Environmental carcinogen benzo[a]pyrene alters neutral lipid storage via a cyp-35A2 mediated pathway in Caenorhabditis elegans.

Polycyclic aromatic hydrocarbons (PAHs), in particular benzo [a]pyrene (BaP), have been identified as carcinogenic components of tobacco smoke. In mammals, the toxicological response to BaP-diol-epoxide is driven by cytochrome P450 (CYP1A1), a pathway which is absent in Caenorhabditis elegans. In contrast, in worms prominently the CYP-35 enzyme family seems to be induced after BaP exposure. In C. elegans, BaP exposure reduces the accumulation of lysosomal neutral lipids in a dose dependent manner and the deletion of cyp-35A2 results in a significant elevation of neutral lipid metabolism. A cyp-35A2:mCherry;unc-47:GFP dual-labelled reporter strain facilitated the identification of three potential upstream regulators that drive BaP metabolism in worms, namely elt-2, nhr-49 and fos-1. This newly described reporter line is a powerful resource for future large-scale RNAi regarding toxicology and lipid metabolism screens.

Caenorhabditis elegans models for striated muscle disorders caused by missense variants of human LMNA.

Striated muscle laminopathies caused by missense mutations in the nuclear lamin gene LMNA are characterized by cardiac dysfunction and often skeletal muscle defects. Attempts to predict which LMNA variants are pathogenic and to understand their physiological effects lag behind variant discovery. We created Caenorhabditis elegans models for striated muscle laminopathies by introducing pathogenic human LMNA variants and variants of unknown significance at conserved residues within the lmn-1 gene. Severe missense variants reduced fertility and/or motility in C. elegans. Nuclear morphology defects were evident in the hypodermal nuclei of many lamin variant strains, indicating a loss of nuclear envelope integrity. Phenotypic severity varied within the two classes of missense mutations involved in striated muscle disease, but overall, variants associated with both skeletal and cardiac muscle defects in humans lead to more severe phenotypes in our model than variants predicted to disrupt cardiac function alone. We also identified a separation of function allele, lmn-1(R204W), that exhibited normal viability and swimming behavior but had a severe nuclear migration defect. Thus, we established C. elegans avatars for striated muscle laminopathies and identified LMNA variants that offer insight into lamin mechanisms during normal development.

A humanized Caenorhabditis elegans model of hereditary spastic paraplegia-associated variants in KLC4.

Hereditary spastic paraplegia (HSP) is a group of degenerative neurological disorders. We identified a variant in human kinesin light chain 4 (KLC4) that is suspected to be associated with autosomal-dominant HSP. How this and other variants relate to pathologies is unknown. We created a humanized Caenorhabditis elegans model in which klc-2 was replaced by human KLC4 (referred to as hKLC4) and assessed the extent to which hKLC4 retained function in the worm. We observed a slight decrease in motility but no nuclear migration defects in the humanized worms, suggesting that hKLC4 retains much of the function of klc-2. Five hKLC4 variants were introduced into the humanized model. The clinical variant led to early lethality, with significant defects in nuclear migration when homozygous and a weak nuclear migration defect when heterozygous, possibly correlating with the clinical finding of late-onset HSP when the proband was heterozygous. Thus, we were able to establish humanized C. elegans as an animal model for HSP and to use it to test the significance of five variants of uncertain significance in the human gene KLC4.

Neisseria gonorrhoeae cervical spine epidural abscess requiring spinal decompression and instrumented fusion.

Disseminated gonococcal infection (DGI) is an uncommon complication of Neisseria gonorrhoeae infection, and typically presents with either a triad of tenosynovitis, dermatitis and polyarthralgia, or with extra-axial large joint septic arthritis. Spinal epidural abscess is a rare manifestation of DGI, with only a few previously reported cases, none of which required placement of metalware into the infected space. Here we report a severe case of isolated N. gonorrhoeae cervical spine epidural abscess necessitating surgical source control (C7/T1 laminectomy and debridement) and metalware placement (C6-T2 posterior instrumented fusion). The case was successfully managed by a combination of surgical intervention followed by six weeks of predominantly oral, targeted antimicrobial therapy.

