Antibody Lineages with Vaccine-Induced Antigen-Binding Hotspots Develop Broad HIV Neutralization.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody.

Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

Longitudinal Analysis Reveals Early Development of Three MPER-Directed Neutralizing Antibody Lineages from an HIV-1-Infected Individual.

Lineage-based vaccine design is an attractive approach for eliciting broadly neutralizing antibodies (bNAbs) against HIV-1. However, most bNAb lineages studied to date have features indicative of unusual recombination and/or development. From an individual in the prospective RV217 cohort, we identified three lineages of bNAbs targeting the membrane-proximal external region (MPER) of the HIV-1 envelope. Antibodies RV217-VRC42.01, -VRC43.01, and -VRC46.01 used distinct modes of recognition and neutralized 96%, 62%, and 30%, respectively, of a 208-strain virus panel. All three lineages had modest levels of somatic hypermutation and normal antibody-loop lengths and were initiated by the founder virus MPER. The broadest lineage, VRC42, was similar to the known bNAb 4E10. A multimeric immunogen based on the founder MPER activated B cells bearing the unmutated common ancestor of VRC42, with modest maturation of early VRC42 intermediates imparting neutralization breadth. These features suggest that VRC42 may be a promising template for lineage-based vaccine design.

A potent and broad neutralization of SARS-CoV-2 variants of concern by DARPins.

We report the engineering and selection of two synthetic proteins-FSR16m and FSR22-for the possible treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. FSR16m and FSR22 are trimeric proteins composed of DARPin SR16m or SR22 fused with a T4 foldon. Despite selection by a spike protein from a now historical SARS-CoV-2 strain, FSR16m and FSR22 exhibit broad-spectrum neutralization of SARS-CoV-2 strains, inhibiting authentic B.1.351, B.1.617.2 and BA.1.1 viruses, with respective IC50 values of 3.4, 2.2 and 7.4 ng ml-1 for FSR16m. Cryo-EM structures revealed that these DARPins recognize a region of the receptor-binding domain (residues 456, 475, 486, 487 and 489) overlapping a critical portion of the angiotensin-converting enzyme 2 (ACE2)-binding surface. K18-hACE2 transgenic mice inoculated with B.1.617.2 and receiving intranasally administered FSR16m showed less weight loss and 10-100-fold lower viral burden in upper and lower respiratory tracts. The strong and broad neutralization potency makes FSR16m and FSR22 promising candidates for the prevention and treatment of infection by SARS-CoV-2.

Increase in CFC-11 emissions from eastern China based on atmospheric observations.

The recovery of the stratospheric ozone layer relies on the continued decline in the atmospheric concentrations of ozone-depleting gases such as chlorofluorocarbons1. The atmospheric concentration of trichlorofluoromethane (CFC-11), the second-most abundant chlorofluorocarbon, has declined substantially since the mid-1990s2. A recently reported slowdown in the decline of the atmospheric concentration of CFC-11 after 2012, however, suggests that global emissions have increased3,4. A concurrent increase in CFC-11 emissions from eastern Asia contributes to the global emission increase, but the location and magnitude of this regional source are unknown3. Here, using high-frequency atmospheric observations from Gosan, South Korea, and Hateruma, Japan, together with global monitoring data and atmospheric chemical transport model simulations, we investigate regional CFC-11 emissions from eastern Asia. We show that emissions from eastern mainland China are 7.0 ± 3.0 (±1 standard deviation) gigagrams per year higher in 2014-2017 than in 2008-2012, and that the increase in emissions arises primarily around the northeastern provinces of Shandong and Hebei. This increase accounts for a substantial fraction (at least 40 to 60 per cent) of the global rise in CFC-11 emissions. We find no evidence for a significant increase in CFC-11 emissions from any other eastern Asian countries or other regions of the world where there are available data for the detection of regional emissions. The attribution of any remaining fraction of the global CFC-11 emission rise to other regions is limited by the sparsity of long-term measurements of sufficient frequency near potentially emissive regions. Several considerations suggest that the increase in CFC-11 emissions from eastern mainland China is likely to be the result of new production and use, which is inconsistent with the Montreal Protocol agreement to phase out global chlorofluorocarbon production by 2010.

Impact of maternal body mass index (BMI) and the challenges of fetal echocardiography.

