A Randomized Phase 2 Trial of Felzartamab in Antibody-Mediated Rejection.

BACKGROUND: Antibody-mediated rejection is a leading cause of kidney-transplant failure. The targeting of CD38 to inhibit graft injury caused by alloantibodies and natural killer (NK) cells may be a therapeutic option. METHODS: In this phase 2, double-blind, randomized, placebo-controlled trial, we assigned patients with antibody-mediated rejection that had occurred at least 180 days after transplantation to receive nine infusions of the CD38 monoclonal antibody felzartamab (at a dose of 16 mg per kilogram of body weight) or placebo for 6 months, followed by a 6-month observation period. The primary outcome was the safety and side-effect profile of felzartamab. Key secondary outcomes were renal-biopsy results at 24 and 52 weeks, donor-specific antibody levels, peripheral NK-cell counts, and donor-derived cell-free DNA levels. RESULTS: A total of 22 patients underwent randomization (11 to receive felzartamab and 11 to receive placebo). The median time from transplantation until trial inclusion was 9 years. Mild or moderate infusion reactions occurred in 8 patients in the felzartamab group. Serious adverse events occurred in 1 patient in the felzartamab group and in 4 patients in the placebo group; graft loss occurred in 1 patient in the placebo group. At week 24, resolution of morphologic antibody-mediated rejection was more frequent with felzartamab (in 9 of 11 patients [82%]) than with placebo (in 2 of 10 patients [20%]), for a difference of 62 percentage points (95% confidence interval [CI], 19 to 100) and a risk ratio of 0.23 (95% confidence interval [CI], 0.06 to 0.83). The median microvascular inflammation score was lower in the felzartamab group than in the placebo group (0 vs. 2.5), for a mean difference of -1.95 (95% CI, -2.97 to -0.92). Also lower was a molecular score reflecting the probability of antibody-mediated rejection (0.17 vs. 0.77) and the level of donor-derived cell-free DNA (0.31% vs. 0.82%). At week 52, the recurrence of antibody-mediated rejection was reported in 3 of 9 patients who had a response to felzartamab, with an increase in molecular activity and biomarker levels toward baseline levels. CONCLUSIONS: Felzartamab had acceptable safety and side-effect profiles in patients with antibody-mediated rejection. (Funded by MorphoSys and Human Immunology Biosciences; ClinicalTrials.gov number, NCT05021484; and EUDRACT number, 2021-000545-40.).

Sensitivity of ocean circulation to warming during the Early Eocene greenhouse.

Multiple abrupt warming events ("hyperthermals") punctuated the Early Eocene and were associated with deep-sea temperature increases of 2 to 4 °C, seafloor carbonate dissolution, and negative carbon isotope (δ13C) excursions. Whether hyperthermals were associated with changes in the global ocean overturning circulation is important for understanding their driving mechanisms and feedbacks and for gaining insight into the circulation's sensitivity to climatic warming. Here, we present high-resolution benthic foraminiferal stable isotope records (δ13C and δ18O) throughout the Early Eocene Climate Optimum (~53.26 to 49.14 Ma) from the deep equatorial and North Atlantic. Combined with existing records from the South Atlantic and Pacific, these indicate consistently amplified δ13C excursion sizes during hyperthermals in the deep equatorial Atlantic. We compare these observations with results from an intermediate complexity Earth system model to demonstrate that this spatial pattern of δ13C excursion size is a predictable consequence of global warming-induced changes in ocean overturning circulation. In our model, transient warming drives the weakening of Southern Ocean-sourced overturning circulation, strengthens Atlantic meridional water mass aging gradients, and amplifies the magnitude of negative δ13C excursions in the equatorial to North Atlantic. Based on model-data consistency, we conclude that Eocene hyperthermals coincided with repeated weakening of the global overturning circulation. Not accounting for ocean circulation impacts on δ13C excursions will lead to incorrect estimates of the magnitude of carbon release driving hyperthermals. Our finding of weakening overturning in response to past transient climatic warming is consistent with predictions of declining Atlantic Ocean overturning strength in our warm future.

PLS3 missense variants affecting the actin-binding domains cause X-linked congenital diaphragmatic hernia and body-wall defects.

