Genome-scale clustered regularly interspaced short palindromic repeats screen identifies nucleotide metabolism as an actionable therapeutic vulnerability in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common malignancy that develops in patients with ataxia-telangiectasia, a cancer-predisposing inherited syndrome characterized by inactivating germline ATM mutations. ATM is also frequently mutated in sporadic DLBCL. To investigate lymphomagenic mechanisms and lymphoma-specific dependencies underlying defective ATM, we applied ribonucleic acid (RNA)-seq and genome-scale loss-offunction clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens to systematically interrogate B-cell lymphomas arising in a novel murine model (Atm-/-nu-/-) with constitutional Atm loss, thymic aplasia but residual T-cell populations. Atm-/-nu-/-lymphomas, which phenotypically resemble either activated B-cell-like or germinal center Bcell-like DLBCL, harbor a complex karyotype, and are characterized by MYC pathway activation. In Atm-/-nu-/-lymphomas, we discovered nucleotide biosynthesis as a MYCdependent cellular vulnerability that can be targeted through the synergistic nucleotidedepleting actions of mycophenolate mofetil (MMF) and the WEE1 inhibitor, adavosertib (AZD1775). The latter is mediated through a synthetically lethal interaction between RRM2 suppression and MYC dysregulation that results in replication stress overload in Atm-/-nu-/-lymphoma cells. Validation in cell line models of human DLBCL confirmed the broad applicability of nucleotide depletion as a therapeutic strategy for MYC-driven DLBCL independent of ATM mutation status. Our findings extend current understanding of lymphomagenic mechanisms underpinning ATM loss and highlight nucleotide metabolism as a targetable therapeutic vulnerability in MYC-driven DLBCL.

Compromised health: Examining growth and health in a late antique Roman infant and child cemetery.

OBJECTIVES: Combining research from infant and child development, public health, anthropology, and history, this research examines the relationship between growth, growth disruption, and skeletal indicators of chronic and/or episodic physiological stress (stress) among juvenile individuals (n = 60) interred at the late antique infant and child cemetery at Poggio Gramignano (PG) (ca. 5th century CE), associated with a rural agricultural community. MATERIALS AND METHODS: Growth disruption-evidenced by decreased long bone length compared to dental age-and stress experience-evidenced by skeletal stress indicators-within these individuals are compared to those within juveniles from a comparative sample (n = 66) from two urban Roman-era cemeteries, Villa Rustica (VR) (0-250 CE) and Tragurium City Necropolis (TCN) (0-700 CE). RESULTS: Results indicate that individuals from PG had significantly smaller femoral lengths-for-age than those from VR and TCN; however, the frequency of skeletal stress indicators was higher among juveniles from VR and TCN. DISCUSSION: These differences in growth and stress experience are likely related to the different biosocial and ecological environments present in these two regions. For the community at PG, internal and external violent conflicts, as well as social, political, and economic turmoil, and subsistence shortages, endemic and epidemic infectious disease, nutritional deficiencies, and inherited or acquired anemia may have synergized to create chronically and/or episodically deleterious conditions for its juveniles.

Synthesis and characterisation of a nucleotide based pro-drug formulated with a peptide into a nano-chemotherapy for colorectal cancer.