OBJECTIVES: Fetal echocardiography is the gold standard modality to detect suspected congenital heart disease (CHD). Accurate diagnosis and subsequent prognosis is even more challenging in the presence of a raised maternal body mass index (BMI). This retrospective study aimed to gain insight into the prevalence of obesity within the cohort of patients referred for fetal echocardiography. STUDY DESIGN/METHODS: Retrospective analysis of all pregnant patients referred to the Scottish National Fetal Cardiology Service between 2015 and 2021 due to a suspected fetal cardiac abnormality and examining the associated trends in maternal BMI and the Scottish Index of Multiple Deprivation (SIMD). RESULTS: BMI data were available for 962 (96.3%) of the 998 patients referred during the study period. Median BMI during the study period was 31. BMI range in the seven-year period was 16-63. There was no association between BMI group and year (P = 0.889). A median of 58% of patients referred were classified as overweight (BMI > 25 kg/m2), and only 37% were reported to have a BMI within normal limits. Referral BMI was relatively consistent in the seven years with no dramatic increase in the obese categories. Mean BMI in SIMD 5 (lowest level of deprivation), was significantly lower (P = 0.001), than in SIMD 1 (highest deprivation). CONCLUSIONS: People of child bearing age should be aware the potential limitations that a raised BMI may have upon diagnostic/screening accuracy impacting subsequent ability to provide accurate fetal cardiac diagnoses and prognostic fetal cardiac imaging.

A kilonova as the electromagnetic counterpart to a gravitational-wave source.

Gravitational waves were discovered with the detection of binary black-hole mergers and they should also be detectable from lower-mass neutron-star mergers. These are predicted to eject material rich in heavy radioactive isotopes that can power an electromagnetic signal. This signal is luminous at optical and infrared wavelengths and is called a kilonova. The gravitational-wave source GW170817 arose from a binary neutron-star merger in the nearby Universe with a relatively well confined sky position and distance estimate. Here we report observations and physical modelling of a rapidly fading electromagnetic transient in the galaxy NGC 4993, which is spatially coincident with GW170817 and with a weak, short γ-ray burst. The transient has physical parameters that broadly match the theoretical predictions of blue kilonovae from neutron-star mergers. The emitted electromagnetic radiation can be explained with an ejected mass of 0.04 ± 0.01 solar masses, with an opacity of less than 0.5 square centimetres per gram, at a velocity of 0.2 ± 0.1 times light speed. The power source is constrained to have a power-law slope of -1.2 ± 0.3, consistent with radioactive powering from r-process nuclides. (The r-process is a series of neutron capture reactions that synthesise many of the elements heavier than iron.) We identify line features in the spectra that are consistent with light r-process elements (atomic masses of 90-140). As it fades, the transient rapidly becomes red, and a higher-opacity, lanthanide-rich ejecta component may contribute to the emission. This indicates that neutron-star mergers produce gravitational waves and radioactively powered kilonovae, and are a nucleosynthetic source of the r-process elements.

Impaired hospitalized patient mobility is associated with nurse injuries.

BACKGROUND: Staff injuries adversely affect the health of staff members as well the ability of health care teams to effectively care for patients. Identifying patients who pose an increased risk of injury may allow for the planning of risk mitigation strategies, but few studies have examined patient factors associated with staff injury risk. AIMS: Examine the relationship between staff injury and patient mobility, which has been linked to other key hospital outcomes. METHODS: Linking occupational health and electronic medical record data, we examined documented patient mobility levels, based on the Activity Measure for Post-Acute Care (AM-PAC) and the Johns Hopkins Highest Level of Mobility (JH-HLM) Scale, on the day prior to injury. In addition, we created a matched cohort of control patients not associated with staff injury to examine the influence of patient mobility on the odds of staff injury. RESULTS: We identified 199 staff injuries associated with 181 patients with 1063 matched controls. Patients had median scores of 11 and 3 on the AM-PAC and JH-HLM, respectively, indicating moderate-severe mobility impairments. In addition, scores in the lowest AM-PAC tertile (6-15) and lowest JH-HLM tertile (1-4) were associated with a 4.46-fold and 2.90-fold increase in the odds of nurse injury, respectively. CONCLUSIONS: These results indicate that moderate-severe mobility impairments are associated with increased risk of nurse injury. Hospitals and clinical care teams should consider documenting mobility routinely and utilizing these values to identify patients who pose an increased risk of nurse injury.