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with an X-linked condition characterized by diaphragm defects, variable anterior body-wall anomalies, and/or facial dysmorphism. Using linkage analysis and exome or genome sequencing, we found that missense variants in plastin 3 (PLS3), a gene encoding an actin bundling protein, co-segregate with disease in all families. Loss-of-function variants in PLS3 have been previously associated with X-linked osteoporosis (MIM: 300910), so we used in silico protein modeling and a mouse model to address these seemingly disparate clinical phenotypes. The missense variants in individuals with CDH are located within the actin-binding domains of the protein but are not predicted to affect protein structure, whereas the variants in individuals with osteoporosis are predicted to result in loss of function. A mouse knockin model of a variant identified in one of the CDH-affected families, c.1497G>C (p.Trp499Cys), shows partial perinatal lethality and recapitulates the key findings of the human phenotype, including diaphragm and abdominal-wall defects. Both the mouse model and one adult human male with a CDH-associated PLS3 variant were observed to have increased rather than decreased bone mineral density. Together, these clinical and functional data in humans and mice reveal that specific missense variants affecting the actin-binding domains of PLS3 might have a gain-of-function effect and cause a Mendelian congenital disorder.

COL11A2 as a candidate gene for vertebral malformations and congenital scoliosis.

Human vertebral malformations (VMs) have an estimated incidence of 1/2000 and are associated with significant health problems including congenital scoliosis (CS) and recurrent organ system malformation syndromes such as VACTERL (vertebral anomalies; anal abnormalities; cardiac abnormalities; tracheo-esophageal fistula; renal anomalies; limb anomalies). The genetic cause for the vast majority of VMs are unknown. In a CS/VM patient cohort, three COL11A2 variants (R130W, R1407L and R1413H) were identified in two patients with cervical VM. A third patient with a T9 hemivertebra and the R130W variant was identified from a separate study. These substitutions are predicted to be damaging to protein function, and R130 and R1407 residues are conserved in zebrafish Col11a2. To determine the role for COL11A2 in vertebral development, CRISPR/Cas9 was used to create a nonsense mutation (col11a2L642*) as well as a full gene locus deletion (col11a2del) in zebrafish. Both col11a2L642*/L642* and col11a2del/del mutant zebrafish exhibit vertebral fusions in the caudal spine, which form due to mineralization across intervertebral segments. To determine the functional consequence of VM-associated variants, we assayed their ability to suppress col11a2del VM phenotypes following transgenic expression within the developing spine. While wildtype col11a2 expression suppresses fusions in col11a2del/+ and col11a2del/del backgrounds, patient missense variant-bearing col11a2 failed to rescue the loss-of-function phenotype in these animals. These results highlight an essential role for COL11A2 in vertebral development and support a pathogenic role for two missense variants in CS.

Fasciclin 2 plays multiple roles in promoting cell migration within the developing nervous system of Manduca sexta.

The coordination of neuronal and glial migration is essential to the formation of most nervous systems, requiring a complex interplay of cell-intrinsic responses and intercellular guidance cues. During the development of the enteric nervous system (ENS) in Manduca sexta (tobacco hornworm), the IgCAM Fasciclin 2 (Fas2) serves several distinct functions to regulate these processes. As the ENS forms, a population of 300 neurons (EP cells) undergoes sequential phases of migration along well-defined muscle pathways on the visceral mesoderm to form a branching Enteric Plexus, closely followed by a trailing wave of proliferating glial cells that enwrap the neurons. Initially, both the neurons and glial cells express a GPI-linked form of Fas2 (GPI-Fas2), which helps maintain cell-cell contact among the pre-migratory neurons and later promotes glial ensheathment. The neurons then switch isoforms, predominantly expressing a combination of transmembrane isoforms lacking an intracellular PEST domain (TM-Fas2 PEST-), while their muscle band pathways on the midgut transiently express transmembrane isoforms containing this domain (TM-Fas2 PEST+). Using intracellular injection protocols to manipulate Fas2 expression in cultured embryos, we found that TM-Fas2 promotes the directed migration and outgrowth of individual neurons in the developing ENS. Concurrently, TM-Fas2 expression by the underlying muscle bands is also required as a substrate cue to support normal migration, while glial expression of GPI-Fas2 helps support their ensheathment of the migratory neurons. These results demonstrate how a specific IgCAM can play multiple roles that help coordinate neuronal and glial migration in the developing nervous system.

Redefining the molecular rejection states in 3230 heart transplant biopsies: Relationships to parenchymal injury and graft survival.

