Elucidating The Pathological Mechanism Of Histone H3.3 Mutations In Neurodevelopment

In eukaryotic cells, octamers of histone proteins intricately organize DNA, forming a macromolecule known as nucleosomes. A nucleosome is comprised of two copies of histone H2A, H2B, H4, and H3. Histone H3.3 (H3.3), a histone variant, is often found at actively transcribed loci. H3.3 plays a role in cellular inheritance as ablation of H3.3 expression leads to loss of active gene states and dysfunction of heterochromatin telomeric structures. H3F3A and H3F3B, the two genes are known to encode H3.3, are expressed in all human cells with higher expression in the gonads and brain. A recent publication detailed H3.3 as the causative gene in a neurodevelopmental disorder with craniofacial abnormalities. The underlying cause of the disease is unknown; however, H3.3 is involved in a wide range of central nervous system functions. This dissertation highlights work that aimed to profile the effects of these germline mutations. We performed a comprehensive screen on pathogenic H3.3 variants utilizing a combination of techniques and methodology, including tissue culture, bottom-up proteomics, and neurodevelopmental assays in Xenopus laevis. We found global alterations to histone post-translational modifications with significant alterations to histone acetylation on histone H2a, H3, and H4 peptides. In addition, performing quantitative proteomics in the 293T cells led us to determine that these mutations affect several cellular processes, such as RNA splicing, cell motility, neurofilament maintenance, folic acid metabolism, and post-synaptic density, all processes known to be dysregulated in other neurological syndromes. Utilizing the model organism Xenopus, we introduced two of the pathogenic variants into embryos. We observed reduced craniofacial cartilage, abnormal head shape, and impaired motility due to kinked tails in the mutant tadpoles. We performed quantitative proteomics on these tadpoles and found pathways related to carbon metabolism and amino acid degradation upregulated. Lastly, our transcriptomic analyses corroborated some of these finds and an upregulation of the TGF-beta signaling pathway in one of the mutants. This work provides the first mechanistic study of these germline H3.3 mutations, introduces pathways dysregulated that can be further studied. Understanding the basic biology of these mutations will shed light on the molecular mechanisms of H3.3 in neurodevelopment

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Elucidating the Pathological Mechanism of Histone H3.3 Mutations in Neurodevelopment

In eukaryotic cells, octamers of histone proteins intricately organize DNA, forming a macromolecule known as nucleosomes. A nucleosome is comprised of two copies of histone H2A, H2B, H4, and H3. Histone H3.3 (H3.3), a histone variant, is often found at actively transcribed loci. H3.3 plays a role in cellular inheritance as ablation of H3.3 expression leads to loss of active gene states and dysfunction of heterochromatin telomeric structures. H3F3A and H3F3B, the two genes are known to encode H3.3, are expressed in all human cells with higher expression in the gonads and brain. A recent publication detailed H3.3 as the causative gene in a neurodevelopmental disorder with craniofacial abnormalities. The underlying cause of the disease is unknown; however, H3.3 is involved in a wide range of central nervous system functions. This dissertation highlights work that aimed to profile the effects of these germline mutations. We performed a comprehensive screen on pathogenic H3.3 variants utilizing a combination of techniques and methodology, including tissue culture, bottom-up proteomics, and neurodevelopmental assays in Xenopus laevis. We found global alterations to histone post-translational modifications with significant alterations to histone acetylation on histone H2a, H3, and H4 peptides. In addition, performing quantitative proteomics in the 293T cells led us to determine that these mutations affect several cellular processes, such as RNA splicing, cell motility, neurofilament maintenance, folic acid metabolism, and post-synaptic density, all processes known to be dysregulated in other neurological syndromes. Utilizing the model organism Xenopus, we introduced two of the pathogenic variants into embryos. We observed reduced craniofacial cartilage, abnormal head shape, and impaired motility due to kinked tails in the mutant tadpoles. We performed quantitative proteomics on these tadpoles and found pathways related to carbon metabolism and amino acid degradation upregulated. Lastly, our transcriptomic analyses corroborated some of these finds and an upregulation of the TGF-beta signaling pathway in one of the mutants. This work provides the first mechanistic study of these germline H3.3 mutations, introduces pathways dysregulated that can be further studied. Understanding the basic biology of these mutations will shed light on the molecular mechanisms of H3.3 in neurodevelopment