TimiRGeN: R/Bioconductor package for time series microRNA-mRNA integration and analysis.

MOTIVATION: The analysis of longitudinal datasets and construction of gene regulatory networks (GRNs) provide a valuable means to disentangle the complexity of microRNA (miRNA)-mRNA interactions. However, there are no computational tools that can integrate, conduct functional analysis and generate detailed networks from longitudinal miRNA-mRNA datasets. RESULTS: We present TimiRGeN, an R package that uses time point-based differential expression results to identify miRNA-mRNA interactions influencing signaling pathways of interest. miRNA-mRNA interactions can be visualized in R or exported to PathVisio or Cytoscape. The output can be used for hypothesis generation and directing in vitro or further in silico work such as GRN construction. AVAILABILITY AND IMPLEMENTATION: TimiRGeN is available for download on Bioconductor (https://bioconductor.org/packages/TimiRGeN) and requires R v4.0.2 or newer and BiocManager v3.12 or newer. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Osteoarthritis year in review: genetics, genomics, epigenetics.

OBJECTIVE: In this review, we have highlighted the advances over the past year in genetics, genomics and epigenetics in the field of osteoarthritis (OA). METHODS: A literature search of PubMed was performed using the criteria: "osteoarthritis" and one of the following terms "genetic(s), genomic(s), epigenetic(s), polymorphism, noncoding ribonucleic acid (RNA), microRNA, long noncoding RNA, lncRNA, circular RNA, RNA sequencing (RNA-seq), single cell sequencing, transcriptomics, or deoxyribonucleic acid (DNA) methylation between April 01, 2020 and April 30, 2021. RESULTS: In total we identified 765 unique publications, which eventually reduced to 380 of relevance to the field as judged by two assessors. Many of these studies included multiple search terms. We summarised advances relating to genetics, functional genetics, genomics and epigenetics, focusing on our personal key papers during the year. CONCLUSIONS: This year few studies have identified new genetic variants contributing to OA susceptibility, but a focus has been on refining risk loci or their functional validation. The use of new technologies together with investigating the cross-talk between multiple tissue types, greater sample sizes and/or better patient classification (OA subtypes) will continue to increase our knowledge of disease mechanisms and progress towards understanding and treating OA.

Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes.

OBJECTIVE: MicroRNA 140 (miR-140) is a chondrocyte-specific endogenous gene regulator implicated in osteoarthritis (OA). As mechanical injury is a primary aetiological factor in OA, we investigated miR-140-dependent mechanosensitive gene regulation using a novel CRISPR-Cas9 methodology in primary human chondrocytes. METHOD: Primary (passage 1/2) human OA chondrocytes were isolated from arthroplasty samples (six donors) and transfected with ribonuclear protein complexes or plasmids using single guide RNAs (sgRNAs) targeting miR-140, in combination with Cas9 endonuclease. Combinations of sgRNAs and single/double transfections were tested. Gene editing was measured by T7 endonuclease 1 (T7E1) assay. miRNA levels were confirmed by qPCR in chondrocytes and in wild type murine femoral head cartilage after acute injury. Predicted close match off-targets were examined. Mechanosensitive miR-140 target validation was assessed in 42 injury-associated genes using TaqMan Microfluidic cards in targeted and donor-matched control chondrocytes. Identified targets were examined in RNAseq data from costal chondrocytes from miR-140-/- mice. RESULTS: High efficiency gene editing of miR-140 (90-98%) was obtained when two sgRNAs were combined with double RNP-mediated CRISPR-Cas9 transfection. miR-140 levels fell rapidly after femoral cartilage injury. Of the top eight miR-140 gene targets identified (P < 0.01), we validated three previously identified ones (septin 2, bone morphogenetic protein 2 and fibroblast growth factor 2). Novel targets included Agrin, a newly recognised pro-regenerative cartilage agent, and proteins associated with retinoic acid signalling and the primary cilium. CONCLUSION: We describe a highly efficient CRISPR-Cas9-mediated strategy for gene editing in primary human chondrocytes and identify several novel mechanosensitive miR-140 targets of disease relevance.

Ultrafast Suppression of the Ferroelectric Instability in KTaO_{3}.