The first-generation Molecular Microscope (MMDx) system for heart transplant endomyocardial biopsies used expression of rejection-associated transcripts (RATs) to diagnose not only T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR) but also acute injury. However, the ideal system should detect rejection without being influenced by injury, to permit analysis of the relationship between rejection and parenchymal injury. To achieve this, we developed a new rejection classification in an expanded cohort of 3230 biopsies: 1641 from INTERHEART (ClinicalTrials.gov NCT02670408), plus 1589 service biopsies added to improve the power of the machine learning algorithms. The new system used 6 rejection classifiers instead of RATs and generated 7 rejection archetypes: No rejection, 48%; Minor, 24%; TCMR1, 2.3%; TCMR2, 2.7%; TCMR/mixed, 2.7%; early-stage ABMR, 3.9%; and fully developed ABMR, 16%. Using rejection classifiers eliminated cross-reactions with acute injury, permitting separate assessment of rejection and injury. TCMR was associated with severe-recent injury and late atrophy-fibrosis and rarely had normal parenchyma. ABMR was better tolerated, seldom producing severe injury, but in later biopsies was often associated with atrophy-fibrosis, indicating long-term risk. Graft survival and left ventricular ejection fraction were reduced not only in hearts with TCMR but also in hearts with severe-recent injury and atrophy-fibrosis, even without rejection.

Natural killer cell functional genetics and donor-specific antibody-triggered microvascular inflammation.

Antibody-mediated rejection (ABMR) is a leading cause of graft failure. Emerging evidence suggests a significant contribution of natural killer (NK) cells to microvascular inflammation (MVI). We investigated the influence of genetically determined NK cell functionality on ABMR development and activity. The study included 86 kidney transplant recipients subjected to systematic biopsies triggered by donor-specific antibody detection. We performed killer immunoglobulin-like receptor typing to predict missing self and genotyped polymorphisms determining NK cell functionality (FCGR3AV/F158 [rs396991], KLRC2wt/del, KLRK1HNK/LNK [rs1049174], rs9916629-C/T). Fifty patients had ABMR with considerable MVI and elevated NK cell transcripts. Missing self was not related to MVI. Only KLRC2wt/wt showed an association (MVI score: 2 [median; interquartile range: 0-3] vs 0 [0-1] in KLRC2wt/del recipients; P = .001) and remained significant in a proportional odds multivariable model (odds ratio, 7.84; 95% confidence interval, 2.37-30.47; P = .001). A sum score incorporating all polymorphisms and missing self did not outperform a score including only KLRC2 and FCGR3A variants, which were predictive in univariable analysis. NK cell genetics did not affect graft functional decline and survival. In conclusion, a functional KLRC2 polymorphism emerged as an independent determinant of ABMR activity, without a considerable contribution of missing self and other NK cell gene polymorphisms.

A dyadic approach to the delineation of diagnostic entities in clinical genomics.

The delineation of disease entities is complex, yet recent advances in the molecular characterization of diseases provide opportunities to designate diseases in a biologically valid manner. Here, we have formalized an approach to the delineation of Mendelian genetic disorders that encompasses two distinct but inter-related concepts: (1) the gene that is mutated and (2) the phenotypic descriptor, preferably a recognizably distinct phenotype. We assert that only by a combinatorial or dyadic approach taking both of these attributes into account can a unitary, distinct genetic disorder be designated. We propose that all Mendelian disorders should be designated as "GENE-related phenotype descriptor" (e.g., "CFTR-related cystic fibrosis"). This approach to delineating and naming disorders reconciles the complexity of gene-to-phenotype relationships in a simple and clear manner yet communicates the complexity and nuance of these relationships.

Precision diagnostics in transplanted organs using microarray-assessed gene expression: concepts and technical methods of the Molecular Microscope® Diagnostic System (MMDx).