Developing an International Multidisciplinary Research Collaboration: Lessons Learned

Background. Improving global health through international research collaboration is increasingly becoming a common pathway of transferring knowledge across cultures. Developing such collaborations, however, is challenging. This presentation will discuss the ongoing process, challenges, and lessons learned for developing a multidisciplinary HIV/AIDS research partnership between American and Tanzanian universities. Process of Partnership. To date, HIV prevention programs in Tanzania have not been entirely successful, and numerous investigators have called for multidisciplinary models. For this reason, an investigator from the Wright State University (WSU) College of Nursing partnered with researchers from WSU\u27s school of medicine and schools of nursing and public health at the Muhimbili University of Health and Allied Sciences (MUHAS) in Tanzania. Initially, two WSU investigators travelled to Tanzania in June 2011 to confer with MUHAS investigators to get support for developing a collaborative research project. The meeting resulted in a WSU-funded needs-assessment project to be conducted in Tanzania in December 2011. Observations and Conclusions. Taking a project to an international level involves practical considerations. Partnership expectations on data sharing and dissemination, leadership and authorship should be made explicit. Understanding all the steps for research approval and the ethical boards involved is also essential. Budgetary issues should be anticipated, as international research is expensive. For example, costs for the local investigators must be covered in the budget because data collection is best accomplished by local researchers, who have no local funding. Other potential barriers such as power outages, unreliable internet access, language differences, and cultural insensitivity should be considered

Developing an International Multidisciplinary Research Collaboration: Lessons Learned

Background. Improving global health through international research collaboration is increasingly becoming a common pathway of transferring knowledge across cultures. Developing such collaborations, however, is challenging. This presentation will discuss the ongoing process, challenges, and lessons learned for developing a multidisciplinary HIV/AIDS research partnership between American and Tanzanian universities. Process of Partnership. To date, HIV prevention programs in Tanzania have not been entirely successful, and numerous investigators have called for multidisciplinary models. For this reason, an investigator from the Wright State University (WSU) College of Nursing partnered with researchers from WSU\u27s school of medicine and schools of nursing and public health at the Muhimbili University of Health and Allied Sciences (MUHAS) in Tanzania. Initially, two WSU investigators travelled to Tanzania in June 2011 to confer with MUHAS investigators to get support for developing a collaborative research project. The meeting resulted in a WSU-funded needs-assessment project to be conducted in Tanzania in December 2011. Observations and Conclusions. Taking a project to an international level involves practical considerations. Partnership expectations on data sharing and dissemination, leadership and authorship should be made explicit. Understanding all the steps for research approval and the ethical boards involved is also essential. Budgetary issues should be anticipated, as international research is expensive. For example, costs for the local investigators must be covered in the budget because data collection is best accomplished by local researchers, who have no local funding. Other potential barriers such as power outages, unreliable internet access, language differences, and cultural insensitivity should be considered

Comparative evaluation of glomerular morphometric techniques reveals differential technical artifacts between focal segmental glomerulosclerosis and normal glomeruli

Abstract Morphometric estimates of mean or individual glomerular volume (MGV, IGV) have biological implications, over and above qualitative histologic data. However, morphometry is time‐consuming and requires expertise limiting its utility in clinical cases. We evaluated MGV and IGV using plastic‐ and paraffin‐embedded tissue from 10 control and 10 focal segmental glomerulosclerosis (FSGS) mice (aging and 5/6th nephrectomy models) using the gold standard Cavalieri (Cav) method versus the 2‐profile and Weibel–Gomez (WG) methods and a novel 3‐profile method. We compared accuracy, bias and precision, and quantified results obtained when sampling differing numbers of glomeruli. In both FSGS and controls, we identified an acceptable precision for MGV of 10‐glomerular sampling versus 20‐glomerular sampling using the Cav method, while 5‐glomerular sampling was less precise. In plastic tissue, 2‐ or 3‐profile MGVs showed greater concordance with MGV when using Cav, versus MGV with WG. IGV comparisons using the same glomeruli reported a consistent underestimation bias with both 2‐ or 3‐profile methods versus the Cav method. FSGS glomeruli showed wider variations in bias estimation than controls. Our 3‐profile method offered incremental benefit to the 2‐profile method in both IGV and MGV estimation (improved correlation coefficient, Lin's concordance and reduced bias). In our control animals, we quantified a shrinkage artifact of 52% from tissue processed for paraffin‐embedded versus plastic‐embedded tissue. FSGS glomeruli showed overall reduced shrinkage albeit with variable artifact signifying periglomerular/glomerular fibrosis. A novel 3‐profile method offers slightly improved concordance with reduced bias versus 2‐profile. Our findings have implications for future studies using glomerular morphometry