We use an x-ray free-electron laser to study the lattice dynamics following photoexcitation with ultrafast near-UV light (wavelength 266 nm, 50 fs pulse duration) of the incipient ferroelectric potassium tantalate, KTaO_{3}. By probing the lattice dynamics corresponding to multiple Brillouin zones through the x-ray diffuse scattering with pulses from the Linac Coherent Light Source (LCLS) (wavelength 1.3 Å and <10 fs pulse duration), we observe changes in the diffuse intensity associated with a hardening of the transverse acoustic phonon branches along Γ to X and Γ to M. Using force constants from density functional theory, we fit the quasiequilibrium intensity and obtain the instantaneous lattice temperature and density of photoexcited charge carriers. The density functional theory calculations demonstrate that photoexcitation transfers charge from oxygen 2p derived π-bonding orbitals to Ta 5d derived antibonding orbitals, further suppressing the ferroelectric instability and increasing the stability of the cubic, paraelectric structure.

Repeated large-scale retreat and advance of Totten Glacier indicated by inland bed erosion.

Climate variations cause ice sheets to retreat and advance, raising or lowering sea level by metres to decametres. The basic relationship is unambiguous, but the timing, magnitude and sources of sea-level change remain unclear; in particular, the contribution of the East Antarctic Ice Sheet (EAIS) is ill defined, restricting our appreciation of potential future change. Several lines of evidence suggest possible collapse of the Totten Glacier into interior basins during past warm periods, most notably the Pliocene epoch, causing several metres of sea-level rise. However, the structure and long-term evolution of the ice sheet in this region have been understood insufficiently to constrain past ice-sheet extents. Here we show that deep ice-sheet erosion-enough to expose basement rocks-has occurred in two regions: the head of the Totten Glacier, within 150 kilometres of today's grounding line; and deep within the Sabrina Subglacial Basin, 350-550 kilometres from this grounding line. Our results, based on ICECAP aerogeophysical data, demarcate the marginal zones of two distinct quasi-stable EAIS configurations, corresponding to the 'modern-scale' ice sheet (with a marginal zone near the present ice-sheet margin) and the retreated ice sheet (with the marginal zone located far inland). The transitional region of 200-250 kilometres in width is less eroded, suggesting shorter-lived exposure to eroding conditions during repeated retreat-advance events, which are probably driven by ocean-forced instabilities. Representative ice-sheet models indicate that the global sea-level increase resulting from retreat in this sector can be up to 0.9 metres in the modern-scale configuration, and exceeds 2 metres in the retreated configuration.

Antiviral Activity and Crystal Structures of HIV-1 gp120 Antagonists.

As part of our effort to discover drugs that target HIV-1 entry, we report the antiviral activity and crystal structures of two novel inhibitors in a complex with a gp120 core. NBD-14204 showed similar antiviral activity against all the clinical isolates tested. The IC50 values were in the range of 0.24-0.9 µM with an overall mean of 0.47 ± 0.03 µM, showing slightly better activity against the clinical isolates than against the lab-adapted HIV-1HXB2 (IC50 = 0.96 ± 0.1 µM). Moreover, the antiviral activity of NBD-14208 was less consistent, showing a wider range of IC50 values (0.66-5.7 µM) with an overall mean of 3 ± 0.25 µM and better activity against subtypes B and D (Mean IC50 2.2-2.5 µM) than the A, C and Rec viruses (Mean IC50 2.9-3.9 µM). SI of NBD-14204 was about 10-fold higher than NBD-14208, making it a better lead compound for further optimization. In addition, we tested these compounds against S375Y and S375H mutants of gp120, which occurred in some clades and observed these to be sensitive to NBD-14204 and NBD-14208. These inhibitors also showed modest activity against HIV-1 reverse transcriptase. Furthermore, we determined the crystal structures of both inhibitors in complexes with gp120 cores. As expected, both NBD-14204 and NBD-14208 bind primarily within the Phe43 cavity. It is noteworthy that the electron density of the thiazole ring in both structures was poorly defined due to the flexibility of this scaffold, suggesting that these compounds maintain substantial entropy, even when bound to the Phe43 cavity.

An evaluation of the Chesapeake Bay management strategy to improve water quality in small agricultural watersheds.