There is a major unmet need for improved accuracy and precision in the assessment of transplant rejection and tissue injury. Diagnoses relying on histologic and visual assessments demonstrate significant variation between expert observers (as represented by low kappa values) and have limited ability to assess many biological processes that produce little histologic changes, for example, acute injury. Consensus rules and guidelines for histologic diagnosis are useful but may have errors. Risks of over- or under-treatment can be serious: many therapies for transplant rejection or primary diseases are expensive and carry risk for significant adverse effects. Improved diagnostic methods could alleviate healthcare costs by reducing treatment errors, increase treatment efficacy, and serve as useful endpoints for clinical trials of new agents that can improve outcomes. Molecular diagnostic assessments using microarrays combined with machine learning algorithms for interpretation have shown promise for increasing diagnostic precision via probabilistic assessments, recalibrating standard of care diagnostic methods, clarifying ambiguous cases, and identifying potentially missed cases of rejection. This review describes the development and application of the Molecular Microscope® Diagnostic System (MMDx), and discusses the history and reasoning behind many common methods, statistical practices, and computational decisions employed to ensure that MMDx scores are as accurate and precise as possible. MMDx provides insights on disease processes and highly reproducible results from a comparatively small amount of tissue and constitutes a general approach that is useful in many areas of medicine, including kidney, heart, lung, and liver transplants, with the possibility of extrapolating lessons for understanding native organ disease states.

Assessing Guidelines on the Need for Colonoscopy After Initial Flexible Sigmoidoscopy in Young Patients With Outlet-Type Rectal Bleeding.

BACKGROUND: Although young-age-of-onset colorectal cancer is increasing in incidence, lack of screening leads to symptomatic presentation, often with rectal bleeding. Because most cancers in patients younger than 50 years are left-sided, flexible sigmoidoscopy is a reasonable way of investigating bleeding in these patients. OBJECTIVE: To predict which patients undergoing flexible sigmoidoscopy for outlet-type rectal bleeding need a full colonoscopy. DESIGN: Findings at colonoscopy were compared with published indications for colonoscopy after flexible sigmoidoscopy, which were as follows: 1) any number of advanced adenomas defined as a tubular adenoma of >9 mm diameter, a tubulovillous or villous adenoma of any size, or any adenoma with high-grade dysplasia; 2) 3 or more tubular adenomas of any size or histology; 3) any sessile serrated lesion; and 4) 20 or more hyperplastic polyps. SETTING: Charity Hospital with volunteer specialists. PATIENTS: Patients were included if they were younger than 57 years, had outlet-type rectal bleeding, and underwent flexible sigmoidoscopy at least to the descending colon followed by colonoscopy with biopsy of all resected lesions. INTERVENTIONS: Flexible sigmoidoscopy and colonoscopy with excision of all removable lesions. MAIN OUTCOME MEASURES: Findings at colonoscopy. RESULTS: There were 66 patients who had a colonoscopy between 5 and 811 days after sigmoidoscopy and also had complete data. There were 43 men and 23 women with a mean age of 39.5 years. Analysis of flexible sigmoidoscopy criteria for finding proximal high-risk lesions on colonoscopy showed a sensitivity of 76.9%, a specificity of 67.9%, a positive predictive value of 37%, a negative predictive value of 92.3%, and an accuracy of 69.7%. LIMITATIONS: A large number of exclusions for inadequate colonoscopy or inadequate data resulted in a reduced patient number in the study. CONCLUSIONS: Our criteria for follow-up colonoscopy based on the findings at initial flexible sigmoidoscopy in young patients with outlet-type rectal bleeding are reliable enough to be used in routine clinical practice, provided this is audited. See Video Abstract. GUA DE EVALUACIN PARA LA NECESIDAD DE COLONOSCOPIA DESPUS DE UNA SIGMOIDOSCOPIA FLEXIBLE INICIAL EN PACIENTES JVENES CON RECTORRAGIA: ANTECEDENTES:Si bien la edad de aparición temprana del cáncer colorrectal está aumentando en incidencia, la falta de pruebas de detección conduce a una presentación sintomática, a menudo con sangrado rectal. Debido a que la mayoría de los cánceres en pacientes menores de 50 años son del lado izquierdo, la sigmoidoscopia flexible es una forma razonable de investigar el sangrado en estos pacientes.OBJETIVO:Predecir qué pacientes sometidos a sigmoidoscopia flexible por rectorragia necesitan una colonoscopia completa.DISEÑO:Los resultados de la colonoscopia se compararon con las indicaciones publicadas para la colonoscopia después de una sigmoidoscopia flexible. Estos fueron: 1. Cualquier número de adenomas avanzados, definidos como un adenoma tubular > 9 mm, un adenoma tubulovelloso o velloso de cualquier tamaño, o cualquier adenoma con displasia de alto grado. 2. Tres o más adenomas tubulares de cualquier tamaño o histología. 3. Cualquier lesión serrada sésil. 4. Veinte o más pólipos hiperplásicos.ENTORNO CLINICO:Hospital de Caridad con especialistas voluntarios.PACIENTES:Menores de 57 años, con rectorragia, sometidos a sigmoidoscopia flexible al menos hasta el colon descendente, seguida de colonoscopia con biopsia de todas las lesiones resecadas.INTERVENCIONES:sigmoidoscopia flexible y colonoscopia con escisión de todas las lesiones removibles.PRINCIPALES MEDIDAS DE VALORACIÓN:Hallazgos en la colonoscopia.RESULTADOS:66 casos a los que se les realizó una colonoscopia entre 5 y 811 días después de la sigmoidoscopia, que también tenían datos completos. 43 hombres y 23 mujeres con una edad media de 39,5 años. El análisis de los criterios de sigmoidoscopia flexible para encontrar lesiones proximales de alto riesgo en la colonoscopia mostró una sensibilidad del 76,9 %, una especificidad del 67,9 %, un valor predictivo positivo del 37 %, un valor predictivo negativo del 92,3 % y una precisión del 69,7 %.LIMITACIONES:Gran número de exclusiones por colonoscopia inadecuada o datos inadecuados que causan un número reducido de pacientes en el estudio.CONCLUSIÓN:Nuestros criterios para la colonoscopia de seguimiento basados en los hallazgos de la sigmoidoscopia flexible inicial en pacientes jóvenes con rectorragia son lo suficientemente confiables para ser utilizados en la práctica clínica habitual, siempre que se audite. (Traducción- Dr. Ingrid Melo ).

