Epigenomic analysis of multilineage differentiation of human embryonic stem cells.

Epigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in nonexpressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation.

Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome.

Differential methylation of the two parental genomes in placental mammals is essential for genomic imprinting and embryogenesis. To systematically study this epigenetic process, we have generated a base-resolution, allele-specific DNA methylation (ASM) map in the mouse genome. We find parent-of-origin dependent (imprinted) ASM at 1,952 CG dinucleotides. These imprinted CGs form 55 discrete clusters including virtually all known germline differentially methylated regions (DMRs) and 23 previously unknown DMRs, with some occurring at microRNA genes. We also identify sequence-dependent ASM at 131,765 CGs. Interestingly, methylation at these sites exhibits a strong dependence on the immediate adjacent bases, allowing us to define a conserved sequence preference for the mammalian DNA methylation machinery. Finally, we report a surprising presence of non-CG methylation in the adult mouse brain, with some showing evidence of imprinting. Our results provide a resource for understanding the mechanisms of imprinting and allele-specific gene expression in mammalian cells.

Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.

The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance.

A pathogenic DYT-THAP1 dystonia mutation causes hypomyelination and loss of YY1 binding.

Dystonia is a disabling disease that manifests as prolonged involuntary twisting movements. DYT-THAP1 is an inherited form of isolated dystonia caused by mutations in THAP1 encoding the transcription factor THAP1. The phe81leu (F81L) missense mutation is representative of a category of poorly understood mutations that do not occur on residues critical for DNA binding. Here, we demonstrate that the F81L mutation (THAP1F81L) impairs THAP1 transcriptional activity and disrupts CNS myelination. Strikingly, THAP1F81L exhibits normal DNA binding but causes a significantly reduced DNA binding of YY1, its transcriptional partner that also has an established role in oligodendrocyte lineage progression. Our results suggest a model of molecular pathogenesis whereby THAP1F81L normally binds DNA but is unable to efficiently organize an active transcription complex.

The Impact of Primary Liver Disease and Social Determinants in a Mixed Donor Liver Transplant Program: A Single-Center Analysis.

Organ allocation in liver transplantation (LT) remains imperfect. Periodic center reviews ensure programs transparently evaluate the impact of practice on access to transplantation, reflecting, in particular, patient (primary disease, social determinants) and program (deceased versus live donation) factors. Adult Ontario residents waitlisted for first LT at Toronto General Hospital from November 2012 to May 2019 were reviewed. Analyses were performed between distance to transplant center, income, education level, population density and primary liver disease, with LT, deceased donor liver transplant (DDLT), living donor liver transplant (LDLT), and delisting. Of 1735 listed patients, 549 were delisted (32%), while 1071 were transplanted (62%), with 819 DDLT recipients (76%) and 252 LDLT recipients (24%), while 115 (7%) remained actively listed at data census. On univariate analysis, DDLT recipients lived 30% closer (median 39.7 versus 60.6 km; P < 0.001), lived in more populous areas (median 8501.0 versus 6868.5 people in a 1-km radius; P < 0.001), and resided in households that annually earned 10% less (median $92,643.17 versus $102,820.89 Canadian dollars; P < 0.001) compared with LDLT recipients. These findings with population density and income differences between DDLT versus LDLT receival remained significant on multivariate modeling even when accounting for primary liver disease. Primary liver disease was a statistically significant factor on multivariate analyses in LT receival (P = 0.001) as well as DDLT versus LDLT receival (P < 0.001). Of patients listed for end-stage liver disease, more patients with autoimmune cholestatic liver diseases received LDLT (34%-41%) than DDLT (27%-30%); this contrasted with patients with noncholestatic diseases LDLT (8%-19%) versus DDLT (37%-59%) receival (P < 0.001). Review of transplant allocation in a large mixed-donor North American liver transplant program demonstrates how patient social determinants and primary liver disease etiology continue to be significantly associated with ultimate transplantation.

Chromatin architecture reorganization during stem cell differentiation.