Recent studies in colorectal cancer patients (CRC) have shown that increased resistance to thymidylate synthase (TS) inhibitors such as 5-fluorouracil (5-FU), reduce the efficacy of standard of care (SoC) treatment regimens. The nucleotide pool cleanser dUTPase is highly expressed in CRC and is an attractive target for potentiating anticancer activity of chemotherapy. The purpose of the current work was to investigate the activity of P1, P4-di(2',5'-dideoxy-5'-selenouridinyl)-tetraphosphate (P4-SedU2), a selenium-modified symmetrically capped dinucleoside with prodrug capabilities that is specifically activated by dUTPase. Using mechanochemistry, P4-SedU2 and the corresponding selenothymidine analogue P4-SeT2 were prepared with a yield of 19% and 30% respectively. The phosphate functionality facilitated complexation with the amphipathic cell-penetrating peptide RALA to produce nanoparticles (NPs). These NPs were designed to deliver P4-SedU2 intracellularly and thereby maximise in vivo activity. The NPs demonstrated effective anti-cancer activity and selectivity in the HCT116 CRC cell line, a cell line that overexpresses dUTPase; compared to HT29 CRC cells and NCTC-929 fibroblast cells which have reduced levels of dUTPase expression. In vivo studies in BALB/c SCID mice revealed no significant toxicity with respect to weight or organ histology. Pharmacokinetic analysis of blood serum showed that RALA facilitates effective delivery and rapid internalisation into surrounding tissues with NPs eliciting lower plasma Cmax than the equivalent injection of free P4-SedU2, translating the in vitro findings. Tumour growth delay studies have demonstrated significant inhibition of growth dynamics with the tumour doubling time extended by >2weeks. These studies demonstrate the functionality and action of a new pro-drug nucleotide for CRC.

The osteogenic and angiogenic potential of microRNA-26a delivered via a non-viral delivery peptide for bone repair.

Bone-related injuries and diseases are among the most common causes of morbidity worldwide. Current bone-regenerative strategies such as auto- and allografts are invasive by nature, with adverse effects such as pain, infection and donor site morbidity. MicroRNA (miRNA) gene therapy has emerged as a promising area of research, with miRNAs capable of regulating multiple gene pathways simultaneously through the repression of post-transcriptional mRNAs. miR-26a is a key regulator of osteogenesis and has been found to be upregulated following bone injury, where it induces osteodifferentiation of mesenchymal stem cells (MSCs) and facilitates bone formation. This study demonstrates, for the first time, that the amphipathic, cell-penetrating peptide RALA can efficiently deliver miR-26a to MSCs in vitro to regulate osteogenic signalling. Transfection with miR-26a significantly increased expression of osteogenic and angiogenic markers at both gene and protein level. Using a rat calvarial defect model with a critical size defect, RALA/miR-26a NPs were delivered via an injectable, thermo-responsive Cs-g-PNIPAAm hydrogel to assess the impact on both rate and quality of bone healing. Critical defects treated with the RALA/miR-26a nanoparticles (NPs) had significantly increased bone volume and bone mineral density at 8 weeks, with increased blood vessel formation and mechanical properties. This study highlights the utility of RALA to deliver miR-26a for the purpose of bone healing within an injectable biomaterial, warranting further investigation of dose-related efficacy of the therapeutic across a range of in vivo models.

Peptide delivery of a multivalent mRNA SARS-CoV-2 vaccine.

Lipid nanoparticles (LNP) have been instrumental in the success of mRNA vaccines and have opened up the field to a new wave of therapeutics. However, what is ahead beyond the LNP? The approach herein used a nanoparticle containing a blend of Spike, Membrane and Envelope antigens complexed for the first time with the RALA peptide (RALA-SME). The physicochemical characteristics and functionality of RALA-SME were assessed. With >99% encapsulation, RALA-SME was administered via intradermal injection in vivo, and all three antigen-specific IgG antibodies were highly significant. The IgG2a:IgG1 ratio were all >1.2, indicating a robust TH1 response, and this was further confirmed with the T-Cell response in mice. A complete safety panel of markers from mice were all within normal range, supported by safety data in hamsters. Vaccination of Syrian Golden hamsters with RALA-SME derivatives produced functional antibodies capable of neutralising SARS-CoV-2 from both Wuhan-Hu-1 and Omicron BA.1 lineages after two doses. Antibody levels increased over the study period and provided protection from disease-specific weight loss, with inhibition of viral migration down the respiratory tract. This peptide technology enables the flexibility to interchange and add antigens as required, which is essential for the next generation of adaptable mRNA vaccines.

POLθ processes ssDNA gaps and promotes replication fork progression in BRCA1-deficient cells.