Natural Killer Cell Presence in Antibody-Mediated Rejection.

Transcript analyses highlight an important contribution of natural killer (NK) cells to microvascular inflammation (MVI) in antibody-mediated rejection (ABMR), but only few immunohistologic studies have quantified their spatial distribution within graft tissue. This study included 86 kidney transplant recipients who underwent allograft biopsies for a positive donor-specific antibody (DSA) result. NK cells were visualized and quantified within glomeruli and peritubular capillaries (PTC), using immunohistochemistry for CD34 alongside CD16/T-bet double-staining. Staining results were analyzed in relation to histomorphology, microarray analysis utilizing the Molecular Microscope Diagnostic System, functional NK cell genetics, and clinical outcomes. The number of NK cells in glomeruli per mm2 glomerular area (NKglom) and PTC per mm2 cortical area (NKPTC) was substantially higher in biopsies with ABMR compared to those without rejection, and correlated with MVI scores (NKglom Spearman's correlation coefficient [SCC] = 0.55, p < 0.001, NKPTC 0.69, p < 0.001). In parallel, NK cell counts correlated with molecular classifiers reflecting ABMR activity (ABMRprob: NKglom 0.59, NKPTC 0.75) and showed a trend towards higher levels in association with high functional FCGR3A and KLRC2 gene variants. Only NKPTC showed a marginally significant association with allograft function and survival. Our immunohistochemical results support the abundance of NK cells in DSA-positive ABMR.

Neuromechanical Differences between Pronated and Supinated Forearm Positions during Upper-Body Wingate Tests.

Arm-cycling is a versatile exercise modality with applications in both athletic enhancement and rehabilitation, yet the influence of forearm orientation remains understudied. Thus, this study aimed to investigate the impact of forearm position on upper-body arm-cycling Wingate tests. Fourteen adult males (27.3 ± 5.8 years) underwent bilateral assessments of handgrip strength in standing and seated positions, followed by pronated and supinated forward arm-cycling Wingate tests. Electromyography (EMG) was recorded from five upper-extremity muscles, including anterior deltoid, triceps brachii lateral head, biceps brachii, latissimus dorsi, and brachioradialis. Simultaneously, bilateral normal and propulsion forces were measured at the pedal-crank interface. Rate of perceived exertion (RPE), power output, and fatigue index were recorded post-test. The results showed that a pronated forearm position provided significantly (p < 0.05) higher normal and propulsion forces and triceps brachii muscle activation patterns during arm-cycling. No significant difference in RPE was observed between forearm positions (p = 0.17). A positive correlation was found between seated handgrip strength and peak power output during the Wingate test while pronated (dominant: p = 0.01, r = 0.55; non-dominant: p = 0.03, r = 0.49) and supinated (dominant: p = 0.03, r = 0.51; don-dominant: p = 0.04, r = 0.47). Fatigue changed the force and EMG profile during the Wingate test. In conclusion, this study enhances our understanding of forearm position's impact on upper-body Wingate tests. These findings have implications for optimizing training and performance strategies in individuals using arm-cycling for athletic enhancement and rehabilitation.