Higher-order chromatin structure is emerging as an important regulator of gene expression. Although dynamic chromatin structures have been identified in the genome, the full scope of chromatin dynamics during mammalian development and lineage specification remains to be determined. By mapping genome-wide chromatin interactions in human embryonic stem (ES) cells and four human ES-cell-derived lineages, we uncover extensive chromatin reorganization during lineage specification. We observe that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we find widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results therefore provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.

A Comparison of Transphyseal Neck-Head Tunneling and Multiple Epiphyseal Drilling on Femoral Head Healing Following Ischemic Osteonecrosis: An Experimental Investigation in Immature Pigs.

BACKGROUND: Two operative procedures are currently advocated to stimulate the necrotic femoral head healing in children with Legg-Calve-Perthes disease: transphyseal neck-head tunneling (TNHT) and multiple epiphyseal drilling (MED). The purpose of this study was to compare the bone healing and physeal function after treatment with TNHT or MED in a piglet model of ischemic osteonecrosis. METHODS: Eighteen piglets were induced with osteonecrosis by surgically placing a ligature tightly around the right femoral neck. One week later, the piglets were assigned to 1 of 3 treatment groups (n=6/group): (1) local nonweight bearing only (NWB), (2) TNHT plus NWB, or (3) MED plus NWB. The unoperated left femoral heads were used as normal controls. The animals were euthanized at 8 weeks after osteonecrosis induction. Histologic, histomorphometric, radiographic, microcomputed tomography (CT), and calcein-labeling assessments were performed. Statistical analysis included a 1-way ANOVA. RESULTS: Micro-CT analyses showed higher femoral head bone volume in the MED group compared with the TNHT and the NWB groups (P<0.01). The MED group had a higher mean trabecular number (P<0.001) and new bone formation (P=0.001) based on calcein-labeling parameters compared with the TNHT and the NWB groups. In addition, the osteoclast number per bone surface was lower in the MED group compared with the NWB group (P=0.001). Histologic and micro-CT assessments of the proximal femoral physis revealed a larger physeal disruption at the site of physeal drilling in the TNHT group compared with the MED group. However, no significant differences in physeal elongation (P=0.61) and femoral neck length (P=0.31) were observed between the treatment groups. CONCLUSIONS: MED produced a higher bone volume and stimulated greater bone formation than the TNHT or the NWB alone. Both procedures did not produce a significant physeal growth disturbance during the study period. CLINICAL RELEVANCE: This preclinical study provides evidence that MED produces more favorable bone healing than the TNHT in a large animal model of Legg-Calve-Perthes disease.

Essential function of NHE8 in mouse retina demonstrated by AAV-mediated CRISPR/Cas9 knockdown.

We studied the role of sodium/proton exchanger 8 (NHE8) in retinal pigment epithelium (RPE) and photoreceptor cells of adult mouse retina by using the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Neisseria meningitidis (Nm). Specific single guide RNAs (sgRNAs) were designed to knockdown the Slc9a8 gene, which encodes the NHE8. Nuclease null NmCas9 and sgRNAs were packaged respectively using adeno-associated viral vector (AAV), and delivered into mouse eyes in vivo by subretinal injection on wild-type mice of about four-week-old when mouse retina is fully developed. Eye samples were collected four weeks after injection for phenotype examination. Real-time PCR analysis demonstrated ∼38% reduction of NHE8 transcripts in retinas injected with AAV-knockdown sgRNA and AAV-Cas9. Loss of photoreceptor cells was found in eyes injected with AAV-knockdown sgRNA and AAV-Cas9 under either the human rhodopsin promoter or the minimal chicken ß-actin promoter, while normal morphology was observed in control eyes injected with AAV-Cas9 and AAV-control sgRNA; immunostaining data showed degenerating photoreceptor cells and RPE cells in eyes injected with knockdown sgRNA and Cas9 AAVs. We further determined that mutant M120K-NHE8 displayed altered intracellular pH regulation in human RPE and primary mouse RPE cells using genetically encoded pH sensor pHluorin and that primary cultured NHE8 mutant RPE cells showed different pH titration curves. These results indicate that NHE8 plays essential function in both RPE and photoreceptor cells. NHE8 dysfunction either in photoreceptor or RPE is sufficient to cause retinal degeneration in adult mice at any age.