Examination of Diazaspiro Cores as Piperazine Bioisosteres in the Olaparib Framework Shows Reduced DNA Damage and Cytotoxicity

Development of poly­(ADP-ribose) polymerase inhibitors (PARPi’s) continues to be an attractive area of research due to synthetic lethality in DNA repair deficient cancers; however, PARPi’s also have potential as therapeutics to prevent harmful inflammation. We investigated the pharmacological impact of incorporating spirodiamine motifs into the phthalazine architecture of FDA approved PARPi olaparib. Synthesized analogues were screened for PARP-1 affinity, enzyme specificity, catalytic inhibition, DNA damage, and cytotoxicity. This work led to the identification of <b>10e</b> (12.6 ± 1.1 nM), which did not induce DNA damage at similar drug concentrations as olaparib. Interestingly, several worst in class compounds with low PARP-1 affinity, including <b>15b</b> (4397 ± 1.1 nM), induced DNA damage at micromolar concentrations, which can explain the cytotoxicity observed in vitro. This work provides further evidence that high affinity PARPi’s can be developed without DNA damaging properties offering potential new drugs for treating inflammatory related diseases

Imaging joint infections using D-methyl-11C-methionine PET/MRI: initial experience in humans.

PurposeNon-invasive imaging is a key clinical tool for detection and treatment monitoring of infections. Existing clinical imaging techniques are frequently unable to distinguish infection from tumors or sterile inflammation. This challenge is well-illustrated by prosthetic joint infections that often complicate joint replacements. D-methyl-11C-methionine (D-11C-Met) is a new bacteria-specific PET radiotracer, based on an amino acid D-enantiomer, that is rapidly incorporated into the bacterial cell wall. In this manuscript, we describe the biodistribution, radiation dosimetry, and initial human experience using D-11C-Met in patients with suspected prosthetic joint infections.Methods614.5 ± 100.2&nbsp;MBq of D-11C-Met was synthesized using an automated in-loop radiosynthesis method and administered to six healthy volunteers and five patients with suspected prosthetic joint infection, who were studied by PET/MRI. Time-activity curves were used to calculate residence times for each source organ. Absorbed doses to each organ and body effective doses were calculated using OLINDA/EXM 1.1 with both ICRP 60 and ICRP 103 tissue weighting factors. SUVmax and SUVpeak were calculated for volumes of interest (VOIs) in joints with suspected infection, the unaffected contralateral joint, blood pool, and soft tissue background. A two-tissue compartment model was used for kinetic modeling.ResultsD-11C-Met was well tolerated in all subjects. The tracer showed clearance from both urinary (rapid) and hepatobiliary (slow) pathways as well as low effective doses. Moreover, minimal background was observed in both organs with resident micro-flora and target organs, such as the spine and musculoskeletal system. Additionally, D-11C-Met showed increased focal uptake in areas of suspected infection, demonstrated by a significantly higher SUVmax and SUVpeak calculated from VOIs of joints with suspected infections compared to the contralateral joints, blood pool, and background (P &lt; 0.01). Furthermore, higher distribution volume and binding potential were observed in suspected infections compared to the unaffected joints.ConclusionD-11C-Met has a favorable radiation profile, minimal background uptake, and fast urinary extraction. Furthermore, D-11C-Met showed increased uptake in areas of suspected infection, making this a promising approach. Validation in larger clinical trials with a rigorous gold standard is still required