Emerging technologies in personalized medicine.

A variety of technologies are emerging to help clinicians provide patient-specific diagnosis and therapies. This special edition of the Molecular Aspects of Medicine is a collection of mini reviews covering a broad range of topics, from systems to model patient variants and discover therapies (Microphysiological systems with patient derived tissue and CRISPR-humanized animal models), to new modalities in diagnostics and therapeutics (Extracellular Vesicles, RNA therapeutics, microbiome and molecular dynamics).

A humanized Caenorhabditis elegans model of Hereditary Spastic Paraplegia-associated variants in kinesin light chain KLC4.

Hereditary spastic paraplegia (HSP) is a group of degenerative neurological disorders. We identified a variant in human kinesin light chain KLC4 that is suspected to be associated with autosomal dominant HSP. How this and other variants relate to pathologies is unknown. We created a humanized C. elegans model where klc- 2 was replaced with human KLC4 and assessed the extent to which hKLC4 retained function in the worm. We observed a slight decrease in motility but no nuclear migration defects in the humanized worms, suggesting that hKLC4 retains much of the function of klc-2 . Five hKLC4 variants were introduced into the humanized model. The clinical variant led to early lethality with significant defects in nuclear migration when homozygous, and a weak nuclear migration defect when heterozygous, possibly correlating with the clinical finding of late onset HSP when the proband was heterozygous. Thus, we were able to establish humanized C. elegans as an animal model for HSP and use it to test the significance of five variants of uncertain significance in the human gene KLC4 . Summary Statement: We identified a variant in KLC4 associated with Hereditary Spastic Paraplegia. The variant had physiological relevance in a humanized C. elegans model where we replaced klc-2 with human KLC4 .

Phenotypic screening models for rapid diagnosis of genetic variants and discovery of personalized therapeutics.

Precision medicine strives for highly individualized treatments for disease under the notion that each individual's unique genetic makeup and environmental exposures imprints upon them not only a disposition to illness, but also an optimal therapeutic approach. In the realm of rare disorders, genetic predisposition is often the predominant mechanism driving disease presentation. For such, mostly, monogenic disorders, a causal gene to phenotype association is likely. As a result, it becomes important to query the patient's genome for the presence of pathogenic variations that are likely to cause the disease. Determining whether a variant is pathogenic or not is critical to these analyses and can be challenging, as many disease-causing variants are novel and, ergo, have no available functional data to help categorize them. This problem is exacerbated by the need for rapid evaluation of pathogenicity, since many genetic diseases present in young children who will experience increased morbidity and mortality without rapid diagnosis and therapeutics. Here, we discuss the utility of animal models, with a focus mainly on C. elegans, as a contrast to tissue culture and in silico approaches, with emphasis on how these systems are used in determining pathogenicity of variants with uncertain significance and then used to screen for novel therapeutics.

Systems to model the personalized aspects of microbiome health and gut dysbiosis.

The human gut microbiome is a complex and dynamic microbial entity that interacts with the environment and other parts of the body including the brain, heart, liver, and immune system. These multisystem interactions are highly conserved from invertebrates to humans, however the complexity and diversity of human microbiota compositions often yield a context that is unique to each individual. Yet commonalities remain across species, where a healthy gut microbiome will be rich in symbiotic commensal biota while an unhealthy gut microbiota will be experiencing abnormal blooms of pathobiont bacteria. In this review we discuss how omics technologies can be applied in a personalized approach to understand the microbial crosstalk and microbial-host interactions that affect the delicate balance between eubiosis and dysbiosis in an individual gut microbiome. We further highlight the strengths of model organisms in identifying and characterizing these conserved synergistic and/or pathogenic host-microbe interactions. And finally, we touch upon the growing area of personalized therapeutic interventions targeting gut microbiome.