Topological domains in mammalian genomes identified by analysis of chromatin interactions.

The spatial organization of the genome is intimately linked to its biological function, yet our understanding of higher order genomic structure is coarse, fragmented and incomplete. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories, and numerous models have been proposed for how chromosomes fold within chromosome territories. These models, however, provide only few mechanistic details about the relationship between higher order chromatin structure and genome function. Recent advances in genomic technologies have led to rapid advances in the study of three-dimensional genome organization. In particular, Hi-C has been introduced as a method for identifying higher order chromatin interactions genome wide. Here we investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution. We identify large, megabase-sized local chromatin interaction domains, which we term 'topological domains', as a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, indicating that topological domains are an inherent property of mammalian genomes. Finally, we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, transfer RNAs and short interspersed element (SINE) retrotransposons, indicating that these factors may have a role in establishing the topological domain structure of the genome.

Progressive degeneration of human neural stem cells caused by pathogenic LRRK2.

Nuclear-architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as ageing. It is therefore plausible that diseases whose manifestations correlate with ageing might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of ageing-associated disorders by focusing on a leucine-rich repeat kinase 2 (LRRK2) dominant mutation (G2019S; glycine-to-serine substitution at amino acid 2019), which is associated with familial and sporadic Parkinson's disease as well as impairment of adult neurogenesis in mice. Here we report on the generation of induced pluripotent stem cells (iPSCs) derived from Parkinson's disease patients and the implications of LRRK2(G2019S) mutation in human neural-stem-cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in nuclear-envelope organization, clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in Parkinson's disease iPSCs and were recapitulated after targeted knock-in of the LRRK2(G2019S) mutation in human embryonic stem cells. Analysis of human brain tissue showed nuclear-envelope impairment in clinically diagnosed Parkinson's disease patients. Together, our results identify the nucleus as a previously unknown cellular organelle in Parkinson's disease pathology and may help to open new avenues for Parkinson's disease diagnoses as well as for the potential development of therapeutics targeting this fundamental cell structure.

Does the frequency of molecular monitoring after tyrosine kinase inhibitor discontinuation affect outcomes of patients with chronic myeloid leukemia?

BACKGROUND: To the authors' knowledge, the optimal frequency of monitoring after tyrosine kinase inhibitor (TKI) discontinuation in patients with chronic myeloid leukemia (CML) has not been established. Data regarding the discontinuation of second-generation TKIs used in first-line treatment or after the failure of first-line treatment with TKIs are limited. Herein, the authors report real-world experience with "reduced frequency" molecular monitoring in patients with CML in all phases who discontinued treatment with imatinib, dasatinib, or bosutinib. METHODS: The records of patients who discontinued TKIs were reviewed. Patients who discontinued TKIs were monitored prospectively on an intended schedule of monthly blood quantitative reverse transcriptase-polymerase chain reaction for BCR-ABL1 for 3 months, quarterly for 12 months, and every 6 months thereafter until loss of major molecular response (MMR). After loss of MMR, the TKI that previously was discontinued was reinitiated. RESULTS: Between January 2010 and September 2015, a total of 24 patients in chronic (21 patients), accelerated (2 patients), and lymphoid blast (1 patient) phase discontinued imatinib (16 patients), dasatinib (5 patients), or bosutinib (3 patients) used in the front-line treatment or beyond. Blood quantitative reverse transcriptase-polymerase chain reaction for BCR-ABL1 was performed 1.3 ± 0.7 times within the first 3 months (24 patients) and 2.7 ± 1.4 times in the following 12 months (18 patients). With a median follow-up of 36.5 months (range, 3.2-67.4 months), the probabilities of treatment-free remission at 1 year and 2 years were 65.7% (95% confidence interval, 55.8%-75.6%) and 59.7% (95% confidence interval, 49.1%-70.3%), respectively. Loss of MMR was observed in 9 patients at a median of 2.8 months (range, 1.8-14.2 months) after discontinuation of TKIs. CONCLUSIONS: With the limitations of a small sample size, the results of the current study demonstrate that less frequent monitoring of BCR-ABL1 does not appear to affect outcomes, and that discontinuation of TKIs used as first-line treatment or beyond after resistance or intolerance to first-line treatment appears feasible. Cancer 2017;123:2482-88. © 2017 American Cancer Society.