Polymerase theta (POLθ) is an error-prone DNA polymerase whose loss is synthetically lethal in cancer cells bearing breast cancer susceptibility proteins 1 and 2 (BRCA1/2) mutations. To investigate the basis of this genetic interaction, we utilized a small-molecule inhibitor targeting the POLθ polymerase domain. We found that POLθ processes single-stranded DNA (ssDNA) gaps that emerge in the absence of BRCA1, thus promoting unperturbed replication fork progression and survival of BRCA1 mutant cells. A genome-scale CRISPR-Cas9 knockout screen uncovered suppressors of the functional interaction between POLθ and BRCA1, including NBN, a component of the MRN complex, and cell-cycle regulators such as CDK6. While the MRN complex nucleolytically processes ssDNA gaps, CDK6 promotes cell-cycle progression, thereby exacerbating replication stress, a feature of BRCA1-deficient cells that lack POLθ activity. Thus, ssDNA gap formation, modulated by cell-cycle regulators and MRN complex activity, underlies the synthetic lethality between POLθ and BRCA1, an important insight for clinical trials with POLθ inhibitors.

Exploring the genetic space of the DNA damage response for cancer therapy through CRISPR-based screens.

The concepts of synthetic lethality and viability have emerged as powerful approaches to identify vulnerabilities and resistances within the DNA damage response for the treatment of cancer. Historically, interactions between two genes have had a longstanding presence in genetics and have been identified through forward genetic screens that rely on the molecular basis of the characterized phenotypes, typically caused by mutations in single genes. While such complex genetic interactions between genes have been studied extensively in model organisms, they have only recently been prioritized as therapeutic strategies due to technological advancements in genetic screens. Here, we discuss synthetic lethal and viable interactions within the DNA damage response and present how CRISPR-based genetic screens and chemical compounds have allowed for the systematic identification and targeting of such interactions for the treatment of cancer.

Analysis of risk factors associated with complications following mandibulectomy and maxillectomy in dogs.

OBJECTIVE: To provide information about complication rates and the risk factors for complications with mandibulectomy and maxillectomy procedures in dogs. ANIMALS: 459 client-owned dogs that underwent a mandibulectomy or maxillectomy between January 1, 2007, and January 1, 2018. PROCEDURES: Inclusion criteria included a complete medical record that contained an anesthesia record, surgical report, available histopathology results, and results of CBC and serum biochemical analysis before surgery. A minimum follow-up of 90 days after surgery was required. RESULTS: 271 complications occurred in 171 of 459 (37.3%) dogs. Eighteen complications were not given a severity description. Of the remaining 253 complications, most were considered minor (157/253 [62.1%]). Multivariable logistic regression analysis revealed that only increased surgical time had a significant (OR, 1.36; 95% CI, 1.12 to 1.54) association with the occurrence of ≥ 1 complication. For each additional hour of surgery, the odds of complications increased by 36%. Preoperative radiation therapy or chemotherapy increased the odds of incisional dehiscence or oral fistula formation (OR, 3.0; 95% CI, 1.3 to 7.2). Additionally, undergoing maxillectomy, compared with mandibulectomy, increased the odds of incisional dehiscence or oral fistula formation (OR, 1.8; 95% CI, 1.1 to 3.1). Two hundred forty-four of 271 (90.0%) complications occurred in the perioperative period (0 to 3 months after surgery). CONCLUSIONS AND CLINICAL RELEVANCE: Compared with mandibulectomy, performing maxillectomy increased the risk for incisional dehiscence or oral fistula formation. Mandibulectomy and maxillectomy had a moderate risk for a complication.

A Cautionary Tale of Etanercept Use in Patients With Toxic Epidermal Necrolysis.

Toxic epidermal necrolysis (TEN) is a severe cutaneous reaction that can be life-threatening. In the United States, there are no established guidelines for the treatment of TEN. Supportive care including fluids and supportive therapies are the current recommendations. Research surrounding TEN involves mostly case studies or small, uncontrolled studies. Recent literature describes the use of tumor necrosis factor blockers in the treatment of TEN with positive results. These case reports describe decreased time to reepithelization, hospital length of stay, and minimal side effects. Conversely, we present three fatalities after the administration of etanercept.