Chesapeake Bay water quality has been a concern since 1970. In rural areas, agriculture is the dominant N and P source, and the voluntary application of best management practices (BMPs) is the primary management tool. Here we test the hypothesis that the current management approach of primarily voluntary, untargeted BMP implementation is insufficient to create detectable, widespread reductions in N, P, and total suspended solid (TSS) concentrations in agricultural watersheds of the Choptank basin, a tributary of Chesapeake Bay. To test this hypothesis, we assessed BMP implementation and sampled water quality on participating farms, at intermediate streams within each watershed, and at watershed outlets of four watersheds from 2013 to 2014. We also present water quality data from 2003 to 2014 at the outlets of 12 additional agricultural and one forested watershed and survey-directed interviews of farmers. By the end of 2014, large numbers of BMPs, both structural and cultural, had been implemented. Of the 16 agricultural watersheds, 50% showed significant decreases in baseflow N, 37.5% showed no changes, and 12.5% showed increasing TN. Baseflow P significantly decreased at just one watershed, increased at one, and remained stable at 14. Stormflow N was similar to baseflow, but stormflow P was 5 times higher than baseflow. These data partially support our hypothesis. Surveys suggested farmers considered themselves responsible for the quality of water leaving their farms, but out-of-pocket cost was the major impediment to further BMP adoption. We suggest that greater outreach and more financial support for farmers to implement BMPs is required to increase the types and densities of BMPs needed to achieve regional water quality goals.

Quantitative assessment of linear noise-reduction filters for spectroscopy.

Linear noise-reduction filters used in spectroscopy must strike a balance between reducing noise and preserving lineshapes, the two conflicting requirements of interest. Here, we quantify this tradeoff by capitalizing on Parseval's Theorem to cast two measures of performance, mean-square error (MSE) and noise, into reciprocal- (Fourier-) space (RS). The resulting expressions are simpler and more informative than those based in direct- (spectral-) space (DS). These results provide quantitative insight not only into the effectiveness of different linear filters, but also information as to how they can be improved. Surprisingly, the rectangular ("ideal" or "brick wall") filter is found to be nearly optimal, a consequence of eliminating distortion in low-order Fourier coefficients where the major fraction of spectral information is contained. Using the information provided by the RS version of MSE, we develop a version that is demonstrably superior to the brick-wall and also the Gauss-Hermite filter, its former nearest competitor.

Biomechanical Features of Reinforced Esophageal Hiatus Repair in a Porcine Model.

Recurrence rates in the laparoscopic repair of the hiatal hernia range from 12% to 59%. Limitation of reinforcement has been principally the risk of adverse events caused by synthetic materials. Biologic and resorbable synthetic materials are valid alternatives. This study compares the host response to all these materials after hiatal hernia repair. A total of 20 Landrace pigs, underwent laparoscopic primary hiatal hernia repair and reinforced with a polypropylene mesh (PROLENE: polypropylene [PP]), an absorbable synthetic scaffold (GOREBIO-A: polyglycolic acid [PGA]), a urinary bladder matrix scaffold, (Gentrix: urinary bladder matrix [UBM]), or without reinforcement, control group (C). Animals were survived for 3 months. Endpoints included gross morphology, biomechanical testing, and histology. Pigs in PP and PGA groups showed fibrosis at the repair site, with robust adhesions. In UBM and C groups, only mild adhesions were found. Load at failure (gr) and stiffness (gr/mm) of PP were higher than C group (PP:2103 ± 548.3 versus C:951.1 ± 372.7, P = 0.02; PP:643.3 ± 301 versus C:152.6 ± 142.7, P = 0.01). PGA and UBM values for both parameters were in between PP and C samples. However, stiffness in UBM was tended to be lower than PP group, and approached a significant difference (643.3 ± 301 versus 243 ± 122.1, P = 0.0536). In UBM group, the histology resembled native tissue. By contrast, PP and PGA groups showed mononuclear infiltrates, fibroencapsulation, necrosis, remnants of mesh, and disorganized tissue that was validated with a histologic score. In this setting, UBM scaffolds showed the most appropriate features for hiatal hernia repair, recovering the tissue properties that can help reduce the possibility of early failure and prevent complications associated with the implanted material.

Observational evidence for interhemispheric hydroxyl-radical parity.