Palliative performance scale and survival among outpatients with advanced cancer.

PURPOSE: Previous studies have examined the association between the Palliative Performance Scale (PPS) and survival duration; however, few have examined patients with incurable cancer in the outpatient setting. In addition to exploring this association further, the purpose of this study was to identify key PPS markers that could serve as triggers to signify the need for key care discussions. METHODS: Study subjects were followed prospectively from the time of referral for a specialist palliative care consultation until death. PPS ratings and survival estimates were determined for each visit. RESULTS: For the final study population of 368 patients, at baseline, the median PPS rating was 60. Overall median and mean survival duration were approximately 4 and 6 months, respectively. Median survival duration for patients with PPS ratings of 70, 60, and 50 were found to be approximately 6, 3, and 2 months, respectively. Twenty-four percent of all survival estimates were found to be accurate. CONCLUSIONS: Given the ongoing challenge of inaccurate survival estimates, this data suggests what may be of greatest clinical utility is to use specific PPS ratings as triggers for key care discussions among patients with incurable and progressive cancer.

Practice Implications of Colonoscopic Investigation of Microscopic Colitis in Patients Above 50 Years of Age Presenting With Chronic Diarrhoea: A Multi-Centre Review.

Background Patients with an unknown cause for chronic diarrhoea will usually undergo a colonoscopy as part of the investigative work-up, and it is acceptable practice for the patients to undergo random biopsies. The optimum number of biopsies has yet to be established. This study investigated the implications of routine random biopsies for diagnosing microscopic colitis in patients 50 years and older who presented with chronic diarrhoea. Methodology A retrospective cohort study of a prospectively maintained internal hospital database across three tertiary teaching hospitals in Perth, Western Australia, on participants >50 years old who presented for an elective colonoscopy to investigate chronic diarrhoea between January 2016 and June 2019. Data was captured from medical records, imaging, colonoscopy, and histopathology reports, and patient follow-up was analysed using SPSS v.29 (IBM Corp., Armonk, NY). Results There were 216 patients, with the majority female (67%) and a mean age of 64.6 (SD±9.9). Microscopic colitis was identified in 7.4% (95% CI = 3.9-10.9%). Most positive biopsies (81.3%) were from the left colon. The median number of biopsies per case was seven (IQR=5). The median procedure duration and scope withdrawal time were 23 and eight minutes, respectively. Most of the procedures were done by a consultant (77%). Bowel was adequately prepped in 76.9% of the cases. Univariate analysis demonstrated that the rate of identification of microcolitis was associated with the number of biopsies taken; microcolitis positivity had a higher mean number of biopsies, 10.8 vs 6.7 (p<0.001). Key complications were a 30-day readmission rate, seven-day re-presentation with acute colitis, post-procedure bleeding, requiring further imaging or angioembolisation and increased length of stay on readmission. Conclusion The prevalence of positive biopsies for microcolitis is low (7.4%). Biopsies during colonoscopy are associated with clinically significant morbidity and health care costs. Most positive biopsies were attained from the left colon. It may be time to standardise practice in investigating microscopic colitis as a cause of chronic diarrhoea in patients > 50 years old.

Single-Center Experiences: A Comparison of Intracorporeal and Extracorporeal Anastomosis Outcomes in Right Hemicolectomy.