Geospatial and Temporal Associations between Increases in Opioid Deaths, Socioeconomics, and Rates of Sexually Transmitted Infections in the Northeast United States 2012-2017.

With the introduction of fentanyl to illegal markets in 2013 and an overall rise in rates of synthetic opioid use, opioid-related deaths have increased significantly. A similar trend has been observed for sexually transmitted infections, homicides, and poor mental health outcomes. In this paper, we explore the spatiotemporal relationship between opioid death rates and sexually transmitted infection (STI) rates in counties from the Northeast region of the United States between the years 2012-2017. We hypothesized that rates for gonorrhea, chlamydia, and human immunodeficiency virus (HIV) would all be positively associated with opioid death rates and that there would be a similar association between the STI rates and later time periods relative to earlier time periods. A negative binomial mixed-effects regression model was employed to assess these associations. Contrary to the study hypothesis, opioid death rates were not found to be significantly associated with the STI rates after accounting for other demographic and socioeconomic variables, with the exception of opioid deaths and gonorrhea in urban counties. Additionally, the regression demonstrated a significant association between infection rate and time period beyond the included socioeconomic variables and opioid deaths. Overall, this study indicates that declining sexual health outcomes may parallel rising opioid death, though both trends may be explained by similar underlying factors related to time period.

Risk Factors of Recurrent Falls Among Older Adults Admitted to the Trauma Surgery Department.

Falls affect more than 29 million American adults ages ≥65 years annually. Many older adults experience recurrent falls requiring medical attention. These recurrent falls may be prevented through screening and intervention. In 2014 to 2015, records for 199 older adult patients admitted from a major urban teaching hospital's emergency department were queried. Open-ended variables from clinicians' notes were coded to supplement existing closed-ended variables. Of the 199 patients, 52 (26.1%) experienced one or more recurrent falls within 365 days after their initial fall. Half (50.0%) of all recurrent falls occurred within the first 90 days following discharge. A large proportion of recurrent falls among older adults appear to occur within a few months and are statistically related to identifiable risk factors. Prevention and intervention strategies, delivered either during treatment for an initial fall or upon discharge from an inpatient admission, may reduce the incidence of recurrent falls among this population.

Synthesis and Evaluation of a Thermoresponsive Degradable Chitosan-Grafted PNIPAAm Hydrogel as a "Smart" Gene Delivery System.

Thermoresponsive hydrogels demonstrate tremendous potential as sustained drug delivery systems. However, progress has been limited as formulation of a stable biodegradable thermosensitive hydrogel remains a significant challenge. In this study, free radical polymerization was exploited to formulate a biodegradable thermosensitive hydrogel characterized by sustained drug release. Highly deacetylated chitosan and N-isopropylacrylamide with distinctive physical properties were employed to achieve a stable, hydrogel network at body temperature. The percentage of chitosan was altered within the copolymer formulations and the subsequent physical properties were characterized using 1H-NMR, FTIR, and TGA. Viscoelastic, swelling, and degradation properties were also interrogated. The thermoresponsive hydrogels were loaded with RALA/pEGFP-N1 nanoparticles and release was examined. There was sustained release of nanoparticles over three weeks and, more importantly, the nucleic acid cargo remained functional and this was confirmed by successful transfection of the NCTC-929 fibroblast cell line. This tailored thermoresponsive hydrogel offers an option for sustained delivery of macromolecules over a prolonged considerable period.

A single cell transcriptional atlas of early synovial joint development.

