a public hospital pediatric clinic. Responsive parenting behaviors were assessed with an observational checklist utilized as part of standard VIP practice at baseline and two follow-up VIP visits. RESULTS: Multilevel models adjusted for baseline sociodemographics (child's sex and age, and maternal education) and time between visits showed that responsive parenting behaviors during parent-child reading and play significantly increased after a single VIP visit (Cohen's d = 0.52, p < 0.05) with additional impact following completion of a second visit (cumulative for 2 visits: d = 0.76, p < 0.05). CONCLUSIONS: A single VIP visit is associated with increased responsive parenting behaviors. Findings support offering VIP widely, regardless of capacity to ensure attendance at multiple visits. IMPACT: This is the first study showing the minimal effective dose of the Video Interaction Project (VIP) for increasing responsive parenting behaviors. Responsive parenting behaviors increased by over 22% following a single VIP visit, with a cumulative increase of 37% following the second visit compared to baseline. Findings have important implications for implementation and scalability of pediatric-based preventive programs that support early relational health through activities such as reading and play.

Fabrication, characterization, and potential applications of re-assembled casein micelles.

Re-assembled casein micelles (rCMs), were formulated in the 1970s as a model system to understand native casein micelles (nCMs) in milk. These early works allowed an understanding of the critical factors involved in the formation of rCMs, such as minerals (citrate, phosphate, and calcium), casein type (αs-, ß-, and κ-casein) and the extent of their phosphorylation. rCMs were also used to understand the effect of treatments such as ethanol, high hydrostatic pressure and heating on the stability and integrity of the micelles. More recently, the applications of rCMs have been investigated, these include their use as a nanocarrier of bioactive molecules and as electrode-bound substrates to monitor chymosin activity by electrochemistry, to cite a few. Moreover, the potential to use rCMs in both food and non-food applications remains to be fully exploited. The advantage of choosing rCMs over nCMs as an encapsulant and a lucrative food ingredient is due to their more efficient preparation and being free from impurities. In this review, we report on the formulation of rCMs, their physico-chemical properties and their behavior under different physico-chemical treatments, along with the applications and challenges of rCMs in food systems and their industrial production as a dairy ingredient.

Patients With High Cardiovascular Risk as Candidates to Bempedoic Acid, After Treatment With Statins, Ezetimibe and PCSK9 Inhibitors: An Estimation and Cost-Effectiveness Analysis.

ABSTRACT: Low-density lipoprotein cholesterol (LDLc) is the lead effector of atherosclerosis and main treatment target. Bempedoic acid is a novel oral drug in the therapeutic armamentarium which is able to reduce LDLc. The objectives of this study were (1) to select the potential patients for administering bempedoic acid such as those with a very high cardiovascular risk in which objectives of LDLc were not achieved despite conventional treatment with PCSK9 inhibitors (PCSK9i) and/or statins and ezetimibe and (2) to estimate the cost-effectiveness of bempedoic acid in different scenarios. The methods used were a multicenter and retrospective study of 652 patients initiating treatment with any PCSK9 inhibitor in 17 different hospitals. Before and on-treatment LDLc cholesterol levels, medical treatments, clinical indication, and baseline characteristics were recorded. The results obtained from 443 subjects in secondary prevention were analyzed. The mean (±) LDLc level at baseline was 142.5 ± 46.4 mg/dL and 61.5 ± 40.5 mg/dL in the follow-up, with a reduction of 55.9% ( P < 0.0001); 71.6% of the patients reached the target of LDL < 55 mg/dL or >50% reduction. Of those patients treated with medium-intensity and low-intensity statins plus PCSK9 inhibitors (with or without ezetimibe), only 5.7% of them were able to reduce LDL below 55 mg/dL and the main LDLc reduction in this group was the lowest (42.9% on average). Patients with TG values >135 mg/dL represented 41.6% of the sample, of which approximately 10% of them were using fibrates. Assuming only LDLc reduction and the UK price, the incremental cost-effectiveness ratio was 88,359€; 83,117€; 82,378€; and 79,015€ for different discount rates. In conclusion, one-third of the patients could achieve the target LDL proposed in the 2019 ESC/EAS guidelines. Approximately 10% of them could also benefit from treating hypertriglyceridemia as indicated in the 2021 ESC guidelines on cardiovascular disease prevention. Patients with medium-intensity and low-intensity statins plus PCSK9i and ezetimibe would be the most benefited. Bempedoic acid could be a not cost-efficacy therapy in all the scenarios, but we need to wait for the CLEAR OUTCOMES Trial results.

Bee Species, Botanical Sources and the Chemical Composition of Propolis from Yucatan, Mexico.

Propolis is used by corbiculated bees to protect the bee hive; it is mostly used to seal cracks, to reduce or prevent microbial growth and to embalm invaders. Different factors have been reported to influence the chemical composition of propolis, including bee species and the flora surrounding the hive. Nevertheless, the majority of the studies are focused on propolis produced by Apis mellifera, while studies on the chemical composition of propolis produced by stingless bees are still limited. In this investigation, the chemical composition of 27 propolis samples collected in the Yucatan Peninsula from A. mellifera beehives, together with 18 propolis samples from six different species of stingless bees, were analyzed by GC-MS. Results showed that lupeol acetate and ß-amyrin were the characteristic triterpenes in propolis samples from A. mellifera, while grandiflorenic acid and its methyl ester were the main metabolites present in samples from stingless bees. Multivariate analyses were used to explore the relationship between bee species and botanical sources on the chemical composition of the propolis samples. Differences in body size and, therefore, foraging abilities, as well as preferences for specific botanical sources among bee species, could explain the observed variation in propolis chemical composition. This is the first report on the composition of propolis samples from the stingless bees Trigona nigra, Scaptotrigona pectoralis, Nannotrigona perilampoides, Plebeia frontalis and Partamona bilineata.

The impact of COVID-19 on the number of births in Yucatan, Mexico.

OBJECTIVE: To analyze the impact of COVID-19 on the number of births in Yucatan, Mexico during 2020 and 2021. MATERIAL AND METHODS: A total of 470 651 live births occurred in Yucatan from January 1st, 2008, to December 31st, 2021, and were included in the analysis. The monthly number of births observed during January 2008-February 2020 was used to describe pre-pandemic trends. Time-series analysis was applied to examine whether the number of births observed from December 2020 (9 months after the beginning of the pandemic) to December 2021 differed from the expected values. Trends in the number of births according to maternal age, parity and education were examined to identify changes differentiated by sociodemographic characteristics. RESULTS: The number of births in 2021 decreased by 18% (5869 births) compared with 2019, which represents a reduction from 12.89 to 12.48 per thousand inhabitants. The observed number of births from December 2020 to July 2021 was significantly lower than the figure expected. April (expected = 2863 vs. observed = 1722), May (expected = 2948 vs. observed = 1990), and June (expected = 2997 vs. observed = 1978) 2021 showed the largest differences between expected and observed values. Then, from August to December 2021, the observed number of births fell within the expected range. Birth decline was slightly more pronounced among mothers between 20 and 29 years of age and in those without previous offspring. CONCLUSION: We provide evidence of birth decline in Yucatan during the COVID-19 pandemic. Birth rate reduction in Yucatan doubled the world average and young women without children were the most affected.