Background Anastomosis formed in minimally invasive laparoscopic right hemicolectomy (LRH) may be achieved intra-corporeally (ICA) or extra-corporeally (ECA). This study compared the return of bowel function and other associated early patient outcomes and morbidity rates after an ICA or ECA in LRH. Methodology The study conducted a single-center retrospective cohort study of elective LRH from January 2021 to September 2023. Patient demographics, surgical techniques, and outcomes were analyzed using IBM SPSS Statistics for Windows, Version 29.0 (IBM Corp., Armonk, NY). Results Ninety participants underwent LRH, and the anastomotic type was evenly distributed - with male patients comprising 53 (58.9%) of the total. The mean age was 64 (standard deviation [SD] ±16.8) years, and the median body mass index (BMI) was 27.0 (interquartile range [IQR] = 7.8). The mean follow-up period was 5.1 (SD ± 6.0) months. Univariate analysis showed that ICA had a shorter time for return of bowel function (P < 0.01). Additionally, ICA was associated with lower pain scores (P < 0.01), low morbidity (P = 0.02), and shorter hospital stays (P = 0.01). When comparing ICA to ECA, no significant difference was observed for procedure duration (P = 0.13), anastomotic leak (AL, P = 1.00), surgical-site infections (P = 0.36), lymph node yield (P = 0.26), and any-cause mortality. Multivariate logistic regression, controlling for statistically insignificant confounding factors, revealed that ECA was significantly and independently associated with increased time to first flatus (odds ratio [OR] 2.3, P = 0.01) and higher average postoperative pain (OR 1.5, P = 0.02) compared to ICA. Conclusions This single-center experience showed that ICA is associated with a quicker return to normal bowel function and low morbidity outcomes. ICA participants were positively associated with clinically relevant and health economics outcomes of shorter hospital stays without significantly adding to the procedure's duration times or compromising principles of oncological resection yield.

Intranasal Delivery of Cell-Penetrating Therapeutic Peptide Enhances Brain Delivery, Reduces Inflammation, and Improves Neurologic Function in Moderate Traumatic Brain Injury.

Following traumatic brain injury (TBI), secondary brain damage due to chronic inflammation is the most predominant cause of the delayed onset of mood and memory disorders. Currently no therapeutic approach is available to effectively mitigate secondary brain injury after TBI. One reason is the blood-brain barrier (BBB), which prevents the passage of most therapeutic agents into the brain. Peptides have been among the leading candidates for CNS therapy due to their low immunogenicity and toxicity, bioavailability, and ease of modification. In this study, we demonstrated that non-invasive intranasal (IN) administration of KAFAK, a cell penetrating anti-inflammatory peptide, traversed the BBB in a murine model of diffuse, moderate TBI. Notably, KAFAK treatment reduced the production of proinflammatory cytokines that contribute to secondary injury. Furthermore, behavioral tests showed improved or restored neurological, memory, and locomotor performance after TBI in KAFAK-treated mice. This study demonstrates KAFAK's ability to cross the blood-brain barrier, to lower proinflammatory cytokines in vivo, and to restore function after a moderate TBI.

Nanosheet Promotes Chronic Wound Healing by Localizing Uncultured Stromal Vascular Fraction Cells.

Objective: To develop an efficacious and efficient method for treating chronic wounds using "nanosheet" that improves the survival and localization of transplanted cells without prior seeding to optimally derive the regenerative potentials of uncultured stromal vascular fraction (SVF) cells. Approach: We propose a method whereby the wound is covered by uncultured SVF cells using the nanosheet [porous poly(d, l,-lactic acid)] (PDLLA) films) designed to hold cells in a single-cell layer. A chronic wound model was created on 12-month-old db/db mice by inflecting a full-thickness skin excision on their dorsum and was subsequently given either no treatment or a treatment with SVF cells alone (with Tegaderm dressing), nanosheet alone, or nanosheet with SVF cells. Results: The placement of the nanosheet improved the grafted cell retention rate at day 10 timepoint by 5 folds, and the wound area was the smallest in the wounds treated with SVF cells plus nanosheet in comparison to the other groups. Collagen deposition and epidermal growth factor were significantly higher in the wound beds treated with SVF cells with the nanosheet, offering some mechanistic insights. Innovation: Porous poly(d, l,-lactic acid acid) (PDLLA) films or "nanosheet" printed on the nanoscale (1-100 nm in thickness) as a cellular scaffold for cytotherapy for the treatment of chronic wounds. Conclusion: The use of the nanosheet is an effective way to improve the transplanted SVF cell retention and accelerate the overall wound closure.