Defining a natural killer cell-enriched molecular rejection-like state in lung transplant transbronchial biopsies.

In lung transplantation, antibody-mediated rejection (AMR) diagnosed using the International Society for Heart and Lung Transplantation criteria is uncommon compared with other organs, and previous studies failed to find molecular AMR (ABMR) in lung biopsies. However, understanding of ABMR has changed with the recognition that ABMR in kidney transplants is often donor-specific antibody (DSA)-negative and associated with natural killer (NK) cell transcripts. We therefore searched for a similar molecular ABMR-like state in transbronchial biopsies using gene expression microarray results from the INTERLUNG study (#NCT02812290). After optimizing rejection-selective transcript sets in a training set (N = 488), the resulting algorithms separated an NK cell-enriched molecular rejection-like state (NKRL) from T cell-mediated rejection (TCMR)/Mixed in a test set (N = 488). Applying this approach to all 896 transbronchial biopsies distinguished 3 groups: no rejection, TCMR/Mixed, and NKRL. Like TCMR/Mixed, NKRL had increased expression of all-rejection transcripts, but NKRL had increased expression of NK cell transcripts, whereas TCMR/Mixed had increased effector T cell and activated macrophage transcripts. NKRL was usually DSA-negative and not recognized as AMR clinically. TCMR/Mixed was associated with chronic lung allograft dysfunction, reduced one-second forced expiratory volume at the time of biopsy, and short-term graft failure, but NKRL was not. Thus, some lung transplants manifest a molecular state similar to DSA-negative ABMR in kidney and heart transplants, but its clinical significance must be established.

Morphologic and Molecular Features of Antibody-Mediated Transplant Rejection: Pivotal Role of Molecular Injury as an Independent Predictor of Renal Allograft Functional Decline.

Current knowledge about the factors correlating with functional decline and subsequent failure of kidney allografts in antibody-mediated rejection (ABMR) is limited. We conducted a cohort study involving 75 renal allograft recipients diagnosed with late ABMR occurring at least 6 months after transplantation. The study aimed to examine the correlation of molecular and histologic features with estimated glomerular filtration rate (eGFR) trajectories and death-censored graft survival. We focused on sum scores reflecting histologic ABMR activity versus chronicity and molecular scores of ABMR probability (ABMRProb), injury-repair response (IRRAT) and fibrosis (ciprob). In multivariable Cox analysis, a Banff lesion-based chronicity index (ci+ct+cg[x2]; hazard ratio per interquartile range [IQR]: 1.97 [95% confidence interval: 0.97 to 3.99]) and IRRAT (1.93 [0.96 to 3.89]) showed the strongest associations with graft failure. Among biopsy variables, IRRAT exhibited the highest relative variable importance and emerged as the sole independent predictor of eGFR slope (change per IQR: -4.2 [-7.8 to -0.6] mL/min/1.73 m2/year). In contrast, morphologic chronicity associated with baseline eGFR only. We conclude that the extent of molecular injury is a robust predictor of renal function decline. Transcriptome analysis has the potential to improve outcome prediction and possibly identify modifiable injury, guiding targeted therapeutic interventions.

Very large release of mostly volcanic carbon during the Palaeocene-Eocene Thermal Maximum.

The Palaeocene-Eocene Thermal Maximum (PETM) was a global warming event that occurred about 56 million years ago, and is commonly thought to have been driven primarily by the destabilization of carbon from surface sedimentary reservoirs such as methane hydrates. However, it remains controversial whether such reservoirs were indeed the source of the carbon that drove the warming. Resolving this issue is key to understanding the proximal cause of the warming, and to quantifying the roles of triggers versus feedbacks. Here we present boron isotope data-a proxy for seawater pH-that show that the ocean surface pH was persistently low during the PETM. We combine our pH data with a paired carbon isotope record in an Earth system model in order to reconstruct the unfolding carbon-cycle dynamics during the event. We find strong evidence for a much larger (more than 10,000 petagrams)-and, on average, isotopically heavier-carbon source than considered previously. This leads us to identify volcanism associated with the North Atlantic Igneous Province, rather than carbon from a surface reservoir, as the main driver of the PETM. This finding implies that climate-driven amplification of organic carbon feedbacks probably played only a minor part in driving the event. However, we find that enhanced burial of organic matter seems to have been important in eventually sequestering the released carbon and accelerating the recovery of the Earth system.