Protein structure-based in-silico approaches to drug discovery: Guide to COVID-19 therapeutics.

With more than 5 million fatalities and close to 300 million reported cases, COVID-19 is the first documented pandemic due to a coronavirus that continues to be a major health challenge. Despite being rapid, uncontrollable, and highly infectious in its spread, it also created incentives for technology development and redefined public health needs and research agendas to fast-track innovations to be translated. Breakthroughs in computational biology peaked during the pandemic with renewed attention to making all cutting-edge technology deliver agents to combat the disease. The demand to develop effective treatments yielded surprising collaborations from previously segregated fields of science and technology. The long-standing pharmaceutical industry's aversion to repurposing existing drugs due to a lack of exponential financial gain was overrun by the health crisis and pressures created by front-line researchers and providers. Effective vaccine development even at an unprecedented pace took more than a year to develop and commence trials. Now the emergence of variants and waning protections during the booster shots is resulting in breakthrough infections that continue to strain health care systems. As of now, every protein of SARS-CoV-2 has been structurally characterized and related host pathways have been extensively mapped out. The research community has addressed the druggability of a multitude of possible targets. This has been made possible due to existing technology for virtual computer-assisted drug development as well as new tools and technologies such as artificial intelligence to deliver new leads. Here in this article, we are discussing advances in the drug discovery field related to target-based drug discovery and exploring the implications of known target-specific agents on COVID-19 therapeutic management. The current scenario calls for more personalized medicine efforts and stratifying patient populations early on for their need for different combinations of prognosis-specific therapeutics. We intend to highlight target hotspots and their potential agents, with the ultimate goal of using rational design of new therapeutics to not only end this pandemic but also uncover a generalizable platform for use in future pandemics.

Clinical variants in Caenorhabditis elegans expressing human STXBP1 reveal a novel class of pathogenic variants and classify variants of uncertain significance.

Purpose: Modeling disease variants in animals is useful for drug discovery, understanding disease pathology, and classifying variants of uncertain significance (VUS) as pathogenic or benign. Methods: Using Clustered Regularly Interspaced Short Palindromic Repeats, we performed a Whole-gene Humanized Animal Model procedure to replace the coding sequence of the animal model's unc-18 ortholog with the coding sequence for the human STXBP1 gene. Next, we used Clustered Regularly Interspaced Short Palindromic Repeats to introduce precise point variants in the Whole-gene Humanized Animal Model-humanized STXBP1 locus from 3 clinical categories (benign, pathogenic, and VUS). Twenty-six phenotypic features extracted from video recordings were used to train machine learning classifiers on 25 pathogenic and 32 benign variants. Results: Using multiple models, we were able to obtain a diagnostic sensitivity near 0.9. Twenty-three VUS were also interrogated and 8 of 23 (34.8%) were observed to be functionally abnormal. Interestingly, unsupervised clustering identified 2 distinct subsets of known pathogenic variants with distinct phenotypic features; both p.Tyr75Cys and p.Arg406Cys cluster away from other variants and show an increase in swim speed compared with hSTXBP1 worms. This leads to the hypothesis that the mechanism of disease for these 2 variants may differ from most STXBP1-mutated patients and may account for some of the clinical heterogeneity observed in the patient population. Conclusion: We have demonstrated that automated analysis of a small animal system is an effective, scalable, and fast way to understand functional consequences of variants in STXBP1 and identify variant-specific intensities of aberrant activity suggesting a genotype-to-phenotype correlation is likely to occur in human clinical variations of STXBP1.

The use of CRISPR to generate a whole-gene humanized MAPT and the examination of P301L and G272V clinical variants, along with the creation of deletion null alleles of ptl-1, pgrn-1 and alfa-1 loci.