Strategies for combating antibiotic resistance in bacterial biofilms.

Biofilms, which are complexes of microorganisms that adhere to surfaces and secrete protective extracellular matrices, wield substantial influence across diverse domains such as medicine, industry, and environmental science. Despite ongoing challenges posed by biofilms in clinical medicine, research in this field remains dynamic and indeterminate. This article provides a contemporary assessment of biofilms and their treatment, with a focus on recent advances, to chronicle the evolving landscape of biofilm research.

ZNL0325, a Pyrazolopyrimidine-Based Covalent Probe, Demonstrates an Alternative Binding Mode for Kinases.

The pyrazolopyrimidine (PP) heterocycle is a versatile and widely deployed core scaffold for the development of kinase inhibitors. Typically, a 4-amino-substituted pyrazolopyrimidine binds in the ATP-binding pocket in a conformation analogous to the 6-aminopurine of ATP. Here, we report the discovery of ZNL0325 which exhibits a flipped binding mode where the C3 position is oriented toward the ribose binding pocket. ZNL0325 and its analogues feature an acrylamide side chain at the C3 position which is capable of forming a covalent bond with multiple kinases that possess a cysteine at the αD-1 position including BTK, EGFR, BLK, and JAK3. These findings suggest that the ability to form a covalent bond can override the preferred noncovalent binding conformation of the heterocyclic core and provides an opportunity to create structurally distinct covalent kinase inhibitors.

Development of a Covalent Inhibitor of c-Jun N-Terminal Protein Kinase (JNK) 2/3 with Selectivity over JNK1.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family, which includes JNK1-JNK3. Interestingly, JNK1 and JNK2 show opposing functions, with JNK2 activity favoring cell survival and JNK1 stimulating apoptosis. Isoform-selective small molecule inhibitors of JNK1 or JNK2 would be useful as pharmacological probes but have been difficult to develop due to the similarity of their ATP binding pockets. Here, we describe the discovery of a covalent inhibitor YL5084, the first such inhibitor that displays selectivity for JNK2 over JNK1. We demonstrated that YL5084 forms a covalent bond with Cys116 of JNK2, exhibits a 20-fold higher Kinact/KI compared to that of JNK1, and engages JNK2 in cells. However, YL5084 exhibited JNK2-independent antiproliferative effects in multiple myeloma cells, suggesting the existence of additional targets relevant in this context. Thus, although not fully optimized, YL5084 represents a useful chemical starting point for the future development of JNK2-selective chemical probes.

A collaborative approach to improving representation in viral genomic surveillance.

The lack of routine viral genomic surveillance delayed the initial detection of SARS-CoV-2, allowing the virus to spread unfettered at the outset of the U.S. epidemic. Over subsequent months, poor surveillance enabled variants to emerge unnoticed. Against this backdrop, long-standing social and racial inequities have contributed to a greater burden of cases and deaths among minority groups. To begin to address these problems, we developed a new variant surveillance model geared toward building 'next generation' genome sequencing capacity at universities in or near rural areas and engaging the participation of their local communities. The resulting genomic surveillance network has generated more than 1,000 SARS-CoV-2 genomes to date, including the first confirmed case in northeast Louisiana of Omicron, and the first and sixth confirmed cases in Georgia of the emergent BA.2.75 and BQ.1.1 variants, respectively. In agreement with other studies, significantly higher viral gene copy numbers were observed in Delta variant samples compared to those from Omicron BA.1 variant infections, and lower copy numbers were seen in asymptomatic infections relative to symptomatic ones. Collectively, the results and outcomes from our collaborative work demonstrate that establishing genomic surveillance capacity at smaller academic institutions in rural areas and fostering relationships between academic teams and local health clinics represent a robust pathway to improve pandemic readiness.