Synovial joint development begins with the formation of the interzone, a region of condensed mesenchymal cells at the site of the prospective joint. Recently, lineage-tracing strategies have revealed that Gdf5-lineage cells native to and from outside the interzone contribute to most, if not all, of the major joint components. However, there is limited knowledge of the specific transcriptional and signaling programs that regulate interzone formation and fate diversification of synovial joint constituents. To address this, we have performed single cell RNA-Seq analysis of 7329 synovial joint progenitor cells from the developing murine knee joint from E12.5 to E15.5. By using a combination of computational analytics, in situ hybridization and in vitro characterization of prospectively isolated populations, we have identified the transcriptional profiles of the major developmental paths for joint progenitors. Our freely available single cell transcriptional atlas will serve as a resource for the community to uncover transcriptional programs and cell interactions that regulate synovial joint development.

Changes of the end plate cartilage are associated with intervertebral disc degeneration: A quantitative magnetic resonance imaging study in rhesus monkeys and humans.

BACKGROUND: The end plate plays an important role in intervertebral disc degeneration progression. The aim of the study was to examine the compositional and structural changes of the end plate with age and to investigate the correlation between end plate and disc degeneration by T1ρ and T2 map magnetic resonance imaging. METHODS: There were 12 young monkeys (6-7 years old), 20 aged monkeys (14-17 years old) and 12 human participants (30-50 years old) in this study. T1ρ or T2 map values of the nucleus pulposus and end plate cartilage were analyzed according to Pfirrmann grades and age. Afterwards, micro computed tomography and histological analysis were used to confirm the end plate changes in monkeys. Pearson's correlation was performed to investigate the relationship between end plate and disc degeneration. RESULTS: In monkeys, T1ρ (r=-0.794, P<0.001) and T2 map values (r=-0.8, P<0.001) of the nucleus pulposus were negatively associated with Pfirrmann grades. Moreover, the T2 map was more suitable than T1ρ for the evaluation of end plate degeneration. Age was an important influence factor of end plate and disc degeneration, which was confirmed by microcomputed tomography, Safranin O/fast green staining, and collagen II staining. The T2 map value of lower end plate degeneration positively correlated with that of the intervertebral discs in monkeys (R2=0.3133, P<0.001) and humans (R2=0.2092, P<0.001). CONCLUSION: This study suggests that the compositional and structural changes of the end plate can be quantitatively evaluated by T2 map. Furthermore, cartilage end plate degeneration is associated with disc degeneration during ageing. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: A better understanding of how the cartilage end plate affects disc degeneration is needed, which may propose a new clinical application using T2 map to evaluate end plate degeneration.

Altered gait mechanics are associated with severity of chondropathy after hip arthroscopy for femoroacetabular impingement syndrome.

BACKGROUND: Suboptimal patient-reported function and movement impairments often persist after hip arthroscopy for femoroacetabular impingement syndrome (FAIS). Individuals with FAIS with preoperative cartilage pathology (ie. chondropathy) demonstrate distinct movement patterns and have worse post-operative outcomes. It is unknown whether the presence of chondropathy after surgery negatively affects movement and function. RESEARCH QUESTION: Do sagittal plane gait mechanics differ based on chondropathy severity following arthroscopy for FAIS? METHODS: A cross-sectional walking gait analysis was performed for 25 participants post-arthroscopy (2.48 ±â€¯1.38y) and 12 healthy controls (HCs). Peak total support moment (TSM) and relative contributions of the hip, knee, and ankle were calculated during loading response. The Hip Osteoarthritis MRI Scoring System was used to categorize the FAIS group into no-mild or moderate-severe chondropathy groups based on 3 T magnetic resonance imaging of their surgical hip. The interactions of group by limb were evaluated for kinetic variables, covaried by gait speed. RESULTS: Groups did not differ based on age, BMI and sex distribution (P ≥ 0.14). 13 participants with FAIS presented with moderate-severe chondropathy and 12 presented with no-mild chondropathy. Participants with moderate-severe chondropathy walked significantly slower than both other groups (P = 0.006) and demonstrated lower peak TSM than those with no-mild chondropathy (P = 0.002). Participants with no-mild chondropathy demonstrated lower hip (61.5 %) and greater ankle (17.7 %) contributions to the TSM on the involved limb compared to the moderate-severe group (hip:73.4 %, P = 0.07; ankle:10.5 %, P = 0.007). SIGNIFICANCE: Slower gait speed alone did not explain the lower TSM strategy in participants with moderate-severe chondropathy. Interestingly, the joint contribution strategy of this group was not different than HCs. Participants with no-mild chondropathy demonstrated a TSM strategy that shifted the demand away from their hip and toward their ankle. Given the small sample size, and large variability in joint strategies, future work needs to examine whether these alterations in gait strategy, with or without advanced chondropathy, impact patient function.

Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography.

Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed following the high yielding Michaelis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5'-deoxythymidine-5'-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an H-phosphonate and then chain extended using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 to -3.1 °C per phosphoroselenolate) when introduced at the 5'-termini of A-form or mixed duplexes or at juxtaposed central loci within a B-form duplex (-1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 Å. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.

Phytoremediation removal rates of benzene, toluene, and chlorobenzene.

Phytoremediation is a sustainable remedial approach, although performance efficacy is rarely reported. In this study, we assessed a phytoremediation plot treating benzene, toluene, and chlorobenzene. A comparison of the calculated phytoremediation removal rate with estimates of onsite contaminant mass was used to forecast cleanup periods. The investigation demonstrated that substantial microbial degradation was occurring in the subsurface. Estimates of transpiration indicated that the trees planted were removing approximately 240,000 L of water per year. This large quantity of water removal implies substantial removal of contaminant due to large amounts of contaminants in the groundwater; however, these contaminants extensively sorb to the soil, resulting in large quantities of contaminant mass in the subsurface. The total estimate of subsurface contaminant mass was also complicated by the presence of non-aqueous phase liquids (NAPL), additional contaminant masses that were difficult to quantify. These uncertainties of initial contaminant mass at the site result in large uncertainty in the cleanup period, although mean estimates are on the order of decades. Collectively, the model indicates contaminant removal rates on the order of 10-2-100 kg/tree/year. The benefit of the phytoremediation system is relatively sustainable cleanup over the long periods necessary due to the presence of NAPL.

Phytoforensics: Trees as bioindicators of potential indoor exposure via vapor intrusion.

Human exposure to volatile organic compounds (VOCs) via vapor intrusion (VI) is an emerging public health concern with notable detrimental impacts on public health. Phytoforensics, plant sampling to semi-quantitatively delineate subsurface contamination, provides a potential non-invasive screening approach to detect VI potential, and plant sampling is effective and also time- and cost-efficient. Existing VI assessment methods are time- and resource-intensive, invasive, and require access into residential and commercial buildings to drill holes through basement slabs to install sampling ports or require substantial equipment to install groundwater or soil vapor sampling outside the home. Tree-core samples collected in 2 days at the PCE Southeast Contamination Site in York, Nebraska were analyzed for tetrachloroethene (PCE) and results demonstrated positive correlations with groundwater, soil, soil-gas, sub-slab, and indoor-air samples collected over a 2-year period. Because tree-core samples were not collocated with other samples, interpolated surfaces of PCE concentrations were estimated so that comparisons could be made between pairs of data. Results indicate moderate to high correlation with average indoor-air and sub-slab PCE concentrations over long periods of time (months to years) to an interpolated tree-core PCE concentration surface, with Spearman's correlation coefficients (ρ) ranging from 0.31 to 0.53 that are comparable to the pairwise correlation between sub-slab and indoor-air PCE concentrations (ρ = 0.55, n = 89). Strong correlations between soil-gas, sub-slab, and indoor-air PCE concentrations and an interpolated tree-core PCE concentration surface indicate that trees are valid indicators of potential VI and human exposure to subsurface environment pollutants. The rapid and non-invasive nature of tree sampling are notable advantages: even with less than 60 trees in the vicinity of the source area, roughly 12 hours of tree-core sampling with minimal equipment at the PCE Southeast Contamination Site was sufficient to delineate vapor intrusion potential in the study area and offered comparable delineation to traditional sub-slab sampling performed at 140 properties over a period of approximately 2 years.