To study important genes involved in Frontotemporal Dementia ( MAPT , GRN and C9orf72 ), we created deletion alleles in the three orthologous genes ( ptl-1 , pgrn-1 , and alfa-1 ). Simultaneously, we replaced the C. elegans ptl-1 gene with the predicted orthologous human MAPT gene, often called whole-gene humanization, which allows direct assessment of conserved gene function, as well as the opportunity to examine consequences of clinical disease-associated patient variations. Each gene was manipulated using a different selection strategy, including a novel strategy using an unc-18 mutation rescue technique. Clinical MAPT ALS/FTD missense variants G272V and P301L were successfully inserted in hMAPT . Neither ptl-1 loss or clinical variants caused neuronal defects in young adult or aged C. elegans , based on examination of glutamatergic phasmid neurons. Yet, we noted decreased survival to day 9 in the P301L hMAPT strain, compared to control strains. Based on these results, we comment on strategies for humanization, including the importance of confirming C. elegans gene predictions and identifying loss of function defects for each gene before embarking on humanization, and we report the creation of strains and a new gene-editing selection strategy that will be useful for future studies.

The effects of burning on isotope ratio values in modern bone: Importance of experimental design for forensic applications.

This study examined preservation of isotope ratio values by comparing isotope composition of bones before and after burning. We analyzed common geoprofiling isotope systems (δ13C, δ15N, δ18O, and 87Sr/86Sr) and lesser studied systems (δ34S and δ88/86Sr) to evaluate if inferences about diet and residence history were altered by the burning process. We used two burn methods: one to simulate previous academic studies using a muffle furnace and one to more closely resemble a house fire or body disposal attempt using open flame. To mimic previous burn studies, ribs and femora from four dry modern human skeletons were heated in a muffle furnace. To resemble a forensic burn situation, fleshed pig ribs from a single geographic location were burned on an open fire both with and without use of a diesel accelerant. Isotope ratios from bone collagen, carbonate, phosphate, and strontium were analyzed. Fleshed pig samples burned in an open fire maintained unaltered isotope ratio values. Dry human samples burned in a muffle furnace maintained unaltered isotope ratio values in most isotope systems, except for δ18O values in carbonate and phosphate, which showed a depletion of 18O at higher temperatures. This research suggests that the isotope composition of fleshed burned bone retains the geoprofiling inferences of unburned bone, at least within the parameters of the open fire burn used in this study. However, oxygen isotopes of carbonate and phosphate from dry bone burned in a muffle furnace do not retain the geoprofiling inferences. This research demonstrates the need for research using an experimental design relevant to a specific burn situation.

A model for the assessment, care, and treatment of suicidal risk within the military intelligence community.

This paper describes the development of a behavioral health and wellness model into the US Army Intelligence and Security Command (INSCOM) to address concerns about suicide within this community. In response to stresses existing within the intelligence community (IC), INSCOM partnered with the Army Public Health Center (APHC) to assess the health and wellbeing of Command personnel. A Community Health Assessment (CHA) survey was conducted (N = 2,704 Soldiers; N = 959 Civilians) that included focus groups across three installations and secondary source data. Six key areas were prioritized: suicide behavior, behavioral health access to care and health promotion, behavioral health stigma and maintaining clearances, workplace environment, sleep health, and overall fitness. Several actions were implemented to address the report's findings and recommendations. A Command Surgeon office was established within INSCOM. An INSCOM Health Assessment and Readiness Team (I-HART) was established. The Deputy Undersecretary of the Army provided support to address suicide within INSCOM by approving 4 highly qualified experts (HQE's) in behavioral health and clinical suicidology to provide research oversight and make recommendations. The Command General approved 8 behavioral health providers. There are planned research efforts within the command focusing on scalable and technology enabled care delivery to improve mental well-being and decrease suicides.

Cognitive Impairment in Adult Inflammatory Bowel Disease: A Systematic Review and Meta-Analysis.