The hydroxyl radical (OH) is a key oxidant involved in the removal of air pollutants and greenhouse gases from the atmosphere. The ratio of Northern Hemispheric to Southern Hemispheric (NH/SH) OH concentration is important for our understanding of emission estimates of atmospheric species such as nitrogen oxides and methane. It remains poorly constrained, however, with a range of estimates from 0.85 to 1.4 (refs 4, 7-10). Here we determine the NH/SH ratio of OH with the help of methyl chloroform data (a proxy for OH concentrations) and an atmospheric transport model that accurately describes interhemispheric transport and modelled emissions. We find that for the years 2004-2011 the model predicts an annual mean NH-SH gradient of methyl chloroform that is a tight linear function of the modelled NH/SH ratio in annual mean OH. We estimate a NH/SH OH ratio of 0.97 ± 0.12 during this time period by optimizing global total emissions and mean OH abundance to fit methyl chloroform data from two surface-measurement networks and aircraft campaigns. Our findings suggest that top-down emission estimates of reactive species such as nitrogen oxides in key emitting countries in the NH that are based on a NH/SH OH ratio larger than 1 may be overestimated.

Accuracy of contrast-enhanced CT and predictive factors for extracapsular spread in unknown primary head and neck squamous cell cancer.

AIM: To determine the accuracy of contrast-enhanced computed tomography (CECT) for nodal extracapsular spread (ECS) and identify predictive radiological signs and clinicopathological features for ECS in unknown-primary head and neck squamous cell cancer (UPHNSCC). MATERIALS AND METHODS: The CECT imaging of patients who underwent primary neck dissection for UPHNSCC during 2011-2015 was analysed. The largest pathological-looking node at each radiologically involved level was evaluated in consensus by two head and neck radiologists. Parameters included longest diameter, margin sharpness, haziness in adjacent fat, necrosis, and loss of fat plane with adjacent structures. Independent assessment was also made regarding the presence/absence of ECS. Findings and clinicopathological parameters were correlated with histopathology. RESULTS: Thirty-one patients with 39 neck levels had metastatic nodal involvement determined on CECT. Confirmed ECS was found at 26 levels in 23 patients. Sensitivity of radiological assessment for ECS by nodal level was 81-85% (95% confidence interval [CI]=65-93%) and specificity 46-54% (95% CI=19-81%); kappa 0.87. On univariate analysis based on the largest involved node per patient, longest diameter being ≥30 mm (p=0.007), haziness in adjacent fat (p=0.023), increasing age (p=0.006), and more advanced pathological nodal status (p=0.027) were statistically significantly associated with ECS. Haziness and increasing age were independent predictors on multivariate analysis (odds ratio [OR]=26.4 and 1.24). CONCLUSION: Expert assessment of ECS on CECT had good sensitivity with excellent interobserver agreement. A longest nodal diameter of ≥30 mm, haziness in the surrounding fat on CECT, advanced pathological nodal status, and advancing patient age were significantly associated with ECS in UPHNSCC patients, findings not previously reported.

Identification of long non-coding RNAs expressed in knee and hip osteoarthritic cartilage.

OBJECTIVE: Long intergenic non-coding RNAs (lincRNAs) are emerging as key regulators in gene expression; however, little is known about the lincRNA expression changes that occur in osteoarthritis (OA). Here we aimed to define a transcriptome of lncRNAs in OA cartilage, specifically comparing the lincRNA transcriptome of knee and hip cartilage. METHOD: RNA-seq was performed on nucleic acid extracted from hip cartilage from patients undergoing joint replacement surgery because of either OA (n = 10) or because of a neck of femur fracture (NOF; n = 6). After transcript alignment, counts were performed using Salmon and differential expression for ENSEMBL lincRNAs determined using DESeq2. Hip RNA-seq lincRNA expression was compared to a knee dataset (ArrayExpress; E-MTAB-4304). ChIP-seq data from ENCODE was used to determine whether lincRNAs were associated with promoters (plncRNA) or unidirectional enhancer-like regulatory elements (elncRNAs). RESULTS: Our analysis of the hip transcriptome identified 1692 expressed Transcripts Per Million (TPM ≥1) Ensembl lincRNAs, of which 198 were significantly (FDR ≤0.05) differentially expressed in OA vs normal (NOF) cartilage. Similar analysis of knee cartilage transcriptome identified 648 Emsembl lincRNAs with 93 significantly (FDR ≤0.05) differentially expressed in intact vs damaged cartilage. In total, 1834 lincRNAs were expressed in both hip and knee cartilage, with a highly significant correlation in expression between the two cartilages. CONCLUSION: This is the first study to use RNA-seq to map and compare the lincRNA transcriptomes of hip and knee cartilage. We propose that lincRNAs expressed selectively in cartilage, or showing differential expression in OA, will play a role in cartilage homoeostasis.