Myhre syndrome is caused by dominant-negative dysregulation of SMAD4 and other co-factors.

Myhre syndrome is a connective tissue disorder characterized by congenital cardiovascular, craniofacial, respiratory, skeletal, and cutaneous anomalies as well as intellectual disability and progressive fibrosis. It is caused by germline variants in the transcriptional co-regulator SMAD4 that localize at two positions within the SMAD4 protein, I500 and R496, with I500 V/T/M variants more commonly identified in individuals with Myhre syndrome. Here we assess the functional impact of SMAD4-I500V variant, identified in two previously unpublished individuals with Myhre syndrome, and provide novel insights into the molecular mechanism of SMAD4-I500V dysfunction. We show that SMAD4-I500V can dimerize, but its transcriptional activity is severely compromised. Our data show that SMAD4-I500V acts dominant-negatively on SMAD4 and on receptor-regulated SMADs, affecting transcription of target genes. Furthermore, SMAD4-I500V impacts the transcription and function of crucial developmental transcription regulator, NKX2-5. Overall, our data reveal a dominant-negative model of disease for SMAD4-I500V where the function of SMAD4 encoded on the remaining allele, and of co-factors, are perturbed by the continued heterodimerization of the variant, leading to dysregulation of TGF and BMP signaling. Our findings not only provide novel insights into the mechanism of Myhre syndrome pathogenesis but also extend the current knowledge of how pathogenic variants in SMAD proteins cause disease.

The Trifecta Study: Comparing Plasma Levels of Donor-derived Cell-Free DNA with the Molecular Phenotype of Kidney Transplant Biopsies.

BACKGROUND: The relationship between the donor-derived cell-free DNA fraction (dd-cfDNA[%]) in plasma in kidney transplant recipients at time of indication biopsy and gene expression in the biopsied allograft has not been defined. METHODS: In the prospective, multicenter Trifecta study, we collected tissue from 300 biopsies from 289 kidney transplant recipients to compare genome-wide gene expression in biopsies with dd-cfDNA(%) in corresponding plasma samples drawn just before biopsy. Rejection was assessed with the microarray-based Molecular Microscope Diagnostic System using automatically assigned rejection archetypes and molecular report sign-outs, and histology assessments that followed Banff guidelines. RESULTS: The median time of biopsy post-transplantation was 455 days (5 days to 32 years), with a case mix similar to that of previous studies: 180 (60%) no rejection, 89 (30%) antibody-mediated rejection (ABMR), and 31 (10%) T cell-mediated rejection (TCMR) and mixed. In genome-wide mRNA measurements, all 20 top probe sets correlating with dd-cfDNA(%) were previously annotated for association with ABMR and all types of rejection, either natural killer (NK) cell-expressed (e.g., GNLY, CCL4, TRDC, and S1PR5) or IFN-γ-inducible (e.g., PLA1A, IDO1, CXCL11, and WARS). Among gene set and classifier scores, dd-cfDNA(%) correlated very strongly with ABMR and all types of rejection, reasonably strongly with active TCMR, and weakly with inactive TCMR, kidney injury, and atrophy fibrosis. Active ABMR, mixed, and active TCMR had the highest dd-cfDNA(%), whereas dd-cfDNA(%) was lower in late-stage ABMR and less-active TCMR. By multivariate random forests and logistic regression, molecular rejection variables predicted dd-cfDNA(%) better than histologic variables. CONCLUSIONS: The dd-cfDNA(%) at time of indication biopsy strongly correlates with active molecular rejection and has the potential to reduce unnecessary biopsies. CLINICAL TRIAL REGISTRATION NUMBER: NCT04239703.

Reduced Sensitivity to Fluopyram in Meloidogyne graminis following Long-Term Exposure in Golf Turf.