BACKGROUND: People living with inflammatory bowel disease (IBD) are exposed to multiple risk factors for cognitive impairment and frequently report cognitive difficulties. However, the presence of cognitive impairment in IBD has not been systematically reviewed. METHODS: Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, we performed a systematic multidatabase search for cross-sectional and longitudinal studies comparing adults with IBD versus healthy controls for domain-specific cognitive function or scores on multidomain cognitive screening tools. For any domain reported by 3 or more studies, we conducted random-effects meta-analysis to calculate the standardized mean difference between groups; lower scores reflected poorer performance. Between-study heterogeneity was assessed using the I2 statistic and study quality assessed using an IBD-modified Newcastle-Ottawa scale. RESULTS: Of 8302 articles screened, 12 studies (n = 687) were included in the qualitative synthesis and 11 in meta-analyses. All studies were cross-sectional. Studies generally excluded people with active IBD and older adults. Despite no significant differences on multidomain screening tools such as the Mini Mental State Examination (-0.27 [95% confidence interval -0.68, 0.08], P = 0.14), people with IBD showed significant deficits compared with healthy controls in attention (standardized mean difference -0.36 [-0.60, -0.12], P = 0.003, I2 = 0%), executive function (standardized mean difference -0.45 [-0.77, -0.13, P = 0.005, I2 = 42.5%), and specifically in working memory (standardized mean difference -0.58 [-0.85, -0.30], P < 0.001, I2 = 0%). Deficits in learning and recall were nonsignificant (P = 0.089) and other domains insufficient for meta-analysis. CONCLUSIONS: People with IBD show deficits in attention and executive function, particularly in working memory, suggesting that cognitive impairment is a potential extraintestinal manifestation of IBD.

Computed Tomography and Magnetic Resonance Imaging are Similarly Reliable in the Assessment of Glenohumeral Arthritis and Glenoid Version.

BACKGROUND: The purpose of this study was to compare the intraobserver and interobserver reliability of CT and T2-weighted MRI for evaluation of the severity of glenoid wear, glenohumeral subluxation, and glenoid version. METHODS: Sixty-one shoulders with primary osteoarthritis had CT and MRI scans before shoulder arthroplasty. All slices were blinded and randomized before evaluation. Two fellowship-trained shoulder surgeons and three orthopaedic surgery trainees reviewed the images to classify glenoid wear (Walch and Mayo classifications) and glenohumeral subluxation (Mayo classification). Glenoid version was measured using Friedman's technique. After a minimum two-week interval, the process was repeated. RESULTS: Intraobserver reliability was good for the CT group and fair-to-good for the MRI group for the Walch, Mayo glenoid, and Mayo subluxation classifications; interobserver reliability was poor for the CT and fair-to-poor for the MRI group. For the measurement of glenoid version, intraobserver reliability was good for the CT and substantial for the MRI group; interobserver agreement was good for both groups. There were no significant differences in reliability between staff surgeons and trainees for any of the classifications or measurements. CONCLUSION: CT and MRI appear similarly reliable for the classification of glenohumeral wear patterns. For the measurement of glenoid version, MRI was slightly more reliable than CT within observers. Differences in training level did not produce substantial differences in agreement, suggesting these systems can be applied by observers of different experience levels with similar reliability.

Platforms for Personalized Polytherapeutics Discovery in COVID-19.

The COVID-19 pandemic entered its third and most intense to date wave of infections in November 2020. This perspective article describes how combination therapies (polytherapeutics) are a needed focus for helping battle the severity of complications from SARS-CoV-2 infection. It outlines the types of systems that are needed for fast and efficient combinatorial assessment of therapeutic candidates. Proposed are micro-physiological systems using human iPSC as a format for tissue-specific modeling of infection, the use of gene-humanized zebrafish and C. elegans for combinatorial drug screens due to the animals being addressable in liquid multi-well formats, and the use of engineered pseudo-typing systems to safely model infection in the transgenic animals and engineered tissue systems.