In recent years, some golf course superintendents in Florida have reported that the turf health is no longer as great, and nematode responses to fluopyram have decreased. The objective of this research was to determine if the mechanism of the reported reduced efficacy was attributable to either: i) enhanced degradation accelerating its breakdown in the soil, or ii) reduced sensitivity to the nematicide in the nematode populations. In a field experiment, soil and nematodes were collected from small plots that had been treated multiple times over four years, for only one year, or never treated. Soil and nematodes were additionally collected from commercial turf sites where either multiple applications of fluopyram had been made for numerous years, or it had never been used. Bioassay experiments found no evidence of enhanced degradation. However, M. graminis collected from small field plots and commercial sites with long-term use of fluopyram were less sensitive to fluopyram in-vitro than those from small plots and commercial sites where fluopyram had not been used. These results indicate that nematicide resistance is a likely cause of reduced fluopyram efficacy on golf-course turf in Florida.

Prediction of HIV-1 protease cleavage site from octapeptide sequence information using selected classifiers and hybrid descriptors.

BACKGROUND: In most parts of the world, especially in underdeveloped countries, acquired immunodeficiency syndrome (AIDS) still remains a major cause of death, disability, and unfavorable economic outcomes. This has necessitated intensive research to develop effective therapeutic agents for the treatment of human immunodeficiency virus (HIV) infection, which is responsible for AIDS. Peptide cleavage by HIV-1 protease is an essential step in the replication of HIV-1. Thus, correct and timely prediction of the cleavage site of HIV-1 protease can significantly speed up and optimize the drug discovery process of novel HIV-1 protease inhibitors. In this work, we built and compared the performance of selected machine learning models for the prediction of HIV-1 protease cleavage site utilizing a hybrid of octapeptide sequence information comprising bond composition, amino acid binary profile (AABP), and physicochemical properties as numerical descriptors serving as input variables for some selected machine learning algorithms. Our work differs from antecedent studies exploring the same subject in the combination of octapeptide descriptors and method used. Instead of using various subsets of the dataset for training and testing the models, we combined the dataset, applied a 3-way data split, and then used a "stratified" 10-fold cross-validation technique alongside the testing set to evaluate the models. RESULTS: Among the 8 models evaluated in the "stratified" 10-fold CV experiment, logistic regression, multi-layer perceptron classifier, linear discriminant analysis, gradient boosting classifier, Naive Bayes classifier, and decision tree classifier with AUC, F-score, and B. Acc. scores in the ranges of 0.91-0.96, 0.81-0.88, and 80.1-86.4%, respectively, have the closest predictive performance to the state-of-the-art model (AUC 0.96, F-score 0.80 and B. Acc. ~ 80.0%). Whereas, the perceptron classifier and the K-nearest neighbors had statistically lower performance (AUC 0.77-0.82, F-score 0.53-0.69, and B. Acc. 60.0-68.5%) at p < 0.05. On the other hand, logistic regression, and multi-layer perceptron classifier (AUC of 0.97, F-score > 0.89, and B. Acc. > 90.0%) had the best performance on further evaluation on the testing set, though linear discriminant analysis, gradient boosting classifier, and Naive Bayes classifier equally performed well (AUC > 0.94, F-score > 0.87, and B. Acc. > 86.0%). CONCLUSIONS: Logistic regression and multi-layer perceptron classifiers have comparable predictive performances to the state-of-the-art model when octapeptide sequence descriptors consisting of AABP, bond composition and standard physicochemical properties are used as input variables. In our future work, we hope to develop a standalone software for HIV-1 protease cleavage site prediction utilizing the linear regression algorithm and the aforementioned octapeptide sequence descriptors.

The novel estrogen receptor modulator STX attenuates Amyloid-ß neurotoxicity in the 5XFAD mouse model of Alzheimer's disease.

Based on previous evidence that the non-steroidal estrogen receptor modulator STX mitigates the effects of neurotoxic Amyloid-ß (Aß) in vitro, we have evaluated its neuroprotective benefits in a mouse model of Alzheimer's disease. Cohorts of 5XFAD mice, which begin to accumulate cerebral Aß at two months of age, were treated with orally-administered STX starting at 6 months of age for two months. After behavioral testing to evaluate cognitive function, biochemical and immunohistochemical assays were used to analyze key markers of mitochondrial function and synaptic integrity. Oral STX treatment attenuated Aß-associated mitochondrial toxicity and synaptic toxicity in the brain, as previously documented in cultured neurons. STX also moderately improved spatial memory in 5XFAD mice. In addition, STX reduced markers for reactive astrocytosis and microgliosis surrounding amyloid plaques, and also unexpectedly reduced overall levels of cerebral Aß in the brain. The neuroprotective effects of STX were more robust in females than in males. These results suggest that STX may have therapeutic potential in Alzheimer's Disease.