Chromatin structure regulates cancer-specific alternative splicing events in primary HPV-related oropharyngeal squamous cell carcinoma.

Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV+ OPSCC) represents a unique disease entity within head and neck cancer with rising incidence. Previous work has shown that alternative splicing events (ASEs) are prevalent in HPV+ OPSCC, but further validation is needed to understand the regulation of this process and its role in these tumours. In this study, eleven ASEs (GIT2, CTNNB1, MKNK2, MRPL33, SIPA1L3, SNHG6, SYCP2, TPRG1, ZHX2, ZNF331, and ELOVL1) were selected for validation from 109 previously published candidate ASEs to elucidate the post-transcriptional mechanisms of oncogenesis in HPV+ disease. In vitro qRT-PCR confirmed differential expression of 9 of 11 ASE candidates, and in silico analysis within the TCGA cohort confirmed 8 of 11 candidates. Six ASEs (MRPL33, SIPA1L3, SNHG6, TPRG1, ZHX2, and ELOVL1) showed significant differential expression across both methods. Further evaluation of chromatin modification revealed that ASEs strongly correlated with cancer-specific distribution of acetylated lysine 27 of histone 3 (H3K27ac). Subsequent epigenetic treatment of HPV+ HNSCC cell lines (UM-SCC-047 and UPCI-SCC-090) with JQ1 not only induced downregulation of cancer-specific ASE isoforms, but also growth inhibition in both cell lines. The UPCI-SCC-090 cell line, with greater ASE expression, also showed more significant growth inhibition after JQ1 treatment. This study confirms several novel cancer-specific ASEs in HPV+OPSCC and provides evidence for the role of chromatin modifications in regulation of alternative splicing in HPV+OPSCC. This highlights the role of epigenetic changes in the oncogenesis of HPV+OPSCC, which represents a unique, unexplored target for therapeutics that can alter the global post-transcriptional landscape.

Epidemiology, Management, and Outcomes of Large and Small Native Joint Septic Arthritis in Adults.

BACKGROUND: Native joint septic arthritis (NJSA) is poorly studied. We describe the epidemiology, treatment, and outcomes of large joint NJSA (LNJSA) and small joint NJSA (SNJSA) in adults at Middlemore Hospital, Auckland, New Zealand. METHODS: This was a coding-based retrospective study of patients ≥16 years old admitted between 2009 and 2014. Prosthetic joint infections were excluded. RESULTS: Five hundred forty-three NJSA episodes were included (302 LNJSA, 250 SNJSA). Only 40% had positive synovial fluid culture. Compared to SNJSA, LNJSA has higher incidence (13 vs 8/100 000 person-years [PY]), occurs in older, more comorbid patients, and is associated with greater rates of treatment failure (23% vs 12%) and mortality, despite longer antibiotic treatment. Total incidence is higher than previously reported (21/100 000 PY), with marked interethnic variation. Incidence rises with age (LNJSA only) and socioeconomic deprivation (LNJSA and SNJSA). Tobacco smokers and males are overrepresented. The most commonly involved joints were knee (21%) and hand interphalangeal (20%). Staphylococcus aureus was the most common pathogen (53%). Mean antibiotic duration was 25 days for SNJSA and 40 days for LNJSA, and the mean number of surgical procedures was 1.5 and 1.6, respectively. Treatment failure was independently associated with LNJSA, age, intra-articular nonarthroplasty prosthesis, and number of surgical procedures. CONCLUSIONS: This is the largest contemporary series of adult NJSA. SNJSA has better outcomes than LNJSA and may be able to be safely treated with shorter antimicrobial courses. Incidence is high, with significant ethnic and socioeconomic variation. Microbiological NJSA case ascertainment underestimates case numbers as it frequently excludes SNJSA.